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Trojan Horse Strategy : synthesis of piperazine-based siderophores

Pauline Loupias 1,*, Alexandra Dassonville-Klimpt 1, Elodie Lohou 1, Nicolas Taudon 2 and Pascal Sonnet 1

1 AGIR, EA 4294, UFR de Pharmacie, Université de Picardie Jules Verne, 1 rue des

Louvels, 80037 Amiens, Cedex 1;

2 Unité de Toxicologie Analytique, Institut de Recherche Biomédicales des Armées,

91223 Brétigny-sur-Orge.

* Corresponding author: pauline.loupias@etud.u-picardie.fr

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Graphical Abstract

Trojan Horse Strategy : Synthesis of piperazine-based siderophores

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N H N O O

chelator chelator

N H O OH HO N O OH N OH O HO O H N O

Iron chelator : A 1,4-disubstituted piperazines MPPS0225 (A; n = 3; m=0)

n n

N H O Cl HO HO R N H

linker-ATB

N N H N N H

O NH O

chelator ATB

n n m m

chelator

NH

ATB

m

B 3,6-disubstituted dioxopiperazines

Gram-negative bacteria’s resistance such as Pseudomonas aeruginosa and the Burkholderia group to conventional antibiotics leads to therapeutic failure. Use of iron transport systems is a promising strategy to overcome resistance phenomenon. These TonB-dependent receptors, essential for the survival of microorganisms, allow specific recognition of ferric siderophore complexes to transport iron within bacteria. Bacteria express different receptors allowing them to recognize endogenous siderophores and xenosiderophores. These specific systems may allow the introduction of antibacterial agents by forming antibiotic-siderophore conjugates or toxic

• complexes. Previous work has shown that piperazine 1,4-dicatechol structures could be

recognized by P. aeruginosa strains. To further investigate this platform, we synthesized iron chelators bearing 3-hydroxypyridin-4-ones and 1,3-dihydroxypyridin-4-one ligands. At the same time, we were interested in the synthesis of a more complex 2,5-dioxopiperazine platform, part

• f the rhodotorulic acid (RA), siderophore of Rhodotorula (pFe = 21,8). A RA synthesis will be

developed as well as the corresponding 3,6-disubstituted analogs. We would like to study the influence, on the iron complexation, of the nitrogenous platform, the presence of stereogenic centers and the nature of the iron ligands. The best siderophores analogs will be highlighted through the evaluation of the siderophore-like potential as well as physicochemical studies of synthesized compounds. Keywords: Trojan Horse; siderophores; iron.

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Abstract

The resistance of bacteria to antibiotics is an emerging phenomenon and a real health problem. ESKAPE multi-drug resistant bacteria are a major problem in hospitals and especially for immunocompromised patients. We are particularly interested in Gram-negative bacteria such as Pseudomonas aeruginosa and Burkholderia group, previously classified in the genus Pseudomonas, which are resistant to antibiotics via a lack of membrane permeability or efflux. The use of bacterial iron transport systems is a promising strategy to overcome this resistance phenomenon by restoring the activity of conventional antibiotics. Iron is a micronutrient necessary for the survival of bacteria. It is essential to many biological processes such as respiration and DNA synthesis. However, the ferric iron is not very bioavailable due to its low solubility in water and sequestration in host-protein. An iron complex could be linked to a conventional antibiotic, as a Trojan Horse strategy, to restore its activity.

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Introduction

ATB Sid-Fe(III)

linker

Figure 1 : Trojan Horse Strategy

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Introduction

1.Page MGP, Dantier C, Desarbre E.. Antimicrob. Agents Chemother. 2010, 54, 2291–2302.

Siderophore : Iron source Receptor ExbD-ExbB-TonB systems External membrane Periplasmic membrane Periplasm ABC Transporter Periplasmic Proteins

Under iron limited conditions, many bacteria then synthesize molecules of low molecular weight called siderophores able to chelate the surrounding iron. These siderophore-Fe (III) complexes are then recognized by specific receptors responsible for bringing the essential iron element to the bacteria. Interestingly, the bacteria is able to recognize its endogenous siderophores but also siderophores synthesized by other bacteria or synthetic siderophores1.

Figure 2 : Siderophore pathway

In particular, P. aeruginosa and B. pseudomallei both possess FptA receptors for the recognition

• f pyocheline, the endogenous siderophore of P. aeruginosa. These two types of bacteria are also

capable of recognizing catecholate siderophores such as cepaciachelin for B. pseudomallei2, and enterobactin for P. aeruginosa3 (Fig. 3). These specific systems may allow the introduction of antibacterial agents by forming antibiotic-siderophore conjugates or toxic complexes such as gallium complexes, in the bacteria.

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OH OH N H O NH O OH OH H N O NH2

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cepaciachelin HN O O O O O OH OH O N H NH O OH OH O O HO HO enterobactin

Figure 3 : Siderophores recognized by B. pseudomallei and

• P. aeruginosa

2.Butt AT.; Thomas MS. Frontliers in Cellular and Infection Microbiology , 2017. 3.J.B. Neilands, T.J. Erickso, W.H. Rastetter. Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12. The Journal of Biological Chemistry. 1981, 256(8), 3831-3832.

Introduction

Results and discussion

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Previous work in the laboratory has shown that piperazine 1,4-dicatechol structures (MPPS0225) can be recognized by strains of Pseudomonas aeruginosa. Bacterial growth has been observed as a function of the ratio MPPS0225/Fe(III) in Medium Minimum Succinate. With a ratio MPPS0225/Fe(III) equal to 1,5, bacterial growth has been observed for DSM1117 strains, producing pyoverdine and pyocheline (Fig. 4).

Figure 4: Representation of bacterial growth (in Medium Minimum Succinate) as a function of the ratio MPPS0225/Fe(III) for DSM1117 strains

MPPS0225/Fe(III) = 1,5

MPPS0225 + FeCl3 2,4 µmol/mL MPPS0225 only FeCl3 2,4 µmol/mL [MPPS0225] Ratio [MPPS0225]/[Fe(III)]

N N H N O OH OH HO OH N H O MPPS0225

Results and discussion

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The same observation was found for PAD07 strains, which don’t produce pyoverdine and

• pyocheline. We can assume that our compound is recognized by the bacteria (Fig. 5) and there is

a competition between MPPS0225 and the endogenous siderophores of P. aeruginosa (Fig. 4).

Figure 5: Representation of bacterial growth (in Medium Minimum Succinate) as a function of the ratio MPPS0225/Fe(III) for PAD07 strains

[MPPS0225] MPPS0225 + FeCl3 2,4 µmol/mL MPPS0225 only Ratio [MPPS0225]/[Fe(III)]

Ratio MPPS0225/Fe(III) = 1,5

N N H N O OH OH HO OH N H O MPPS0225

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Figure 6 illustrates the determination of the stoichiometry of the complex Fe(III)-MPPS0225 at pH = 5.70 using the JOB plot method. We can see the evolution of the ligand-to-metal charge- transfer (LMCT) absorption at 550 nm up to a 0.58 molar fraction of ligand, corresponding to an iron(III)/MPPS0225 2:3 stoichiometry. At physiological pH this stoichiometry is kept.

Figure 6 : Determination of the stoichiometry of the complex MPPS0225-iron(III)

Results and discussion

To complete these results, MPPS0225 has been synthesized for physicochemicals studies like the pFe measurement. In order to further investigate this piperazine platform, we have synthesized iron chelators bearing 3-hydroxypyridin-4-ones ligands, bioisosteres of catechol groups. The bidentate ligands precursors 3 and 4 were synthesized with para-methoxybenzyl (PMB) as a protective group in order to be coupled with the 1,4-bis(3-aminopropyl)piperazine 1.

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N N

chelator chelator

3 3

N N

H2N NH2

3 3

+ bidentate ligands precursors

1

Figure 7 : Retrosynthesis of 1,4 disubstituted piperazines

Results and discussion

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Figure 8: Synthesis of the bidendate ligands precursors

O HO OH OH O PMBO OPMB OPMB K2CO3 TBAI PMB-Cl acetone 70% NaOH dioxane quant. O HO OPMB OPMB 2 3 O OH O O OPMB O K2CO3 PMB-Cl DMF 65% 4

The bidentate ligands precursors 3 and 4 were synthesized with para-methoxybenzyl (PMB) as a protective group with a 70% and 65% yield.

Results and discussion

As mentioned before, 3 and 4 were synthesized to be coupled with the 1,4-bis(3- aminopropyl)piperazine 1. The common hydrogenation step was optimized and carried out using a H-cube system generating hydrogen by electrolysis of water. MPPS0225 and 7 were, respectively, obtained with a 35% and 30% yield.

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Figure 9: Synthesis of MPPS0225 and the hydroxypyridinone analog

N N H N O OH OH HO OH N H O MPPS0225 N N N N O O OH OH 7 NH2 N N H2N

3 3

O OPMB O NaOH EtOH/H2O 40% 4 N N N N O O OPMB OPMB 6 O HO OPMB OPMB 3 EDCI HOBt DCM 45% N N H N O OPMB OPMB PMBO OPMB N H O 5 H2,Pd(OH)2 /C MeOH 90% H2,Pd(OH)2 /C MeOH 90% 1

Results and discussion

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At the same time, we were interested in the synthesis of a more complex 2,5-dioxopiperazine platform, with analogs of the rhodotorulic acid (RA), a siderophore recognized by Gram-negative bacteria, showing an interesting iron affinity (pFe = 21,8). Different ways to obtain RA have been described4 but the one we propose should be more efficient. Indeed, it is a convergent strategy which could lead to the synthesis of analogs thanks to asymmetric alkylations of a key intermediate carrying two cleavable chiral inductors (13). RA should be obtained in ten steps from the commercially available (S)-phenylglycinol (Fig. 10).

Ph NH2 OH H N O O N H N N

3 3

OH OH O O RA (S)-phenylglycinol N Ph OH O O N Ph HO 13 N Ph OTBDMS O O N Ph TBDMSO 11

Figure 10: Retrosynthesis of rhodotorulic acid (RA)

4.a) Y. Isowa, T. Takashima, M. Ohmori, H. Kurita, M. Sato, K. Mori. Bulletin of the chemical society of Japan, 1972, 45, 1467-1471. b) T. Fujii, Y. Hatanaka. Tetrahedron. 1973, 29, 3825-3831. c) J. Wiedmer, W. Keller-Schierlein. Helevtica Chimica Acat. 1969, 52(2), 388-396. d) M. Nakao, S. Fukayama, S. Kitaike, S. Sano. Heterocyles, 2015, 90(2), 1309-1316.

Results and discussion

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Figure 11: Synthesis of rhodotorulic acid

Ph NH2 N Ph OTBDM S Cl O O OMe N Ph OTBDMS O O N Ph TBDMSO OH N Ph OH O O N Ph HO NH Ph OTBDMS O OMe N Ph OH O O N Ph HO O OMe Et3N THF, rt 75% Cl O Et3N Cl Ph NH2 OTBDMS Ph NH2 OTBDMS TBDMSCl imidazole DCM, rt quant. DCM, rt 60% MeOH, reflux 60% TBAF DCM, rt 70% 12 8 9 10 11 N Ph OH O O N Ph HO OH HO

3 3

2 steps N Ph OH O O N Ph HO N N

3 3

OBn Ac Ac OBn H N O O N H N N

3 3

OH OH O O RA 2 steps Br 8 13 Br LiHMDS HMPA THF -10 to -78 °C 20%

At this time, we synthesized the dioxopiperazine 13 in 6 steps from the (S)-phenylglycinol. This

• ne was protected with TBDMSCl to afford 8 which was alkylated in presence of methyl 2-

bromoacetate to afford the secondary amine 9. This product was engaged in an amidification reaction to give 10 which was cyclized in presence of 8 to dioxopiperazine 11. Then, the TBDMS protective group was cleaved with TBAF to afford 12. Thanks to the (S)-phenylglycinol chiral inductors, the cis-dialkylation product 13 was obtained in a 4% global yield.

Results and discussion

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We managed to synthesize two 1,4-disubstituted piperazines, MPPS0225 and 7 in, respectively 3 and 4 steps with 35% and 30% yields. The RA synthesis has been carried out until the formation

• f a key intermediate 13 in 6 steps with a 4% yield.

As for the dicatechol siderophore analogs, MPPS0225, we will first study the bacterial recognition

• f synthesized chelators 7 and RA by measuring the siderophore-like effect and evaluate their

ability to complex iron by physicochemical methods. The siderophores having shown the best capacity for recognition and complexation of iron will then be linked to an antibiotic, via a cleavable spacer or not depending on whether the target of the antibiotic is periplasmic or

• cytoplasmic. The antibacterial activity of these conjugates (or complexes) will then be evaluated

and compared to the antibiotic alone to estimate the vectorization capacity of the siderophore. In parallel, cytotoxicity studies will also be conducted on these compounds to determinate their therapeutic index.

Conclusions

We would like to thank the DGA and the Haut de France region for their financial support.

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Acknowledgments