Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
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Synthesis and antibacterial activity of catecholate–ciprofloxacin conjugates Sylvain Fardeau a, Alexandra Dassonville-Klimpt a, Nicolas Audic b, André Sasaki a,b, Marine Pillon a, Emmanuel Baudrin a, Catherine Mullié a, Pascal Sonnet a,⇑ a b
Laboratoire de Glycochimie, des Antimicrobiens, et des Agroressources, CNRS FRE 3517, UFR de Pharmacie, Université de Picardie Jules Verne, F-80037 Amiens, France Pharmamens, 9 avenue Théophile Gautier, 75016 Paris, France
a r t i c l e
i n f o
Article history: Received 21 February 2014 Revised 9 May 2014 Accepted 29 May 2014 Available online xxxx Keywords: Antibacterial Pseudomonas Siderophore Trojan horse strategy Iron chelator–ciprofloxacin conjugate
a b s t r a c t The development of an efficient route to obtain artificial siderophore–antibiotic conjugates active against Gram-negative bacteria is crucial. Herein, a practical access to triscatecholate enterobactin analogues linked to the ciprofloxacin along with their antibacterial evaluation are described. Two series of conjugates were obtained with and without a piperazine linker which is known to improve the pharmacokinetics profile of a drug. A monocatecholate–ciprofloxacin conjugate was also synthesized and evaluated. The antibacterial activities against Pseudomonas aeruginosa for some conjugates are related to the iron concentration in the culture medium and seem to depend on the bacterial iron uptake systems. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Today, multi-antibiotic resistance among pathogenic bacteria is evolving and expanding to the point of that it becomes a global public health problem. Pseudomonas aeruginosa is an opportunistic pathogen, particularly infecting humans with compromised natural defenses. It is also well known as a nosocomial pathogen. In intensive care units, P. aeruginosa is responsible for up to 10% of all hospital-acquired infections and ranks among the top five organisms causing nosocomial infections1 as well as chronic lung infections in cystic fibrosis patients.2,3 The low permeability of its outer membrane added to the propensity of this bacterium to form biofilms further increase its immunity to external attacks, thus leading to a decreased activity of certain classes of antibiotics, such as aminoglycosides, fluoroquinolones or b-lactams. Moreover, this bacterium can become resistant through multiple mechanisms (change in membrane permeability, decrease in intracellular drug concentration, antibiotic deactivation) and achieve clinical resistance. P. aeruginosa is indeed very difficult to eradicate because of its intrinsic resistance coupled with an adaptive one to a wide variety of classical antibiotics. Since few new classes of antibiotics have emerged in the last 20 years, restoring classical antibiotic
⇑ Corresponding author. Tel.: +33 (0) 322827494; fax: +33 (0) 322827469. E-mail address:
[email protected] (P. Sonnet).
activities is an attractive strategy to fight bacterial infectious diseases. Combating the reduction of intracellular antibiotic concentration in bacteria by the development of efficient drug delivery processes is a promising approach.4 Pseudomonas, like most living organisms, needs iron for its growth and survival. Indeed, iron is involved in many key metabolic processes such as the Krebs cycle, oxidative phosphorylation or electron transport.5 Soluble Fe(II) is oxidized to Fe(III) which forms ferric hydroxo complexes and precipitates inducing a low free Fe(III) concentration between 1018–1024 mol/L, in the bacterial environment. This low free Fe(III) concentration is below the required level to sustain bacterial life, which was evaluated as at least 106 mol/L.6 To survive in iron-deficient media, bacteria can synthesize low molecular weight molecules called siderophores which possess a high affinity for ferric iron and are able to scavenge bound iron from their environment due to their superior binding strength.7 Among the siderophores produced by P. aeruginosa are pyoverdines (primary siderophores) and pyochelin (secondary siderophore), whose specific receptors are located on the external membrane of the bacterium (Fig. 1). To facilitate the penetration of quinolones into the bacterial cells and improve the access of the antibiotics to their target, Abdallah, Mislin and co-workers8 have synthesized pyoverdine or pyochelin–quinolone conjugates. Their results have shown that these natural siderophores facilitate the transport of antibiotics into bacterial cells generating the corresponding siderophores.
http://dx.doi.org/10.1016/j.bmc.2014.05.067 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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S. Fardeau et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx L-Thr8 NH2 + H 2N
H
N H
+
HN O O
N H
O D-Ser3
NH
H N
H
N OH O H OH
O
N H
OH
OH
O
O H N H H
O
OH NH OH
N H H
HO O
RO
O
OH
H N
OH
H N
COOH COOH O O
RO
O
S H
Pyochelin
O
NH
H
H
RO
O
N H
N
L-OHOrn6
OH
O
OH
S
N
COOH H
N
L-Thr7
O O
O
N H O
O
HN H
O
HO
H
Type I Pyoverdine
OH
HN HO
OH H H N
O
NH
O
D-Ser1 HO
O
L-OHOrn4 L-Lys5
L-Arg2
NH
RO O
O
NH
OH
H N
H
S
S
R = H, Ac
O
N
RO
OH
N
N O
H 2N
COOH
RO
Enterobactin
GR69153
Triscatecholate siderophore
Figure 1. Molecular structures of natural and synthetic siderophores.
Thus, the iron uptake pathways could be used in a Trojan horse strategy to counter the low outer membrane permeability of P. aeruginosa. However, this strategy is of limited practical application since each P. aeruginosa strain produces its own pyoverdine along with its specific transporter. Moreover, very few cross-feedings were observed between these different pyoverdines and their corresponding transporters. Furthermore, the pyoverdine synthesis is difficult and limits its utilization.9 However, P. aeruginosa can acquire iron through the uptake of xenosiderophores.4 For example, P. aeruginosa is able to use enterobactin, a siderophore produced by Escherichia coli.10 Recently, Miller et al.11 have synthesized a simplified enterobactin-like siderophore since enterobactin is a triscatecholate siderophore that possesses no site suitable for drug conjugation (Fig. 1). The corresponding conjugates with ampicillin and amoxicillin, compared to the parent drugs, exhibited significantly enhanced in vitro antibacterial activities against Gram-negative species, especially against P. aeruginosa. The authors have shown that these catecholate conjugates use the
bacterial iron uptake systems to penetrate in the bacterium. Many others siderophore-antibiotic conjugates were described in the literature and the catechol or hydroxamate siderophore analogues are the most efficient to combat P. aeruginosa [4andrefcited]. Indeed, it was shown that only one catechol group can be sufficient to lead to very active conjugates.12 Most easy to synthesize, they are known to use the outer membrane proteins Cir and Fiu to enter into E. coli13 and PfeA and Pir A into P. aeruginosa.14 For example, the monocatecholate conjugates GR69153, shown in Figure 1, possess an in vitro activity equivalent to or exceeding those of ceftazidime and ceftriaxone against Gram-negative bacilli12d and is transported across the active iron transport system.12e With the aim of developing more potent and better targeted antibiotics by following the same Trojan horse strategy, we report below the synthesis and the antibacterial activity of new series of catecholate–ciprofloxacin conjugates 1–3 against P. aeruginosa (Fig. 2). Triscatecholate–ciprofloxacin conjugates 1 and 2 were assembled with a metabolizable ester linker to optimize the
O F
O
COOH
HO
F H N
HO
N
O
O
O
O
N
N HO
n = 1, 2 ou 3 NH
HO O HO
NH
N
N
n
HO
COOH
N
OH O
3
1a (n=1) 1b (n=2) 1c (n=3)
O
HO HO
HO
F
H N
COOH
O
O
O
N N
O HO
O
N
N
N
NH HO O
NH
2
HO HO
Figure 2. Molecular structures of catecholate-ciprofloxacin conjugates 1–3.
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S. Fardeau et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
interaction of ciprofloxacin with its cytoplasmic target. Indeed, the DNA gyrase is more sensitive to steric hindrance than periplasmic targets like transpeptidases.4,15 An additional piperazine has been incorporated into the structure of conjugate 2 to study its influence on the activity and the pharmacokinetic properties. Finally, the importance of the spacer and of a single catechol group on the bacterial recognition and the activity was shown by the study of monocatecholate-ciprofloxacin 3.
Finally, the best result was obtained by combination of triamine 5 with benzoyl fluoride 6 previously prepared by activation of benzoic acid 12 with cyanuric fluoride. Thus, triamide 13 was afforded in 83% yield. The removal of the silyl group was achieved with tetrabutylammonium fluoride to provide alcohol 4 in 95% yield. Three steps were necessary to prepare triscatecholate–ciprofloxacin conjugates 1a–c (Scheme 3). First, the preparation of chloroesters 14a–c was carried out successfully by using the suitable acyl chloride compound. Second, the treatment of these compounds 14a–c with ciprofloxacin by refluxing in acetonitrile in the presence of potassium carbonate gave the protected conjugates 15a–c with moderate yields (9–31%). And finally, the benzyl protective groups were removed by hydrogenolysis over Pearlman’s catalyst to obtain the fully deprotected siderophore analogues 1a–c in excellent yield. Thus, compounds 1a–c were synthesized in eight steps with an overall yield of 2–4% according to the length of the arm spacer (n = 1–3). The synthesis of compound 2 that has an additional piperazine, required five steps starting from tripod 4 as shown in Scheme 4. A direct oxidation of alcohol 4 to acid 16 was investigated using the Jone’s reagent or potassium permanganate. Unfortunately, these reactions proceeded poorly, giving the corresponding carboxylic acid along with several decomposition products. The best result has been obtained with a two steps synthesis. Alcohol 4 was initially treated with sodium hypochlorite in the presence of TEMPO as catalyst and the resulting aldehyde was transformed into acid 16 in 90% yield using a mixture of sodium chlorite and sulfamic acid. The access to amide 17 from 16 turned to be difficult. The preparation of the N-hydroxysuccinimide (NHS) active ester of 16 was tried by treating 16 and NHS with dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) but no peptide was isolated after addition of 2-(piperazin-1-yl)
2. Results and discussion 2.1. Chemistry As shown in Scheme 1, the access to the compounds 1 and 2 required the synthesis of their common alcohol intermediate 4 that was prepared by a peptide coupling between triamine 5 and three molecules of benzoyl fluoride 6. Compound 5 was obtained in a five step synthesis according to the previously described procedure by Imbert et al.16 whereas benzoyl fluoride 6 was synthesized by activation of 2,3-dihydroxybenzoic acid 7 with cyanuric fluoride. The synthetic pathway to obtain common alcohol intermediate 4 is depicted in Scheme 2. Esterification of 2,3-dihydroxybenzoic acid 7 with methanol in the presence of a catalytic amount of sulfuric acid, followed by benzylation of the hydroxyl groups gave compound 11 in quantitative yield. Saponification of ester 11 with lithium hydroxide led to the corresponding acid 12 in 99% yield. Two classical ways were investigated to connect three benzoic acids 12 with protected triamine 5: (i) activation of acid 12 in situ with peptide coupling agents and (ii) preparation of the corresponding acid halide. No coupled product 13 was isolated with the first approach. Triamide 13 was obtained in 25% yield when triamine 5 was treated with the corresponding acid chloride of 12.
O F
COOH
HO H N
HO
O
O
H
N
N
N
O
9
n
O
8
n = 1, 2 ou 3
HO NH HO O
O
N
H 2N 10
1a (n=1) 1b (n=2) 1c (n=3)
NH
BnO
HO
H N
BnO
HO
TBDMSO COF
O
OH
OBn
O
+
BnO O
NH 2
H 2N
NH BnO
NH
4
H 2N
5
OBn 6
BnO BnO COOH OH O
HO
F
H N
HO
COOH
O
O
O
N N
O HO
O
N
OH 7
N
N
NH HO O
NH
2
HO HO
Scheme 1. Synthesis of triscatecholate–ciprofloxacin conjugates 1–2.
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S. Fardeau et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx COOH OH
COOMe OBn
(a), (b) 100%
OH
OBn
7
COOH OBn
(c) 99 %
11
H N
(d)
O BnO
H 2N
(e) OBn
NH 2
H 2N
10
NH
OBn +
8
BnO O
NH
83%
13, R = TBDMS (f), 95%
BnO
6
5
H 2N
OR
O
COF
ref 11
9
H N
BnO
12 TBDMSO
O
BnO
84 %
OBn
4, R = H
BnO
Scheme 2. Preparation of alcohol 4. Reagents and conditions: (a) H2SO4, MeOH, reflux, 4 h; (b) BnBr, K2CO3, acetone, reflux, 12 h; (c) LiOH, THF, reflux, 24 h; (d) cyanuric fluoride, Et3N, CH2Cl2, 20 °C, 4 h; (e) Et3N, CH2Cl2, 20 °C, 14 h; (f) TBAF 1 N, THF, reflux, 10 h.
O F BnO
BnO
H N
BnO
BnO
O
O
O
OH
O (a)
NH BnO O BnO
85-95%
NH
BnO O
4
BnO
NH RO O
14a-c BnO BnO
N
N n
O 9-31%
NH
O
RO
(b)
NH
N
O
O Cl n
BnO
H N
RO
O
O
BnO
COOH
RO
H N
RO
NH
15a-c, R = Bn (c) 97-99%
1a-c, R = H
RO
Scheme 3. Synthesis of triscatecholate–ciprofloxacin conjugates 1a–c. Reagents and conditions: (a) ClCO(CH2)nCl, Et3N, CH2Cl2, 20 °C, 8 h (n = 1, 2 or 3); (b) ciprofloxacin, K2CO3, CH3CN, reflux, 3 days; (c) H2, Pd(OH)2/C, MeOH, 20 °C, 4 days.
ethanol. Then, the formation of the oxybenzotriazole ester of 16 was carried out using HOBt/EDCI without success. Finally, the peptide coupling reagent benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate or PyBOP was used in the presence of triethylamine to afford amide 17 in 49% yield. The clivable linker was formed by esterification of 17 with the 2-chloroacetyl chloride. The substitution of the resulting chloroester 18 with ciprofloxacin led to the protected conjugate 19 with poor yield (31%). Then, the benzyl groups were cleanly removed to afford triscatecholateciprofloxacin conjugate 2 in good yield. This compound 2 was synthesized in ten steps with overall yield of 6% from 2,3-dihydroxybenzoic acid 7. To have references other than native ciprofloxacin for the biological studies, compounds 20 and 21 were synthesized in two steps from ciprofloxacin in 39% yield (Scheme 5) 17. The synthesis of monocatecholate–ciprofloxacin conjugate 3 used the same starting product and requires three steps. After the protection of the ciprofloxacin acid function, the corresponding protected ciprofloxacin 22 was successfully coupled with the previously described benzoyl fluoride 6 to afford the corresponding amide 23. Finally, after the cleavage of the benzyl groups, the monocatecholate-ciprofloxacin conjugate 3 was obtained in 76% overall yield. 2.2. Antibacterial activity P. aeruginosa represents one of the major challenges in the treatment of hospital-acquired infections. Fluoroquinolones such as ciprofloxacin remain as one of the potentially useful antibiotic classes in the treatment of P. aeruginosa nosocomial infections. Unfortunately, P. aeruginosa has developed resistance to fluoroquinolones by several mechanisms such as (i) mutations of their targets (topoisomerases) in bacteria, (ii) overexpression of naturally occurring efflux-pumps, especially those belong to the ResistanceNodulation-Division (RND) family.18 Catecholate–ciprofloxacin conjugates 1–3 represent an interesting possibility to restore a high
antibiotic concentration within the bacterial cell, mandatory for an optimal antibacterial activity, through their specific recognition by the active iron uptake pathways. The conjugates 1–3, 20–21 and ciprofloxacin as a control were tested for their antibacterial activity against both a P. aeruginosa susceptible strain (DSM 1117) and a resistant strain (AM 85), as shown in Table 1. The influence of the iron concentration on the antibacterial activity was evaluated by conducting assays in an iron-rich medium (Mueller Hinton or MH) as well as in an irondeficient medium (Succinate Minimum Medium or SMM) with and without the addition of 1 lM FeCl3. In the iron-rich medium, conjugates 1b, 2 and 3 were found to be inactive at concentrations up to 128 lg/mL for the resistant strain. The triscatecholate conjugates 1a and 1c showed a moderate activity against the susceptible strain, with MICs of 8 and 64 lg/mL, respectively (Table 1). However, in both cases, this activity was lower than that of the unconjugated ciprofloxacin. This could be related to the low solubility of the conjugates in aqueous media and/or the absence of hydrolysis of the ester linker in the extracellular medium.8c Unfortunately, the latter hypothesis could not be proven as a classical LC–MS analysis failed to identify either the native molecules or the free ciprofloxacin moiety of our compounds in the culture medium. The ciprofloxacin derivative acid 21 displayed an antimicrobial activity similar to that of the unmodified ciprofloxacin, showing that the functionalization by a carboxylic acid to subsequently graft catechols did not affect the biological activity of ciprofloxacin. Consequently, it is possible to obtain antibacterial active ciprofloxacin derivatives from hydrolysis by esterase of triscatecholate conjugates 1–2. However, when the carboxylic function was esterified, as in compound 20, the antimicrobial activity significantly decreased, reaching the same MIC as the one witnessed for compound 1a (Table 1). We can suppose that the low antibacterial activity of 1–2 and 20 could be due to the fact that the ester linker was not or partially hydrolyzed in the extracellular media. The best antibacterial activity was observed
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BnO
BnO
BnO
H N
BnO
H N
BnO
O O NH O
NH
N
N H O
16
BnO
N
NH 17
BnO
BnO
O
RO H N
BnO O
H N
RO O
BnO
O
N N
RO
NH
O
N O
BnO
19, R = Bn
RO
O
18
N
NH
31% NH
N N
O
RO
(e)
COOH
O
O
N
N H O
F
O
OBn O
OH
BnO
BnO
(d)
BnO
NH
O 4
OBn O
49%
BnO
BnO
72%
O (c)
90%
BnO
O
BnO
NH
H N
BnO OH
O
(a), (b)
BnO BnO
O
O
OH
(f), 99%
Cl
2, R = H
RO
BnO
Scheme 4. Synthesis of triscatecholate–ciprofloxacin conjugate 2. Reagents and conditions: (a) NaClO 2.2 N, KBr, TEMPO, TBAB, CH2Cl2/H2O (1:1, v/v), 0 °C, 2 h; (b) NaClO2, NH2SO3H, THF/H2O (1:1, v/v), 20 °C, 8 h; (c) 2-(piperazin-1-yl)ethanol, PyBOP, Et3N, DMF, 20 °C, 8 h; (d) ClCOCH2Cl, Et3N, CH2Cl2, 20 °C, 8 h; (e) ciprofloxacin, K2CO3, CH3CN, reflux, 3 days; (f) H2, Pd(OH)2/C, MeOH, 20 °C, 4 days.
O
O
O
F
OH
N
46% MeOOC
HN
O F
OH
(a)
N
N
O
F
OH
(b)
N
N
84%
N
HOOC
N
N
20
ciprofloxacin
O
21
(c) 86% O
O
F
BnO
F
CO2 Bn
F
OBn O
CO2 Bn
(d)
+ N
N
N
91%
HN
6
OBn O
22
N
N
BnO
23
O CO2 H
F (e) 98%
N
N
N
HO OH
3
O
Scheme 5. Synthesis of ciprofloxacin derivatives 20 and 21 and monocatecholate–ciprofloxacin 3. Reagents and conditions: (a) BrCH2COOMe, Et3N, KI, DMF, 90 °C, 5 h; (b) H2SO4, CH3COOH/H2O (1.5:1, v/v), 150 °C, 3 h; (c) (i) Boc2O, Et3N, MeOH, 20 °C, 4 h, (ii) BnBr, Et3N, CH3CN, reflux, 14 h, (iii) HCl 4 N, THF, 20 °C, 10 h, (iv) NaHCO3; (d) Et3N, CH2Cl2, 20 °C, 6 h,; (e) H2, Pd(OH)2/C, MeOH, 20 °C, 4 days.
Table 1 Minimum inhibitory concentrations (lg/mL) of catecholate–ciprofloxacin conjugates 1–3 Compound 1a
1b
1c
2
3
20
21
Ciprofloxacin
P. aeruginosa DSM 1117 Mueller–Hinton Succinate minimum medium Succinate minimum medium + FeCl3 (1 lM)
8 8 4
>128 >128 64
64 64 32
>128 >128 >128
>128 32 16
8 16 16
0.25 0.125 0.125
0.25 0.25 0.25
P. aeruginosa AM 85 Mueller–Hinton Succinate minimum medium Succinate minimum medium + FeCl3 (1 lM)
>128 >128 >128
>128 >128 >128
>128 >128 >128
>128 >128 >128
>128 64 32
>128 >128 >128
16 16 16
16 16 32
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S. Fardeau et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
with this triscatecholate 1a in which the linker between the ciprofloxacin moiety and the metabolizable ester is the shortest. By taking into account the molecular weight difference between 1a and ciprofloxacin, the concentration of ciprofloxacin released in the medium by the cleavage of the spacer arm of 1a would be closed to 0.5 lg/mL, against the MIC of 0.25 lg/mL witnessed for the unconjugated ciprofloxacin. The introduction of a piperazine linker (conjugate 2) did not provide any gain in biological activity compared to 1. Moreover, none of the conjugates showed any activity against the resistant strain AM 85 in these iron-rich conditions. In iron-deficient culture conditions (SMM), conjugates 1a–c and 2 showed an activity against P. aeruginosa DSM 1117 similar to that observed in iron-rich conditions. Interestingly, the addition of 1 lM FeCl3 to the SMM enhanced the activity of compounds 1a and 1c by two fold and enabled the determination of a MIC of 64 lg/mL for compound 1b, which was inactive at up to 128 lg/ mL in iron-rich culture conditions. The addition of 1 lM FeCl3 in SMM should support the formation of 1:1 complexes between iron and triscatecholates 1a–c and would explain the difference in activity. Indeed, this 1:1 stoichiometry is favorized for this kind of triscatecholate, as seen for enterobactin or its synthetic triscatechol analogs CaCAM or TRENCAM, under the pH conditions used in our culture medium (7 < pH < 7.3).16 This gain in activity under iron-rich conditions could be explained by the vectorization of ciprofloxacin by conjugate–iron complexes 1 through their uptake by the siderophore transporters of P. aeruginosa. Indeed, it was not witnessed for compound 20, in which the catechol iron-chelating does not appear (Table 1). Unfortunately, conjugates 1 and 2 were inactive against the AM 85 strain. In SMM, conjugate 3 became active against both P. aeruginosa strains, with a MIC of 32 lg/mL. On the resistant strain, conjugate 3 had a MIC close to that of the unconjugated ciprofloxacin. In compound 3, the artificial catecholate siderophore and ciprofloxacin were connected by a stable linker. The molecule was there presumably more stable than the corresponding metabolizable ester linker in conjugates 1–2. When FeCl3 (1 lM) was added to SMM, both conjugate 3 and ciprofloxacin showed a similar antibacterial activity against AM 85. This result could be attributed to a more effective transport of the corresponding conjugate-Fe complexes through iron uptake pathways.
mixed H2O/DMSO solvent mixture (with a 0.2 mole fraction of DMSO). Note that the measured pH values in this medium cannot be directly compared to that in pure water media due to a decreased permittivity when DMSO is added. To demonstrate the interaction of ciprofloxacin with iron, we followed the titration of a ciprofloxacin/iron 1:3 mixture using UV–visible spectrophotometry (Fig. 3). Starting at a pH = 3.3, we observe a band around 450 nm which can be ascribed to the ligand to metal charge transfert (LMCT) band of a complex between ciprofloxacin and iron(III). As the pH increases, we can see an evolution of the spectra with the appearance of an isosbestic point at 442 nm characteristic of an equilibrium between two different Fe/CIP complexes (Fig. 3a). Thus, the LMCT band shifts from 450 nm to about 430 nm. This latter band was previously reported as related to the Fe(CIP)3 species.20 As the pH is further increased, the band intensity decreases towards a complete absence of absorption above 400 nm at pH = 11 (Fig. 3b). A full description of the equilibria taking place within this system is beyond the scope of this paper and will be reported later. The important facts here are that we can characterize the formation of an interaction between iron(III) and the ciprofloxacin through the examination of absorption bands around 430–450 nm. Keeping this in mind, we examined the behavior of compound 3. This latter could interact with iron(III) both through the catecholate and the ciprofloxacin moieties. As these two chelating units
0.6
(a) 0.5
Absorbance
6
0.4 0.3 0.2 pH= 3.3 0.1 0
2.3. Hemolytic activity
2.4. Complexation studies While the tris-catecholates ligands complex easily iron under neutral pH conditions, it could be more ambiguous for compound 3. Furthermore, ciprofloxacin by itself can interact with iron(III) though the formation of 1:1,19 1:2 and 1:320 complexes. In that case, the complexation occurs as a bidentate ligand through the keto and carboxylate functions.21 It was thus important for us to check if the catechol moiety played a role in the iron(III) complexation under the tested conditions, through a comparison of the behavior of ciprofloxacin and conjugate 3. To ensure a good dissolution of both compounds, these experiments were realized in a
400
440
480
520
560
Wavelength (nm) 1
(b) 0.8
Absorbance
To provide a first assessment of the potential toxicity of our products, an evaluation of their hemolytic activity was carried out. This activity was below 10% of that of the saponin positive control for compounds 1a to c, as well as ciprofloxacin, compounds 3 and 20 at concentrations up to 384 lg/mL. The compounds 2 and 21 displayed a hemolytic activity close to 40% but only for the highest tested concentration (384 lg/mL), well above MIC values for the P. aeruginosa susceptible strain for 21. Therefore, a direct toxicity on the cell membrane does not seem to be generated at useful concentrations for the more active compounds.
pH= 6.8
0.6
0.4
pH= 6.8
0.2
0
pH= 11.0 400
440
480
520
Wavelength (nm) Figure 3. Evolution of the UV–visible spectra of a 1:3 solution of iron(III) and ciprofloxacin as a function of pH in a H2O/DMSO medium (x = 0.2 DMSO) at 25 °C (a) 3.3 < pH < 6.8 and (b) 6.8 < pH < 11.
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are separated by a piperazine ring, we can identify with which part of the molecule the complexation occurs by looking at their independent LMCT bands. At pH = 3, the spectrum of compound 3 presents two absorption domains within the 360–800 nm range: at about 450 nm the interaction of iron with the fluoroquinolone and at 680 nm the LMCT band related to the catechol22 (Fig. 4). This means that both chelating units are used for the complexation. However, as soon pH increases, we notice the decrease of the intensity of the two bands concomitantly with the appearance of a new band at 625 nm. During this process, three isosbestic points (423 nm, 510 nm and 703 nm) are evidenced. At pH = 4, the band at 680 nm is solely observed. Keeping in mind that at this pH, normally ciprofloxacin iron interaction should lead to the band around 430–450 nm, it means that above pH = 4 only the catechol is used for the complexation of iron. From these results, we can propose that there is an evolution from a mixed iron ciprofloxacin/catechol complex towards a bis-catechol one. Such behavior does not come at of the blue since the pFe values of the ligands depend on the pH value. To illustrate this, we plotted
0.9
Absorbance
0.8 0.7 0.6 0.5 0.4 0.3 0.2 400
450
500
550
600
650
700
750
800
Wavelength (nm) Figure 4. Evolution of the UV–visible spectra of a 1:3 solution of iron(III) and compound 3 as a function of pH in a H2O/DMSO medium (x = 0.2 DMSO) at 25 °C, 3.0 < pH < 4.1.
45 40
(e)
35 (b)
pFe
30 25
(c)
20 (a)
(d)
10 5
pFe versus pH for ciprofloxacin and three different catechol using thermodynamic date from the lit.19,22–24 (Fig. 5). At low pH, the chelation is thus more likely to occur through ciprofloxacin rather than through the catecholate. However, whatever the catechol nature, there is a pH value above which the complexation by this group is preferred to ciprofloxacin. This is indeed what we observed for compound 3. We have thus unambiguously demonstrated that at physiological pH (well above pH = 4 in H2O/DMSO) iron(III) is complexed by compound 3 and that it occurs solely through the catechol moiety. Above pH = 4 (not shown here), the further shift of the LMCT band towards lower wavelength values can be consistent with the formation of the tris-catecholate iron(III) complex according to previous studies of different iron(III) catechol systems.23 Moreover, as illustrated by curve (e) on Figure 5, the pFe value of triscatecholates (we took TRENCAM as a ligand closed to the chelating moiety of compounds 1a–c) is much higher at physiological pH, namely pFe = 27.8, than ciprofloxacin and thus at pH = 7.4 the complexation is realized only through the formation of a 1:1 iron(III):triscatecholate complex.24 3. Conclusion
1
15
7
4
6
8
10
12
pH (in pure water) Figure 5. Evolution of pFe versus pH for (a) ciprofloxacin, (b) Tiron, (c) dihydroxyphenylacetic acid and (d) catechol (e) TRENCAM (calculated using thermodynamic data from the lit.19,22–24 for [Fe3+] = 106 M and [Ligand] = 105 M).
We report the synthesis and the antibacterial activity of new catecholate–ciprofloxacin conjugates 1–3 as artificial siderophore conjugates. The conjugates 1a–c or 2 with a tripodal backbone were obtained respectively in 2–4% and 6% yields from the same key alcohol intermediate 4. The monocatecholate conjugate 3 was afforded in three steps in 75% overall yield. Relative to the parent ciprofloxacin drug, conjugates 1–3 exhibited moderate activities on both P. aeruginosa susceptible and resistant ciprofloxacin strains. The triscatecholate 1a and 1c showed a moderate activity against the CIP susceptible strain DSM 1117, with MICs of 8 and 64 lg/mL, respectively. The ciprofloxacin derivative acid 21 showed the same activity against Pseudomonas that the ciprofloxacin. This result validates our approach by showing that it is possible to obtain antibacterial active ciprofloxacin derivatives from hydrolysis by esterase of triscatecholate conjugates 1–2. The low antibacterial activity of 1–2 and 20 could be due to an absence or a partially hydrolysis of the ester linker in the extracellular medium. The addition of 1 lM FeCl3 to the SMM enhanced their antibacterial activities, presumably due to a better recognition of their corresponding iron complexes by the iron uptake transporters in P. aeruginosa. The gain in activity for 1a–c, contrary for compound 20, under iron-rich conditions could be explained by the vectorization of ciprofloxacin by conjugateiron complexes 1 through their uptake by the siderophore transporters of P. aeruginosa. The conjugate 3 was found to be the most active conjugate against the P. aeruginosa resistant strain. We have demonstrated that at physiological pH iron(III) is complexed by compound 3 and that it occurs solely through the catechol moiety. Moreover, the pFe value of triscatecholates is much higher at physiological pH than ciprofloxacin, the complexation is realized only through the formation of a 1:1 iron(III)/triscatecholate complex. To further develop Trojan horse catecholate-antibiotic conjugates, the choice of the nature of the catecholate and of the spacer arm appear essential. 4. Experimental 4.1. Chemistry All commercially available products were used without further purification unless otherwise specified. All solvents were dried
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via literature procedures when reactions required anhydrous conditions or used without further purification. Flash column chromatography purifications were carried out on silica gel (Kieselgel 60, 40–63 lm, 230–400 mesh ASTM, Merck). Analytical TLC were performed on precoated silica gel 60 F254 plates (Merck) and the compounds were visualized under a UV light (254 nm) and with ethanolic phosphomolybdic acid. Melting points were determined on a Stuart SMP3 apparatus and reported uncorrected. 1H and 13C NMR spectra were recorded using Bruker 600, 500 or 300 MHz spectrometers. Chemical shifts are reported in parts per million (d, ppm) and the signals are quoted as s (singlet), bs (broad singlet), d (doublet), bd (broad doublet), dd (doublet of doublet), dt (doublet of triplet), t (triplet), bt (broad triplet), q (quartet), bq (broad quartet), m (multiplet). J values are given in Hertz. Signals assignments were made using HMBC, HSQC, COSY, and NOESY experiments when necessary. High-resolution mass spectra (electrospray in positive mode—ESI+) were recorded on a Micromasswaters Q-TOF Ultima apparatus. 4.1.1. 4-(3-Aminopropyl)-4-((tert-butyldimethylsilyloxy)methyl) heptane-1,7-diamine (5) Compound 5 was synthesized according to the literature protocol.16 1 H NMR (600 MHz, CDCl3) d 0.28 (s, 6H), 0.57 (s, 9H), 0.88– 1.03 (m, 12H), 2.31 (m, 6H), 3.03 (s, 2H); 13C NMR (150 MHz); 5.8, 17.6, 25.7, 26.5, 30.9, 39.0, 42.5, 66.2; 1H and 13C NMR data were in agreement with the lit.16 HRMS-ESI (m/z): [M+H]+ calcd for C17H42N3OSi: 332.3097, found 332.3124. 4.1.2. Methyl 2,3-bis(benzyloxy)benzoate (11) 2,3-Dihydroxybenzoic acid 7 (10 g, 65 mmol) was dissolved in MeOH (200 mL) in the presence of concentrated H2SO4 (10 mL). After 4 h of refluxing, the solvent was evaporated under reduced pressure. The resulting solid was dissolved in water and K2CO3 was added until pH 7. The aqueous layer was extracted three times with EtOAc. The combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved with acetone (300 mL). K2CO3 (40.3 g, 290 mmol) and benzyl bromide (18.5 mL, 150 mmol) were added and the mixture was stirred at reflux for 12 h. The volatiles were removed and the resulting oil was purified by silica gel column chromatography with gradient elution (cyclohexane to cyclohexane/EtOAc 8:2) to provide the desired product 11 as a white solid (22 g, quantitative). Characterization of 11 matches a previous description of the same compound prepared by another method;25 1H NMR (300 MHz, CDCl3) d 3.69 (s, 3H), 5.15 (s, 2H), 5.18 (s, 2H), 7.08–7.19 (m, 2H), 7.34– 7.49 (m, 11H); 13C NMR (75 MHz, CDCl3) d 51.5, 71.2, 76.3, 116.6, 123.0, 124.1, 127.3–128.3, 127.9, 135.6, 147.4, 150.6, 169.6; HRMS-ESI (m/z): [M+H]+ calcd for C22H24O4: 349.1440, found 349.1422. 4.1.3. 2,3-Bis(benzyloxy)benzoic acid (12) Lithium hydroxide (4.3 g, 180 mmol) was added to a solution of 11 (22 g, 60 mmol) in THF/H2O (220 mL; 10:1 v/v). After 24 h of refluxing, THF was evaporated under reduced pressure. The residue was dissolved in water (100 mL) and the resulting solution was acidified to pH 2 by addition of 6 M aqueous hydrochloric acid. This aqueous layer was extracted with CH2Cl2, dried over Na2SO4, filtered and concentrated in vacuo to obtain 12 (19.8 g, 99%) as a white solid. Characterization of 12 matches a previous description of the same compound prepared by another method;25 1H NMR (300 MHz, CDCl3) d 5.22 (s, 2H), 5.28 (s, 2H), 7.11–7.21 (m, 2H), 7.34–7.49 (m, 11H); 13C NMR (75 MHz, CDCl3) d 72.0, 76.6, 116.6, 123.0, 124.1, 127.3–128.3, 127.9, 135.6, 147.4, 150.6, 172.5; HRMS-ESI (m/z): [M+H]+ calcd for C21H22O4: 335.1283, found 335.1282.
4.1.4. 2,3-Bis(benzyloxy)benzoyl fluoride (6) Cyanuric fluoride or 2,4,6-trifluoro-1,3,5-triazine (0.72 mL, 8.7 mmol) was added dropwise to a solution of 12 (5 g, 15 mmol) and Et3N (1.41 mL, 10 mmol) in dry CH2Cl2 (50 mL). After 4 h of stirring at 20 °C, the mixture was quenched with water. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to afford 6 (4.2 g, 84%) as a white solid. Mp: 59 °C; 1H NMR (300 MHz, CDCl3) d 5.18 (s, 2H), 5.25 (s, 2H), 7.11–7.41 (m, 13H); 13C NMR (75 MHz, CDCl3) d 71.8, 76.1, 116.8, 123.1, 124.2, 127.3–128.3, 127.9, 135.6, 147.4, 150.6, 181.2. 4.1.5. N,N0 -(4-(3-(2,3-Bis(benzyloxy)benzamido)propyl)-4(((tert-butyldimethylsilyloxy)methyl)heptane-1,7-diyl)bis(2,3bis(benzyloxy)benzamide) (13) Benzoyl fluoride 6 (4.8 g, 14.4 mmol) and Et3N (2.2 mL, 16 mmol) were added to a solution of 5 (1.33 g, 4 mmol) in CH2Cl2 (80 mL). The solution was vigorously stirred at 20 °C for 14 h. The volatiles were evaporated and the crude product was purified by column chromatography with gradient elution (CH2Cl2 to CH2Cl2/ MeOH 98:2). 13 (4.3 g, 83%) was isolated as a yellow oil. 1H NMR (600 MHz, CDCl3) d 0.01 (s, 6H), 0.85 (s, 9H), 1.03–1.18 (m, 12H), 3.11 (s, 2H), 3.23 (m, 6H), 5.09 (s, 6H), 5.13 (s, 6H), 7.13 (m, 6H), 7.29–7.49 (m, 30H), 7.78 (dd, J = 6.3, 3.2 Hz, 3H), 7.96 (m, 3H); 13C NMR (150 MHz, CDCl3) d 5.8, 17.8, 22.9, 25.5, 30.7, 38.9, 40.2, 66.7, 70.9, 76.3, 116.6, 123.0, 124.1, 127.3–128.3, 127.9, 136.1, 146.5, 151.4, 164.6; HRMS-ESI (m/z): [M+H]+ calcd for C80H90N3O10Si: 1280.6395, found 1280.5461. 4.1.6. N,N0 -(4-(3-(2,3-Bis(benzyloxy)benzamido)propyl)-4(hydroxymethyl)heptane-1,7-diyl)bis(2,3-bis(benzyloxy) benzamide) (4) A 1 M solution of TBAF in THF (5 mL, 5 mmol) was added to a mixture of 13 (1.2 g, 1 mmol) in THF (50 mL). After 10 h of refluxing, the solvent was removed under pressure. The resulting residue was purified by silica gel column chromatography (CH2Cl2/MeOH 97:3) to afford 4 (1.1 g, 95%) as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.05–1.24 (m, 12H), 3.23 (s, 2H), 3.35 (m, 6H), 5.04 (6H), 5.12 (s, 6H), 7.13 (m, 6H), 7.22–7.49 (m, 30H), 7.85 (m, 3H), 8.05 (m, 3H); 13C NMR (150 MHz, CDCl3) d 23.0, 31.0, 38.9, 40.8, 66.5, 70.4, 75.8, 116.5, 123.0, 124.0, 127.1–128.9, 127.8, 136.1, 146.6, 151.5, 164.6; HRMS-ESI (m/z): [M+H]+ calcd for C74H76N3O10: 1166.5531, found 1166.5394. 4.1.7. 5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl) pentyl 2-chloroacetate (14a) 2-Chloroacetylchloride (31 lL, 0.396 mmol) was added dropwise to a solution of 4 (330 mg, 0.283 mmol) and Et3N (47 lL, 0.340 mmol) in dry CH2Cl2 (9 mL). After 8 h of stirring to 20 °C, the mixture was quenched with water. The volatiles were removed under reduced pressure and the crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 98:2) to give 14a (325 mg, 94%) as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.14– 1.38 (m, 12H), 3.48 (m, 6H), 3.74 (s, 2H), 3.98 (s, 2H), 5.10 (s, 6H), 5.15 (s, 6H), 7.18 (m, 6H), 7.23–7.52 (m, 30H), 7.88 (m, 3H), 8.01 (m, 3H); 13C NMR (150 MHz, CDCl3) d 24.1, 31.7, 39.2, 40.6, 40.7, 66.6, 71.5, 76.2, 117.5, 124.1, 125.0, 127.1–128.9, 128.4, 137.1, 148.6, 152.5, 165.5, 168.2; HRMS-ESI (m/z): [M+H]+ calcd for C76H76ClN3O11: 1242.5257, found 1242.5164. 4.1.8. 5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl)pentyl 3-chloropropanoate (14b) According to the same procedure described for 14a, 3-chloropropanoyl chloride was reacted with 4 (330 mg, 0.283 mmol) to obtain 14b (300 mg, 89%), after purification by column chromatography, as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.17–1.36 (m,
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12H), 2.66 (t, J = 6.6 Hz, 2H), 3.56 (m, 6H), 3.62 (t, J = 6.6 Hz, 2H), 3.81 (s, 2H), 5.04 (s, 6H), 5.12 (s, 6H), 7.13 (m, 6H), 7.22–7.49 (m, 30H), 7.85 (m, 3H), 8.05 (m, 3H); 13C NMR (150 MHz, CDCl3) d 23.0, 31.0, 38.9, 39.4, 40.6, 40.8, 66.5, 70.4, 116.5, 123.0, 124.0, 127.1–128.9, 127.8, 136.1, 146.6, 151.5, 164.6, 168.4; HRMS-ESI (m/z): [M+H]+ calcd for C77H78ClN3O11: 1256.5403, found 1256.5321. 4.1.9. 5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl) pentyl 4-chlorobutanoate (14c) According to the same procedure described for 14a, 4-chlorobutanoyl chloride was reacted with 4 (330 mg, 0.283 mmol) to obtain 14c (320 mg, 85%), after purification by column chromatography, as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.17–1.66 (m, 14H), 2.59 (m, 2H), 3.57 (m, 6H), 3.81 (s, 2H), 3.89 (s, 2H), 5.09 (s, 6H), 5.17 (s, 6H), 7.14 (m, 6H), 7.21–7.48 (m, 30H), 7.82 (m, 3H), 7.98 (m, 3H); 13C NMR (150 MHz, CDCl3) d 22.9, 26.4, 28.3, 30.9, 38.9, 40.6, 48.7, 66.5, 70.4, 75.8, 116.5, 123.0, 124.0, 127.1– 128.9, 127.8, 136.1, 146.6, 151.5, 164.6, 169.4; HRMS-ESI (m/z): [M+Na]+ calcd for C78H80ClNaN3O11: 1292.5379, found 1292.5419. 4.1.10. 7-(4-(2-(5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3 bis(benzyloxy)benzamido)propyl)pentyloxy)-2oxoethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydro quinoline-3-carboxylic acid (15a) A mixture of 14a (200 mg, 0.16 mmol), ciprofloxacin (265 mg, 0.8 mmol) and K2CO3 (110 mg, 0.8 mmol) in dry acetonitrile (5 mL) was refluxed for 3 days. The suspension was filtered and the solution was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (CH2Cl2/ MeOH 90:10) to provide 15a (76 mg, 31%) as a beige powder. Mp: 261 °C; 1H NMR (600 MHz, CDCl3) d 1.17–1.62 (m, 16H), 2.59 (m, 4H), 2.96 (m, 4H), 3.19 (s, 2H), 3.41 (m, 6H), 3.68 (m, 1H), 4.01 (s, 2H), 5.02 (s, 6H), 5.14 (s, 6H), 7.21 (m, 6H), 7.29–7.61 (m, 32H), 7.73 (m, 3H), 8.07 (m, 3H), 8.66 (m, 1H); 13C NMR (150 MHz, CDCl3) d 7.6, 23.4, 31.8, 36.0, 38.1, 40.3, 45.7, 53.6, 59.6, 68.0, 71.3, 76.7, 102.1, 109.3, 112.2, 115.3, 115.9, 122.8, 124.5, 127.9, 127.1–128.9, 133.2, 135.8, 144.2, 147.5, 152.6, 164.2, 165.3, 170.4, 173.8; HRMS-ESI (m/z): [M+Na]+ calcd for C93H93FN6NaO14: 1559.6631, found 1559.6849. 4.1.11. 7-(4-(3-(5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl)pentyloxy)-3oxopropyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydro quinoline-3-carboxylic acid (15b) According to the same procedure described for 15a, 14b was reacted with ciprofloxacin (315 mg, 0.95 mmol) to obtain 15b (80 mg, 26%), after purification by column chromatography, as a white powder. Mp: 258 °C; 1H NMR (600 MHz, CDCl3) d 1.09– 1.54 (m, 16H), 2.51–2.92 (m, 10H), 3.32 (s, 2H), 3.58 (m, 6H), 3.94 (m, 3H),5.09 (s, 6H), 5.21 (s, 6H), 7.28 (m, 6H), 7.34–7.65 (m, 32H), 7.77 (m, 3H), 8.11 (m, 3H), 8.52 (m, 1H); 13C NMR (150 MHz, CDCl3) d 7.4, 23.1, 32.5, 35.8, 38.1, 38.8, 38.6, 40.9, 54.0, 60.2, 68.1, 72.1, 76.2, 102.7, 109.7, 112.9, 116.1, 116.4, 122.5, 124.9, 127.2, 126.7–129.5, 133.8, 135.6, 144.4, 146.4, 156.1, 164.1, 165.8, 171.1, 176.3; HRMS-ESI (m/z): [M+H]+ calcd for C94H96FN6O14: 1551.6969, found 1551.6892. 4.1.12. 7-(4-(4-(5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl)pentyloxy)-4oxobutyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydro quinoline-3-carboxylic acid (15c) According to the same procedure described for 15a, 14c was reacted with ciprofloxacin (397 mg, 1.2 mmol) to obtain 15c (33 mg, 9%), after purification by column chromatography, as a
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brown powder. Mp: 259 °C; 1H NMR (600 MHz, CDCl3) d 1.12– 1.75 (m, 18H), 2.59 (m, 8H), 2.96 (m, 4H), 3.19 (s, 2H), 3.41 (m, 6H), 3.68 (m, 1H), 5.02 (s, 6H), 5.14 (s, 6H), 7.21 (m, 6H), 7.29– 7.61 (m, 32H), 7.73 (m, 3H), 8.07 (m, 3H), 8.66 (m, 1H); 13C NMR (150 MHz, CDCl3) d 7.2, 23.3, 25.5, 30.5, 31.7, 36.3, 38.2, 40.4, 45.8, 53.8, 55.3, 59.5, 71.4, 76.8, 102.4, 109.6, 112.3, 115.5, 116.0, 122.7, 124.6, 128.1, 127.1–128.9, 133.3, 135.8, 143.9, 147.1, 152.3, 164.0, 165.3, 170.5, 174.3; HRMS-ESI (m/z): [M+Na]+ calcd for C95H97FN6NaO14: 1587.6944, found 1587.6749. 4.1.13. 1-Cyclopropyl-7-(4-(2-(5-(2,3-dihydroxybenzamido)-2,2bis(3-(2,3-dihydroxy benzamido)propyl)pentyloxy)-2oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydro quinoline3-carboxylic acid (1a) A suspension of compound 15a (40 mg, 0.026 mmol) and Pd(OH)2/C (10% wt, 5 mg) in MeOH was stirred under an hydrogen atmosphere for 4 days at 20 °C. The catalyst was filtered through a Celite pad and washed with MeOH. The filtrate was concentrated to give 1a (25 mg, 98%) as a white powder. Mp : 257 °C; 1H NMR (600 MHz, DMSO-d6) d 1.07–1.41 (m, 16H), 2.21–2.76 (m, 8H), 3.02 (s, 2H), 3.34 (m, 6H), 3.54 (m, 1H), 3.94 (s, 2H), 7.18 (m, 6H), 7.37–7.41 (m, 2H), 7.61 (m, 3H), 7.91 (m, 3H), 8.41 (m, 1H); 13 C NMR (150 MHz, DMSO-d6) d 7.5, 23.4, 31.9, 36.4, 38.5, 40.6, 46.0, 53.9, 59.9, 68.4, 102.6, 109.8, 112.6, 115.5, 116.2, 123.8, 124.9, 127.2, 133.6, 144.7, 147.7, 153.0, 164.3, 165.7, 170.8, 173.2; HRMS-ESI (m/z): [M+Na]+ calcd for C51H57FN6NaO14: 1019.3814, found 1019.3846. 4.1.14. 1-Cyclopropyl-7-(4-(3-(5-(2,3-dihydroxybenzamido)-2,2bis(3-(2,3-dihydroxy benzamido)propyl)pentyloxy)-3oxopropyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydro quinoline-3-carboxylic acid (1b) According to the same procedure described for 1a, 15b (60 mg, 0.038 mmol) gave 1b (36 mg, 97%) as a beige powder. Mp : 254 °C; 1 H NMR (600 MHz, DMSO-d6) d 1.24–1.61 (m, 16H), 2.47–2.83 (m, 10H), 3.11 (s, 2H), 3.32 (m, 6H), 3.69 (m, 3H), 7.26 (m, 6H), 7.28– 7.39 (m, 2H), 7.64 (m, 3H), 8.01 (m, 3H), 8.50 (m, 1H); 13C NMR (150 MHz, DMSO-d6) d 7.7, 23.5, 31.9, 35.9, 37.3, 38.2, 38.3, 40.4, 53.9, 59.8, 102.4, 109.6, 112.5, 115.6, 115.9, 122.9, 124.3, 127.5, 134.6, 144.6, 147.7, 152.8, 163.5, 165.5, 170.7, 175.2; HRMS-ESI (m/z): [M+Na]+ calcd for C52H59FN6NaO14: 1033.3971, found 1033.4079. 4.1.15. 1-Cyclopropyl-7-(4-(4-(5-(2,3-dihydroxybenzamido)-2,2bis(3-(2,3-dihydroxy benzamido)propyl)pentyloxy)-4oxobutyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydro quinoline-3-carboxylic acid (1c) According to the same procedure described for 1a, 15c (20 mg, 0.013 mmol) gave 1c (13 mg, 99%) as a beige powder. Mp: 261 °C; 1 H NMR (600 MHz, DMSO-d6) d 1.11–1.71 (m, 18H), 2.66–2.87 (m, 12H), 3.27 (s, 2H), 3.52 (m, 6H), 3.81 (m, 1H), 7.39 (m, 6H), 7.30– 7.42 (m, 2H), 7.62 (m, 3H), 8.11 (m, 3H), 8.49 (m, 1H); 13C NMR (150 MHz, DMSO-d6) d 7.6, 23.4, 25.4, 30.9, 31.8, 36.0, 38.1, 40.3, 45.7, 53.6, 55.2, 59.6, 102.1, 109.3, 112.2, 115.3, 115.9, 122.8, 124.5, 126.9, 133.2, 144.2, 147.5, 152.6, 163.8, 165.3, 170.4, 174.3; HRMS-ESI (m/z): [M+Na]+ calcd for C53H61FN6 NaO14:1047.4127, found 1047.4184. 4.1.16. 5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl) pentanoic acid (16) To a solution of KBr (5.5 mg, 0.05 mmol), TBAB (2 mg, 0.006 mmol) and TEMPO (2.3 mg, 0.015 mmol) in water (2 mL) was added a solution of 4 (116 mg, 0.1 mmol) in CH2Cl2 (2 mL). An aqueous solution of sodium hypochlorite 2.2 N (91 lL, 0.2 mmol) was added and the resulting biphasic mixture was stirred vigorously at 0 °C for 2 h. The reaction was quenched to an
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aqueous solution of sodium thiosulfate (20 mL) and was extracted to CH2Cl2. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The resulting solid was dissolved in THF/H2O (10 mL, 1:1, v/v) and sulfamic acid (19 mg, 0.2 mmol) followed by sodium chlorite (18 mg, 0.2 mmol) were added. After 8 h of stirring at 20 °C, THF was evaporated and the aqueous layer was extracted to CH2Cl2. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (CH2Cl2/MeOH 95:5) to give 16 (106 mg, 90%) as yellow oil. 1H NMR (600 MHz, CDCl3) d 1.07–1.26 (m, 12H), 3.23 (m, 6H), 5.06 (s, 6H), 5.11 (s, 6H), 7.15 (m, 6H), 7.19–7.53 (m, 30H), 7.61 (m, 3H), 7.84 (m, 3H); 13C NMR (150 MHz, CDCl3) d 22.9, 30.3, 39.6, 48.6, 69.4, 74.8, 115.4, 121.6, 124.0, 126.2–127.8, 127.9, 135.2, 145.2, 150.3, 165.7, 179.2; HRMS-ESI (m/z): [M+H]+ calcd for C74H74N3O11: 1180.5323, found 1180.5321. 4.1.17. N,N-(4-(3-(2,3-Bis(benzyloxy)benzamido)propyl)-4-(4(2-hydroxyethyl)piperazine-1-carbonyl)heptane-1,7diyl)bis(2,3-bis(benzyloxy)benzamide) (17) A mixture of 16 (120 mg, 0.1 mmol), PyBOP (104 mg, 0.2 mmol) and Et3N (167 lL, 1.2 mmol) in dry DMF (10 mL) was stirred at 20 °C for 15 min. 2-(Piperazin-1-yl)ethanol (36 mg, 0.2 mmol) was added and the mixture was stirred for 8 h at 20 °C. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (CH2 Cl2/MeOH/NH4OH 95:5:0.1) to afford 17 (62 mg, 49%) as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.11–1.34 (m, 12H), 2.54 (m, 6H), 3.31–3.42 (m, 12H), 5.01 (s, 6H), 5.15 (s, 6H), 7.13–7.55 (m, 36H), 7.63 (m, 3H), 7.95 (m, 3H); 13C NMR (150 MHz, CDCl3) d 23.3, 31.4, 40.5, 44.4, 49.1, 56.1, 58.2, 59.0, 70.6, 75.7, 113.2, 122.6, 125.1, 127.6–128.7, 129.4, 136.3, 146.2, 151.7, 166.1, 176.8; HRMS-ESI (m/z): [M+H]+ calcd for C80H86N5O11: 1292.6324, found 1292.6322. 4.1.18. 2-(4-(5-(2,3-Bis(benzyloxy)benzamido)-2,2-bis(3-(2,3bis(benzyloxy)benzamido)propyl)pentanoyl)piperazin-1yl)ethyl 2-chloroacetate (18) According to the same procedure described for 14a, 2-chloroacetyl chloride was reacted with 17 (110 mg, 0.085 mmol) to obtain 18 (84 mg, 72%) as a red oil. 1H NMR (600 MHz, CDCl3) d 1.07–1.53 (m, 12H), 2.58 (m, 8H), 3.29–3.45 (m, 8H), 4.19 (m, 4H) 5.06 (s, 6H), 5.13 (s, 6H), 7.19–7.65 (m, 36H), 7.76 (m, 3H), 8.01 (m, 3H); 13C NMR (150 MHz, CDCl3) d 23.3, 31.4, 40.5, 44.4, 48.3, 56.1, 58.2, 62.2, 70.6, 75.7, 113.2, 122.6, 125.1, 127.6–128.7, 129.4, 136.3, 146.2, 151.7, 166.8, 167.8, 177.4; HRMS-ESI (m/z): [M+H]+ calcd C82H87ClN5O12: 1368.6040, found 1368.6051. 4.1.19. 7-(4-(2-(2-(4-(5-(2,3-Bis(benzyloxy)benzamido)-2,2bis(3-(2,3-bis(benzyloxy) benzamido)propyl)pentanoyl)piperazin-1-yl)ethoxy)-2oxoethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (19) According to the same procedure described for 15a, 18 was reacted with ciprofloxacin (397 mg, 1.2 mmol) to obtain 19 (123 mg, 31%), after purification by column chromatography, as a yellow oil. 1H NMR (600 MHz, CDCl3) d 1.06–1.61 (m, 16H), 2.39– 2.61 (m, 10H), 3.22–3.73 (m, 17H), 4.07 (m, 2H), 5.08 (s, 6H), 5.14 (s, 6H), 7.13–7.69 (m, 38H), 7.83 (m, 3H), 8.08 (m, 3H), 8.42 (m, 1H); 13C NMR (150 MHz, CDCl3) d 7.6, 23.3, 30.9, 35.8, 40.5, 46.4, 49.4, 53.1, 54.6, 56.3, 57.0, 59.2, 61.6, 71.6, 76.4, 103.2, 108.5, 111.8, 114.1, 115.1, 121.7, 124.1, 127.8–129.7, 129.3, 132.7, 136.3, 143.1, 145.8, 151.3, 164.8, 166.8, 167.8, 169.4, 176.1; HRMS-ESI (m/z): [M+H]+ calcd for C99H104FN8O15: 1663.7605, found 1663.7605.
4.1.20. 1-Cyclopropyl-7-(4-(5-(2,3-dihydroxybenzamido)-2,2bis(3-(2,3-dihydroxy benzamido)propyl)pentanoyl)piperazin-1yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2) According to the same procedure described for 1a, 19 (30 mg, 0.018 mmol) gave 2 (18 mg, 99%) as a yellow oil.1H NMR (600 MHz, DMSO-d6) d 1.03–1.54 (m, 16H), 2.32–2.64 (m, 10H), 3.18–3.68 (m, 17H), 3.82 (m, 2H), 7.06–7.31 (m, 8H), 7.73 (m, 3H), 7.89 (m, 3H), 8.51 (m, 1H); 13C NMR (150 MHz, DMSO-d6) d 7.6, 23.6, 31.3, 36.2, 40.9, 47.7, 50.1, 53.5, 54.8, 57.2, 57.9, 60.1, 61.9, 109.1, 110.4, 115.2, 115.9, 121.3, 124.6, 130.1, 134.0, 143.6, 145.0, 152.6, 165.7, 165.8, 168.2, 169.4, 175.8; HRMS-ESI (m/z): [M+H]+ calcd for C57H68FN8O15: 1123.4788, found 1123.4789. 4.1.21. 1-Cyclopropyl-6-fluoro-7-(4-(2-methoxy-2oxoethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid (20) To a solution of ciprofloxacin (1.0 g, 3.02 mmol) in DMF (15 mL) was added methyl bromoacetate (0.692 g, 4.53 mmol), triethylamine (0.816 mL) and potassium iodide (0.752 g, 4.53 mmol) under an argon atmosphere. The mixture was stirred at 90 °C. After 5 h, the reaction mixture was poured into water (15 mL). The resulting precipitate was filtered off, washed with water and recrystallised from methanol. Compound 20 was obtained as a yellow powder (0.557 g, 46%). Mp: 226 °C; 1H NMR (300 MHz, DMSOd6) d 1.20 (m, 2H), 1.33 (m, 2H), 2.77 (t, 4H, J = 4.7 Hz), 3, 42–345 (m, 5H), 3.66 (s, 3H), 7.59 (d, 1H, 4JHF = 7.5 Hz), 7.92 (d, 1H, 3 JHF = 13.4 Hz), 8.69 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 11.1, 39.4, 52.9, 54.7, 55.1, 61.5, 109.9, 114.3, 114.6, 119.6, 142.7, 148.1, 151.5, 153.6, 161.1, 173.9, 179.9, 181.4; HRMS-ESI (m/z): [M+Na]+ calcd for C20H22FN3NaO5: 426.1441, found 426.1453. 4.1.22. 7-(4-(carboxymethyl)piperazin-1-yl)-1-cyclopropyl-6fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (21) To a solution of 20 (0.500 g, 1.24 mmol) in CH3COOH/H2O (4.2 mL, 1.5:1 v/v) was added concentrated sulfuric acid (0.24 mL). The mixture was stirred at 150 °C for 3 h and poured into water. The residue was filtered off, washed successively with water and methanol and dried. Compound 21 was afforded as a white powder (0.406 g, 84%). Mp : 266 °C (258–261 °C lit.17) 1H NMR (300 MHz, DMSO-d6) d 1.21 (m, 2H), 1.35 (m, 2H), 3.11 (m, 4H), 3, 49 (m, 4H), 3.68 (m, 2H), 3.85 (m, 1H), 7.59 (d, 1H, 4 JHF = 7.4 Hz), 7.93 (d, 1H, 3JHF = 13.4 Hz), 8.67 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 11.1, 39.4, 51.1, 54.9, 60.1, 110.2, 114.4, 114.7, 122.5, 142.6, 147.6, 151.7, 154.7, 158.0, 169.3, 172.6, 179.8; HRMS-ESI (m/z): [M+H]+ calcd for C19H21FN3O5: 390.1465, found 390.1452. 4.1.23. Benzyl 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)1,4-dihydro quinoline-3-carboxylate (22) To a suspension of ciprofloxacin (331 mg, 1 mmol) and Boc2O (260 mg, 1.2 mmol) in dioxane (5 mL) was added Et3N (167 lL, 1.2 mmol). The reaction was stirred at 20 °C for 4 h and the volatiles were removed. The solid was dissolved in CH3CN (8 mL) and benzyl bromide (144 lL, 1.2 mmol) followed by addition of K2CO3 (165 mg, 1.2 mmol) were added. The resulting mixture was heated at reflux for 14 h. The solution was cooled to 20 °C and the resulting white solid was filtered off and rinsed successively with water and cyclohexane. After drying, the powder was dissolved in THF (5 mL) and HCl 4 N (0.5 mL) was added. The solution was stirred at 20 °C for 10 h, basified with an aqueous solution of NaHCO3 and the solvent was evaporated. 20 (345 mg, 81%) was obtained as a yellow oil and was reacted immediately in the next step. 1H NMR (500 MHz, DMSO-d6) d 1.10–1.28 (m, 4H), 3.09 (m, 4H), 3.23 (m, 4H), 3.41 (m, 1H), 4.94 (s, 2H), 7.35–7.52 (m, 6H), 8.03 (s, 1H), 8.52 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 7.7, 35.7, 45.7, 51.3, 65.8, 103.0, 111.4, 111.5, 115.6, 127.3–128.9,
Please cite this article in press as: Fardeau, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.067
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133.6, 135.9, 141.9, 147.3, 152.6, 164.9, 174.3; HRMS-ESI (m/z): [M+H]+ calcd for C24H25FN3O3: 422.1880, found 422, 1859. 4.1.24. Benzyl 7-(4-(2,3-Bis(benzyloxy)benzoyl)piperazin-1-yl)1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3carboxylate (23) A solution of 22 (211 mg, 0.5 mmol), benzoyl fluoride 6 (198 mg, 0.6 mmol) and Et3N (83 lL, 0.6 mmol) in dry CH2Cl2 (20 mL) was stirred at 20 °C for 6 h. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography (CH2Cl2/MeOH 95:5). 21 (344 mg, 91%) was afforded as a yellow powder. Mp = 251 °C; 1H NMR (600 MHz, CDCl3) d 1.17–1.62 (m, 4H), 2.59 (m, 4H), 2.96 (m, 4H), 3.68 (m, 1H), 5.02 (s, 2H), 5.14 (s, 4H),7.21 (m, 2H), 7.29–7.61 (m, 17H), 7.73 (m, 1H), 8.66 (s, 1H); 13C NMR (150 MHz, CDCl3) d 7.7, 36.0, 45.7, 53.6, 71.3, 76.7, 102.1, 109.3, 112.2, 115.3, 115.9, 122.8, 124.5, 127.9, 127.1–128.9, 133.2, 135.8, 144.2, 147.5, 152.6, 164.2, 165.3, 173.8; HRMS-ESI (m/z): [M+Na]+ calcd for C45H40FN3 NaO6: 760.2799, found 760.2799. 4.1.25. 1-Cyclopropyl-7-(4-(2,3-dihydroxybenzoyl)piperazin-1yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3) According to the same procedure described for 1a, 23 (100 mg, 0.13 mmol) gave 3 (59 mg, 98%) as a white solid. Mp : 252 °C; 1H NMR (300 MHz, DMSO-d6) d 1.14–1.65 (m, 4H), 2.62 (m, 4H), 3.01 (m, 4H), 3.74 (m, 1H), 7.26 (m, 2H), 7.34–7.56 (m, 2H), 7.78 (m, 1H), 8.42 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 7.7, 36.3, 45.8, 53.9, 102.2, 109.4, 112.2, 115.3, 115.8, 122.7, 124.4, 127.9, 133.2, 144.1, 147.5, 152.6, 164.3, 165.5, 174.0; HRMS-ESI (m/z): [M+H]+ calcd for C24H23FN3O6: 468.1571, found 467.1479. 4.2. Antibacterial activity 4.2.1. Bacterial strains The following strains were used for testing antibacterial susceptibility: Pseudomonas aeruginosa DSM 1117 as a reference strain, susceptible to ciprofloxacin (Deutsche Sammlung für Mikroorganismen, Braunschweig, Germany) and P. aeruginosa AM 85, a clinical strain formerly identified as resistant to ciprofloxacin through an efflux mediated mechanism.18 4.2.2. In vitro susceptibility Bacteria were grown overnight at 35 °C in Tryptic Soy Broth and streaked on Tryptic Soy Agar (TSA) (AES, Bruz, France). From these isolation plates, inocula were prepared according to CLSI recommendations and the broth microdilution technique carried out as advised using drug concentrations, ranging from 0.0625 to 128 lg/mL, obtained from serial two-fold dilutions of a stock solution of each product prepared in DMSO with gentle heating (40 °C) and sonication for 10 min.26 Ciprofloxacin (Sigma–Aldrich) was used as control in each series of experiments. The MIC was determined as the highest dilution at which wells remained clear. Both cation-adjusted Mueller–Hinton broth at pH 7.3 (AES) and Succinate Minimum Medium (SMM, pH = 7.0), a medium virtually deprived of iron,27 were used to evaluate the MIC of our products. Products 1a–c, 2 and 3 were tested in the latter medium with and without the addition of 1 lM FeCl3 (Sigma–Aldrich), to evaluate their antibacterial activity under iron-enriched and iron-deprived conditions, respectively. 4.3. Hemolytic activity The assay was adapted from the protocol described by Taniyama et al.28 Briefly, human red blood cells were centrifuged (1000 g, 10 min), the pellet washed three times with TRIS buffer (TRIS 10 mmol.l1, NaCl 150 mmol.l1, pH 7.4) and finally diluted
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at 0.5% (v/v) in the same buffer. Experiments were conducted in triplicate in 96-well plates. Saponin 1% (m/v) was used as a positive hemolysis control (100% hemolysis), DMSO and TRIS buffer were used as negative controls. 15 lL of the tested molecule (concentration range: 0.19–384 mg/mL) or control solution were added to 235 lL of the 0.5% (v/v) red blood cell suspension. In addition to the catechol-associated compounds synthesized in this study, ciprofloxacin was used as comparative agents. Plates were incubated at 37 °C for 1 h and then centrifuged (1000 g, 10 min). Then, 100 lL of supernatant were retrieved from each well for optical density measurement at 405 nm. The percentage of hemolytic activity was calculated using the saponin mean absorbance at 405 nm as the 100% hemolysis value and the negative control absorbance at 405 nm as blank. The drug concentration inducing a 50% hemolysis (HC50) was then deduced from the dose-response curves. 4.4. Complexation studies The measurements were realized in a H2O/DMSO mixture with a 0.2 molar fraction of DMSO. The pH measurements and the potassium hydroxide additions were performed using a CRISON pH-burette 24 automatic titrator. 0.1 M HCl and 0.1 M KOH were prepared in the H2O/DMSO solvent mixture by dilution of standard commercial solution and titrated by borax and potassium hydrogenphatalate solutions, respectively. The combined CRISON 52-02 glass electrode used for pH measurements was calibrated through titrations of the 0.1 M HCl solution by potassium hydroxide in H2O/DMSO. The apparent ionization constant was found at pQc = 15.54(5). Iron(III) solutions were obtained by dissolution of iron(III) chloride and titrated by EDTA. During the titration, the temperature was maintained constant at 25.0 °C using a thermostated circulating bath and nitrogen was bubbled through the solution. The spectrophotometric measurements were realized using a Jasco V630-bio UV–visible spectrophotometer. 5. Notes The authors declare no competing financial interest. Acknowledgments The authors thank the DGA for financial support of this study. S.F. was the recipient of a Grant from DGA (Direction Générale de l’Armement, Ministère de la Défense, France) and Pharmamens. M.P. was a recipient of the Grant from the French government. References and notes 1. Trautmann, M.; Lepper, P. M.; Haller, M. Am. J. Infect. Control 2005, 33, S41. 2. Lyczak, J. B.; Cannon, C. L.; Pier, G. B. Microbes Infect. 2000, 2, 1051. 3. Rowe, S. M.; Stacey, M. D.; Miller, B. S.; Sorscher, E. J. N. Engl. J. Med. 1992, 2005, 352. 4. Mislin, G. L. A.; Schalk, I. J. Metallomics 2014, 6, 408. 5. (a) Briat, J. F. J. Gen. Microbiol. 1992, 138, 2475; (b) Fardeau, S.; Mullié, C.; Dassonville-Klimpt, A.; Audic, N.; Sonnet, P. In Microbiology Book Series-2011; Méndez-Vilas, A. Eds.; 2011; Vol. 2, Formatex:Badajoz, Spain, pp. 695–705. 6. (a) Raymond, K. N.; Dertz, E. A.; Kim, S. S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3584; (b) Ferguson, A. D.; Ködding, J.; Walker, G.; Bös, C.; Coulton, J. W.; Diederichs, K.; Braun, V.; Welte, W. Structure 2001, 9, 707. 7. Hider, R. C.; Kong, X. Nat. Prod. Rep. 2010, 27, 637. 8. (a) Hennard, C.; Truong, Q. C.; Desnottes, J. F.; Paris, J. M.; Moreau, N. J.; Abdallah, M. A. J. Med. Chem. 2001, 44, 2139; (b) Rivault, F.; Liebert, C.; Burger, A.; Hoegy, F.; Abdallah, M. A.; Schalk, I. J.; Mislin, G. L. Bioorg. Med. Chem. Lett. 2007, 17, 640; (c) Noël, S.; Gasser, V.; Pesset, B.; Hoegy, F.; Rognan, D.; Schalk, I. J.; Mislin, G. L. Org. Biomol. Chem. 2011, 9, 8288. 9. Mashiach, D. R.; Meijler, M. M. Org. Lett. 2013, 15, 1702. 10. Cornelis, P.; Dingemana, J. Front. Cell. Infect. Microbiol. 2013, 3, 1. 11. Ji, C.; Miller, P. A.; Miller, M. J. J. Am. Chem. Soc. 2012, 134, 9898. 12. (a) Wanatabe, N.-A.; Nagasu, T.; Katsu, K.; Kitoh, K. Antimicrob. Agents Chemother. 1987, 31, 497; (b) Weissberger, B. A.; Abruzzo, G. K.; Fromtling, R. A., et al. J. Antibiot. 1989, 42, 795; (c) Valiant, M. E.; Gilfillan, E. C.; Gadebusch,
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Please cite this article in press as: Fardeau, S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.067