Recovery of the Antibiotic Ac- Tivity Against Resistant Bacteri- Al Strains by Selective Guanidi- Nylation of the 3″-Methylami
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Zárate SG, et al., J Mod Chem Sci 2017, 1: 002 HSOA Journal of Modern Chemical Sciences Research Article Their potent bactericidal activity relies on binding specifically to the decoding A site of RNA in the bacterial small ribosomal subunit, thus Recovery of the Antibiotic Ac- interfering with protein synthesis [2,3]. These compounds are widely tivity against Resistant Bacteri- used in the treatment of serious infections caused by gram negative/ gram positive bacteria, although their clinical use is associated with al Strains by Selective Guanidi- dose-limiting nephrotoxicity and ototoxicity [4,5]. Worse yet, many of the aminoglycoside antibiotics originally identified are no longer nylation of the 3″-Methylamino clinically useful as they are compromised by bacterial resistance mechanisms [6]. Group of Gentamicin Acquired resistance to aminoglycoside antibiotics can occur via Sandra G Zárate1,2, Agatha Bastida1, Andrés G Santana1* and three different mechanisms: Mutation of the ribosomal target, reduced Julia Revuelta1* permeability for the antibiotics, and enzymatic modification of the drugs, thus leading to inactivation. From a clinical point of view, the 1Departmento de Química Bioorgánica, Instituto de Química Orgánica General (CSIC), Juan de la Cierva, Spain most relevant source of resistance is the enzymatic inactivation of the drugs by modification of its amine or hydroxyl groups. Antibiotic 2 Facultad de Tecnología-Carrera de Ingeniería Química, Universidad Mayor Modifying Enzymes (AMEs) can be broadly classified as N-Acetyl- Real y Pontificia de San Francisco Xavier de Chuquisaca, Sucre, Bolivia transferases (AACs), O-Adenyl Transferases (ANTs) and O-Phos- photransferases (APHs). Each family involves enzymes that catalyse the same reactions but with different regioselectivity and substrate specificity (Figure 1). With this in mind, and given the urgent demand for the discovery of new antibiotics to overcome the resistance to these compounds, Abstract considerable effort has been devoted to designing new semisynthetic aminoglycoside antibiotics that are immune to inactivation [7]. The Aminoglycoside antibiotics are highly potent, wide-spectrum intrinsic potency of aminoglycosides makes them excellent candi- bactericidals. Unfortunately, their use in clinical practice has been dates to explore new ways to avoid bacterial resistance and diminish seriously limited as a result of their toxicity and susceptibility to enzy- toxicity. Our group has been actively involved in the design of new matic inactivation. Herein, we describe the synthesis and biological antibiotics that are not susceptible to inactivation by AMEs. With this properties of a new gentamicin derivative (C1a and C2 gentamicin components). The selective modification of N-3″ in this antibiotic not purpose, different design strategies have been employed (Figure 1) only maintains an antimicrobial activity similar to the parent amino- [8]. glycoside but also against resistant strains expressing several AMEs (AAC (6′)-Ib, APH(2″), ANT(6) and APH(3′)). Introduction Ever since their discovery in the mid XX century, aminoglycoside antibiotics have become one of the go to treatments for bacterial in- fections. The first congener of this family, streptomycin was isolated in 1944 and soon after, several members of this class of compounds were also reported [1]. From a chemical point of view, aminoglyco- sides are water soluble polycationic pseudo-oligosaccharides consti- tuted by amino sugars attached to a cyclitol by glycosidic linkages. *Corresponding authors: Julia Revuelta, Departmento de Química Bioorgánica, Instituto de Química Orgánica General (CSIC) Juan de la Cierva, 3. 28006-Ma- drid, Spain, Tel: +34 912587487; E-mail: [email protected] Andrés G Santana, Departmento de Química Bioorgánica, Instituto de Química Orgánica General (CSIC) Juan de la Cierva, 3. 28006-Madrid, Spain, Tel: +34 91 2587455; E-mail: [email protected] Citation: Zárate SG, Bastida A, Santana AG, Revuelta J (2017) Recovery of Figure 1: Representation of the different aminoglycoside systems (a- the Antibiotic Activity against Resistant Bacterial Strains by Selective Guanidi- g) designed and synthesized by our group to prevent AME inactivation. nylation of the 3″-Methylamino Group of Gentamicin. J Mod Chem Sci 1: 002. Addition of a substituent at the N-3″ position of kanamycin A (strategy highlighted with a red square) is well tolerated by the rRNA and, par- Received: July 11, 2017; Accepted: September 12, 2017; Published: Septem- ticularly, guanidinylation of this position prevents the inactivation of ber 26, 2017 the antibiotic by AMEs. Citation: Zárate SG, Bastida A, Santana AG, Revuelta J (2017) Recovery of the Antibiotic Activity against Resistant Bacterial Strains by Selective Guanidinylation of the 3″-Methylamino Group of Gentamicin. J Mod Chem Sci 1: 002. • Page 2 of 4 • These derivatives included changes in the distribution of the ami- Synthesis of Compounds 3a-3b no groups (Figure 1a-c) [9], conformational restriction of the drug 1,3,2′,6′,3″-(Cbz) -gentamicin C (3a), 1,3,2′,6′,3″-(Cbz) -genta- (Figure 1d) [10], the simultaneous incorporation of kanamycin and 5 1 5 micin C (3b) and 1,3,2′,6′,3″ -(Cbz) -gentamicin C (3c). ribostamycin fragments within the same antibiotic scaffold (Figure 1a 5 2 1e) [11], aminoglycoside dimerisation (Figure 1f) [12] and the intro- To a well stirred solution of gentamicin C1 (2a), C1a (2b) and C2 duction of bulky substituent’s properly positioned to interfere with (2c) (ratio = 25:35:40) free base (0.3 g, 0.64 mmol) in a saturated ° drug recognition (Figure 1g) [13]. Unfortunately, despite their struc- aqueous solution of Na2CO3 (4 ml) at 0 C was added a solution of tural diversity, these compounds are substrates of the AMEs to some CbzCl (benzyl chloroformate) (0.65 g, 3.84 mmol) in acetone (1.5 extent and some of them even lost their antibiotic activity. More re- ml) drop by drop. The mixture was vigorously stirred for 2 hours at cently, we have demonstrated that the A site shows a clear tolerance this temperature and then 8 hours at room temperature. Subsequently, for modification at the N-3″ position of the aminoglycosides [14,15] the solvent was removed under reduced pressure and the residue was and that the guanidinylation of this position maintains the antibiotic pulverized in a mortar. This solid was added to an aqueous solution activity against aminoglycoside 6′-acetyl-transferase and 4′-nucleoti- of HCl (1M) until neutralization and the formed solid product was dyl-transfersase-expressing strains (Figure 1h) [16,17]. filtered and dried under vacuum, obtaining the mixture of compounds 3a-c (0.36 g, 50%). MS-API-ES: 1148 (M+H)+ (3a), 1134 (M+H)+ Herein, we propose the synthesis and evaluation of the new genta- + micin derivatives 1a and 1b with the aim of expanding the usefulness (3b), 1120 (M+H) (3c). of our strategy, demonstrating its broad applicability against other re- 1,3,2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C1 (4a) sistance enzymes. Gentamicin is a mixture of various congeners: C1, 1,3,2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C1a (4b) C1a and C2 (Figure 2). Structural differences between them are minor, 1,3,2′,6′,-(Cbz)4-2″,3″-carbamate-gentamicin C2 (4c) differing only by a methyl or a hydrogen substitution in two R groups To a stirred solution of the mixture of compounds 3a-c (0.3g, 0.32 (R or R ) on the purpurosamine residue. This mixture has been the 1 2 mmol) in a mixture of 1,4-dioxane/H O (30 ml/10 ml) was added an only aminoglycoside antibiotic used to date in clinic and recently it 2 aqueous solution of NaOH (2.5M). The mixture was stirred at 50°C has demonstrated utility for the treatment of sepsis caused by diverse for 24 hours, after which was added an aqueous solution of HCl (1M) strains of MRSA bacteria [18]. The MIC of gentamicin (2a-c) typical- until pH 10. The mixture was concentrated under reduced pressure ly ranges from 6 to 48 mg/ml. However, many strains have acquired and subsequently water was added (25 ml). The obtained suspension aminoglycoside resistance genes that encode various AMEs, which was filtered and the solid was dried under vacuum. Finally, the resi- eventually result in very high resistance to gentamicin (MICs usually due was purified through a column chromatography (CHCl /MeOH/ higher than 200 mg/ml). The most clinically relevant of these genes 3 NH OH, 8:2:0.2 → 6:3:1), yielding the mixture of products 4a, 4b are aac (6′), aac(2′), aph(2″) and ant(2″). 4 and 4c as a white solid (60%). MS-API-ES: 1040 (M+H)+ (4a), 1026 (M+H)+ (4b), 1012 (M+H)+ (4c). 1,3,2′,6′-(Cbz)4-gentamicin C1a (5a), 1,3,2′,6′-(Cbz)4-gentamicin C2 (5b) and 1,3,2′-(Cbz)3-gentamicin C1 (6). To a solution of free base gentamicin C1, C1a and C2 (2a-c) (ratio = 25:35:40) (0.3 g, 0.64 mmol) in DMSO (4 ml) at 0°C was added N-benzyloxycarbonyl succinimide (0.52 g, 2.11 mmol). The mixture was stirred vigorously for 2 hours at room temperature. Subsequently, Et O was added (100 ml), observing the formation of a colorless oil. Figure 2: (Left) the structures of gentamicin C1, C1a and C2 components 2 (2a-c) are shown, indicating the positions susceptible to enzymatic mod- After solvent decantation the residue was purified by resin chroma- ification. tography column Amberlite™ IRA-120-H+ (5 g), yielding a mixture of compounds 5a and 5b (0.17 g, 37%) when the resin was eluted with Materials and Methods a 0.5M solution of NH4OH in 1,4-dioxane/H2O (1:1) and compound 6 (73 mg, 13%) when the concentration of NH4OH was increased to Gentamicin (2a-c) was purchased from Sigma Aldrich. All the re- 1M.