AMINOGLYCOSIDES ACTIVE AGAINST RESISTANT BACTERIA
Overexpression of aminoglycoside modifying enzymes from resistant bacteria is the most commonly encountered mode of resistance [1-3]. several aminoglycoside-modifying enzymes have been identified catalyzing a wide range of modifications including acetylation, phosphorylation, and adenylation, which introduce functional groups on the aminoglycosides that prevent the binding to the targeted site of rRNA making the bacteria resistant to their action. These enzymes are classified as aminoglycoside phosphotransferases (APHs), aminoglycoside acetyltransferases (AACs), and aminoglycoside nucleotidyltransferases (ANTs). More than 50 different isoforms of these enzymes have been clinically isolated with differences in their ability to modify various aminoglycosides.
Many aminoglycoside derivatives have
been developed with the goal of evading the action of aminoglycoside modifying
enzymes, to achieve new products which retain activity against
aminoglycoside-resistant bacteria. Examples include methylation at the amino
group where acetylation may take place, substitution of hydroxy group with
fluoride to avoid the action of APHs or ANTs, and deoxygenation of
One of the most prevalent modifying enzymes is APH(3 ), catalyzes phosphorylation at the 3 -OH of both neomycin and kanamycin classes of aminoglycosides, producing phosphorylated adduct incapable of binding toward the ribosomal target (Figure 1). Dideoxygenation at 3 and 4 positions has been proved to be effective against APH(3 ) [4-13]. This approach has led to the syntheses and discovery of tobramycin [14-15], 48,49 arbekacin , 50 and similar aminoglycosides.
Figure 1. Phosphorylation of kanamycin and neomycin classes of aminoglycosides by APH(3 ).
The most widely used methods for 3 ,4 -dideoxygenation on aminoglycosides involve converting a trans-diol into leaving groups like dimesylate, followed by zinc-mediated elimination [17-18]. An efficient synthesis of 3 ,4 -dideoxykanamycin B (arbekacin), is based on the conversion of the trans-3 ,4 -diol into benzylsulfonates followed by elimination using NaI (Scheme 1) . Several syntheses use the kanamycin scaffold, there are very few examples of deoxygenation on neomycin class . The syntheses of both classes of antibiotics usually start from kanamycin or neomycin, limiting the options for introducing novel structural motifs at other positions of aminoglycosides.
Scheme 1. Synthesis of 3' ,4' -dideoxykanamycin
An improved synthesis for the preparation of 3 ,4 -dideoxy-1,3,2 ,6 -tetraazidoneamine, 6 (Scheme 2) jhas also been reported. The dideoxygenation can be carried out in the presence of azido groups. The syntheses of 3 ,4 -dideoxygenation products of both classes of aminoglycosides have been accomplished utilizing 6 as the common scaffold (Scheme 3) . Direct glycosylation of 6 with thioglycosides gives 3 ,4 -dideoxygenated kanamycin derivatives, while regioselective protection of O-6 hydroxyl group of 6, followed by glycosylation with trichloroacetimidate donors, gives 3 ,4 -dideoxy pyranmycin derivatives.
Scheme 2. Synthesis of 3',4' -dideoxyneamine acceptor.
Scheme 3. Synthesis of 3',4'-dideoxy kanamycin B and pyranmycin like aminoglycosides.
The synthesized 3',4'-dideoxyaminoglycosides were assayed against aminoglycoside susceptible and resistant strains of E. coli (Table 1). Both compound 9 and 12 showed activity against APH(3) resistant bacteria, but were less active against AAC(6)/APH(2) resistant strains.
TABLE 1. MIC of 3',4'-Dideoxyaminoglycosides 9 and 12
Modification the N-1 position of the 2-deoxystreptamine in kanamycin or neomycin like antibiotics, is one of the most effective methods for achieveing activity against aminoglycoside resistant bacteria. This strategy has led to the development of semi-synthetic amikacin analogues characteerized by the presence of (S)-4-amino-2-hydroxybutyryl (AHB) group at N-1 position.
The synthesis of kanamycin and neomycin like aminoglycosides bearing a modification at N-1 position can be achieved via enzymatic [20-22] or chemical methods [23-31]. Several compounds bearing an AHB group at N-1 have been reported: the synthesis of neomycin derivatives bearing AHB at N-1 has been achieved using a regioselective carbamate cyclization followed by hydrolysis (Figure 2) [25, 31]. Compounds, 13 and 15, with ring IV attached to the O-2 were much less active than those with ring IV attached to O-3 (Table 2). The presence of an AHB group at N-1 on the neomycin scaffold increase the activity against resistant E. coli. All four analogs are ineffective against P. aeruginosa.
Figure 2. Neomycin derivatives at N1.
Table 2. MIC mg/mL of neomycin derivatives at N1.
Chemical modification of butirosin with AHB at N-1 has led to the synthesis of several derivatives (Figure 3) . These compounds manifested better activity than butirosin A, especially compound 18 against P. aeruginosa (Table 3). Compounds with deoxygenation at C-3 preserve their activity against bacteria equipped with APH while butirosin, which still has its 3 -OH, is inactive.
Figure 3. Ribostamycin analogs
TABLE 3. MIC of ribostamycin analogs
The regioselective functionalisation of kanamycin with AHB at N-1 has been achieved taking advantage of metal chelation (Scheme 4) . using a similar approach, kanamycin derivatives have been prepared (Figure 4) . The antibacterial activity of kanamycin derivatives with AHB at N-1(amikacin like), is similar to amikacin regardless of the presence of 3'-deoxygenation (Table 4). The derivatives are less effective against AAC(6 ). One interesting finding is the difference between 3',4'-dideoxygenation and 3'-deoxygenation, in fact the first modification makes the compounds inactive against ANT(2 ) resistant bacteria, while the latter retains the antibacterial activity. Moreover, 3'-deoxyamikacin appears to be more active against bacteria expressing with APH(3 )-I than APH(3 )-III. Compound 38 bearing a 6-Cl was equally active though it proved more toxic.
Scheme 4. Synthesis of kanamycin
Figure 4. kanamycin analogs.
TABLE 4. MIC (mg/mL) of Synthesized Kanamycin Derivatives bearing AHB at N-1
Fluoro-substituted derivatives of amikacin have been obtained by direct functionalisation (Scheme 5) . Taking advantage of metal-chelation and steric hindrance 5-OH was selectively fluorinated. Compounds 132 and 133 showed similar trends of antibacterial activity (Table 5). However, the activities of amikacin derivatives 46 and 47 against E. coli (ANT(2 )) were found to be much better than those reported in Table 4 regardless of the sites of deoxygenation. These fluorosubstituted amikacin analogsshowed weaker activity against AAC(6 ), even when AHB was attached at N-1.
Scheme 5. Fluorinated amikacin
TABLE 5. MIC (mg/mL) of fluoro amikacin derivatives
A number of
functional groups at the N-1 position have been introduced to explore the SAR of
amikacin like series (Figure 5) .
Figure 5. Kanamycin derivatives at N-1.
TABLE 6. MIC (mg/mL) of Kanamycin derivatives at N-1
Other functionalities at the N-1 position, such as a 2-aminoethanesulfonyl group, have also been attempted (Figure 6) . The introductionof this functional group provided similar activity against E. coli, which is expected from an amikacin-type aminoglycoside (Table 7). However, compounds, 60–63, appeared to be much less active against P. aeruginosa including (APH(3 )-I). The result could be attributed to the observed relative low permeability of P. aeruginosa.
Figure 6. Amikacin and buritosin derivatives at N-1.
TABLE 7. MIC (mg/mL) of Amikacin and Butirosin Analogs
The reactivity of N-1 azido group has
been significantly increased by introducing di-(4-chlorobenzoyl) at the O-5 and
O-6 positions, affording the synthesis of intermediate, 65 (Scheme 6).64
Glycosylation at either O-5 or O-6 position followed by attachment of the AHB
side chain generates pyranmycin and kanamycin analogs modified at N-1 position.
The N-1-modified aminoglycosides were tested against aminoglycoside susceptible
and resistant strains of E. coli. All the synthesized aminoglycosides
showed activity against both resistant strains (Table 8). One of the synthesized
kanamycin analogs, compound 69 is more active in vitro against APH(3 )-I
than the clinically used
Scheme 6.Synthesis of pyranmycin and kanamycin B analogs
TABLE 7. MIC (mg/mL) of Pyranmycin and Kanamycin B analogs
3'-Deoxygenated Aminoglycoside: Tobramycin analogs
Synthesis methods for the direct modification of tobramycin have been reported . Tobramycin (or nebramycin) differs from kanamycin B only in 3'-deoxy, which makes tobramycin immune from the action of APH(3 ). A library of tobramycin analogs bearing various functionalities at O-5 has been reported (Scheme 7). In general, these derivatives resulted less active than tobramycin (Table 8). Interestingly, compounds 74 and 76 showed activity against P. aeruginosa (ATCC 27853) (MIC = 12.5 mg/mL).
Scheme 7. Synthesis of tobramycin analogs
TABLE 8. MIC (mg/mL) of tobramycin analogs
Several neamine dimers were then synthetised and their antibacterial activity and capability to resist or inhibit the aminoglycoside modifying enzymes was tested (Scheme 8) [34-36]. These neamine dimers displayed antibacterial activity with some compound being very potent (Table 9). In addition, these dimers have also showed inhibitory effect against AAC(6 )-APH(2 ).
Scheme 8. Neamine dimers
TABLE 9. MIC (mg/mL) of Neamine Dimers
Two neamine/nebramine dimers were synthesized and assayed (Figure 7) [35-36] showing improved activity against various clinically isolated strains of P. aeruginosa (Table 10). Dimers of kanamycin and neomycin have also been synthesized but microbiological data have not been reported [37-38].
Figure 7. Neamine dimers
TABLE 10. MIC (mg/mL) of Neamine Dimers
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