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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
hydroxy groups at the modification sites.

3',4'-Dideoxygenation

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 [16], 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) [19]. Several syntheses use the kanamycin scaffold, there are very few examples of deoxygenation on neomycin class [20]. 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)[21] . 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

Compounds E. coli (TG1) E. coli (TG1)

(AAC(6 )/APH(2 ))

E. coli (TG1)

(APH(3 )-I)

  Amikacin 1 1 0.5
Kanamycin B 4 Inactive 32
Ribostamycin 2 16 Inactive
Butirosin  0.5 0.5 0.5
12 8 4 4
9 8 Inactive 4


N-1 Modification

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.

Bacterial Strains Paromomycin 13 14 15 16

S. aureus

0.39 6.25 3.12 100 3.12

B. subtilis

<0.2  0.78 <0.2 3.12 <0.2

E. coli K-12

3.12 50 12.5 >100 6.25

E. coli (APH(3)-APH(5))

>100 >100 >100 >100 6.25

P. aeruginosa A3

12.5 100 50 >100 100
M. smegmatis 0.39 3.12 6.25 100 1.56

Chemical modification of butirosin with AHB at N-1 has led to the synthesis of several derivatives (Figure 3) [20]. 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

Bacterial strains  Butirosin A Gentamycin 17 18 19 20 21 22
S. aureus (APH) 50 3.1 25 3.1 6.3 3.1  6.3
E. coli 4.4 1.6 6.2 2.2
E. coli (APH) 200 200 200     12.5 3.1 25 25
P. aeruginosa 200 25 200 6.3 25 12.5 25
P. aeruginosa GNT
(resistant strain)
31.3 126 10.0 3.9 12.5   6.3 6.3

The regioselective functionalisation of kanamycin with AHB at N-1 has been achieved taking advantage of metal chelation (Scheme 4) [5]. using a  similar approach,  kanamycin derivatives have been prepared (Figure 4) [27]. 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

Bacterial Strains amikacin 35 36 37 38 39 40 41 42 43
S. aureus   1.56 3.13 3.13 3.13 0.78 <0.2 <0.2 0.39  0.39 0.78
S. aureus (ANT(4 )) 1.56 6.25 12.5 6.25 0.78 1.56 3.13 6.25 12.5 6.25
E. coli 0.78 12.5 >100 25 3.13 0.78 0.39 1.56  0.78 1.56
E. coli (AAC(6 )) 100 >100 >100 >100 >100 >100 >100 >100 >100 >100
E. coli (APH(3 )-I) 1.56 12.5 12.5     6.25  3.13 0.78 0.78 1.56 0.78 3.13
E. coli (ANT(2 )) 3.13 >100 >100 >100 >100 1.56 1.56  3.13 3.13 6.25
P. aeruginosa 3.13 12.5 6.25 3.13 3.13 0.78 1.56 1.56 3.13 3.13
P. aeruginosa
(APH(3 )-I)
 
6.25 12.5 25 12.5 50 12.5 12.5  12.5 25 12.5
P. aeruginosa
(AAC(6 ))
 
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100
P. aeruginosa
(AAC(3))
 
12.5 25 >100 25 12.5 12.5 12.5 25 50 12.5
P. aeruginosa
(APH(3 )-III)
 
12.5 >100 >100 >100 50 12.5  100 25 100 25

Fluoro-substituted derivatives of amikacin have been obtained by direct functionalisation (Scheme 5) [28]. 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

Bacterial Strains  arbekacin amikacin 46 47
S. aureus <0.2 <0.2 <0.2 <0.2
S. aureus (ANT(4 )) 0.78 25 50 6.25
E. coli 0.39 <0.2 0.39 0.39
E. coli (AAC(6 )) >100 50 50 12.5
E. coli (APH(3 )-I) 0.78 0.78 0.39 0.78
E. coli (ANT(2 ))    0.39 6.25 0.78 1.56
P. aeruginosa <0.2 0.78 0.39 <0.2
P. aeruginosa (APH(3 )-II) 1.56 3.12 1.56 0.78
P. aeruginosa (AAC(6 )) 3.12 25 >100 6.25

A number of different functional groups at the N-1 position have been introduced to explore the SAR of amikacin like series (Figure 5) [32].
Among these derivatives, compounds 49, 50, and 59 display superior activity (Table 6). The presence of 2-OH with S-configuration on the side chain plays a critical role. Interestingly, compound 59, is more active than amikacin and has a shorter chain with rspect to the normal AHB scaffold. Compounds 54 and 59 showed also activity against P. aeruginosa (ATCC 27853) (IC50 6.25 mg/mL). Unfortunately, both compounds are inactive against resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA) (ATCC 33591).

Figure 5. Kanamycin derivatives at N-1.

TABLE 6. MIC (mg/mL) of Kanamycin derivatives at N-1

Compounds S. aureus

(ATCC 13709)

 E. coli

(ATCC 25922)

Compounds S. aureus

(ATCC 13709)

 E. coli

(ATCC 25922)

Kanamycin A 1.22.5 2.55 52 >10 510
Kanamycin B  0.30.6 1.22.5 53 >10 >10
Tobramycin  0.30.6 0.61.2 54  2.55 2.55
Amikacin 1.22.5 1.22.5 55 >10  2.55
Paromomycin  1.22.5 2.55 56  2.55 >10
48  >10 >10 57  >10 >10
49  1.22.5 510 58 2.55 >10
50 1.22.5 2.55 59  0.61.2 0.61.2
51  >10 2.55      

Other functionalities at the N-1 position, such as a 2-aminoethanesulfonyl group, have also been attempted (Figure 6) [30]. The introductionof this functional group provided similar activity against E. coli, which is expected from an amikacin-type aminoglycoside (Table 7). However, compounds, 6063, 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

Bacterial strains kanamycin B 60 61 62 63
S. aureus 0.39 6.25 6.25 <0.39 3.12
E. coli K-12 0.78 1.56  3.12 1.56 12.5
E. coli (APH(3 )-I) Inactive 1.56 3.12 1.56 6.25
E. coli (ANT(2 ))    12.5 3.12 6.25 3.12  50
P. aeruginosa 50 12.5 3.12 1.56 6.25
P. aeruginosa (APH(3 )-I) 100 >100 25 25 >100
P. aeruginosa (AAC(3)) >100 >100 25 25 >100

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
amikacin.

Scheme 6. Synthesis of pyranmycin and kanamycin B analogs

TABLE 7. MIC (mg/mL) of Pyranmycin and Kanamycin B analogs

 Compound E.coli TG1 E.coli TG1

(APH(3 )-I)

E.coli TG1

(AAC(6 )/APH(2 ))

Butirosin 0.5 0.25
Ribostamycin 2 Inactive 8
67 4 4 4
Amikacin 1 0. 5 1
Kanamycin 4 Inactive Inactive
68 4 2 2
69 0.25 1

3'-Deoxygenated Aminoglycoside: Tobramycin analogs

Synthesis methods for the direct modification of tobramycin have been reported [33]. 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

Compounds S. aureus

(ATCC 13709)

E. coli

(ATCC 25922)

Compounds S. aureus

(ATCC 13709)

E. coli

(ATCC 25922)

Kanamycin A 1.22.5 2.55 72  >10 2.55
Kanamycin B 0.30.6 1.22.5 73 >10 510
Tobramycin 0.30.6 0.61.2 74  >10 2.55
Amikacin 1.22.5 1.22.5 75 510 12
Paromomycin 1.22.5 2.55 76 2.55 0.61
Neamine >10 >10      

Dimeric compounds

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

Compounds E. coli

(ATCC 25922)

Compounds E. coli

(ATCC 25922)

Compounds E. coli

(ATCC 25922)

Compounds E. coli

(ATCC 25922)

Neamine 50100 Kanamycin 12.5 81 12.525 85 6.25
Neomycin 3.1 Paromomycin 6.25 82 100 88 31
Gentamicin 1.6 Tobramycin 3.1 83 100 90 125
Ribostamycin 12.5 80 2550 84 12.5    

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

Bacterial strain Resistance Tobramycin 82 96 97
S. aureus (ATCC 33591) MRSA    >64 16 16
P. aeruginosa (strain 609) Resistant 128 8 4
P. aeruginosa (strain 663) Resistant 128 8 8
P. aeruginosa (PAE−NUH05) Resistant 100 6.3 3.1
P. aeruginosa (PAE−NUH06) Resistant   100 12.5 6.3
P. aeruginosa (27 EN) Resistant 4 1
P. aeruginosa (98 EN) Resistant 16 0.5
P. aeruginosa (89 EN) Resistant 64 4

 


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