Apralogs: Apramycin 5- O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant

Article abstract of DOI:10.1021/jacs.9b11601

Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-β-d-ribofuranosides, 5-O-β-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.

Full text of DOI:10.1021/jacs.9b11601

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Article
Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved
Antibacterial Activity and Ribosomal Selectivity and Reduced
Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant
Jonathan C. K. Quirke, Parasuraman Rajasekaran, Vikram A Sarpe, Amr Sonousi, Ivan
Osinnii, Marina Gysin, Klara Haldimann, Qiao-Jun Fang, Dimitri Shcherbakov, Sven N.
Hobbie, Su-Hua Sha, Jochen Schacht, Andrea Vasella, Erik C. Böttger, and David Crich
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b11601 • Publication Date (Web): 02 Dec 2019
Downloaded from pubs.acs.org on December 5, 2019
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Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved
Antibacterial Activity and Ribosomal Selectivity and Reduced
Susceptibility to the Aminoacyltranserferase (3)-IV Resistance
Determinant.
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a,b,c,d,†
a,c,d,†
a,c,d,†
d,†,‡
Jonathan C. K. Quirke,
Parasuraman Rajasekaran,
Vikram A Sarpe,
Amr Sonousi,
e
e
e
f
e
Ivan Osinnii, Marina Gysin, Klara Haldimann, Qiao-Jun Fang, Dimitri Shcherbakov, Sven N.
Hobbie, Su-Hua Sha, Jochen Schacht, Andrea Vasella, Erik C. Böttger, and David Crich.
e
f
g
h,*
e,*
a,b,c,d,*
a) Department of Pharmaceutical and Biomedical Sciences, University of Georgia, 250 West Green Street, Athens, GA
0602, USA
3
b) Department of Chemistry, University of Georgia, 140 Cedar Street, Athens, GA 30602, USA,
c) Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
d) Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, MI 48202, USA
e) Institute of Medical Microbiology, University of Zurich, Gloriastrasse 28, 8006 Zürich, Switzerland
f) Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Walton Research
Building, Room 403-E, 39 Sabin Street, Charleston, SC 29425, USA
g) Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan, 1150 West Medical
Center Drive, Ann Arbor, Michigan 48109, USA
h) Organic Chemistry Laboratory, ETH Zürich, Vladimir-Prelog-Weg 1-5/10, 8093 Zürich, Switzerland
ABSTRACT: Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics
characterized by a mono-substituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because
of its unusual structure apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the
aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all
aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make
apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of
multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind we describe the design, synthesis,
and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the
antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes, and of overcoming the rare
aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented
mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-β-D-ribofuranosides, 5-O-β-D-
eryrthofuranosides and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays
with wild-type bacterial and humanized bacterial ribosomes, and extensive antibacterial assays with wild-type and resistant Gram-
negative bacterial carrying either single or multiple resistance determinants. Ex-vivo studies with mouse cochlear explants confirm
the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used
to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in-vivo.
†
These authors contributed equally.
‡
Current address: Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo
11562, Egypt.
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Introduction
Apramycin 1 (Figure 1), originally known as nebramycin factor 2 and produced by Streptomyces
tenebrarius, is an atypical 2-deoxystreptamine (DOS) aminoglycoside antibiotic (AGA) first
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reported by workers at Eli Lilly, and patented for use in veterinary medicine, where it continues
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to find application. Structurally, apramycin is characterized by an unusual eight-carbon dialdose
in the form of a bicyclic hemiacetal that is linked by glycosidic bonds to the 4-position of 2-
deoxystreptamine and, via an unusual α,β-1,1’-linked disaccharide motif, to 4-amino-4-deoxy-α-
D-glucopyranose.6, 7 The antibacterial activity of apramycin and its unusual bicyclic dialdose motif
spurred several synthetic studies8-11 culminating in a total synthesis by the Tatsuta group in 1984.12
Most work in the area, however, has focused on derivatization and modification of the natural
product with the goal of improving antibacterial activity. Thus, the effect of limited modifications
at the 5-, 6-, 3’-, N1-, N2’-, N7’-, N4’’-, O8’- and O6’’-positions,12-27 and most recently of double
2
8
and triple modifications at the 5-, 6-, and 4’’-positions, have been widely described.
The unusual structure of apramycin is such that it is not modified by the majority of the
aminoglycoside-modifying enzymes (AMEs),21, 29-32 with the exception of the aminoglycoside N-
3
0
acetyl transferase AAC(3)-IV. The AAC(3) N-acetyltransferases are present in various isoforms
and constitute a primary mechanism of resistance to AGAs.33-38 Because of its structure, the
antibiotic activity of apramycin is not thwarted by the presence of the ribosomal methyltransferases
(
RMTases) acting on N7 of G1405 in the drug binding pocket of the bacterial ribosome,21, 30, 39
4
0
whose presence blocks the action of all AGAs in current clinical practice, including the recently
introduced plazomicin.41-43 Consequently, apramycin displays broad spectrum activity against a
wide range of Gram-negative and Gram-positive pathogens in vitro and in-vivo, including drug-
resistant pathogens carrying the most prevalent AGA resistance determinants.30-32, 39, 44-50
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OH
O
H2N
HO
OH
6'
NH
O
HO
O
O
H2N
H2N
O
NH2
HO
5
6
1
OH
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Figure 1. Structure of apramycin
Using a series of cell-free translation assays with wild-type and humanized bacterial ribosomes,51-
5
3
we have discovered that apramycin enjoys excellent selectivity for prokaryotic over eukaryotic
and especially over the mitochondrial and A1555G mutant mitochondrial ribosomes,21, 54 whose
inhibition is considered to be the root cause of AGA-induced ototoxicity.38, 52, 55-57 This was
reinforced by ex-vivo cochlear explant and in-vivo ototoxicity studies in the guinea pig model,
which testified to a low ototoxic potential of apramycin.21
The ready availability by fermentation, lack of susceptibility to most common resistance
determinants, and low levels of ototoxicity displayed in the guinea pig model combine to make
apramycin an attractive candidate for use in the clinic to combat multidrug resistant infections
including carbapenem-resistant enterobacteriaceae (CREs) and other ESKAPE pathogens.58
Apramycin is also an excellent substrate on which to base the development of improved next-
generation AGAs. Such a compound would ideally i) not suffer from susceptibility to the AAC(3)-
IV resistance determinant, ii) display improved activity levels compared to the parent, and iii)
retain excellent selectivity between prokaryotic and eukaryotic ribosomes predictive of reduced
ototoxicity. With this in mind, we began such a program several years ago reporting first on the
2
7
45, 54
importance of the 6’-hydroxyl group on activity, since when several groups,
but most notably
that of Kirby,31, 32, 39, 46-50, 59 have drawn attention to the potential of apramycin as a substrate for
the development of a next-generation AGA. We now report on an extensive program of work
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conducted with these aims in mind, and culminating in a series of derivatives at the 5-position of
apramycin that display all of the requisite characteristics.
Results and Discussion
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Design
Apramycin derives its antibacterial activity from binding to the decoding A site in helix 44 of the
3
0S subunit of the bacterial ribosome in the same manner as both the more common 4,5-
disubstituted DOS series of AGAs and the isomeric 4,6-series that find current application in the
clinic, exemplified by paromomycin 2 and gentamicin 3, respectively.21, 60-63 Alternative modes
of binding of apramycin to the bacterial ribosome, demonstrated by crystallographic and NMR
studies with short sequences of nucleotides,64, 65 we consider to be of only minor relevance in view
of the importance of the 6’-hydroxy group and its axial location on the bicyclic system.27
Consideration of the structures of apramycin, paromomycin, and gentamicin suggested that
appendage of an aminosaccharide or disaccharide to the 5-position of apramycin would provide a
derivative that benefits from the largely electrostatic attraction for the decoding A site that rings
6
6
III and IV are known to provide for the 4,5-series AGAs, without incurring susceptibility to the
RMTs acting on G1405. Further, consideration of the existing data on AGA susceptibility to
resistance determinants suggested that substitution at the 5-position might afford protection against
the AAC(3)-IV from which apramycin suffers, as typical 4,5-AGAs are little targeted by AAC(3)
N-acetyltransferases.35 We elected not to target the apramycin 6-position for derivatization, and
so not to take advantage of the extra affinity provided by ring III of the 4,6-AGAs, as we
anticipated that such compounds would fall victim to the A1405 RMTs, whose effectiveness arises
from blocking the direct hydrogen bond between ring III of the 4,6-series and A1405 N7. More
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pertinently, we were encouraged by the 1981 report of Abe and coworkers that 5-O-β-D-
ribofuranosylation of the apramycin derivative 4, providing 5 after deprotection, did not incur any
reduction of antibacterial activity (Figure 2).15
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R2
OH
O
NHR1
O
HO
HO
I
H2N
NH2
H2N
2
H N
O
II
O
HO
5''
O
NH2
HO
NH2
O
III
OH
O
H2N
O
OH
IV O
HO
O
OH
HN
OH
OH NH2
3
, gentamicin C1A, C1 and C2
1
2
1 2
(
R R = H = C1A, R R
=
Me = C1,
2, paromomycin
R1 = H, R2 = Me = C2)
OAc
O
OH
O
EtO2CHN
AcO
H2N
OAc
HO
OH
NAc
O
NH
O
AcO
HO
O
O
O
O
NHCO2Et
H2N
O
EtO2CHN
H2N
O
O
O
NHCO2Et
OTHP
NH2
HO
HO
OH
HO OH
4
5
Figure 2. Structures of paromomycin, the gentamicins, and apramycin derivatives 4 and 5
Synthesis
The apramycin derivative 6 was prepared in three straightforward steps from apramycin
as described previously.27 Consistent with previous reports on the regioselective
derivatization of 4-O-monosubstituted derivatives of 2-deoxystreptamine at the 6-
position,15, 67, 68 treatment of 6 with a controlled amount of acetic anhydride in pyridine
gave the 5,2’’,3’’-6’’-tetra-O-acetate 7 in 61% yield (Scheme 1). Likewise, controlled
exposure of 6 to benzoyl chloride in pyridine afforded the corresponding tetrabenzoate 8
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in 86 % yield. These efficient four-step preparations of selectively protected apramycin
mono-ols for use in functionalization at the 5-position compare favorably to the methods
employed in the synthesis of 4 by Abe and coworkers.15
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OH
O
O
N3
HO
O
N
O
HO
O
O
N3
N3
6
O
N3
HO
OH
Ac2O, py,
or BzCl, py
OR
O
O
N3
RO
O
N
O
RO
O
O
N3
N3
7: R = Ac, 61%
: R = Bz, 86%
O
N3
HO
8
OR
Scheme 1. Preparation of the Selectively Protected Apramycin Derivatives 7 and 8.
Alcohols 7 and/or 8 were then subjected to glycosylation by a series of glycosyl donors, prepared
as described in the Supporting Information, to give the glycosides described in Table 1. Of note,
the paromobiosyl donor 10 was readily prepared by Lewis acid-mediated cleavage of perazido-
peracetyl paromomycin in the presence of 4-thiocresol, adapting methods described earlier by
Hanessian, Swayze, and Wong and coworkers,69-71 followed by the adjustment of protecting
groups and oxidation levels. All donors carried ester protecting groups at the 2-position so as to
benefit from stereodirecting neighboring group participation, and correspondingly high levels of
selectivity were observed in most cases. The anomeric configuration of the newly introduced
1
3
glycosidic bonds was assigned based on the chemical shift of the anomeric carbon in the C NMR
spectra consistent with established rules (Table 1).72
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Table 1. Glycosylation Reactionsa,b
OX
O
O
N3
XO
O
N
O
XO
O
O
N3
N3
O
N3
O
7: X = Ac, 8: X = Bz
R
1
1
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OX
Glycosylation
R = H
R = glycosyl
13C NMR
δ C1'''
R, Yield,
Donor
Conditions
Selectivity (β:α)
AcO
O
AcO
O
O
CCl3
BF
3 2
.OEt ,
NH
106.1
0
ºC, 4 h
AcO OAc
AcO OAc
18, 95%,
9

: = 9:1
PMBzO
O
PMBzO
O
S
pTol
O
PMBzO
N3
N3
PMBzO
N3
N3
Tf
rt, 6 h
2
O,
O
OPMBz
1
06.8
O
O
OPMBz
O
OPMBz
OPMBz
10
19,
48%

only
BnO
BnO
OPNB
O
O
BF
0 ºC, 40 h
3
.OEt
2
,
α: 102.8
β: 106.9
N3
N3
O
OPNB
O
OPNB
20, 43%
:  1:1.15
BnO
11

BnO
OPNB
O
O
BF
0 ºC, 40 h
3
.OEt
2
,
α: 102.9
β: 107.2
BnO
BnO
O
OH
O
2
OPNB
1
21, 25%
 = 1:1
O
O
O
CCl3
3 2
BF .OEt ,
-78 ºC, 4 h
NH
1
06.4
BzO OBz
BzO OBz
22, 50%
1
3

only
O
O
OAc
BF .OEt ,
0 ºC, 2 h
3
2
O
OAc
107.1
O
OAc
4b
23,b 34%
1

only
N3
N3
O
O
SPh
NIS, AgOTf
α: 102.5
β: 106.7
-20 - 0 ºC, 6 h
PNBO OPNB
PNBO OPNB
24, 78%
15

 = 1:0.6
Cbz
N
Cbz
N
N3
N3
O
O
O
CCl3
BF
3 2
.OEt ,
1
1
06.9
07.0
NH
-30 ºC, 6 h
BnO OAc
5, 93%
BnO OAc
2
16

only
Cbz
Cbz
N
N3
N
N3
O
O
O
CCl3
BF
3 2
.OEt ,
NH
-30 ºC, 6 h
BnO OAc
BnO OAc
26, 94%
17
 only
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5
5
6
a) Unless otherwise noted the tetraacetate 7 was employed as glycosyl acceptor; b) In this example the
tetrabenzoate 8 was employed as glycosyl donor.
The allyl ether of glycoside 23 was further derivatized by treatment with catalytic osmium
tetroxide in the presence of N-methyl morpholine N-oxide (NMO) according to the Van Rheenan
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
3
protocol to give the corresponding diol as a mixture of diastereomers. Oxidative cleavage with
7
4
sodium metaperiodate on silica gel then afforded the corresponding aldehyde 27 in 97% yield
over two steps that was immediately subjected to reductive amination in the presence of sodium
cyanoborohydride75 to give, after saponification of the esters and carbamate, the desired
aminoalkyl ethers. Finally, hydrogenolysis followed by chromatography over Sephadex C25 and
lyophilization of aqueous acetic acid provided the fully deprotected AGAs 28-32 in the form of
their peracetate salts suitable for assay (Table 2).
Table 2. Post-Glycosylation Modifications and Deprotection of Erythrosyl Apramycin
Derivatives
OBz
O
O
N3
BzO
O
i) Amine, AcOH, 0 C
N
O
O
ii) NaBH CN, rt
3
BzO
O
O
iii) NaOH, 80 °C
N3
O
N3
iv) Pd(OH) , H , 50 psi
OH
2
2
O
N3
O
O
OBz
RHN
X
O
OAc
2
2
8: R = H
9: R = CH2CH2OH
30: R = CH2CH2NH2
i) OsO4, NMO
ii) NaIO4, silica, 97%
over two steps
23, X = CH2
27, X = O
31: R = CH2CH2CH2NH2
2: R = CH2CH2CH2NMe2
3
Yield from
Amine
Product
27
18%
3%
BnNH
NCH CH
NCH CH
NCH CH CH
NCH CH CH
2
28
29
30
31
32
H
2
2
2
OH
NH
NHCbz
NMe
H
2
2
2
2
18%
3%
H
2
2
2
2
H
2
2
2
2
2
8%
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Initial attempts to prepare a series of acyclic variants of the above 5-O-glycosyl apramycin
derivatives were thwarted by migration of the ester between the 5- and 6-positions under a variety
of conditions for the installation of an allyl ether, resulting in complex reaction mixtures. The
optimum conditions employed silver oxide and allyl iodide when a clean ether could be isolated
in 59 and 78% yield in the acetyl and benzoyl series, respectively. Unfortunately, extensive 2D
NMR experiments (Supporting Information) revealed ester migration to have taken place and the
products to be the 6-O-allyl ethers 33 and 34 (Scheme 2). Although this ester migration with
derivatization complicated the synthesis of the desired derivatives at the 5-position, it may prove
useful in future work at the 6-position; in this spirit the peracetate 33 was converted to the
dihydroxyl propyl and hydroxyethyl derivatives 35 and 36 suitable for eventual deprotection as set
out in Scheme 2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OR
O
O
N3
N3
RO
O
O
N3
N
O
CH2=CHCH2I,
Ag2O, CH2Cl2
RO
RO
O
O
O
N3
N3
O
HO
N3
33, R = Ac, 59%
34, R = Bz, 78%
7
8
, R = Ac
, R = Bz
OR
NMO, OsO4 (cat)
OAc
O
O
N3
N3
AcO
O
N
O
AcO
O
RO
N3
O
O
i) NaIO4,
ii) NaBH4
O
N3
N3
O
HO
N3
OH
AcO
O
35, 71%
36, 52%
HO
Scheme 2. Allylation and Subsequent Derivatization of Apramycin at the 6-Position.
Fortunately, the lower reactivity of the 5-OH group apparent in the preparation of the esters 7 and
8
(Scheme 1) was also operative under more basic conditions, such that benzylation of 6 with
sodium hydride and a controlled amount of benzyl bromide in DMF such that the requisite
,2’’,3’’,6’’-tetra-O-benzyl ether 37 could be isolated in 38% yield along with 23% of the
4
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
perbenzyl ether 38 (Scheme 3). The location of the free-alcohol in 37 was readily apparent
following acetylation to give 39 and inspection of its 1 and 2D NMR spectra. Allylation of 37
with sodium hydride and allyl iodide then provided the 5-O-allyl ether 40, while treatment with
sodium hydride and 3-bromopropylamine hydrogen bromide gave the aminopropyl ether 41, in 72
and 32% yield, respectively (Scheme 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
O
O
N3
HO
NaH, BnBr
DMF
O
N
O
HO
O
O
N3
N3
O
6
HO
N3
OH
OBn
O
N3
BnO
O
O
N
O
BnO
O
O
N3
N3
O
N3
RO
OBn
37, R = H, 38% + 38, R = Bn, 23%
Ac2O,
py
NaH,
CH2=CHCH2I
NaH,
Br(CH2)3NH2.HBr
N3
N3
N3
O
O
O
O
N3
O
N3
N3
AcO
OBn
9, 68%
OBn
OBn
H2N
3
40, 72%
41, 32%
Scheme 3. Partial Benzylation of Apramycin Giving Rise to 5-O-Alkyl Derivatives.
Hydroboration of 40 with oxidative workup gave the 3-hydroxypropyl ether 42 in 58% yield, while
reaction with NMO and catalytic osmium tetroxide afforded an inseparable mixture of
diastereomeric diols 43 in 78% yield. This mixture of diols was treated with 2,4,6-
triisopropylbenzenesulfonyl chloride in pyridine to give the corresponding chromatographically
separable sulfonates 44 and 45 in 66% combined yield. The sulfonate group was displaced from
the individual diastereomers with sodium azide to afford derivatives 46 and 47 in 81 and 67%
yield, respectively. Further, diol 43 was cleaved with sodium metaperiodate to give the aldehyde
4
8, which upon reduction with sodium borohydride delivered alcohol 49 in 60% yield over two
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steps. Alternatively, aldehyde 48 was subjected to reductive amination with acetic acid and
sodium cyanoborohydride to give compounds 50, 51, and 52 in 74, 55, and 67% yield over two
steps, respectively (Scheme 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OBn
O
N3
BnO
O
O
N
O
BnO
O
O
N3
N3
O
i) BH3.THF
N3
O
40
OBn
O
ii) H2O2, NaOH
NMO,
OsO4 (cat)
N3
O
N3
HO
N3
N3
O
O
HO
OBn
HO
OBn
42, 58%
43, 78%
i-Pr3C6H2SO2Cl, py
NaIO4
N3
N3
O
O
N3
HO
RO
O
O
N3
OBn
O
OBn
48
4
4, R = Trisyl, 31%
NaN3,
DMF
NaBH4
46, R = N3, 81%
+
N3
Amine,
NaBH3CN,
HOAc
N3
O
O
O
N3
HO
N3
HO
RO
O
OBn
OBn
49, 60% over two steps
45, R = Trisyl, 20%
47, R = N3, 67%
NaN3,
DMF
N3
O
O
N3
RHN
OBn
5
0, R = CH2Ph, 74% over two steps
51, R = (CH2)2NHCbz, 55% over two steps
2, R = (CH2)3N3, 67% over two steps
5
Scheme 4. Preparation of 5-O-alkyl apramycin derivatives.
Finally, in order to assign the configuration of the diastereomeric azido alcohols alcohols 46 and
4
7, and by extrapolation the precursor diols 44 and 45, alcohol 37 was alkylated with the
enantiomerically pure triflate 53, obtained by triflation of commercial S-isopropylidene glycerol,
giving 54 in 85% yield. Cleavage of the acetonide, followed by selective sulfonylation then gave
4
5 (Scheme 5) with the S-configuration in the pendant chain, whose NMR spectra correlated with
the more polar of the two isomers prepared in Scheme 4.
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OH
O
O
N3
HO
O
N
O
HO
O
O
N3
N3
O
HO
3
7
N3
OBn
NaH, DMF
N3
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N3
OBn
O
O
O
OTf
54, 85%
53
i) 80% AcOH, rt
ii) i-Pr3CH2SO2Cl, py
N3
O
O
HO
N3
OBn
TrisylO
4
5, 45%
Scheme 5. Alternative Preparation of S-45.
All glycosides and 6-O-ethers were deprotected by a sequence of saponification, followed
by either hydrogenolysis or Staudinger reduction of the azides as detailed in the
Supporting Information. Final purification was achieved by ion exchange chromatography
over Sephadex C25 followed by lyophilization from acetic acid to give the final compounds
in the form of their peracetate salts (Table 3). In the case of the 5-O-ethers the sequence
of saponification and hydrogenolysis was reversed, such that the benzyl ethers and
azides were removed before saponification (Table 3).
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Table 3. Deprotection of Apramycin 5- and 6-O-Derivatives
O
OX
OH
O
O
N3
XO
O
H2N
HO
Conda,b,c
OH
N
O
NH
O
XO
HO
O
O
O
O
N3
NH2
N3
O
RO
H2N
O
RO
N3
NH2
OR'
OR'
Subs
Product,
Subs
49
Product,
Yield
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Yield
HO
O
HO
1
1
8
9
65, 12%
HO OH
55, 69%
HO
O
OH
OH
NH2
HO
OH
43
O
O
66, 14%
H2N
OH
56, 35%
HO
O
H2N
67, 36%
20α
50
41
H2N
O
OH
5
7 85%
HO
O
H2N
2
0β
H2N
68, 19%
O
OH
57, 95%
HO
O
OH
H2N
2
1
2
4
46
47
51
HO
O
OH
69, 11%
5
8, 48%
O
OH
H2N
2
HO OH
7
0, 18%
59, 71%
H2N
O
H2N
N
H
2
2
2
7
1, 12%
HO OH
6
0, 24%
H
N
H2N
O
H2N
N
H
5
6
52
33
7
2, 7%
HO OH
6
1, 66%
H
N
H2N
O
7
3, 75%
HO OH
62, 72%
HO
74, 63%
40
42
36
35
6
3, 20%
OH
HO
6
HO
4, 10%
75, 39%
a) X = R' = Ac, R = glycoside: (i) NaOH or Ba(OH)
2
3 2
; (ii) PMe , NaOH or H ,Pd/C; (iii) AcOH; b) X = R' =
Bn, R = alkyl: (i) H
Pd/C; (iii) AcOH
2
, Pd(OH)
2
; (ii) Ba(OH)
2
; (iii) AcOH; c) X = R = Ac, R' = alkyl: (i) NaOH; (ii) H ,
2
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5
6
Activity and Selectivity at the Drug Target
To assess the influence of modification on activity at the level of the target, all compounds were
tested for their ability to inhibit the production of luciferase in cell-free translation assays with
isolated bacterial ribosomes. To determine the selectivity for bacterial over eukaryotic ribosomes
parallel screening was conducted against a series of engineered bacterial ribosomes carrying the
complete thirty base pair decoding A site of the human mitochondrial ribosome, its A1555G
mutant, and the human cytoplasmic ribosome (Figure 3, Table 4).51, 52, 76-78 Selectivity for
inhibition of the bacterial over mitochondrial ribosomes is particularly important as it has been
demonstrated that inhibition of the mitochondrial ribosome in the cochlear hair cells, where AGAs
have been shown to persist up to thirty days after administration in spite of their otherwise rapid
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
79
52, 55
clearance from the body in the urine, is a primary cause of AGA-induced ototoxicity.
Further, inhibition of the mutant A1555G mitochondrial ribosome is known to be a cause of
hypersusceptibility to AGA-induced ototoxicity in genetically predisposed subjects.53, 77, 80
Selectivity for inhibition of the bacterial over the mitochondrial ribosomes is consequently a
feature that is predictive of reduced ototoxicity.21, 54, 81, 82 Selectivity over the cytoplasmic
ribosome on the other hand is viewed as indicative of low levels of systemic toxicity. For ease of
comparison, in Table 4 and all subsequent tables, compounds are grouped into four distinct sets
according to the type of modification: ribofuranosyl; erythrofuranosyl; 5-O-alkyl; and 6-O-alkyl
(Figure 4).
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Decoding A sites of prokaryotic and eukaryotic ribosomes. The bacterial AGA
binding pocket is boxed. The bacterial numbering scheme is illustrated for the AGA
binding pocket. Changes from the bacterial ribosome binding pocket are coloured green.
The A1555G mutant conferring hypersusceptibility to AGA ototoxicity is coloured red.
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5
-O-ribofuranosyl series
5-O-erythrofuranosyl series
5-O-alkyl series
OH
O
OH
OH
O
H N
O
H N
2
2
H N
2
HO
OH
HO
OH
HN
O
HO
OH
HN
O
HN
O
HO
HO
HO
O
O
O
O
O
O
H N
H N
2
2
H N
H N
2
H N
2
2
H N
O
2
O
R1
O
NH2
R2
NH2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
NH2
O
O
O
O
R1
OH
OH
OH
1
2
6
3: R = Me, R = H
O
OH
R2
O
OH
R1
1
2
64: R = OH, R = H
1
2
6
5: R = CH OH, R = H
2
1
2
59: R1 = H
28: R1 = (CH ) NH
1
2
5
5
5
5
6
6
6
5: R = OH; R = H
66: R = CH OH, R = OH
2
1
2
1
2
6: R = OH; R = 2,6-diaminoidopyranosyl
67: R = NH , R = H
2
2
2
2
1
2
1
1
2
7: R = OH; R = (CH ) NH
8: R = OH; R = (CH ) OH
29: R = (CH ) NH(CH ) OH
68: R = CH NH , R = H
2 2
2
2
2
2 2
2 2
1
2
30: R1 = (CH ) NH(CH ) NH
1
2
2
2
2 2
2 2
2
69: R = CH NH , R = OH (S-isomer)
2 2
1
2
1
1
2
0: R = NH ; R = H
31: R = (CH ) NH(CH ) NH
70: R = CH NH , R = OH (R-isomer)
2 2
2
2 2
2 3
2
1
2
1
1
2
1: R = NH(CH ) NH ; R = H
32: R = (CH ) NH(CH ) NMe
71: R = NH(CH ) NH , R = H
2 2 2
2
2
2
2 2
2 3
2
1
2
1
2
2: R = NH(CH ) NH ; R = H
72: R = NH(CH ) NH , R = H
2
3
2
2 3
2
6
-O-alkyl series
OH
O
OH
O
OH
O
HO
HO
H N
2
H N
NH
2
2
HO
OH
HO
OH
HN
3'
H N
2
O
HN
HO
HO
HO
H2N
O
O
HO
H N
2
O
O
O
O
O
5
''
NH2
H N
O
2
O
O
O
OH
NH2
H N
2
H N
2
HO
H N
2
H N
2
O
III
OH
O
O
NH2
NH2 H2N
O
O
OH
O
OH OH
R2
O
O
OH
R1
O
HO OH
77: ribostamycin
O
OH NH2
76: lividomycin B
1
2
HO
73: R = Me; R = H
NH2
1
2
57
74: R = OH; R = H
(
3'-deoxyparomomycin)
1
2
7
5: R = CH OH; R = OH
2
Figure 4. 5-O-Ribofuranosyl, 5-O-Erythrofuranosyl and 5-O-Alkyl Series of Apramycin Derivatives Studied, and the
Comparators Lividomycin B and Ribostamycin.
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a,b
Table 4. Antiribosomal Activities and Selectivities (IC , µM).
50
Antiribosomal Activity
Selectivity
wt
Mit13
A1555G
Cyt14
Mit13
A1555G
Cyt14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Apramycin
0.11±0.02
127±29
134±42
130±3.5
1123
1186
1155
Ribosyl series
5
5
5
6
0.16
0.12
0.35
0.08
0.31
0.12
0.046
439
2.7
192
79
904
272
0.49
285
30
456
475
22
257
253
1227
81
2815
22
1745
4
3045
184
735
3173
3992
679
5
5
7α
7β
549
994
2940
949
4152
814
288
1483
916
3261
58
60
6
113
191±67
109
150±60
1
133±38
2898
±
0.008
6
2
0.09
80
72
61
917
820
696
Erythrosyl
series
5
28
29
9
0.11
0.02
0.14
329
90
180
208
12
65
474
194
495
2931
4471
1285
1010
1857
573
464
397
4230
9646
3536
2400
3
0
0.030
30±0.5
11.9±3.6
72±14
±
0.006
0.07
0.05
3
3
1
2
46
21
7.4
4.8
204
95
657
403
106
92
2914
1827
5
-O-alkyl series
63
64
1.8
0.35
nd
299
nd
nd
nd
nd
nd
755
nd
nd
nd
1798
1084
1120
2155
nd
nd
nd
6
6
6
6
6
5
6
7
8
9
0.60
0.21
0.11
0.21
2393
434
99
327
124±32
nd
nd
nd
100
210
152±46
3998
2077
906
1553
3100
375
119
236
86±23
nd
914
998
3813
0.040±0.0
1
3
7
71
72
0
0.10
0.10
0.07
158
103
86
145
132
85
112
91
63
1565
1053
1235
1428
1357
1227
1110
938
902
6
-O-alkyl series
7
7
7
3
4
5
2.50
0.32
0.57
449
650
403
829
396
566
646
655
463
179
2055
707
331
1254
992
258
2071
812
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a) All values are single point determinations. For apramycin, 30, 61, and 69 the mean and
standard deviation of 3-5 determinations were assessed as these compounds were selected
for further ototoxicity studies. b) Selectivities are obtained by dividing the eukaryotic by the bacterial
values.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Consideration of the 5-O-(D-ribofuranosyl) series of compounds (55, 56, 57α, 57β, 58, 60, 61, and
6
2) indicates that the β-D-ribofuranosyl modification (55) provokes a relatively insignificant
reduction in inhibitory activity of the bacterial ribosome, but a 3-4-fold reduction in inhibition of
all three eukaryotic ribosomes, leading to significantly increased selectivity. This observation is
consistent with work from the Fridman lab, in which the installation of a β-D-ribofuranosyl moiety
at the 5-position of 4,6-AGAs led to an increase in selectivity for bacterial over cytosolic ribosomes
without any increase in antibacterial activity.83 The antibacterioribosomal activity in this series
can be improved by the incorporation of a further basic amino group in the form of an aminoethyl
appendage at the ribose 3-position (57β) but, surprisingly, not by addition of a paromobiosyl
moiety with two basic amino groups (56) to the same locus. The antibacterioribosomal activity in
this series also can be increased by the placement of a single basic amino group at the ribose 5-
position (60). Extrapolating from this modification, the appendage of an ethylene diamine and to
a lesser extent a propylene diamine moiety to the ribose 5-position affords compounds 61 and 62,
which both display increased antibacterioribosomal activity compared to apramycin. With respect
to selectivity, the single basic amino group attached to the ribofuranosyl ring of 57β moderates the
effect of the ribofuranosyl ring itself on selectivity over the mitochondrial ribosomes (wild-type
and mutant) but does not detract from selectivity over the cytosolic ribosome. The two basic amino
groups of the paromobiosyl moiety of 56 result in significantly increased activity against all three
eukaryotic ribosomes leading to an across the board notable reduction in selectivity. In addition
to their increased antibacterioribosomal activity compounds 60 and 62 display moderately
increased activity against the mutant mitochondrial ribosomes, such that they display overall
comparable selectivity to apramycin itself. In contrast, for 61 increased antibacterioribosomal
activity comes without a comparable increase in activity for the eurkaryotic ribosome, resulting in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
an excellent selectivity, particularly against the mitochondrial ribosomes. The relatively low
selectivity of the 5-amino-5-deoxy-ribosyl derivatives 60 and 62 for the bacterial over the
cytoplasmic ribosome is due to increased inhibition of the latter, consistent with the influence of
the 5’’-amino-5’’-deoxy modification on a series of ribostamycin compounds under consideration
for the treatment of read-through diseases.84 The contrast in selectivity between the homologs 61
and 62must arise from differences in basicity of the monoprotonated diamine moiety, with the
additional methylene group in 62 better insulating the second amine from the electron-withdrawing
effect of the ammonium group.85
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
It is noteworthy that the 5-O-β-D-erythrofuranosyl apramycin derivative 59, which lacks the
hydroxymethyl side chain of the cognate ribofuranosyl series, retains the excellent selectivity of
the ribofuranosyl analog 55 and displays antibacterioribosomal activity that is comparable with
the parent. This observation indicates that the potential inter-residue hydrogen bond from the 5’’-
hydroxy group to the 2’-amino group in 55, inferred by extrapolation from the X-ray crystal
structures of numerous 4,5-AGAs in complex with h44 of the bacterial decoding A site60, 86, 87 is
of little consequence. As in the ribofuranosyl series, the appendage of a monobasic aminoethyl
moiety to the erythrofuranose 3-position results in a compound 28 with increased activity against
the bacterial and eukaryotic ribosomes such that excellent selectivity is retained overall.
Replacement of the aminoethyl moiety in 28 by a hydroethylaminoethyl group (29) or by
aminoalkylaminoethyl groups (30-32) did not result in any further increase in activity or favorable
change in selectivity.
In contrast to the appendage of a D-erythro- or D-ribofuranosyl ring with the correct β-anomeric
configuration, the introduction of a simple alkyl (63), hydroxyethyl (64), or hydroxypropyl chain
(65) to the 5-position of apramycin has a detrimental effect on the ability to inhibit the bacterial
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ribosome. The placement of a dihydroxypropyl chain (66) or an aminoethyl (67) or aminopropyl
chain (68) however causes little or no loss of activity. The incorporation of a 3-amino-2-hydroxy
propyl ether at the 5-position results in an increase in antibacterioribosomal activity in a
configuration dependent manner, with the S-isomer 69 displaying 2-3-fold better activity than the
R-isomer 70 – presumably the result of a favorable hydrogen bonding interaction in the more active
isomer. As the same modification causes little change in the inhibition of the eukaryotic
ribosomes, the S-isomer exhibits notably improved selectivity over the parent apramycin. The
incorporation of a dibasic ethylenediamine (71) and especially a propylenediamine (72) residue
results in a modest increase in antibacterioribosomal activity, without detriment to selectivity. The
incorporation of simple propyl (73), hydroxyethyl (74) and dihydroxypropyl (75) chains at the 6-
position leads to a significant reduction in activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Antibacterial Activity Against Wild-type Bacterial Strains
All compounds were assayed for activity against a panel of wild-type Gram negative pathogens
(Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii,
Pseudomonas aeruginosa) obtained from the diagnostic department of the Institute of Medical
Microbiology, at the University of Zurich. All compounds were also assayed for activity against
a Gram positive Methicillin-resistant Staphylococcus aureus (MRSA) strain and an
aminoglycoside-resistant MRSA strain from the same source (Table 5). For the most part all
compounds displayed antibacterial activity in these screens consistent with their inhibition of the
bacterial ribosome discussed above. Thus, multiple compounds had MIC values similar to and in
some instances even increased MIC activity compared to the parent apramycin across the range of
pathogens screened. A notable exception to this general trend was Pseudomonas aeruginosa for
which markedly lower activity was seen in the ribo- and erythrofuranosyl series, with the exception
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of those compounds with mono- or dibasic chains at the ribose 5-position (60-62) or at the
erythrose 3-position (30 and 31). These compounds also mostly enjoyed increased MIC activities
comparable to apramycin across the whole panel of pathogens tested. In the acyclic ether series
of compounds the 3-amino-2-hydroxypropyl ethers of apramycin (69 and 70) displayed excellent
activity across the panel of strains, including P. aeruginosa, as did the diaminoethers 71 and 72.
In particular, it is noteworthy that 69 with the 3-amino-2S-hydroxy propyl ether at the 5-position
of apramycin consistently displayed increased activity across the entire panel of pathogens as
compared to the parent apramycin.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Table 5. Antibacterial Activities (MIC, μg/mL)a
Species
E. coli
K. pneu. Enterob. A. baum.
P.
aerug.
MRSA
MRSA
Features
WT
WT
WT
WT
WT
WT
APH(2')
ANT(4')-I
AAC(6')-I
AG042
8
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Strain
AG001
4
AG215
1-2
AG290
2-4
AG225
4
AG220
4
AG038
4
Apramycin
Ribosyl series
55
56
4
2
2
1
4
2
16
1-2
8
1-2
1-2
4
16
4
64
16
64
16-32
>64
4
4-8
2-4
64
2-4
16
2-4
32
2
64
2
16
8
32
nd
8
5
5
5
6
6
6
7α
7β
8
0
1
2
16
2-4
32
4
2
2-4
4
1-2
4
1-2
1
2
16
4-8
16
4-8
4-8
4
4
2
2
Erythrosyl series
5
2
2
3
3
3
9
8
9
0
1
2
8-16
1-2
2-4
1-2
2
4
0.5
2
1
1
4
2-4
4
2-4
1-2
1
8
4
8
4-8
4
4
32
16
32
4-8
8
16-32
2
8
1-2
2
2
16
4-8
8-16
4
4
2-4
1-2
0.5
16
5-O-alkyl series
6
6
6
6
6
6
3
4
5
6
7
8
32
8
16
8
4
8
16
2-4
8
4
1-2
2
64-128
16
16
64
16
32
16
4
8
4
4
8
64
16-32
64
16
4
8
2-4
4
32-64
8-16
32
8-16
2
4
1-2
4
64
8-16
16-32
nd
4
4-8
4-8
1
4
8-16
2-4
2
2-4
2-4
2
2-4
4
69
70
2
1
2
2-4
2
4-8
4-8
4
7
7
1
2
1
2
4
4
4
6
-O-alkyl series
7
7
75
3
4
64-128
16
32
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
64-128
64
64
a) All values were determined in duplicate using twofold dilution series
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Antibacterial Activity Against Resistant Bacterial Strains
In view of the already outstanding profile of apramycin in the face of common AMEs, we
concentrated on assessments of activity against a panel of clinical E. coli isolates carrying
genes for two isoforms of AAC(3), of which only AAC(3)-IV is commonly considered to
act on apramycin (Table 6).30, 32, 88, 89 Because several of the analogs are 5-O-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ribofuranosyl derivatives of apramycin, and because phosphorylation at the 5’’-site (the
primary alcohol of the ribofuranosyl side chain) by the promiscuous APH(3’)-I isoform90-
95
of the APH(3’)s is an established mechanism of resistance in the 4,5-series AGAs,96
we also screened for activity against isolates carrying the different APH(3’) isoforms
(Table 6). The 4,5-AGAs ribostamycin, paromomycin and lividomycin B were included as
positive controls.
Consistent with the insensitivity of the 4,5-AGAs ribostamycin and paromomycin to
AAC(3) resistance determinants, the appendage of a β-D-ribofuranosyl ring to the 5-
position of apramycin, as in 55, 56, 57β, 58, 61 and 62, largely restores activity in the
presence of AAC3-IV. This effect is maximal for 56 and 57β, carrying the full
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paromomycin rings III and IV disaccharide, or a 3-O-(2-aminoethyl)-β-D-ribofuranosyl ring
at the 5-position, respectively, neither of which suffer any significant loss of activity.
Together with the observation that 61 and 62 carrying dibasic amino groups at the
ribofuranosyl 5-position are not as effective as 56 and 57β in overcoming AAC(3)-IV, this
indicates that at least one correctly-placed basic amino group is required in addition to
the β-D-ribofuranosyl ring to fully compensate for the presence of AAC(3)-IV. In contrast
to the beneficial influence of the 3-O-(2-aminoethyl)-β-D-ribofuranosyl ring (57β) the
corresponding 3-O-(2-aminoethyl)-β-D-erythrofuranosyl derivative 28 suffers a 16-fold
loss of activity in the presence of AAC(3)-IV indicating that, in addition to the correctly
placed aminoethyl chain, the hydroxymethyl ribose side chain is important in blocking the
action of this AME. The positive influence of the hydroxymethyl group in 57β is
presumably related to its conformation-limiting hydrogen bond to N2’ anticipated from
structural studies of the 4,5-AGAs.61, 83, 97, 98. Whatever the reason for the loss of activity
in the presence of AAC(3)-IV on going from the ribofuranoside 57β to the corresponding
erythrofuranoside 28, it may be largely overcome by the replacement of the monobasic
side chain in 28 by a dibasic one as in the erythrofuranosides 30-32. All of the acyclic
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 26 of 57
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-O-alkyl ethers of apramycin fall victim to the AAC(3)-IV resistance determinant
indicating that the conformational restriction and/or steric bulk of the furanose ring in the
ribo and erythrofuranosyl series is an important factor in overcoming this class of AME.
As expected on the basis of the resilience shown by apramycin itself, none of the
derivatives studied suffered a significant loss of activity in the presence of the AAC(3)-IId
isoform. As the antiribosomal activity of the 6-O-alkyl series of compounds (Table 3) and
their MIC values against the standard panel of strains was disappointingly low, they were
not profiled against the resistant strains.
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Turning to the APH(3’) AMEs, the 5-O-ribosylated derivatives of apramycin 55 and 57β
both showed a slight 2-4-fold reduction in activity in the presence of strains carrying
APH(3’)-I, reflecting the ability of this AME to phosphorylate the 5’’-primary hydroxyl
group. However, these compounds clearly are poor substrates as the reduction in activity
does not match that seen with the 5-O-parombiosyl derivative 56 (8-16-fold) or with the
classical substrate lividomycin B (>32-fold). Replacement of the 5’’-hydroxy group in this
series of compounds by an amino group restores full activity in the presence of APH(3’)-
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Page 27 of 57
Journal of the American Chemical Society
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I as indicated by 57β and 62. As expected, none of the 5-O-erythrosyl apramycin
derivatives, 59, and 28-32, nor any of the apramycin 5-O-alkyl derivatives 63, 64, and 67-
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72, experienced a notable loss of activity in the presence of this AME. None of the
compounds tested showed any significant loss of activity in the presence of the APH(3’)-
IIa isoform when compared to the parent apramycin.
Overall, in the 5-O-β-D-ribofuranosyl series of compounds, derivatives 57β, 61 and 62
are optimal showing little or no loss of activity in the presence of the AAC(3)-IV and
APH(3’)-Ia AMEs, while in the erythrofuranosyl series 30-32 show the most promise.
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Table 6. Antibacterial Activities (MIC, mg/L) Against E. coli Strains with Acquired
AAC(3)and APH(3’) Resistancea
Resistance
WT
AAC(3)-IId
AAC(3)-IV
APH(3’)-I
APH(3’)-IIa
Determinant
0
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7
8
9
0
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9
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5
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7
8
9
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Strain
AG001
AG170
4-8
AG173
128-256
4-8
AG164
4-8
AG166
4-8
Apramycin
Ribostamycin
Paromomycin
Lividomycin B
4
4-8
2
4-8
>256
>256
>256
>256
>256
4-8
4
2-4
4-8
4-8
16
Ribosyl series
5
5
4
2
8
4-8
32
2
16-32
2
8-16
8-16
16
8
2-4
16
2
56
5
7α
7β
16
2-4
16
4
128
4
5
4
5
8
16
nd
2
32-64
nd
8-16
nd
8
60
nd
4
6
6
1
2
2
16
2
2-4
4
16
2-4
2-4
Erythrosyl series
59
8-16
1-2
2-4
1-2
2
16
1
128-256
8
8
2
2
2
3
8
9
0
16-32
64
1
4
4-8
1-2
1-2
1-2
4
4
8
2
31
2-4
1
4
4
3
2
1-2
4-8
2-4
5
-O-alkyl series
63
32
8
64
8
>128
128
>128
>64
128
128
16-32
64
32
8
32
4-8
16
nd
2-4
2-4
4
6
6
6
4
5
6
16
8
16
nd
2
16
nd
2
67
4
6
6
7
8
9
0
8
4
4
2
4
2
4-8
4-8
4
4
4
4
71
4
64
4
4
7
2
4
32
nd
8
a) All values were determined in duplicate using twofold dilution series
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Finally, we assessed activity against a series of clinical E. coli isolates carrying two or
more resistance determinants including the AAC(3)s, the APH(3’)s, and the RMTs (Table
1
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7). While the combination of AAC(3)-IV and APH(3’)-I is detrimental to most compounds,
it is noteworthy that 57β, and 61 in the 5-O-ribosyl series are only affected to a minor
extent by this combination. Similarly, compounds 30-32 in the erythrosyl series show only
modest reductions in activity in the presence of this combination of AMEs. The majority
of compounds is not affected by any of the various isoforms of AAC(3)-II, APH(3’),
AAC(6’) or by the ribosomal methyltransferase RmtB, even when present in combination.
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