Structure-Based Design and Synthesis of Apramycin-Paromomycin Analogues: Importance of the Configuration at the 6′-Position and Differences between the 6′-Amino and Hydroxy Series

Article abstract of DOI:10.1021/jacs.7b07754

The preparation of a series of four analogues of the aminoglycoside antibiotics neomycin and paromomycin is described in which ring I, involved in critical binding interactions with the ribosomal target, is replaced by an apramycin-like dioxabicyclo[4.4.0]octane system. The effect of this modification is to lock the hydroxymethyl side chain of the neomycin or paromomycin ring I, as part of the dioxabicyclooctane ring, into either the gauche-gauche or the gauche-trans conformation (respectively, axial or equatorial to the bicyclic system). The antiribosomal activity of these compounds is investigated with cell-free translation assays using both bacterial ribosomes and recombinant hybrid ribosomes carrying eukaryotic decoding A site cassettes. Compounds substituted with an equatorial hydroxyl or amino group in the newly formed ring are considerably more active than their axial diastereomers, lending strong support to crystallographically derived models of aminoglycoside-ribosome interactions. One such bicyclic compound carrying an equatorial hydroxyl group has activity equal to that of the parent yet displays better ribosomal selectivity, predictive of an enhanced therapeutic index. A paromomycin analog lacking the hydroxymethyl ring I side chain is considerably less active than the parent. Antibacterial activity against model Gram negative and Gram positive bacteria is reported for selected compounds, as is activity against ESKAPE pathogens and recombinant bacteria carrying specific resistance determinants. Analogues with a bicyclic ring I carrying equatorial amino or hydroxyl groups mimicking the bound side chains of neomycin and paromomycin, respectively, show excellent activity and, by virtue of their novel structure, retain this activity in strains that are insensitive to the parent compounds.

Full text of DOI:10.1021/jacs.7b07754

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Article
Structure-Based Design and Synthesis of Apramycin-Paromomycin
Analogues. Importance of the Configuration at the 6#-Position
and Differences Between the 6#-Amino and Hydroxy Series.
Appi Reddy Mandhapati, Guanyu Yang, Takayuki Kato, Dimitri Shcherbakov,
Sven N. Hobbie, Andrea Vasella, Erik C. Böttger, and David Crich
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07754 • Publication Date (Web): 11 Sep 2017
Downloaded from http://pubs.acs.org on September 11, 2017
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StructureꢀBased Design and Synthesis of Apramycinꢀ
Paromomycin Analogues. Importance of the Configuration at
the 6′ꢀPosition and Differences Between the 6′ꢀAmino and
Hydroxy Series.
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Appi Reddy Mandhapati,a Guanyu Yang,a Takayuki Kato,a Dimitri Shcherbakov,b Sven N.
Hobbie,b Andrea Vasella,c,* Erik C. Böttger,b,* and David Crich.a,*
a) Department of Chemistry, Wayne State University, Detroit, MI 48202, USA; b) Institute of Medical
Microbiology, University of Zurich, 8006 Zurich, Switzerland; c) Organic Chemistry Laboratory, ETH
Zurich, 8093 Zurich, Switzerland.
Email: dcrich@chem.wayne.edu
ABSTRACT: The preparation of a series of four analogues of the aminoglycoside antibiotics neomycin and paromomycin
is described in which ring I, involved in critical binding interactions with the ribosomal target, is replaced by an
apramycinꢀlike dioxabicyclo[4.4.0]octane system. The effect of this modification is to lock the hydroxymethyl side chain
of the neomycin or paromomycin ring I, as part of dioxabicyclooctane ring, into either the gaucheꢀgauche or the gaucheꢀ
trans conformation (respectively axial or equatorial to the bicyclic system). The antiribosomal activity of these
compounds is investigated with cellꢀfree translation assays using both bacterial ribosomes and recombinant hybrid
ribosomes carrying eukaryotic decoding A site cassettes. Compounds substituted with an equatorial hydroxyl or amino
group in the newly formed ring are considerably more active than their axial diastereomers lending strong support to
crystallographicallyꢀderived models of aminoglycosideꢀribosome interactions. One such bicyclic compound carrying an
equatorial hydroxyl group has activity equal to that of the parent, yet displays better ribosomal selectivity, predictive of an
enhanced therapeutic index. A paromomycin analog lacking the hydroxymethyl ring I side chain is considerably less
active than the parent. Antibacterial activity against model Gram negative and Gram positive bacteria is reported, for
selected compounds, as is activity against ESKAPE pathogens and recombinant bacteria carrying specific resistance
determinants. Analogues with a bicyclic ring I carrying equatorial amino or hydroxyl groups mimicking the bound side
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chains of neomycin and paromomcyin, respectively, show excellent activity and by virtue of their novel structure retain
this activity in strains that are insensitive to the parent compounds.
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Introduction.
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Multidrugꢀresistant infectious diseases present a significant and ever increasing threat to society
and demand the development of new generations of antibiotic agents.1ꢀ4 One approach to the
development of new and improved antibiotics is the reinvestigation of existing drug classes,
taking advantage of the power of modern synthetic organic chemistry5 and of the accrued
knowledge of the mechanisms of action of the antibiotics and of their inactivation by resistance
determinants. The aminoglycoside antibiotics (AGAs) are particularly attractive in this light as
both their mechanism of antibacterial action and the common causes of resistance to them are
wellꢀunderstood.6ꢀ9 Advantages of the AGAs as parents for optimization include their broad
spectrum of activity, their nonꢀallergenic character, and their lack of effect on the host intestinal
microbiome.9 In addition, AGAs have physicochemical properties consistent with suggested
guidelines for the development of new antibacterial compounds,10 and following decades of use
in the clinic, their PK, PD and ADME properties are wellꢀunderstood and largely predictable.9
Finally, existing AGAs are available on a large scale by fermentation, and their chemistry is wellꢀ
documented,11ꢀ12 making them good candidates for optimization by chemical modification.
Indeed, these factors underlie the development of plazomicin,9,13ꢀ14 a new generation semiꢀ
synthetic AGA currently in phase three clinical trials, and of related compounds15 designed to
circumvent most current mechanisms of AGA resistance.
In addition to widespread resistance, clinical use of existing AGAs is limited by nephrotoxicity
and ototoxicity (drugꢀinduced hearing loss).9,16ꢀ17 Previous studies suggested that increased
antibacterial activity frequently comes at the cost of increased toxicity, particularly when
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achieved by the introduction of additional basic amines, thereby highlighting the difficulty of the
problem.18ꢀ20 Fortunately, nephrotoxicity can be adequately managed and rendered effectively
reversible by regimes based on the use of a single daily dose.21ꢀ22 Ototoxicity on the other hand
is irreversible and affects up to 20% of the patient population,16,23 with estimates as high as 37%
in patients taking AGAs for prolonged periods.24 Ototoxicity arises through damage to cochlea
hair cells, which take up AGAs through mechanotransducer channels,25ꢀ26 and in which AGAs
persist for days after administration in contrast to the rapid clearance of AGAs from the body as
a whole.9,16ꢀ17 In the general patient population ototoxicity occurs in a sporadic doseꢀdependent
manner; an aggravated form occurs in genetically susceptible individuals and is linked to
mutations in mitochondrial rRNA, especially the transition mutation A1555G in the Aꢀsite of the
mitoribosomal small subunit.27 Genetically susceptible individuals constitute approximately 1 in
500 of the native European population, and it has been suggested that children be screened for
genetic susceptibility before treatment with AGAs.28ꢀ29
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Clearly, future generations of AGAs must be engineered address the issue of toxicity, in
particular ototoxicity, and to overcome resistance mechanisms, without compromising
antibacterial activity. Optimization of AGAs for enhanced activity and reduced ototoxicity is
aided by detailed knowledge of the drug binding site: the bacterial decoding A site in helix 44 of
the small ribosomal subunit,30ꢀ33 and the analogous human decoding A site17,25,34ꢀ36 whose
complexation with AGAs is a significant cause of ototoxicity (Figure 1).
In our laboratories, optimization of ribosomal selectivity is guided by cellꢀfree translation assays
based on a set of wild type bacterial and mutant recombinant ribosomes carrying single point
mutations either in the decoding A site characteristic of the eukaryotic ribosomes, or on hybrid
ribosomes incorporating complete eukaryotic decoding A site cassettes.37 Indeed, the use of such
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mutant and hybrid ribosomes led us to the unusual monosubstituted 2ꢀdeoxystreptamine AGA
apramycin 1, with its bicyclic ring I, as a first generation AGA with excellent selectivity for the
bacterial ribosome over the human mitochondrial and cytosolic ribosomes, that is essentially
devoid of ototoxicity in the guinea pig model.38ꢀ3940 Synthetic derivatives of the 4,5ꢀAGA
paromomycin 2 substituted in ring I at the 4′ꢀposition or at both the 4′ꢀ and 6′ꢀpositions, designed
to mimic apramycin, also showed reduced affinity for the mitochondrial and cytosolic ribosomes.
Animal models confirm the expected significantly reduced ototoxicity of selected paromomycin
derivatives.41ꢀ42 However, except for certain 4′ꢀOꢀglycosyl derivatives,43 this was accompanied
by reduced antibacterial activity.
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Figure 1.43 Decoding A sites of prokaryotic and eukaryotic ribosomes. The 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.
We report on the structureꢀbased design of an improved paromomycin derivative, incorporating
both an apramycinꢀlike bicyclic ring I and a conformationallyꢀrestricted hydroxylmethyl group
ideally poised to mimic the paromomcyin A1408 interaction. In contrast to previous
conformationallyꢀrestricted AGA analogues,44ꢀ46 this derivative retains the full antibacterial
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activity of the parent and displays exquisite selectivity for the bacterial over the eukaryotic
mitochondrial and cytosolic ribosomes.
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Design.
XꢀRay crystallographic studies of both the 4,5ꢀ and 4,6ꢀdisubstituted 2ꢀdeoxystreptamine classes
of aminoglycosides bound to the decoding A site of the bacterial 30S ribosomal subunit and its
truncated models reveal a pseudoꢀbase interaction, pairing N1 and N6 of A1408 with the 6′ꢀ
substituent (OH in paromomycin 2 or NH2 in neomycin 3) and the ring I oxygen (O5′) of the
AGA.30,41,47ꢀ48 In these structures the ring I side chain invariably adopts the gauche,trans (gt)
conformation (Figure 2)49 in the pseudoꢀbase pair. We classify this ring I gt conformationꢀA1408
interaction as type I pseudoꢀbase pair (Figure 3a) to distinguish it from the apramycin ring Iꢀ
A1408 interaction,38,50 here called a type II pseudoꢀbase pair (Figure 3b and 3c), in which the 6′ꢀ
hydroxy group takes up the gauche,gauche (gg) conformation.
gg
tg
gt
O
O
O
H3N
HO
HO
H3N
H3N
HO
HO
HO
HO
O
O
O
XH
XH
XH
X = OH, gg:gt:tg ~ 60:40:0
X = NH3+, gg:gt:tg ~ 10:90:0
Figure 2. Side Chain conformations of Ring I and estimated populations based on methyl 2ꢀ
aminoꢀ2ꢀdeoxyꢀαꢀ
D
ꢀglucopyranoside and methyl 2,6ꢀdiaminoꢀ2,6ꢀdideoxyꢀαꢀDꢀglucopyranoside.
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O
O
H3N
H3N
N
N
HO
HO
HO
N
N
H2N
N
H2N
HO
N
O
O
N
XH
N
H
X
A: Type I pseudo-base pair
B: Type II pseudo-base pair
Paromomycin X = O
+
Neomycin X = NH2
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O
H3N
N
N
N
H2N
O
O
O
N
H2N
H
O
C: Type II pseudo-base pair
as imposed in apramycin
Figure 3. Type I and II Pseudoꢀbase Pairs
Previous work with a series of 4′ꢀOꢀalkyl paromomcyin derivatives revealed a chain length of 2
or 3 carbons in the appended alkyl group to afford the optimum reduction in mitoribosomal
activity with the least loss of antibacterioribosomal activity.42 In the 4′,6′ꢀOꢀalkylidene
paromomycin derivatives studied the ethylidene derivative 4 had a better profile than the
comparable methylidene derivative lacking the terminal methyl group, again suggesting a 2
carbon chain on O4′ to be optimal.42 Therefore, in designing further apramycinꢀparomomycin
hydrids we opted to construct a transꢀdioxadecalinꢀlike structure carrying a methyl substituent
designed to overlay the ethylidene acetal 4. We further reasoned that the antibacterial activity of
these new derivatives would be enhanced by the incorporation of a hydroxyl group at the 6′ꢀ
position so as to permit complete pseudoꢀbase pair formation with A1408. Finally, the
introduction of additional basic amines to increase antibacterial activity owing to anticipated
problems with correspondingly increased toxicity.18ꢀ20 These considerations led us to 5 and 6 as
targets, with the axial isomer 5 mimicking the gg conformation of the paromomycin side chain
(type II pseudoꢀbase – apramycin interaction) and the equatorial isomer 6 mimicking the gt
conformation (type I pseudoꢀbase interaction). We also targeted the 6′ꢀamino analogs 7 and 8 in
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the hope of harnessing the neomycinꢀlike increased activity resulting from the extra amino
group.
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Consideration of the differences in geometry of the Type I and Type II pseudoꢀbase pairs (Figure
3) lead us to reꢀevaluate the effect of deoxygenation at the 6′ꢀposition in paromomycin. Thus, 6′ꢀ
deoxyparomomycin 9 and 6′ꢀdeoxyꢀ6′ꢀfluoroparomomycin 10, previously only investigated for
their antibacterial activity,51 were also screened for activity and selectivity at the target level with
the cellꢀfree translation assays. Seeking to remove any possibly unfavorable hydrophobic
interactions in the 9ꢀribosome complexes, we also prepared and investigated the novel 6′ꢀ
deshydroxymethyl paromomycin derivative 11.
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X
O
Y
O
O
O
O
HO
H2N
HO
H2N
H2N
H2N
O
O
NH2
O
O
NH2
HO
O
O
HO
OH
OH
H2N
OH
O
H2N
OH
O
O
OH
OH
O
OH NH2
OH NH2
4
5: X = OH, Y = H
6: X = H, Y = OH
7: X = H, Y = NH2
8: X = NH2, Y = H
X
O
HO
HO
H2N
H2N
O
O
NH2
O
HO
OH
H2N
OH
O
9: X = CH3
10: X = CH2F
11: X = H
OH
O
OH NH2
Synthesis.
Synthetic work began with the fully protected paromomycin derivative 12,52 which on treatment
with toluenesulfonic acid in methanol afforded the known diol 13.52 Controlled selective
oxidation of the primary alcohol in 13 with catalytic tetramethylpiperidinoxyl (TEMPO) and
bis(acetyoxy)iodobenzene53 then gave an unstable aldehyde 14, which on exposure to
allyltributylstannane and boron trifluoride etherate54 in dichloromethane at 0 oC gave the R and
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Sꢀisomers of 15 in 30% and 28%, respectively, for the two steps. Transacetalization of one
isomer with benzaldehyde dimethylacetal gave the benzylidene acetal 16, whose all equatorial
configuration was established by nuclear Overhauser correlations between the three axial protons
on the upper rim thereby identifying its immediate precursor as the Rꢀalcohol 15R (Scheme 1).
The diastereoisomer 15S was not converted to the corresponding benzylidene acetal under the
same mild conditions.
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Scheme 1. Synthesis of Alcohols 15R and 15S
Treatment of alcohol 15R with Nꢀbromosuccinimide in acetonitrile at 0 oC gave the desired
cycloetherification product 18 in 38% yield along with two byproducts 19 and 20 in 25 and 20%
yield, respectively (Scheme 2). The bicyclic derivative 18, whose relative configuration rests on
coupling constant analysis and the observation of nuclear Overhauser effects around the newly
formed ring, arises from NBS attack on the Reꢀface of the alkene 15R to give the cyclic
bromonium ion antiꢀ17 followed by nucleophilic ring closure with the 4′ꢀhydroxy group. The
diastereoisomeric product 21 arising from NBS attack on the Siꢀface of 15R with subsequent sixꢀ
membered ring formation was not observed. The two rearranged products 19 and 20 are formed
by initial attack of the pyranosyl ring oxygen on the cyclic bromonium ions antiꢀ and synꢀ17,
respectively, with subsequent pyranoside to furanoside rearrangement. Related rearrangements
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arising from intramolecular nucleophilic attack of pyranoside ring oxygens on cyclic bromonium
ions have been described.55ꢀ56 The relative configurations of the two rearranged products 19 and
20 were determined by extensive NMR spectroscopic studies following deprotection.
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Scheme 2. Cycloetherification of Alcohol 15R
Bromoetherification of the diastereoisomeric homoallylic alcohol 15S did not give the desired
fused bicyclic system 22 in any measureable yield, but afforded several rearranged products
similar to 19 and 20, as judged by the inspection of their NMR spectra, which were not pursued
further. Accordingly, 22 was obtained by oxidation of 18 with the Dess Martin periodinane57
followed by sodium borohydride reduction (Scheme 3). Alcohols 18 and 22 were then converted
to the corresponding inverted azides 23 and 24 by reaction with trifluoromethanesulfonic
anhydride followed by displacement with sodium azide (Scheme 3).
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Scheme 3. Preparation of Alcohol 22 and Azides 23 and 24
5
6
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Hydrogenolysis of alcohols 18 and 22, and of azides 23 and 24 over Pd/C in aqueous dioxane
and acetic acid, followed by filtration through Sephadex and lyophilization from aqueous acetic
acid gave the target compounds 5ꢀ8, respectively, in the form of their acetate salts (Scheme 4).
Analogous treatment of the furanosides 19 and 20 gave compounds 25 and 26, whose relative
configurations were established by extensive NMR studies as described in the supporting
information. 6′ꢀDeoxyparomomycin 9 and 6′ꢀdeoxyꢀ6′ꢀfluoroparomomycin 10 were obtained in
the form of their trifluoroacetate salts as described previously, then converted to their acetate
salts by Sephadex chromatography and lyophilization from aqueous acetic acid.
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Scheme 4. Deprotection of Bicyclic Paromomycin Analogs 5ꢀ8 and of Rearranged Products 25
and 26.
Aprosamine 27 was obtained in admixture with 4ꢀaminoglucose by heating apramycin 1 in 4N
HCl as described previously.58 Treatment of this mixture with benzyloxycarbonyl chloride and
sodium carbonate enabled separation of the tetra carbamate 28 in 60% overall yield, from which
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a pure sample of aprosamine 27 was obtained in the form of an anomeric mixture by
hydrogenolysis (Scheme 5). Reaction of 28 with the dimethylacetal of cyclohexanecarbaldehyde
in DMF with catalytic toluenesulfonic acid gave 77% of the acetal 29, which on exposure to
acetic anhydride in pyridine was converted to the equatorial anomeric acetate 30 in 86% yield.59
Dess Martin oxidation57 followed by reduction with sodium borohydride then afforded the 6′ꢀ
epiaprosamine derivative 31 in 41% yield, from which 6′ꢀepiaprosamine 32 was obtained by a
three step deprotection sequence in 90% overall yield (Scheme 5).
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1
4N HCl,
OH
OH
CbzN
HO
CbzCl, MeOH,
H2O, Na2CO3
O
NH
O
O
HO
O
NHCbz
CbzHN
Pd(OH)2/C, H2,
dioxane, AcOH, 63%
O
NHCbz
NH2
H2N
O
HO
OH
NH2
28, 60%
HO
OH
27
C6H11CH(OMe)2,
TsOH, DMF
Y
CbzN
HN
Z
XO
OH
HO
O
O
O
O
NHCbz
O
CbzHN
NH2
H2N
O
O
HO
NHCbz
O
NH2
OH
32, 90%
i) AcOH, H2O,
ii) NaOMe, MeOH,
iii) Pd(OH)2/C,
,
29: X = H, Y = OH, Z = H, 77%
Ac2O, py
30: X = Ac, Y = OH, Z = H, 86%
31: X = Ac, Y = H, Z = OH, 41%
i) DMP
ii) NaBH4,
MeOH
dioxane, AcOH
Scheme 5. Synthesis of Aprosamine 27 and its 6′ꢀEpimer 32.
Reduction of the benzylidene acetal 1252 with borane dimethyl sulfide and dibutylboron triflate60
gave the paromomycin derivative 33, which was oxidized with catalytic TEMPO, potassium
bromide, and bleach61 to the uronic acid 34 in 79% yield. Application of Barton′s
decarboxylation reaction,62 using tertꢀdodecanethiol63 as hydrogen atom source under white light
photolysis conditions, gave the fully protected 6′ꢀdeshydroxymethyl derivative 35 in 55% yield.
This classical radical chain decarboxylation was significantly more effective on this substrate
than more recent photocatalytic decarboxylation reactions64ꢀ65 relying on electron transfer chain
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7
8
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reactions, possibly because of the presence of multiple azide groups. Hydrogenolysis over
palladium hydroxide in a mixture of dioxane, water, and acetic acid followed by lyophilization
then gave 11 (Scheme 6).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 6. Synthesis of the 6′ꢀdeshydromethyl paromomycin derivative 11.
Antiribosomal and Antibacterial Activity.
All compounds were screened in cellꢀfree assays for their ability to inhibit translation by
bacterial wildꢀtype and recombinant hybrid ribosomes carrying complete A site cassettes of the
human mitochondrial (Mit13), mitochondrial A1555G, and cytoplasmic ribosomes (Cyt14). For
all compounds except the rearranged products 25 and 26, which showed no significant activity,
the experimental data together with those of the comparators 1ꢀ4, 6′ꢀepiapramycin 3650 and 6′ꢀ
deoxyapramycin 3750 are reported in Table 1.37 The antibacterial activity of all compounds was
determined against clinical isolates of methicillinꢀresistant strains of the Gramꢀpositive
bacterium Staphylococcus aureus and of the Gramꢀnegative bacteria Escherichia coli and
Pseudomonas aeruginosa as reported in Table 2, with the exception of the inactive compounds
25 and 26. The more active compounds 6 and 7 were also screened for activity against the
ESKAPE pathogens Klebsiella pneumoniae (K. pneumoniae), Enterobacter cloacae (E.
cloacae), and Acinetobacter baumannii (A. baumannii) (Table 3). In order to determine
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susceptibility to common resistance determinants compounds 6 and 7 additionally were screened
for activity against wild type and recombinant E coli strains carrying defined resistance
determinants (Table 4).38
10
11
12
13
14
15
16
Table 1. Antiribosomal Activities (IC50, ꢁg/mL) and Selectivities.a
IC50, ꢁg/mL
Selectivity
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound
bacterial Mit13
A1555G Cyt14
Mit 13 A1555G Cyt 14
Paromomycin 2
0.03
0.02
0.07
0.74
>20
50.64
1.62
5.52
0.22
26.73
10.58
17.12
55.89
90.01
103.05
ꢀ
1688
81
184
11
353
856
798
122
<5.1
ꢀ
Neomycin 3
Apramycin 1
60.97
871
168
<5.1
1886
411
11593
14
382
61
6′ꢀEpiapram. 36
124.21 45.08
101.15 85.26
226.38 76.97
193.33 213.00
231.85 11.82
6′ꢀDeoxyapram. 37
<4.3
641
453
591
2.3
4.5
ꢀ
0.12
0.47
0.02
0.08
0.37
1.99
>10
4
169.16
15.07
12.03
10.31
ꢀ
360
753
150
28
5
6
1.15
0.18
7
2.99
1.66
8.1
8
Aprosamine 27
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
32
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5
6
7
8
9
0.09
0.06
0.23
166.16 93.23
107.56 92.51
152.14
80.06
42.88
1846
1792
389
1036
1542
165
1690
1334
186
9
10
11
89.43
37.95
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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Table 2. Antibacterial Activities (MIC, ꢁg/mL)
MRSA
E coli
P aeruginosa
AG038 AG039 AG042 AG044
AG001 AG055 AG003
AG031 AG032 AG033 AG086
Compound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Paromomycin 2
Neomycin 3
4
>256
128
8
>256
128
8
4ꢀ8
0.5ꢀ1
16
16ꢀ32
8ꢀ16
16
8
8ꢀ16
4
>128
>128
>128
>128
0.5ꢀ1
8
4
32
8
ꢀ
32ꢀ64
>128
>128
Apramycin 1
8
8ꢀ16
32
8
ꢀ
8
ꢀ
4
ꢀ
6′ꢀEpiapramycin 36
32ꢀ64
64
64
32ꢀ64
>128
32
32
32
>128
6′ꢀDeoxyapramycin 37 >128
>128
64
>128
32
>128
>128
≥128
8
>128
ꢀ
ꢀ
ꢀ
ꢀ
64ꢀ128
32
ꢀ
4
5
6
7
8
64ꢀ128
64ꢀ128
8
32
32ꢀ64
8
16ꢀ32
8
32
>128
32
>128
16ꢀ32
32
>128
>128
≥128
>128
>128
>128
≥128
>128
8ꢀ16
4
4
8
4
4
2
2
2
2
32
>128
>128
128
128
>128
>128
>128
>128
128
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5
6
7
8
9
64ꢀ128
>128
32
64ꢀ128
>128
16ꢀ32
16ꢀ32
64
Aprosamine 27
32ꢀ64
>128
16
32ꢀ64
>128
>128
>128
>128
32
32
32ꢀ64
>128
16ꢀ32
16ꢀ32
64
32
32
32
32ꢀ64
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
9
8
8
8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
16
32
10
11
16
64
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Table 3. Antibacterial Activities (MIC, ꢁg/mL) Against ESKAPE Pathogens
5
6
7
K. pneumoniae
A. baumannii E. cloacae
8
9
(AG215)
(AG225)
(AG290)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Paromomycin 2
1
0.5
2
2
1ꢀ2
4
2
1
4
2
1
Neomycin 3
Apramycin 1
2
4
6
7
0.5ꢀ1
4
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Table 4. Antimicrobial Data Against Wild Type and Engineered Strains of E coli Carrying Specific Resistance Determinants for Neomycin
5
6
and Selected Derivatives (MIC, ꢁg/mL)a.
7
8
9
Strain
BM13
AG007
AG105
AG009
AG036
AG037
AG103
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
(AG006)
Resistance
Mechanism
ꢀ
AAC(3) AAC(2′) AAC(6′) ANT(4′, 4′′) APH(3′, 5′′) ArmA
Gentamicinb
Kanamycin Ab
Tobramycinb
Apramycin 1
Neomycin 3
Paromomycin 2
6
0.5
1ꢀ2
0.5
4
32
nd
nd
8
32
1ꢀ2
nd
4
nd
128
64
8
0.5
16
16
4
0.5
>128
>128
>128
2
>256
1
4
1
4
2
8
32
256
2ꢀ4
2
>256
>256
64
0.5
4
2ꢀ4
2
4ꢀ8
8
2ꢀ4
2ꢀ4
2ꢀ4
8ꢀ16
4
4
1ꢀ2
4
2
32ꢀ64
2
7
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a) All values were determined in duplicate using two dilution series. b) These AGAs were used to verify the resistance phenotype
type of the recombinant strains.
7
8
9
NH2
Me
NH2
O
HO
X
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
H2N
O
Y
O
NH2
HO
H2N
HO
H2N
O
O
NH2
HO
O
O
HO
OH
O
NH2
X = Y = OH. kanamycin A
X = H, Y = NH2, tobramycin
HO
Me
OH
HN
Me
gentamicin
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Discussion
6
7
8
9
Comparison of the antibacterioribosomal activities of paromomycin and of its bicyclic
derivatives, the ethylidene acetal 4 and the 6′ꢀhydroxylated analogues 5 and 6, reveals the
essential role of a 6′ꢀhydroxyl group in the correct orientation for tight binding to the bacterial
decoding A site (Table 1). Thus, the equatorial 6′ꢀOH group in the conformationally locked
bicyclic ring I of 6 occupies the identical space to the 6′ꢀhydroxyl group of paromomycin in the
gt conformation as found in its complex with the decoding A site (Figure 3A, type I pseudoꢀbase
pair).30,41 Furthermore, the advantage gained from imposing the gt conformation is sufficient to
overcome any detrimental effect on binding of the additional ring and the concomitant loss of the
hydrogen bond between the paromomycin 4′ꢀOH and the backbone phosphate linking G1491 to
A1492.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the analogous 6′ꢀamino series, 7 with the enforced equatorial amino group is more active than
the axial analog 8, consistent with the 6′ꢀamino AGA neomycin binding to the decoding A site
with its sideꢀchain in the gt conformation (Table 1). Nevertheless, the difference in IC50 values
of the two 6′ꢀamino epimers 7 and 8 (4ꢀ5 fold) is strikingly less than that of the two 6′ꢀhydroxy
derivatives 5 and 6 (23 fold). Furthermore, unlike the 6′ꢀequatorial hydroxy derivative 6, which
has comparable activity to the parent paromomycin, the 6′ꢀequatorial amino derivative 7 is some
four times less active toward the bacterial ribosome than the parent neomycin.
The differences between the 6′ꢀhydroxy and 6′ꢀamino series can be largely attributed to the
differing ground state conformations of the ring one side chains in 6′ꢀhydroxy and 6′ꢀ
aminopyranosides (Figure 2) and the corresponding energetic penalties paid on binding to the
decoding A site in the gt conformation. Thus, adopting literature data on the side chain
conformations of methyl 2ꢀaminoꢀ2ꢀdeoxyꢀαꢀ
D
ꢀglucopyranoside and of methyl 2,6ꢀdiaminoꢀ2,6ꢀ
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ꢀglucopyranoside,49,66 the hydroxymethyl and protonated aminomethyl side chains
3
4
dideoxyꢀαꢀ
D
5
6
7
8
of paromomycin 2 and neomycin 3 are approximately 60:40:0 and 10:90:0 mixtures,
respectively, of the gg, gt and tg conformers. Thus, on binding to the decoding A site in the gt
conformation, paromomycin pays a penalty for restricting the side chain to a higher energy
conformer. Artificially locking the paromomycin side chain into the required gt conformer
eliminates this penalty and results in the highly active compound 6. Conversely, no such gain is
obtained on locking the neomycin side chain in the already largely predominant gt conformation
and the 6′ꢀequatorial amino derivative 7 consequently does not achieve the same level of affinity
for the bacterial ribosome as the parent. Clearly, other factors such as changes in relative
solvation levels may also contribute to the observed pattern of activity changes.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 6 with the equatorial 6′ꢀhydroxy group displays outstanding selectivity for the
bacterial ribosome over the eukaryotic mitochondrial ribosome and good selectivity over the
A1555G mitochondrial mutant ribosome found in individuals genetically predisposed to
ototoxicity.67ꢀ68 These differences in affinity for the prokaryotic and mitochondrial ribosomes
result from differing interactions of the βꢀface of ring I with the bases at the bottom of the
decoding A site. Thus, on the one hand, the G1491=C1409 base pair at the foot of the bacterial
site, in which G1491 interacts with the βꢀface of the ligand ring I by a CHꢀπ interaction,
compensates for any loss of affinity due to the introduction of the hydrophobic bicyclic moiety.
On the other hand, the mitochondrial ribosome, with its two consecutive non WatsonꢀCrick pairs
(C1491•C1409 and A1490•C1410), and consequently looser binding site, compensates much less
effectively for the hydrophobic nature of the bicyclic ring I. With only one nonꢀcognate base
pair (C1491•C1409) in the floor of the decoding A site, the mitochondrial A1555G hybrid
ribosome binds 6 with intermediate affinity between those of the bacterial and wildꢀtype
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5
6
7
8
9
mitochondrial ribosomes. In the 6′ꢀamino series both the equatorial isomer 7 and the axial
isomer 8, like the parent neomycin, strongly inhibit the mitochondrial ribosome and its A1555G
mutant, reflecting the presence of the additional basic amino group.69
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The A1408G mutation in the eukaryotic cytoplasmic decoding A site permits the formation of a
complementary pseudoꢀbase pair of the AGA ring I oxygen and the 6′ꢀhydroxy group with N1
and N2, respectively, of G1408 that is analogous to the A1408=ring I pseudoꢀbase pair in the
bacterial ribosomeꢀAGA complex. Not surprisingly, therefore, 6 with the equatorial hydroxy
group binds more tightly to the cytoplasmic ribosome than does 5 with the axial hydroxy group.
Nevertheless, because of its high affinity for the bacterial ribosome, 6 still shows excellent
selectivity over the cytosolic ribosome. In contrast, the A1408G mutation does not
accommodate a pseudoꢀbase pair with ring I of the 6′ꢀamino AGAs owing to the repulsion
between the base and the AGA 6′ꢀamine.70 This discordance accounts for the much lower
affinity of neomycin and its bicyclic analogs 7 and 8 for cytoplasmic ribosome compared to the
either mitochondrial or bacterial ribosomes. The comparable affinity of both the axial amino
derivative 8 and its equatorial isomer 7 for the cytoplasmic ribosome presumably reflects the
inability of either isomer to take part in an effective pseudo base interaction with G1408.
Overall, with the exception of the interaction of the 6′ꢀaminoꢀderivatives 7 and 8 with the
cytoplasmic ribosome, it is clear that the rigid bicyclic analogues with equatorial 6′ꢀhydroxyl and
6′ꢀamino groups, 6 and 7 respectively, bind more tightly to the ribosomal decoding A site than
their axial analogues 5 and 8. This observation is consistent with gt conformation of the AGA
ring I side in crystallographically observed AGA ribosome complexes and the preferential
formation of a type I pseudoꢀbase pair (Figure 3A). This situation is to be contrasted with that of
the natural bicyclic AGA apramycin 1 and its synthetic 6′ꢀepiꢀisomer 36. In this series, as
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previously reported,50 apramycin, with the axial 6′ꢀhydroxy group, binds more effectively to the
ribosome than the 6′ꢀepimer with its equatorial hydroxy group. Crystallographic studies reveal
that apramycin binds to the complete bacterial decoding A site in the typical manner,38 except for
the type II pseudo base pair between its ring I oxygen and axial 6′ꢀhydroxy group, and A1408 N1
and N6. The same situation pertains with aprosamine 27, lacking the terminal 4ꢀaminoglucosyl
of apramycin, and 6′ꢀepiaprosamine 32 (Table 2). The exact reasons for the preference of an
axial 6′ꢀhydroxy group in the pseudoꢀbase pair between apramycin and the decoding site A1408
in the apramycin series as opposed to the equatorial 6′ꢀhydroxy group in the bicyclic
paromomycin analogs have yet to be elucidated. It is clear from the relative activities of
aprosamine 27 and its isomer 32, however, that they involve the 7′ꢀmethylamino group of
apramycin and its interaction with the 6′ꢀhydroxyl group and the phosphate bridge between
A1492 and A1493, and are not related to the presence of the extra 4ꢀaminoglucosyl ring.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Turning to the 6′ꢀdeoxy compounds 9 and 10, the antibacterioribosomal activity of
paromomycin, with its Type I pseudoꢀbase pair interaction is much less affected by
deoxygenation than that of apramycin, whose 6′ꢀdeoxy derivative 3650 is essentially devoid of
activity (Table 1). Removal of the hydrophobic methyl group from 9 to give 11 results in a
significant loss of activity against the bacterial ribosome (Table 1), but less than the simple
removal of the 6′ꢀhydroxy group from apramycin. Evidently, the 6′ꢀhydroxy group is a more
critical part of the interaction of apramycin with the bacterial decoding A than it is in
paromomycin. It is striking that 11, like 6′ꢀdeoxyapramycin 37, suffers a significantly smaller
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5
6
reduction in activity toward the hybrid ribosomes carrying the eukaryotic binding sites, than it
does toward the bacterial ribosome resulting in a marked loss of selectivity (Table 1).
7
8
9
The antibacterial activities of the bicyclic derivatives 5, 6, 7, and 8 against clinical isolates of
methicillinꢀresistant strains of the Gramꢀpositive bacterium Staphylococcus aureus and of the
Gramꢀnegative bacterium Escherichia coli reflect the trends observed at the target level, with the
exception that 6 and 7 with the equatorial 6′ꢀhydroxy and amino groups, respectively show
moderately increased activity against E. coli in comparison to the parent compounds. The
corresponding axial isomers 5 and 8 are significantly less active, again stressing the importance
of configuration at the 6′ꢀposition. As with all previous 4′ꢀderivatives of paromomycin prepared
in our laboratories,41,43,71 and comparable to apramycin, compounds 5, 6 and 7 show good
activity against two clinical strains of MRSA that are resistant to the parent paromomycin. This
increase in activity arises from the ability to block the resistance mechanism of these two MRSA
strains, which most likely arises from drug modification by either ANT(4′) or APH(3′)
aminoglycoside modifying enzymes. With respect to the four clinical strains of the Gramꢀ
negative bacterium Pseudomonas aeruginosa tested, paromomycin itself is inactive and
neomycin only moderately active against two of the four. Bicyclic compounds 6 and 7, with
their equatorial hydroxyl and amino groups respectively, show modest neomycinꢀlike activity
against two of the four strains but this activity is still inferior to that exhibited by apramycin.
The bicyclic compounds 6 and 7 were also screened for activity against clinical isolates of the
ESKAPE pathogens K pneumoniae, E cloacae, and A baumannii and were found to have
activities comparable to the comparators paromomycin, apramycin and neomycin (Table 3).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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5
6
7
8
9
Consistent with their antibacterioribosomal activities the 6′ꢀdeoxy, 6′ꢀdeoxyꢀ6′ꢀfluoro and 6′ꢀ
(deshydroxymethyl) paramomycin derivatives 9, 10, and 11 show moderately reduced activity
against the two paromomycinꢀsusceptible strains of MRSA and against E coli.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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When screened against a series of recombinant E coli strains carrying specific resistance
determinants the structural modifications inserted into compounds 6 and 7 were found to be more
active than the parents paromomycin and neomycin in the presence of ANT(4′,4′′) and, to a
lesser extent, APH(3′,5′′) and AAC(6′) AMEs (Table 4). The ability of 6 and 7 to thwart the
action of the ANT(4,4′′) class of AMEs clearly arises from the absence of a derivatizable 4′ꢀ
hydroxy group, while the enhanced activity in the presence of APH(3′,5′′) is presumably a
function of the reduced accessibility of the 3′ꢀhydroxy group. A combination of conformational
constraint and steric shielding of the 6′ꢀhydroxy and 6′ꢀamino groups in 6 and 7, respectively,
accounts for the increased activity in the presence of AAC(6′). As expected,72 compounds 6, 7
and the parents paromomycin and neomycin are little affected by AAC3 and AAC2′ AMEs
(Table 4). In addition, as is characteristic for members of the 4,5ꢀseries of deoxystreptamine
aminoglycosides neither 6 nor 7 show any loss of activity in the presence of the ribosomal
methyl transferase ArmA73ꢀ76 that, in contrast, completely inhibits the activity of all 4,6ꢀAGAs
including the current lateꢀstage clinical candidate plazomicin.77
Conclusion. Designed on the basis of analogy with apramycin and previous experience with 4′ꢀ
Oꢀsubstituted analogs of parmomycin, the structurally novel compound 6 was synthesized and
shown to exhibit enhanced inhibitory activity of the bacterial ribosome with respect to the parent
paromomycin. Semisynthetic AGA 6 displays comparable antibacterial activity to the parent
against clinical strains of E coli and MRSA and enhanced activity against recombinant E coli
carrying the AAC(4′,4′′), APH(3′,5′′), and AAC(6′) resistance determinants. Additionally, 6
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shows reduced binding to eukaryotic mitochondrial and cytosolic ribosomes, which is predictive
of an enhanced therapeutic index. These results clearly demonstrate that existing readily
available AGAs can be modified to overcome common resistance mechansims and reduce
common clinical side effects, without significant loss of antibacterial activity. The enhanced
bacterioribosomal activity of 6 is demonstrated to be a function of the equatorial hydroxyl group
at the 6′ꢀposition, thereby providing support for the crystallographicallyꢀderived models of AGAꢀ
ribosome interactions. Importantly, these results demonstrate that aminoglycosides with
increased ribosomal activity can be designed without the need for the incorporation of additional
basic amines and the concomitant increases in toxicity.
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Supporting Information. Full experimental details and copies of the H and C NMR spectra
of all new compounds. The Supporting Information is available free of charge on the ACS
Publications website at http://pubs.acs.org.
Acknowledgment. We thank the NIH (AI123352), the University of Zurich and Wayne State
University for support of this work, and Professor Patrice Courvalin of the Institut Pasteur for
recombinant strains of E coli. We acknowledge support from the NSF (MRIꢀ084043) for the
purchase of the 600 MHz NMR spectrometer in the Lumigen Instrument Center at Wayne State
University.
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