Toward overcoming staphylococcus aureus aminoglycoside resistance mechanisms with a functionally designed neomycin analogue

Article abstract of DOI:10.1021/ml200202y

Deoxygenation of the diol groups in rings A and D of neomycin in combination with the introduction of an N1-(l)-HABA group in the 2-deoxystreptamine subunit (ring B) leads to a novel and potent antibiotic (1) with activity against strains of S. aureus car

Full text of DOI:10.1021/ml200202y

LETTER
pubs.acs.org/acsmedchemlett
Toward Overcoming Staphylococcus aureus Aminoglycoside Resistance
Mechanisms with a Functionally Designed Neomycin Analogue
Stephen Hanessian,*,† Alexandre Giguꢀere,† Justyna Grzyb,† Juan Pablo Maianti,† Oscar M. Saavedra,†
James B. Aggen,‡ Martin S. Linsell,‡ Adam A. Goldblum,‡ Darin J. Hildebrandt,‡ Timothy R. Kane,‡
Paola Dozzo,‡ Micah J. Gliedt,‡ Rowena D. Matias,‡ Lee Ann Feeney,‡ and Eliana S. Armstrong‡
†Department of Chemistry, Universitꢁe de Montrꢁeal, C.P. 6128, Succ. Centre-Ville, Montrꢁeal, Quebec H3C 3J7, Canada
‡Achaogen Inc., 7000 Shoreline Court, Suite 371, South San Francisco, California 94080, United States
S Supporting Information
b
ABSTRACT: Deoxygenation of the diol groups in rings A and D of neomycin in
combination with the introduction of an N1-(L)-HABA group in the 2-deoxystreptamine
subunit (ring B) leads to a novel and potent antibiotic (1) with activity against strains of
S. aureus carrying known aminoglycoside resistance determinants, as well as against an
extended panel of Methicillin-resistant S. aureus isolates (n = 50). Antibiotic 1 displayed
>64 fold improvement in MIC50 and MIC90 against this MRSA collection when
compared to the clinically relevant aminoglycosides amikacin and gentamicin. The
synthesis was achieved in six steps and 15% overall yield.
KEYWORDS: Aminoglycoside, antibiotics, deoxygenation, MRSA, enzymatic modifica-
tion, resistance, neomycin analogues
taphylococcus aureus is a common commensal inhabitant of
the human bacterial flora, colonizing approximately one third
broad-spectrum antibacterial properties.6,7 Nevertheless, their use as
anti-infectives is minimal due to their high susceptibility to multiple
modifying enzymes (Figure 1).6,13ꢀ15
S
of the population’s nostrils.1,2 These Gram-positive facultative
anaerobic cocci are responsible for conditions ranging from minor
infections of skin and soft tissues to systemic illnesses such as
pneumonia, meningitis, osteomyelitis, endocarditis, wound infec-
tions, bacteremia, and sepsis with high morbidity and mortality.1
The advent of antibiotics has had a major impact on the
treatment of such infections for several decades. However, the
appearance and dissemination of methicillin-resistant (or multi-
drug-resistant) S. aureus (MRSA) strains, which became prevalent
in hospitals and later in community settings, with a current
estimated asymptomatic reserve of 2.3 million people in the U.S.
(0.8% of the population) and a potentially higher worldwide
percentage,2 present a major hurdle for anti-infective therapy.
Reliance on vancomycin as a last resort has generated selective
pressure for the proliferation of vancomycin resistant S. aureus
strains (VISA and VRSA).3 The current toll of MRSA infections in
the U.S. surpasses that of HIV/AIDS, with approximately 100,000
yearly infections and 20,000 associated losses of lives.4,5
The most prominent members of clinically used aminoglyco-
sides belong to the 4,6-disubstituted 2-deoxystreptamine class,
including gentamicin, tobramycin, amikacin, isepamicin, and
arbekacin. However, their continued use has resulted in the
emergence of S. aureus strains armored with a range of intracel-
lular modifying enzymes,13ꢀ16 prevalently 40-O-nucleotidyltrans-
ferase, ANT(40)-I, 30/500-O-phosphotransferase, APH(30/500)-
III, and the bifunctional 200-O-phosphotransferase and 60-N-
acetyltransferase, APH(200)/AAC(60), whose respective target
preferences are shown in Figure 1.13ꢀ16 As a result, semisynthetic
members of the 4,6-disubstituted 2-deoxystreptamine class, such
as arbekacin, were developed to overcome the inactivating action
of a subset of the aforementioned enzymes and the different
isoforms prevalent in other pathogens.16,17 In this regard, it
should be noted that no new aminoglycoside antibiotic has been
introduced since the early 1980s.6,17
.
Deoxygenation methodologies have been an active area of
investigation in aminoglycoside antibiotic semisynthesis with the
hope to evade the action of resistance enzymes, in particular the
ring-A 30,40-diol of the 2-deoxystreptamine classes.18ꢀ23 Among
Aminoglycoside antibiotics are powerful broad-spectrum antibio-
tics which target a rRNA helix at the mRNAꢀtRNA decoding center
of the bacterial 30S ribosomal subunit,6ꢀ8 affecting their bactericidal
action by inducing translation inaccuracy and inhibition.9ꢀ12
The class of 4,5-disubstituted 2-deoxystreptamine aminoglyco-
sides, including butirosin, paromomycin, and neomycin (Figure 1),
share a common mode-of-action, ribosomal binding site, and powerful
Received: August 18, 2011
Accepted: September 15, 2011
Published: September 29, 2011
r
2011 American Chemical Society
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LETTER
Figure 1. Representative members of the 2-deoxystreptamine aminoglycoside classes characterized by 4,6-disubstitution (amikacin and gentamicin C1)
or 4,5-disubstitution (neomycin B). Arrows indicate positions targeted by modifying enzymes prevalent in S. aureus (black, complete resistance; gray,
medium resistance; white and crossed, evaded). See Supporting Information file for complete tabulation of aminoglycoside antibiotic structures.
Scheme 1. Application of the GareggꢀSamuelsson Modification of the TipsonꢀCohen Reaction for Tetradeoxygenation in the
Paromomycin and Neomycin Seriesa
a Reagents: (a) TBSOTf, 2,6-lutidine, DCM, 0 °C; (b) PPh3, imidazole, triiodoimidazole, toluene, MeCN, reflux; (c) HF py, py; (d) H2, Pd(OH)2/C,
3
AcOH/H2O (4:1); (e) TrCl, cat. DMAP, py, 70 °C; (f) H2, Pd(OH)2/C, 1 N HCl, MeOH.
the subclasses, 4,5-disubstituted congeners have remained com-
paratively difficult to access in semisynthetic efforts, mainly due to
lengthy protocols involving extensive protecting and functional
group manipulations.18ꢀ24 Classic examples of N1-substitution
have been performed on early semiprotected intermediates rely-
ing on selective reactivity among the secondary amines.6,19,24
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LETTER
Scheme 2. Chemoselective N1-Deprotection of Tetradeoxygenated Paromomycin and Neomycin Intermediates via N1,O6-
Oxazolidinones, for Subsequent Introduction of N1-HABA Groupsa
a Reagents: (a) LiOH, H2O/DMF, RT; (b) EDC, DIPEA, γ-N-Cbz-L-HABA; (c) H2, Pd(OH)2/C, AcOH/H2O (4:1); (d) DDC, Et3N, γ-N-Cbz-L-
HABA; (e) H2, Pd(OH)2/C, 1 N HCl, MeOH.
Alternative strategies have also led to the synthesis of amphiphilic
aminoglycosides with excellent antibacterial profiles.23 Herein
we report the synthesis of a highly potent aminoglycoside (1)
derived from neomycin B with antibacterial activity against
strains of S. aureus expressing modifying enzymes and an
extensive clinical MRSA collection. The key design features
consisted of the implementation of a known but seldom used
deoxygenation protocol and N1-amide substitution, exempli-
fied by structure 1.
The synthetic effort began with the optimization of the
GareggꢀSamuelsson reaction, a modification of the classic
TipsonꢀCohen olefination by diol deoxygenation.25,26 The
substrates were paromomycin and neomycin B intermediates
2 and 5, suitably protected with per-N-benzyloxycarbonyl
groups and whose primary alcohols were capped by silylation
and tritylation, respectively (Scheme 1). The resulting inter-
mediates retained six secondary free alcohols, four of which
featured trans-diol configurations. Engaging these intermedi-
ates with triphenylphosphine, imidazole, and triiodoimidazole
in a refluxing 3:1 mixture of toluene and acetonitrile led to their
reliable tetradeoxygenation, affording 3 and 6 in good yield and
purity (Scheme 1).25ꢀ27
globally deprotected by acid and Pearlman’s hydrogenation to
afford N1-HABA-30,40,3000,4000-tetradeoxy-neomycin 7. The final
analogues were purified by benchtop silica gel column chroma-
tography with solvent mixtures of chloroform, methanol, and
ammonia liquor, providing pure final products as free bases, which
werethereaftertransformed to acetate saltsfor convenientspectro-
scopic analysis and handling.28
In the case of butirosin, it has been shown that the (L)-α-
hydroxy-γ-aminobutyric amide (L-HABA) chain naturally found
on the antibiotic provides an enhancement and broadening of
the antibacterial spectrum compared to its simple congener
ribostamycin.6,17 This intricate side-chain has a close fit within
the distorted H44 helix behind the bases A1492 and A1493
involved in mRNA decoding.10,29 Presumably, most aminoglycoside
modifying enzymes have not evolved active sites able to recog-
nize this unique modification in butirosin.6,16,17 Hence, semisyn-
thetic introduction of a N1-amide moiety in the 4,6-disubstituted
2-deoxystreptamine class has provided the aforementioned
benchmark drugs, such as amikacin, arbekacin, and isepamicin
(Figure 1).6,17
Herein, we took advantage of the simplified reactivity of
intermediates 3 and 6 to effect the chemoselective deprotection
of N1 via N1,O6-oxazolidinones.6,19,29,30 Notably, the cyclic
carbamate system can be selectively hydrolyzed in the presence
of carboxybenzylamino groups with mild aqueous base, liberat-
ing N1 for amide coupling. Our procedure of choice was to
expose intermediates 3 and 6 to a solution of LiOH in DMF for
24 h to liberate the C3-amino groups, in comparable yield
to two-step procedures (Scheme 2).19ꢀ24,29,30 These inter-
mediates were subsequently acylated under standard peptide
Under these conditions, the solitary alcohols at positions 6 and
200 in intermediates 2 or 5 were unreactive toward iodination, and a
common minor byproduct sporadically detected was comprised of
vinyliodide intermediates.25ꢀ27 The silyl groups atpositions 60 and
500 were removed with hydrogen fluoride to afford 3, followed by
standard hydrogenation using Pearlman’s catalyst in a slightly
acidified solution to yield 30,40,3000,4000-tetradeoxy-paromomycin
(4). The analogous 500-O-trityl neomycin intermediate 6 was
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LETTER
Table 1. Minimum Inhibitory Concentration of Tetradeoxygenated Antibiotic Analogues and Controls (μg/mL)a
S. aureus strain
Amk
Gent
NeoB
Par
H-Par
Ribo
Btr
4
9
7
1
ATCC 29213
APH(30/500)-III
ANT(40)-I
4
8
0.5
0.5
0.5
2
2
8
8
2
1
ND
8
0.5
32
0.5
1
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>32
>32
>32
ND
4
64
0.5
>64
0.5
1
APH(200)/AAC(60)
64
>64
>32
>32
16
>64
2
a Abbreviations: Amk, amikacin; Gent, gentamicin C complex; NeoB, neomycin B; Par, paromomycin; H-Par, N1-HABA paromomycin;
Ribo, ribostamycin; Btr, butirosin (N1-HABA ribostamycin); ATCC, American Type Colony Collection; APH, aminoglycoside phosphotransferase;
ANT, aminoglycoside nucleotidyltransferase; AAC, aminoglycoside N-acetyltransferase; ND, not determined.
Table 2. MRSA Collection MIC50/90 (μg/mL)
yield). We are optimistic that carefully designed antibiotics, such
as 1, can help in the fight against multidrug resistant S. aureus and
warrant further investigations of structureꢀactivity-relationships
and therapeutic index.
n = 50
Amk
Gent
1
MIC50
MIC90
32
>32
>32
0.25
0.5
>64
’ ASSOCIATED CONTENT
S
coupling conditions with γ-N-Cbz protected L-HABA to afford 8
and 10 (Scheme 2). Global deprotection was effected using
Pearlman’s hydrogenation under acidic conditions, thereby
affording the novel antibiotics N1-HABA-30,40,3000,4000-tetra-
deoxy-paromomycin (9), and N1-HABA-30,40,3000,4000-tetra-
deoxy-neomycin (1), in overall yields unmatched by previous
efforts with these frameworks.18ꢀ24
The novel tetradeoxy analogues, 4, 7, 9, and 1 (Table 1), were
tested against a panel of susceptible and aminoglycoside resistant
S. aureus strains and compared to a series of naturally occurring
aminoglycosides and the clinically relevant antibiotics gentami-
cin and amikacin.31,32
The novel tetradeoxygenated paromomycin analogues 4 and 9
were found to be 2ꢀ4-fold less active than their neomycin
analogues 7 and 1 (Table 1). This loss of activity may be attributed
to both loss of charge at position 60 (e.g., paromomycin vs
neomycin B) and slight disruption of the particular L-idose sugar
motif of ring D (e.g., neomycin B vs ribostamicin).33
Supporting Information. General methods, experimen-
b
tal procedures, HPLC purity reports, and H NMR and 13C
NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: (514) 343-6738. Fax: (514) 343-5728. E-mail:
stephen.hanessian@umontreal.ca.
’ ACKNOWLEDGMENT
We acknowledge Achaogen Inc., San Francisco, for financial
assistance, Fonds de Recherche en Santꢁe du Quꢁebec (FRSQ)
for a fellowship to J.P.M., and continued support from the
Natural Sciences and Engineering Research Council of Canada
(NSERC).
For both the paromomycin (4 vs 7) and neomycin (9 vs 1)
analogues, a 2ꢀ4-fold increase in activity against wild-type
S. aureus is observed in the presence of the N1-HABA substitution.
Effects of similar magnitude have been observed for the naturally
derived butirosin and ribostamicin pair.6,17
Although tetradeoxy-neomycin analogue 7 effectively frus-
trates the action of ANT(40)-I, it appears to be a target of the
two additional aminoglycoside-resistance determinants of
S. aureus, namely APH(30/500)-III, likely acting on the 500-hydro-
xyl group,34,35 and the 60N-acetyltransferase activity of the bifunc-
tional enzyme APH(200)/AAC(60).13ꢀ16 In contrast, tetradeoxy
analogue 1 was active against all three strains tested (Table 1).
In the light of these results, N1-HABA-30,40,3000,4000-tetradeoxy-
neomycin (1), was tested against a collection of 50 MRSA clinical
isolates (Table 2). The MIC50 and MIC90 values obtained for 1
showed a >64 fold increase in activity compared to the currently
clinically employed amikacin and gentamicin.
In conclusion, we have demonstrated a six step synthesis of
N1-HABA-30,40,3000,4000-tetradeoxy-neomycin (1), and its activity
against an extended panel of clinical MRSA strains. Our semisyn-
thetic strategy relied on a novel application of the chemoselective
GareggꢀSamuelsson deoxygenation reaction to aminoglycosides
for the first time, allowing the combination of N1-modification
via oxazolidinones for efficient access to promising antibiotic
analogues in excellent overall yields (6 steps for 1, 15% overall
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