V. Nizet, Y. Tor et al.
MED
scaffold an ideal starting point for the development of new,
potentially more effective antibiotics.[12] Indeed, semisynthetic,
second-generation aminoglycosides such as amikacin have
proven very successful in the clinic.[9] With this in mind, we set
out to make minor structural modifications to selected mem-
bers of these known antibiotics in an effort to retain or even
improve upon their affinity for the 16S A-site, while potentially
decreasing their susceptibility to the most prevalent modes of
bacterial deactivation.
using di-tert-butyl dicarbonate. The single primary alcohol of
(Boc)5tobramycin (17) was then selectively converted to a steri-
cally demanding sulfonate by treatment with 2,4,6-triisopropyl-
benzenesulfonyl chloride (TPSCl) in pyridine.[12f] Reflux in meth-
anolic ammonia then afforded 6’’-deoxy-6’’-amino(Boc)5tobra-
mycin (19). This three-step process, converting primary alco-
hols into amines, has been previously used in our laboratory
for the synthesis of other modified aminoglycosides.[14] Treat-
ment of the single free amine with 1,3-di-Boc-2-(trifluorome-
thylsulfonyl)guanidine in the presence of triethylamine gave
fully Boc-protected guanidino-aminoglycoside, 6’’-deoxy-6’’-
guanidino(Boc)7tobramycin (20). Acidic deprotection of all Boc
groups using a one to one mixture of trifluoroacetic acid (TFA)
and tri-iso-propyl silane (TIPS) in dichloromethane, followed by
HPLC purification, afforded the analytically pure 6’’-deoxy-6’’-
guanidinotobramycin (2).
A slightly different approach was employed for the synthesis
of 6’’-deoxy-6’’-guanidinoapramycin (16). Care had to be taken
in preparing the sulfonate to prevent activation of multiple hy-
droxy groups. This was achieved by using fewer equivalents of
the sulfonyl chloride (Step b, Scheme 2), in comparison with
the other aminoglycosides. In addition, unlike other aminogly-
coside derivatives, 6’’-deoxy-6’’-triisopropylbenzylsulfonyl(Boc)5-
apramycin (22) was found to degrade in refluxing methanolic
ammonia. Instead, it was converted to the amino intermediate
via a two-step process wherein the sulfonyl functionality was
first substituted for an azide using sodium azide and then sub-
sequently reduced in a palladium-catalyzed hydrogenation to
give 6’’-deoxy-6’’-guanidino(Boc)7apramycin (23).
Here we disclose the synthesis of a small focused library of
aminoglycoside derivatives selectively modified at one or two
positions. We strategically replace amine or hydroxy functional-
ities with a guanidine group in tobramycin, amikacin, kanamy-
cin A, neomycin B neamine, paromomycin, and apramycin (Fig-
ure 1).[12a,c] Most of the newly synthesized guanidino-aminogly-
cosides displayed enhanced affinity for the ribosomal A-site,
the biological target of the parent derivatives, as determined
by an in vitro fluorescence resonance energy transfer (FRET)-
based binding assay. The results of antibacterial tests on a di-
verse collection of regular and resistant strains illustrate that
certain analogues exhibit improved potency against resistant
strains, including MRSA. An amikacin analogue shows particu-
lar promise with activities greater or equal to those of the
parent antibiotic in the majority of strains tested.
Results
Design strategy
The bacterial A-site is a highly discriminating RNA target that is
not tolerant of major structural changes to its cognate li-
gands.[12n,13] Therefore, we decided to selectively and strategi-
cally functionalize aminoglycosides at positions that are less
likely to perturb binding and, at the same time, are synthetical-
ly accessible.[12a,c] A relatively small modification, which would
retain or enhance the overall charge of the RNA-targeting anti-
biotic, could be achieved by replacing a hydroxy or amine
group with a guanidine functionality. In contrast to amines,
the planar guanidine functional group is highly basic and can
participate in well-defined directional hydrogen bonds. To
probe this strategy, we derivatized several aminoglycoside anti-
biotics, including neamine, kanamycin A, tobramycin, paromo-
mycin, neomycin B, amikacin, and apramycin, by converting se-
lected primary alcohols into guanidine groups, or turning an
existing aminomethyl group into the corresponding guanidine
derivative. We hypothesized that beyond yielding a greater af-
finity for the A-site, functional group changes at some sites
could potentially lead to decreased recognition by aminoglyco-
side-modifying enzymes, one of the major bacterial resistance
mechanisms.
A third protocol was used for selectively converting amino-
methyl groups in aminoglycosides to the corresponding guani-
dine derivatives, relying on their higher nucleophilicity com-
pared with the other more sterically hindered amines. Treat-
ment of unprotected tobramycin with sub-stoichiometric
quantities of 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine
followed by deprotection in a one to one mixture of TFA in di-
chloromethane provided desired derivative 3 (Scheme 3). Simi-
lar protocols were applied to amikacin and neamine. Note, all
guanidino-aminoglycoside derivatives were first converted to
their free-base form by exposure to a strong basic anion (OHÀ)
exchange resin (Monosphere 550A, Dowex) prior to their eval-
uation in any A-site binding assays or antibacterial experi-
ments.
Affinity for the bacterial 16S A-site RNA construct
To determine the affinity of all derivatives to the bacterial 16S
A-site, we used a modified version of a FRET-based assay that
was previously developed in our lab.[15] In this assay, a coumar-
in–aminoglycoside conjugate placeholder binds to a Dy-547-la-
beled 16S A-site construct. Coumarin acts as a FRET donor to
its matched Dy-547 acceptor. The affinity of unlabeled ligands
for the A-site can be measured in a competition experiment,
where the compound of interest is titrated in and displaces
the coumarin–aminoglycoside placeholder, resulting in a de-
creased sensitized acceptor emission. Different coumarin–ami-
Synthesis
A general synthetic approach for the conversion of aminogly-
coside primary alcohols to guanidinium groups is illustrated
using tobramycin (1) as an example (Scheme 1). First, all
amines were globally tert-butyloxycarbonyl (Boc)-protected
1238
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ChemMedChem 2012, 7, 1237 – 1244