Dimeric aminoglycosides as antibiotics

Article abstract of DOI:10.1002/anie.200353225

Double duty: Dimeric aminoglycoside antibiotics were designed by linking neamine or nebramine cores with various tethers differing in length and composition. The resulting dimeric aminoglycosides displayed high affinity for the 16S A site of ribosomal RNA (a representation of the complex is shown here) and were effective against various aminoglycoside-resistant bacterial strains.

Full text of DOI:10.1002/anie.200353225

Communications
Drug Design
Dimeric Aminoglycosides as Antibiotics**
Fabio Agnelli, Steven J. Sucheck,* Kenneth A. Marby,
David Rabuka, Su-Lan Yao, Pamela S. Sears,
Fu-Sen Liang, and Chi-Huey Wong*
Aminoglycosides are an important class of natural products
with antimicrobial activity against Gram-positive and Gram-
negative bacteria.[1] They have important clinical applications
in cases of opportunistic infections accompanying AIDS,
cystic fibrosis, and cancer. They are primarily used against
infections caused by Gram-negative bacteria, particularly
Pseudomonas aeruginosa, which are the leading cause of
morbidity and mortality in patients having cystic fibrosis
(CF). However, the rapid emergence of aminoglycoside-
resistant bacterial strains is limiting their efficacy and making
the discovery of structurally novel and more potent classes of
semisynthetic antibiotics a pressing necessity.[2]
Aminoglycosides exert their antibacterial action by selec-
tively binding to the prokaryotic ribosome, and thereby
impeding the synthesis of bacterial proteins.[3] In particular, 2-
deoxystreptamine aminoglycosides (Figure 1) are known to
selectively bind to the decoding A site on the 16Ssubunit of
the ribosomal RNA, causing inhibition of translation and thus
misreading of the genetic code.[4–10]
Structural studies modeling the interaction between neo-
mycin-class aminoglycosides and the 16SA site of E. coli
showed that multiple contacts occur between the major
groove of the RNA and rings I and II of the antibiotic.[11–15]
This conserved pseudodisaccharide motif is the essential
pharmacophore responsible for most of the specificity of
these interactions, while the rest of the molecule has a less
well-defined role. Using surface plasmon resonance (SPR)
and an immobilized 27-mer RNA construct (AS-wt), we
discovered that neamine (the pseudodisaccharide corre-
sponding to rings I and II of neomycin B, Figure 1) binds
the oligonucleotide in a 2:1 stoichiometry, with a Kd of about
10 mm at each binding site. Relying on a favorable divalent
interaction with the RNA target, we then constructed
[*] Dr. S. J. Sucheck, K. A. Marby, D. Rabuka, Dr. S.-L. Yao, Dr. P. S. Sears
Optimer Pharmaceuticals, Inc.
10110 Sorrento Valley Road, Suite C
San Diego, CA 92121 (USA)
Fax: (+1)858-909-0737
E-mail: ssucheck@optimerpharma.com.
Dr. F. Agnelli, F.-S. Liang, Prof. C.-H. Wong
Department of Chemistry and Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2409
E-mail: wong@scripps.edu
[**] Supported by the NIH (grants GM 58439 to C.-H.W. and SBIR 1 R43
AI056617-01 to S.J.S.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or fromthe author.
1562
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200353225
Angew. Chem. Int. Ed. 2004, 43, 1562 –1566

Angewandte
Chemie
monitored while structural modifications were introduced
into compound 1 without modifying the tether length. At the
same time the relative distance between the two neamine
cores was systematically probed by using “unobtrusive”
polyglycol tethers between 9 and 14 atoms in length, with
the expectation that the activity would reach a maximum at
the optimal distance between the two sites.
Compounds 5 and 9 were both prepared by an extension
of the previously reported procedure,[16] starting from per-
azido perbenzyl derivatives of neamine and nebramine,
respectively (see the Supporting Information for the exper-
imental details). Compound 15 was derived from tobramycin
(X = H, Figure 2) but displays a 4,5- (neomycin-type) linking
pattern. In this case, after the diazo-transfer reaction, the a-d-
glucopyranosyl bond of perazido tobramycin was cleaved by
reaction with refluxing 1m H2SO4 in methanol (Scheme 1).
The perazido nebramine so obtained was dissolved in 1:1
mixture of toluene and acetonitrile, placed on a rotary
evaporator, and allowed to stir at 508C under reduced
pressure with cyclohexanone dimethoxy ketal, in the presence
of a catalytic amount of p-toluenesulfonic acid, to afford 10.
Subsequent benzylation of the free hydroxy group at C4’ and
hydrolysis of the ketal afforded diol 11. Treatment of the diol
with tributyltin oxide followed by alkylation with BOMCl
selectively freed the hydroxy group at C5’ for further
manipulation. Compound 12 was then alkylated with (2R)-
(À)-glycidyl tosylate, followed by treatment with 0.5 equiv-
alents of N,N’-dibenzyl-1,4-diaminobutane. Staudinger reduc-
tion and catalytic hydrogenation finally gave deprotected
dimer 15.
The influence of the length and hydrophilicity of the
linker was assessed by preparing two series of compounds:
one with C2-symmetric hydrophilic polyglycol tethers and the
other with hydrophobic polymethylene tethers, as
shown in Figure 3. The polyglycol linkers were
obtained starting from the corresponding diallyl
ethers, either by ozonolysis/borohydride reduction or
hydroboration/oxidation reaction sequences, followed
by standard tosylation conditions. The remaining link-
ers 17c and 17 f were prepared by tosylation of
commercially available tetra- and pentaethylene gly-
cols, respectively. When a solution of the ditosylates 17
and two equivalents of perazido perbenzyl neamine 2
in DMF was treated with an excess of NaH, the
reaction smoothly proceeded to afford protected
dimers 18a–f (Scheme 2). Surprisingly, when the cor-
responding bromides, iodides, and triflates were used,
very sluggish reactions ensued. The order of addition of
the reagents also proved to be crucial: preformation of
the neamine alkoxide, followed by slow addition of the
electrophile resulted in a complex mixture of products.
A two-step catalytic hydrogenation finally afforded the
deprotected dimers 19a–f in 51–72% yields.[18] The
more hydrophobic compounds 19g and 19h were
obtained by similar routes, starting from commercial
1,10-decenediol and 1,12-dodecenediol, respectively.
When the series of polyglycol dimers 19a–f was
Figure 1. Structures of some representative aminoglycoside antibiotics
of the 2-deoxystreptamine class.
neamine dimers. It was determined that 1 (5-linked with a
(CH2)4 tether) binds to AS-wt with a 1:1 stoichiometry,
displaying a Kd of about 40 nm (Figure 2).[16]
Although this binding affinity is better than that of
neomycin B, it is still about 250 times lower than the
approximate theoretical value.[17] Since no direct structural
information about the details of the divalent interaction is
available, a rigorous rational-design approach was not
possible. In order to improve the affinity of our dimers for
RNA, we focused on three possible points of diversity: ring I
of the pseudodisaccharide, the linker attachment points, and
the linking domain. RNA affinity and antibiotic activity were
tested by means of SPR against immobilized AS-wt
RNA, binding constants in the 1–8 mm range were
Figure 2. Structures, binding constants (Kd), and minimum inhibitory concen-
trations (MIC) for the neamine and nebramine dimers.
Angew. Chem. Int. Ed. 2004, 43, 1562 –1566
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1563

Communications
Figure 3. Structures, binding constants (Kd), and minimum inhibitory con-
centrations (MIC) for neamine dimers with hydrophylic (19a–f) and hydro-
phobic (19g,h) tethers of increasing length. No obvious correlation is appa-
rent between tether length and binding affinity.
Scheme 1. Reagents and conditions: a) TfN3, CuSO4, TEA, MeOH,
CH2Cl2, H2O; b) H2SO4, MeOH, reflux; c) cyclohexanone dimethyl
ketal, pTsOH, PhMe, MeCN, 508C, 20 mm Hg; d) BnBr, NaH, DMF;
e) HCl, MeOH; f) SnO2, PhMe, reflux; g) BOMCl, Bu4NI; h) (R)-(À)-
glycidyl tosylate, NaH, DMF; i) N,N’-dibenzyl-1,4-diaminobutane,
EtOH, 70–858C; j) PMe3, NH4OH, H2O, THF; k) H2, Pd(OH)2/C, H2O/
AcOH. BOMCl=benzyloxymethyl chloride, DMF=dimethylformamide,
pTsOH=p-toluenesulfonic acid, TEA=triethylamine, Tf=trifluorosul-
fonyl.
found, with no obvious dependence of Kd on tether length
(Figure 3). The antimicrobial activity was tested versus three
standard, antibiotic-sensitive strains: Staphylococcus aureus
(ATCC 29213), Escherichia coli (ATCC 25922), and Pseudo-
monas aeruginosa (ATCC 27853). The activity of the com-
pounds was also weak, in some cases similar to that of
neamine, but generally weakening with increasing tether
length. A possible explanation for these results might reside
in the high hydrophilicity of the linker. In order to test this
hypothesis, two representative tether lengths (10 and 12
atoms) were chosen, and the more hydrophobic dimers 19g
and 19h were synthesized. When tested against AS-wt RNA,
they displayed Kd values and activities analogous to those of
their polyether-linked counterparts.
Scheme 2. Reagents and conditions: a) 17a–h (0.6 equiv), NaH, DMF;
b) H2, Pd(OH)2/C, THF, MeOH; c) H2, Pd(OH)2/C, aq HCl (1 equiv),
MeOH, H2O.
The diaminobutane-linked dimers shown in Figure 2
possessed significant affinity and antimicrobial activity, and
all displayed a binding stoichiometry of 1:1 for the RNA–
antibiotic complex. Surprisingly, compound 5, lacking the two
N-methyl groups in the tether with respect to 1, displayed
slightly lower affinities and activities (about threefold) than
its predecessor. These data might suggest that this additional
hydrogen-bond-donating capability is not a fundamental
1564
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 1562 –1566

Angewandte
Chemie
Table 2: MIC [mgmLÀ1] of compounds 9 and 15 against a panel of
biofilm-producing P. aeruginosa fromCF clinical isolates.
feature for binding and that the basicity of the two nitrogen
atoms might play a role in determining such properties.
The Kd values of the two nebramine-derived dimers 9 and
15 were comparable to those of the neamine-derived com-
pounds (in the 150 nm range), showing very little contribution
of the 3’-hydroxy group to binding. The position of the anchor
points for the two dimers (4,6 tobramycin-like for 9 and 4,5
neomycin-like for 15) seems to play no significant role in the
RNA recognition. In spite of these similarities, the antimi-
crobial activity for the two compounds differed by a factor of
four against E. coli. These findings, and in particular direct
comparison of compounds 1, 5, 15, 19d, and 19h, show that
the contribution of the linker to binding cannot be neglected
and that a more subtle balance of interactions governs the
binding affinities of these small ligands.
P. aeruginosa
Resistance
Tobramycin
9
15
27 EN
64 EN
24 EN
25 EN
90 EM
98 EM
89 EM
sensitive
sensitive
sensitive
sensitive
resistant
resistant
resistant
4
8
4
2
32
64
>64
>64
4
1
4
2
4
4
0.5
4
8
64
16
64
>64
fold, Table 3). A possible explanation of this finding might
reside in the increased positive charge density that these
molecules bear with respect to neomycin B. However, a
The antimicrobial activity of the diaminobutane-linked
dimers was further investigated against different panels of
aminoglycoside-sensitive and -resistant P. aeruginosa strains
obtained from CF clinical isolates. The neamine-derived
compounds 1 and 5 and the nebramine-derived dimer 9
possess activity levels comparable to (and in some cases
greater than) tobramycin, which is considered the current
gold standard for treatment of CF infections (Table 1). A very
Table 3: Kd [mm] and selectivity profile of neomycin B and compounds 1
and 5 for the 16S A site (expressed as the ratio of the binding constants,
in parentheses) in RNAs of interest.
RNA[a]
Neomycin B
1
5
16S A site
0.2
0.04
0.14
Bcr-Abl
PAX3-FKHR
HIV-FSS
HIV-PAS mRNA
HCV-IIb IRES
HCV-IIId IRES
E. coli TG mRNA
2 (10)
0.8 (4)
1.6 (8)
2 (10)
2.9 (14)
2.9 (14)
2.8 (14)
0.55 (14)
1.1 (27)
1.1 (27)
1.2 (30)
0.58 (14)
1.3 (32)
1.6 (40)
0.46 (3)
1.4 (10)
1.9 (13)
1.7 (12)
0.83 (6)
1.8 (13)
2.7 (19)
Table 1: MIC values [mgmLÀ1] of tobramycin and compounds 1 and 5
against S. aureus ATCC 33591 (MRSA) and P. aeruginosa clinical isolates.
OrganismResistanycien
Tobram
1
5
S. aureus
resistant
>64
8
16
16
[a] Arbitrary abbreviations used. HIV-FSS: HIV frameshift signal; HIV-
PAS: HIV protease active site mRNA; HCV-IIb IRES: HCV IRES domain
IIb; HCV-IIId IRES: HCV IRES domain IIId; E. coli TG: E. coli trans-
glycosilase mRNA. See the Supporting Information for RNA sequences.
ATCC 33591 (MRSA)
P. aeruginosa
sensitive
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
8
8
4
8
(strain 034)[a]
P. aeruginosa
128
128
>200
50
8
(strain 609)[a]
P. aeruginosa
8
(strain 663)[a]
P. aeruginosa
generally significant increase in selectivity for the 16SA site
for compound 1 (and, to a lesser extent, compound 5) reveals
that more subtle factors govern the specificity of the amino-
glycoside–RNA interaction. Indeed, design of more potent
and selective binders remains the major challenge for future
research in the field of aminoglycoside antibiotics.
In conclusion, the neamine and nebramine dimers syn-
thesized in this study represent a new class of aminoglycoside
antibiotics that are active against several aminoglycoside-
resistant bacterial strains and may show promise for the
treatment of P. aeruginosa infections. In order to fully
elucidate the structural details of the dimeric interaction
and further improve the affinity, potency, and specificity of
newly designed aminoglycoside antibiotics, additional studies
are currently underway.
12.5
6.3
6.3
6.3
6.3
12.5
6.3
(PAE_NUH01)[b]
P. aeruginosa
6.3
6.3
3.1
3.1
6.3
(PAE_NUH02)[b]
P. aeruginosa
50
(PAE_NUH03)[b]
P. aeruginosa
100
100
100
(PAE_NUH04)[b]
P. aeruginosa
(PAE_NUH05)[b]
P. aeruginosa
(PAE_NUH06)[b]
[a] Strains obtained fromthe Foothills Medical Centre, Calgary, AB,
Canada. [b] Strains obtained fromNational University Hospital, National
University of Singapore.
Received: November 3, 2003 [Z53225]
common complication in CF-related P. aeruginosa infections
is biofilm formation. Interestingly, when tested against a panel
of biofilm-producing P. aeruginosa strains from CF isolates,
the tobramycin-derived compound 15 was revealed to be
uniquely effective and in many cases more potent than
tobramycin itself (Table 2).
Keywords: aminoglycosides · antibiotics · carbohydrates ·
.
drug design · RNA recognition
Further SPR analysis of 1 and 5 interacting with other
RNA targets of interest showed that both compounds are
more active than neomycin B against the 16SA site (1.5- to 5-
[1] M. K. Kim, D. P. Nicolaou, Infect. Dis. Ther. 2002, 28, 125.
[2] For recent work on aminoglycosides see: B. Llano-Sotelo, E. F.
Azucena, L. P. Kotra, S. Mobashery, C. S. Chow, Chem. Biol.
Angew. Chem. Int. Ed. 2004, 43, 1562 –1566
www.angewandte.org
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1565

Communications
2002, 9, 455; J. Haddad, S. Vakulenko, S. Mobashery, J. Am.
Chem. Soc. 1999, 121, 11922; J. Haddad, L. P. Kotra, B. Llano-
Sotelo, C. Kim, E. F. Azucena, M. Liu, S. B. Vakulenko, C. S.
Chow, S. Mobashery, J. Am. Chem. Soc. 2002, 124, 3229; S.
Hanessian, M. Tremblay, A. Kornienko, N. Moitessier, Tetrahe-
dron 2001, 57, 3255; S. Hanessian, M. Tremblay, E. E. Swayze,
Tetrahedron 2003, 59, 983; S. Hanessian, A. Kornienko, E. E.
Swayze, Tetrahedron 2003, 59, 995; N. W. Luedtke, Q. Liu, Y.
Tor, Biochemistry 2003, 42, 1391; K. Michael, H. Wang, Y. Tor,
Bioorg. Med. Chem. 1999, 7, 1951; S. R. Kirk, Y. Tor, Bioorg.
Med. Chem. 1999, 7, 1979; S. R. Kirk, N. W. Luedtke, Y. Tor, J.
Am. Chem. Soc. 2000, 122, 980; N. W. Luedtke, T. J. Baker, M.
Goodman, Y. Tor, J. Am. Chem. Soc. 2000, 122, 12035; Y. Wang,
R. R. Rando, Chem. Biol. 1995, 2, 281H. Wang, Y. Tor, Angew.
Chem. 1998, 110, 117; Angew. Chem. Int. Ed. 1998, 37, 109.
[3] H. Umezawa, I. R. Hooper, Aminoglycoside Antibiotics,
Springer, New York, 1982.
[4] J. H. Cate, M. M. Yusupov, G. Z. Yusupova, T. N. Earnest, H. F.
Noller, Science 1999, 285, 2095.
[5] W. M. Clemons, J. L. C. May, B. T. Wimberly, J. P. McCutcheon,
M. S. Capel, V. Ramakrishnan, Nature 1999, 400, 833.
[6] D. Moazed, H. F. Noller, Nature 1987, 327, 389.
[7] P. Nissen, J. Hansen, N. Ban, P. B. Moore, T. A. Steitz, Science
2000, 289, 920.
[8] F. Schlunzen, R. Zarivach, J. Harms, A. Bashan, A. Tocilj, R.
Albrecht, A. Yonath, F. Franceschi, Nature 2001, 413, 814.
[9] B. T. Wimberly, D. E. Brodersen, W. M. Clemons, R. J. Morgan-
Warren, A. P. Carter, C. Vonrhein, T. Hartsch, V. Ramakrishnan,
Nature 2000, 407, 327.
[10] M. M. Yusupov, G. Z. Yusupova, A. Baucom, K. Lieberman,
T. N. Earnest, J. H. D. Cate, H. F. Noller, Science 2001, 292, 883.
[11] D. Fourmy, M. I. Recht, S. C. Blanchard, J. D. Puglisi, Science
1996, 274, 1367.
[12] D. Fourmy, M. I. Recht, J. Puglisi, J. Mol. Biol. 1998, 277, 347.
[13] S. R. Lynch, R. L. Gonzalez, J. D. Puglisi, Structure 2003, 11, 43.
[14] Q. Vicens, E. Westhof, Chem. Biol. 2002, 9, 747.
[15] Q. Vicens, E. Westhof, Structure 2002, 10, 647.
[16] Other dimers reported in the literature were based on a
monomer with 1:1 stoichiometric binding, thus only slightly
improving the affinity. See ref. [2] and S. J. Sucheck, A. L. Wong,
K. M. Koeller, D. D. Boehr, K. A. Draker, P. Sears, G. D. Wright,
C. H. Wong, J. Am. Chem. Soc. 2000, 122, 5230.
[17] Assuming independent binding events for each site, the total
binding energy for a dimerically linked molecule DGT should be
the sum of the binding energies for each monomer, DG1 and
DG2, plus the contribution of the linker DGL. DGL includes both
the free energy of the linker interaction with the RNA and also a
term that accounts for the reduced entropy change of binding
due to the tethering of the monomers. If the effect of the linker is
ignored in a first approximation, the resulting Kd should
therefore be in the picomolar range.
[18] A single step deprotection using 20% Pd(OH)2/C (Degussa
type) in 1:1 AcOH/H2O under H2 could also be achieved when
fresh catalyst was added after the reaction appeared to stop.
1566
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 1562 –1566