Rational design of azepane-glycoside antibiotics targeting the bacterial ribosome

Article abstract of DOI:10.1016/j.bmcl.2003.11.028

RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.

Full text of DOI:10.1016/j.bmcl.2003.11.028

Bioorganic & Medicinal Chemistry Letters 14 (2004) 713–718
Rational design of azepane-glycoside antibiotics targeting the
bacterial ribosome
Sofia Barluenga,a,y Klaus B. Simonsen,a Ethel S. Littlefield,a Benjamin K. Ayida,a
Dionisios Vourloumis,a Geoffrey C. Winters,a Masayuki Takahashi,a Sarah Shandrick,b
Qiang Zhao,b Qing Hanb and Thomas Hermannb,*
aDepartment of Medicinal Chemistry, Anadys Pharmaceuticals, Inc., 9050 Camino Santa Fe, San Diego, CA 92121, USA
bDepartments of RNA Biochemistry and Structural Chemistry, Anadys Pharmaceuticals, Inc., 9050 Camino Santa Fe,
San Diego, CA 92121, USA
Received 8 September 2003; revised 5 November 2003; accepted 14 November 2003
Abstract—RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which
participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been
used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic
scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of
Staphylococcus aureus, including aminoglycoside-resistant strains.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
cosides and fragments thereof constitute ideal starting
points for the design and synthesis of novel RNA
binders and antibiotics. In previous approaches towards
novel aminoglycosides, the 2-deoxystreptamine (2-
DOS)15,16 and glucosamine fragments17À19 (Fig. 1a)
have been derivatized as individual moieties and in
context of the neamine20À23 and paromamine24,25 scaf-
folds. Rational design of RNA-targeted novel amino-
glycosides is now feasible based on three-dimensional
structures, which were recently determined for com-
plexes of the bacterial decoding-site RNA26À29 and
whole ribosomal subunits.7,30 Synthetic aminoglyco-
sides may achieve binding affinity and specificity for the
decoding-site RNA target, and antibacterial potency
comparable to the natural antibiotics, however, poten-
tially without being compromised by resistance
mechanisms specific for the natural products.
The bacterial ribosome is a key target for many anti-
biotics,1 among them natural aminoglycosides such as
paromomycin, neomycin B, and neamine (Fig. 1a).
Aminoglycoside antibiotics interact specifically with the
decoding-site ribosomal RNA (rRNA) (Fig. 1b)2À4 and
disrupt functional protein synthesis by interfering with
mRNA-decoding fidelity.5À7 Upon binding, aminogly-
cosides displace two key adenine residues from the deep
groove of the decoding-site rRNA. The ensuing con-
formational change in the adenines, which are involved
in contacts to the mRNA–tRNA hybrid, leads to
reduced discrimination against noncognate tRNAs and
therefore decreased translational accuracy.6,7 The effi-
cacy of aminoglycosides as antibiotics is compromised
by undesirable pharmacological profiles and resistance
development.8,9 Their capacity to bind with high affinity
to the bacterial decoding site and several other RNA
targets,10 however, renders them a lead paradigm in
RNA molecular recognition.11À14 Therefore, aminogly-
In our efforts to develop novel antibiotics, we have sys-
tematically studied aminoglycoside mimetics targeted at
the bacterial decoding-site RNA.16,18,19,25 Here, we
describe the design and synthesis of a series of azepane–
glycosides, in which a substituted heterocycle has been
introduced to mimic the unique spatial arrangement of
key functional groups in 2-DOS (Fig. 1c–e), a uni-
versally conserved scaffold of natural aminoglycoside
antibiotics. Moreover, we report the in vitro and biolo-
Keywords: Antibiotics; RNA; Structure-based design.
* Corresponding author. Tel.:+1-858-530-3659; fax:+1-858-527-1539;
e-mail:
Present address: Laboratoire de Chimie Organique et Bioorganique,
Institut de Science et d’Ingenierie Supramoleculaires, 8 rue Gaspard
Monge, 67000 Strasbourg, France.
y
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.028

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S. Barluenga et al. / Bioorg. Med. Chem. Lett. 14 (2004) 713–718
porated into the azepane ring, thereby modulating the
amine basicity. Conformational analysis showed that the
azepane ring in 1 adopts preferentially a conformation
that projects the amino groups at similar vectors as in 2-
DOS while optimizing the potential interaction surface of
the cyclic system (Fig. 1d). A hydroxy group at the 4-
position, and in anti-configuration relative to the adjacent
exocyclic amino functionality, provided a handle for the
attachment of a glucosamine sugar. A hydroxymethyl
group next to the endocyclic secondary amine could
potentially dock into the binding site of a structurally
conserved water molecule at the deep-groove edge of the
U1406/U1495 base pair (Fig. 1e). The hydroxymethyl
functionality provided also an attachment site for further
substitutions of the azepane-glycoside core scaffold.
3. Synthesis
The key step for the synthesis of the 4,5-disubstituted
azepanes relied on
a
ring-closing metathesis
reaction31À33 which gave access to the seven-membered
ring containing a double bond as a handle for further
derivatization. The development of improved catalysts34
which tolerate a broad range of functionalities within
the molecule, has rendered olefin metathesis the reaction
of choice for the construction of medium-sized
rings.35,36 We planned the preparation of the 4-hydroxy-
5-azido-azepane 10 from alkene 5 by epoxidation fol-
lowed by azide opening of the intermediate 7. Alkene 5
would arise in the forward direction by metathesis of
the olefinic groups in intermediate 4. It was anticipated
that the rigid nature of the cyclic carbamate in 4 would
accelerate the metathesis reaction by providing a favor-
able pre-orientation of the olefin moieties.
Figure 1. (a) Natural aminoglycoside antibiotics such as paromomycin
and neomycin B are derived from paromamine and neamine, which
share the 2-deoxystreptamine (2-DOS) and glucosamine cores, both of
which are involved in RNA molecular-recognition. (b) Secondary
structure of the bacterial decoding-site RNA. (c) Design concept of an
azepane-glycoside ligand, 1, for the decoding-site RNA, derived from
coupling of 6-aminoglucosamine with a 5-aminoazepane scaffold as 2-
DOS mimetic. (d) Energy-minimized solution conformation of the
designed azepane-glycoside 1 (yellow) superimposed on paromamine
(blue). Note that the exocyclic 3-amino functionality of paromamine is
replaced by the endocyclic secondary amine in the azepane ring, and
the 2-hydroxymethyl substituent in the azepane derivative coincides
with a water molecule hydrogen-bonded to the 4-hydroxy group of
paromamine in the RNA complex. (e) Model of the azepane–glycoside
1 (yellow) docked to the three-dimensional structure7,30 of the bacter-
ial decoding-site RNA in complex with paromomycin (blue; only par-
omamine core shown). RNA bases are in dark, sugar phosphate
backbone in light grey with phosphate groups emphasized in magenta.
A water molecule participating in the non-Watson–Crick U1406/
U1495 base pair26,27 and interacting with a 2-DOS hydroxy group is
shown as blue sphere. The flipped-out adenine residues 1492 and 1493,
and the unpaired adenine 1408 are shown in green.
Synthesis of the glycosylic acceptors 8–11 commenced
with the preparation and functionalization of alkene 5
as outlined in Figure 2. The precursor for the metath-
esis, 4, was prepared in five steps from l-allylglycine (2).
Esterification of 2 followed by protection of the amine
and subsequent ester reduction produced a mixture of
the cyclic carbamate 3 and the expected primary alco-
hol, which was completely converted to 3 upon base
treatment. Alkylation of 3 with 4-bromo-1-butene furn-
ished 4 in good overall yields. DMF was essential as a
cosolvent in this alkylation step to prevent decomposi-
tion of 4-bromo-1-butene during the prolonged reaction
time necessary for completion. Exposure of 4 to 1st
generation Grubbs catalyst31,32 afforded 5 as the only
detectable product in 85% yield. Complete consump-
tion of 4 to produce 5 was observed within 10 min, even
at relative high substrate concentration (0.2 M), sup-
porting the importance of pre-orientation of the react-
ing olefins in the metathesis step. Epoxidation of 5
produced a 2:1 mixture of diastereomeric epoxides,
which were separated by column chromatography on
deactivated silica gel, to avoid decomposition during the
purification, to afford the pure isomers (syn-6, and anti-7)
in acceptable yields. Azide opening of the epoxide iso-
mers 6 and 7 produced the acceptors 8–11 in overall
good yields. The anti relationship of epoxide 7 and the
configuration of azido alcohol 9 were unequivocally
gical testing of the synthetic azepane–glycosides for
their inhibitory potency against bacterial protein synth-
esis and cell growth.
2. Design of the azepane-glycosides
The azepane scaffold as a mimetic of 2-DOS was
designed by molecular modeling (see Experimental)
based on the crystal structure7,30 of paromomycin com-
plexed with the bacterial decoding site (Fig. 1). The
design of the seven-membered heterocycle in 1 (Fig. 1c)
was aimed at achieving the distinct spatial arrangement
of the 2-DOS amino groups, one of which was incor-

S. Barluenga et al. / Bioorg. Med. Chem. Lett. 14 (2004) 713–718
715
Figure 3. Synthesis of azepane-glycoside derivatives of 1; Reagents
and solvents: (a) Me3P, THF; (b) Pd(OH)2, H2, AcOH; (c) KOH,
EtOH; (d) HCHO, NaCNBH3, ZnCl2, MeOH; (e) Me3P, THF; (f)
Pd(OH)2, H2, AcOH; (g) Me3P, THF; (h) CbzCl, NaHCO3, H2O/
dioxane; (i) 19!20: (i) MsCl, pyridine, (ii) LiCl, DMF, (iii) Pd(OH)2,
H2, AcOH; 19!21: (i) DAST, CH2Cl2, (ii) Pd(OH)2, H2, AcOH;
19!22: (i) MsCl, pyridine, (ii) NaN3, DMF, (iii) Pd(OH)2, H2, AcOH,
(iv) Me3P, THF. Ms, methanesulfonyl; DAST, (diethylamido)sulfur
trifluoride.
Figure 2. Synthesis of the active azepane-glycoside isomers; Reagents
and solvents: (a) SOCl2, MeOH; (b) CbzCl, NaHCO3, H2O/dioxane;
(c) LiAlH4, THF; (d) NaH, THF; (e) 4-bromo-1-butene, Cs2CO3,
DMF/THF; (f) Grubbs catalyst, CH2Cl2; (g) 1,1,1-trifluoroacetone,
oxone; (h) NaN3, EtOH/H2O; (i) 12 or 13, NIS, TfOH; (j) KOH,
EtOH; (k) Me3P, THF, H2O; (l) Pd(OH)2, H2, AcOH. Ph, phenyl; Bn,
benzyl; Cbz, benzyloxycarbonyl; THF, tetrahydrofuran; DMF, N,N-
dimethylformamide; Grubbs catalyst, benzylidine-bis(tricyclohexyl-
phosphine)dichlororuthenium; oxone, potassium peroxymonosulfate;
NIS, N-iodosuccinimide; TfOH, trifluorosulfonic acid.
4. Biological activity and structure–activity relationship
The synthesized azepane-glycosides were tested for their
activity as inhibitors of bacterial in vitro translation and
growth, as well as for their toxicity against eukaryotic
cells, and for binding to the bacterial decoding-site
RNA target (see Experimental).
determined by single crystal X-ray structure analysis
(data not shown).
The azepane-glycoside 1 and its diastereomer 23 were
inhibitors of bacterial in vitro translation at low micro-
molar concentrations (Table 1 and Fig. 4). While both
compounds showed antibacterial activity against the
Gram-positive Staphylococcus aureus, the isomer 23 was
more potent (MIC=64 and 32 mg/mL for 1 and 23,
respectively). Importantly, both azepane-glycosides, 1
and 23, retained their antibacterial potency against
aminoglycoside-resistant S. aureus (MIC=64 and 32
mg/mL for 1 and 23, respectively, against multi-drug-
resistant BAA40 and BAA44 strains). Neither 1 or 23
inhibited growth of the Gram-negative Escherichia coli
at 64 mg/mL or interfered with proliferation of eukary-
otic cells up to the highest concentration tested (150 mg/
mL), corresponding to a CC50 >320 mM.
Synthesis of the final products is outlined exemplarily
for compounds 1 and 14 (Fig. 2). Glycosylation of the
inseparable 1:2 mixture of 10 and 11 with donors 12 or
1318,19,21 provided the a-pseudo-disaccharides as the
only detectable anomers, which were separated by col-
umn chromatography to afford the pure regioisomers.
A three-step deprotection protocol completed the
synthesis of 1 and 14. Specifically, the carbamate func-
tionality was base-cleaved to produce the hydroxy-
methyl azido-azepane which was reduced to the
corresponding amine (see compound 29, Table 1) and
finally hydrogenolyzed to furnish 1 and 14 in overall
good yields. The remaining diastereomers 23–28 were
synthesized following the same protocol starting either
from pure l-, d-, or racemic allylglycine.
Modeling studies suggested that although the azepane
scaffold adopts distinct conformations in the diaster-
eomers 1 and 23, the latter isomer could be docked to
the decoding-site target in a similar fashion as the ori-
ginally designed compound 1. The diastereomer 24,
which contains the enantiomeric azepane scaffold of 1,
showed no significant potency against bacterial transla-
tion or growth, in line with its weak affinity for the
decoding-site RNA. The affinity of 1 and 23 for binding
to the decoding site RNA (Fig. 4) was at similar levels in
the low micromolar range, supporting the proposed
mode of action for these translation inhibitors as
ligands of the bacterial rRNA target.
The synthesis of derivatives of 1 (Fig. 3) used inter-
mediate 15 which was obtained by coupling of 10 and
12. Carbamate 16 was prepared from 15 by a two-step
deprotection sequence. Carbamate cleavage of 15 pro-
duced the corresponding hydroxymethyl analogue 17,
which was converted to the N-methyl analogue 18 by
reductive amination followed by a two-step deprotec-
tion protocol. Compound 17 was converted to 19 in two
steps, which was further reacted with DAST to produce 21
after deprotection. Mesylation of 19 followed by chloride
or azide displacement gave rise, after deprotection, to 20
and 22, respectively.

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S. Barluenga et al. / Bioorg. Med. Chem. Lett. 14 (2004) 713–718
Table 1. Structure–activity relationships for synthetic azepane-
glycosidesa
Structure
Compd #
IVT IC50 [mM]/RNA IC50 [mM]b
MIC [mg/mL] (E. coli/S. aureus)
Figure 4. Dose–response curves and IC50 values calculated thereof for
the azepane-glycoside 1 showing (a) binding of the ligand to the bac-
terial decoding-site RNA in a fluorescence assay, and (b) inhibition of
bacterial in vitro translation by the compound. The binding curve was
obtained as an average of two titrations and fitted to a single-site
model, resulting in a Hill-coefficient of 0.95. The translation inhibition
curve was calculated by averaging over six independent dose–response
experiments. Error bars indicate ꢁ1s.
translation, however, likely due to interference with a
ribosomal target distinct from the decoding site, as
binding to the latter target was not detectable. None of
the regioisomers 25–28 inhibited bacterial growth at
concentrations ꢀ64 mg/mL. Modeling studies indicated
that, in such regioisomers, the hydroxymethyl sub-
stituent interferes sterically with the docking of the 5-
amino-azepane scaffold to the decoding-site RNA.
Change of the amino sugar moiety in 1 to the hydroxy
derivative in 14 resulted in a more than 10-fold loss of
activity in the translation assay, comparable to the dif-
ference in potency between the natural aminoglycosides
neamine and paromamine,4,18 the corresponding 2-DOS
derivatives of these sugars. Consequently, the com-
pound 14 did not inhibit bacterial growth. The impor-
tance of the sugar moiety for RNA target recognition
by the azepane-glycosides was further underlined by the
finding that the benzylated derivative 29 was completely
inactive in both the translation and growth inhibition
assays.
a The diastereomers 23 and 24, regioisomers 25–28, and derivatives 16,
18, and 20–22 contained the same amino-glucosamine moiety shown
for the designed isomer 1.
b IC50 values are means of six replicate experiments for each com-
pound (ꢁ10%).
5. Conclusion
Exchange of the 2-hydroxymethyl group in 1 to a halo-
gen or amino group in the azepane-glycoside, like in the
derivatives 20, 21, and 22, or methylation of N1 in 18,
reduced the activity of these compounds as translation
inhibitors by more than 10-fold, eventually leading to
loss of antibacterial potency. None of these derivatives
retained binding to the decoding-site RNA, indicating
the involvement of both the 2-hydroxymethyl and N1
amino groups in target recognition. According to the
design of the azepane scaffold (Fig. 1c–e), the amino
hydrogen atom of N1 is required for a hydrogen-bonding
interaction with the decoding-site RNA. The residual
inhibitory activity of 18, 20, and 21 in the translation
assay might be due to nonspecific action on a ribosomal
target other than the decoding site. The carbamate deri-
vative 16 was completely inactive in both the bacterial
translation and growth inhibition assays, further sup-
porting the importance of the free hydroxymethyl and the
amine functionality for interactions with the target.
A rational ligand discovery approach has been used to
design and synthesize novel azepane-glycosides that
bind to the bacterial decoding-site RNA, and thereby
inhibit bacterial translation in vitro. Two of the
designed ligands showed moderate antibacterial activity
against S. aureus and retained this activity against ami-
noglycoside-resistant strains. A small number of iso-
mers and derivatives has been synthesized and tested,
revealing a preliminary structure–activity relationship of
the azepane-glycosides and underlining the importance
of stereochemical design for antibiotic activity with only
the initially designed stereoisomers as inhibitors of in
vitro translation and bacterial growth. The activity of
the azepane-glycosides against aminoglycoside-resistant
strains demonstrates that designed scaffolds can be used
to mimic the biological activity of natural antibiotics,
while being sufficiently distinct from the natural pro-
ducts to escape some resistance mechanisms. The result
substantiates the importance of the bacterial decoding
site as a valuable target for future development of novel
antibiotics that may share many of the positive char-
acteristics of the aminoglycoside antibacterials, such as
Among the regioisomers (25–28) of 1, the azepane–glyco-
sides 25 and 28 showed moderate inhibition of bacterial

S. Barluenga et al. / Bioorg. Med. Chem. Lett. 14 (2004) 713–718
717
potency and bacteriocidality, without being compro-
mised by the widespread pathogen resistance against
these drugs.
dye by T-cells.42 After cells were incubated with series of
compound concentrations for 72 h, XTT solution was
added and fluorescence read at 450 nm and 650 nm. The
50% cytotoxic concentration (CC50) was defined as the
compound concentrations required to reduce by 50%
the number of viable cells.
6. Experimental
6.1. Molecular modelling
Acknowledgements
Compound design and docking was performed using
atom coordinates of the 30S ribosomal subunit–amino-
glycoside complexes7,30 and crystal structures of syn-
thetic RNA constructs containing the bacterial
decoding-site internal loop (Q. Zhao, T. Hermann,
unpublished results). Preferred conformations of the
azepane heterocycle and the azepane-glycosides were
explored by molecular dynamics simulations and energy
minimization following established protocols.37,38
We thank Dr. D. Wall and Ms. J. Froehlich for deter-
mining MIC values, Ms. V. Banh and Dr. K. Steffy for
help with the eukaryotic in vitro translation and cyto-
toxicity assays. This work was supported in part by
National Institutes of Health grant AI51104 to T.H.
References and notes
6.2. Determination of RNA target binding
1. Gale, E. F.; Cundliffe, E.; Renolds, P. E.; Richmond,
M. H.; Waring, M. J. The Molecular Basis of Antibiotic
Action; John Wiley and Sons: London, 1981.
2. Moazed, D.; Noller, H. F. Nature 1987, 327, 389.
3. Purohit, P.; Stern, S. Nature 1994, 370, 659.
4. Wong, C.-H.; Hendrix, M.; Priestley, E. S.; Greenberg,
W. A. Chem. Biol. 1998, 5, 397.
5. Ramakrishnan, V. Cell 2002, 108, 557.
6. Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol. Biol.
1998, 277, 333.
Compounds were tested for binding to the decoding-site
target using an RNA fluorescence assay which determines
the binding affinity of a ligand based on its ability to flip
out the flexible adenines A1492 and A1493 in a model oli-
gonucleotide (compare Fig. 1). The assay thus returns a
true measure of the potency of a compound to bind speci-
fically to the decoding-site internal loop and to induce a
conformational response comparable to that triggered by
natural aminoglycoside antibiotics. Complete experi-
mental details of the assay will be reported separately.
7. Ogle, J. M.; Brodersen, D. E.; Clemons, W. M.; Tarry,
M. J.; Carter, A. P.; Ramakrishnan, V. Science 2001, 292,
897.
8. Wright, G. D.; Berghuis, A. M.; Mobashery, S. In Resol-
ving the Antibiotic Paradox: Progress in Understanding
Drug Resistance and Development of New Antibiotics;
Rosen, B. P., Mobashery, S., Eds.; Plenum: New York,
1998; pp 27–69.
6.3. Determination of translation inhibition
To assess potency of compounds as translation inhibi-
tors, a coupled in vitro transcription–translation assay
was carried out as previously described.16,25
9. Kotra, L. P.; Haddad, J.; Mobashery, S. Antimicrob.
Agents Chemother. 2000, 44, 3249.
10. Schroeder, R.; Waldsich, C.; Wank, H. EMBO J. 2000,
19, 1.
6.4. Determination of bacterial growth inhibition
The antibacterial activity of compounds was evaluated
for Escherichia coli (strain ATCC-25922) and Staphylo-
coccus aureus (ATCC-25923) by determining the mini-
mal inhibitory concentration (MIC).39 Two-fold
dilutions ranging from 64 to 0.03 mg/mL were tested in
triplicate. The MIC was determined as the lowest com-
pound concentration that prevented cell growth after 18
h of incubation at 37 ꢂC. The same protocol was used to
test aminoglycoside-resistant pathogens, including
methicillin-resistant Staphylococcus aureus (ATCC
strain numbers BAA40 and BAA44, respectively, which
are Portuguese and Iberian clones of MRSA)39À41 which
carry multi-drug-resistance against decoding-site bind-
ing aminoglycosides, such as gentamicin, neomycin, and
other antibiotics, including macrolides, ampicillin, ery-
thromycin, penicillin, tetracycline, methicillin, oxacillin,
and spectinomycin.
11. Tor, Y.; Hermann, T.; Westhof, E. Chem. Biol. 1998, 5,
R277.
12. Hermann, T.; Westhof, E. Biopolymers Nucleic Acid Sci-
ences 1999, 48, 155.
13. Hermann, T. Angew. Chem., Int. Ed. Engl. 2000, 39, 1890.
14. Hermann, T. Biochimie 2002, 84, 865.
15. Ding, Y.; Hoftstadler, S. A.; Swayze, E. E.; Griffey, R. H.
Org. Lett. 2001, 3, 1621.
16. Vourloumis, D.; Takahashi, M.; Winters, G. C.; Simon-
sen, K. B.; Ayida, B. K.; Barluenga, S.; Qamar, S.; Shan-
drick, S.; Zhao, Q.; Hermann, T. Bioorg. Med. Chem.
Lett. 2002, 12, 3367.
17. Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm,
C.; Greenberg, W. A. J. Am. Chem. Soc. 1998, 120, 8319.
18. Vourloumis, D.; Winters, G. C.; Takahashi, M.; Simon-
sen, K. B.; Ayida, B. K.; Shandrick, S.; Zhao, Q.; Her-
mann, T. ChemBioChem. 2003, 4, 879.
19. Simonsen, K. B.; Ayida, B. K.; Vourloumis, D.; Winters,
G. C.; Takahashi, M.; Shandrick, S.; Zhao, Q.; Hermann,
T. ChemBioChem. 2003, 4, 886.
20. Alper, P.; Hendrix, M.; Sears, P.; Wong, C.-H. J. Am.
Chem. Soc. 1998, 120, 1965.
6.5. Determination of cytotoxicity
The eukaryotic cytotoxicity of compounds was assessed
in a standard proliferation assay measuring the mito-
chondrial reduction of XTT into an orange formazan
21. Greenberg, W. A.; Priestley, E. S.; Sears, P. S.; Alper,
P. B.; Rosenbohm, C.; Hendrix, M.; Hung, S.-C.; Wong,
C.-H. J. Am. Chem. Soc. 1999, 121, 6527.

718
S. Barluenga et al. / Bioorg. Med. Chem. Lett. 14 (2004) 713–718
22. Nunns, C. L.; Spence, L. A.; Slater, M. J.; Berrisford, D. J.
Tetrahedron Lett. 1999, 40, 9341.
Polymerization, 2nd Ed.; Academic Press: San Diego,
1997.
23. Haddad, J.; Kotra, L.; Llano-Sotelo, P. B.; Kim, C.;
Azucena, E. F.; Liu, M.; Vakulenko, S. B.; Chow, C. S.;
Mobashery, S. J. Am. Chem. Soc. 2002, 124, 3229.
24. Hanessian, S.; Tremblay, M.; Kornienko, A.; Moitessier,
N. Tetrahedron 2001, 57, 3255.
25. Simonsen, K. B.; Ayida, B. K.; Vourloumis, D.; Takaha-
shi, M.; Winters, G. C.; Barluenga, S.; Qamar, S.; Shan-
drick, S.; Zhao, Q.; Hermann, T. ChemBioChem. 2002, 3,
1223.
26. Vicens, Q.; Westhof, E. Structure 2001, 9, 647.
27. Vicens, Q.; Westhof, E. Chem. Biol. 2002, 9, 747.
28. Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D.
Science 1996, 274, 1367.
29. Russell, R. J. M.; Murray, J. B.; Lentzen, G.; Haddad, J.;
Mobashery, S. J. Am. Chem. Soc. 2003, 125, 3410.
30. Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Mor-
gan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V.
Nature 2000, 407, 340.
34. Trnka, T. M.; Grubbs, R. H. Acc. Chem. Res. 2001, 34,
18.
35. Furstner, A. Angew. Chem., Int. Ed. Engl. 2000, 39, 3012.
¨
36. Nicolaou, K. C.; Winssinger, N.; Pastro, J.; Ninkovic, S.;
Sarabia, F.; He, Y.; Vourloumis, D.; Yang, Z.; Li, T.;
Giannakakou, P.; Hamel, E. Nature 1997, 387, 268.
37. Hermann, T.; Westhof, E. J. Mol. Biol. 1998, 276, 903.
38. Hermann, T.; Westhof, E. J. Med. Chem. 1999, 42, 1250.
39. National Committee for Clinical Laboratory Standards. In
Approved Standard M7-A5: Methods for Dilution Anti-
microbial Susceptibility Tests for Bacteria That Grow
Aerobically, 5th Edition (Wayne, PA: National Committee
for Clinical Laboratory Standards). 2000, 327, 389–394.
40. de Lencastre, H.; Cuoto, I.; Santos, I.; Melo-Cristino, J.;
Torres-Pereira, A.; Tomasz, A. Eur. J. Clin. Microbiol.
Infect. Dis. 1994, 13, 64.
41. Sanches, I. S.; Ramirez, M.; Troni, H.; Abecassis, M.;
Padua, M.; Tomasz, A.; de Lencastre, H. J. Clin. Micro-
biol. 1995, 33, 1243.
42. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.;
Boyd, M. R. Cancer. Res. 1988, 48, 4827.
31. Grubbs, R. H.; Chang, S. Tetrahedron 1998, 54, 4413.
32. Grubbs, R. H.; Miller, S. J.; Fu, G. C. Acc. Chem. Res.
1995, 28, 446.
33. Ivin, K. J.; Mol, J. C. Olefin Metathesis and Metathesis