Structure-based design, synthesis, and A-site rRNA cocrystal complexes of functionally novel aminoglycoside antibiotics: C2″ ether analogues of paromomycin

Article abstract of DOI:10.1021/jm061200+

A series of 2″-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit μM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses.

Full text of DOI:10.1021/jm061200+

2352
J. Med. Chem. 2007, 50, 2352-2369
Structure-Based Design, Synthesis, and A-Site rRNA Cocrystal Complexes of Functionally
Novel Aminoglycoside Antibiotics: C2′′ Ether Analogues of Paromomycin
Stephen Hanessian,*† Janek Szychowski,† Susanta Sekhar Adhikari,† Guillermo Vasquez,‡ Pachamuthu Kandasamy,†
Eric E. Swayze,‡ Michael T. Migawa,‡ Ray Ranken,‡ Boris Franc¸ois,§ Julia Wirmer-Bartoschek,§ Jiro Kondo,§ and
Eric Westhof§
Department of Chemistry, UniVersite´ de Montre´al, C. P. 6128, Succ. Centre-Ville, Montre´al, P. Q., Canada, H3C 3J7, Isis Pharmaceuticals,
2292 Faraday DriVe, Carlsbad, California 92008, and Institut de Biologie Mole´culaire et Cellulaire UPR9002 CNRS, UniVersite´ Louis
Pasteur, 15 rue Rene´ Descartes, 67084 Strasbourg Cedex, France
ReceiVed October 14, 2006
A series of 2′′-O-substituted ether analogues of paromomycin were prepared based on new site-selective
functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in
the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed
with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new
analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of
S. aureus. Single digit µM MIC values were obtained against E. coli, with some of the ether appendages
containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia
protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity,
while controls with neomycin and kanamycin were toxic at lower doses.
Introduction
Aminoglycoside antibiotics have occupied a time-honored
position in the hierarchy of antibacterial agents for over half a
century.1 Since the discovery of streptomycin in 1944,2 several
members of this class of polycationic pseudo-oligosaccharides
have been used in clinical practice.3 Gentamicin, tobramycin,
and amikacin are representatives of the more commonly used
aminoglycosides, especially for Gram-negative infections. Al-
though the aminoglycosides exhibit potent bactericidal activity,
their widespread use has been compromised by dose-related
nephrotoxicity and ototoxicity.4 For these and other reasons,
aminoglycoside therapy is confined to a hospital environment,
Figure 1. Structures and simulated bioactive conformations of
and i.v. administration requires careful monitoring. As with other
antibiotic regimens, their use as the primary treatment of life-
threatening infections has also been curtailed due to the rapid
emergence of resistant strains of bacteria.3-5 This has instigated
extensive research efforts on several fronts in search of new,
or chemically modified, antibiotics that can delay or avoid
acquired resistance by pathogenic bacteria.
Although a large number of antibiotic entities have been
introduced in the market during the past two decades, very few
if any new aminoglycosides have been reported, except for
alternative formulations. For example, tobramycin is available
as an effective treatment for the prevention of infection in cystic
fibrosis patients as an inhaler formulation.6 The intrinsic potency
of aminoglycosides makes them excellent candidates to explore
new ways to overcome bacterial resistance and diminish toxicity.
In this paper, we report our efforts in the design and synthesis
of modified analogues of paromomycin and the discovery of a
new paradigm toward the interaction of functionally diverse
ether appendages at C2′′ within the A-site subunit.
paromomycin, tobramycin, and dibekacin.
Although its structure and stereochemistry has been known for
many decades,7 its use in clinical practice has been limited due
to its poor therapeutic index. It’s closest analogue, neomycin
2, is commonly used in ointment preparations for external use.8
Like all other aminoglycosides possessing a hydroxyl group at
C3′ of ring I, paromomycin is subject to phosphorylation by
O-phosphoryltransferases, rendering it devoid of activity.5,9
Although deoxygenation at this site, as in tobramycin 3 and
dibekacin 4, prevents modification by these enzymes, other
modes of enzymatic inactivation exist (N-acetyltransferases,
O-adenyltransferases)3,5 that eventually diminish the efficacy
of aminoglycosides as antibacterials. Added to these natural
defense mechanisms by resistant bacteria are other hurdles to
overcome such as reduced permeability,10 efflux pumps,11 and
target modification12 within bacteria. Despite these major
obstacles, potential solutions can be envisaged thanks to the
important contributions in understanding the mode of antibacte-
rial action of aminoglycosides.13 They inhibit protein biosyn-
thesis by disrupting the fidelity of tRNA selection step involved
in the binding process to the A-site of bacterial ribosome 16S
RNA. Codon misreading in this highly conserved sequence of
nucleotides affects translation and recognition of the mRNA
sequence, leading to aberrant translation and interruption in
protein synthesis.
Paromomycin 1, belongs to the 4,5-disubstituted class of
2-deoxystreptamine-containing aminoglycosides (Figure 1).
* To whom correspondence should be addressed. Phone: 514-343-6738.
Fax: 514-343-5728. E-mail: stephen.hanessian@umontreal.ca.
† Universite´ de Montre´al.
‡ Isis Pharmaeuticals.
§ Universite´ Louis Pasteur.
10.1021/jm061200+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/26/2007

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2353
Figure 2. Paromomycin C2′′ ethers complexed for X-ray study. Left: superposition of paromomycin (blue) with tobramycin (green) from their
respective X-ray complexes; Right: structures of active C2′′ ethers (6-8) cocrystallized with A-site oligonucleotides.
The relevance of these elegant biochemical studies was
rendered all the more important with seminal contributions
toward the elucidation of the molecular structure of the
ribosome,14 especially the high-resolution crystal structures of
bacterial ribosomal 30S particles complexed to aminoglyco-
sides.15 The crystal structures of aminoglycoside complexes with
the decoding A-site of the bacterial ribosome 16S RNA have
been solved with excellent resolution (2.4-2.54 Å).16 Studies
of aminoglycoside-RNA complexes have also been reported
by biochemical,17 spectroscopic,18 mass spectrometric,19 and
NMR techniques.20 Recent insights into molecular modeling
have provided further tools to probe such interactions in silico.21
Design
Westhof and co-workers24 have shown that rings I and II of
paromomycin 1 and tobramycin 3 occupy the same site, and
they are virtually superimposable in their respective X-ray
cocrystal structures in the RNA A-site (Figure 2). Inspection
of the perspective diagrams led us to the realization that the
introduction of aminoalkyl ethers at C2′′ (or C6) hydroxyl
groups, hitherto unexplored sites in the paromomycin frame-
work, might lead to analogues with a hybrid function. Prelimi-
nary modeling showed that a priori, such C2′′ ethers containing
a basic group derived from paromomycin could reach the space
occupied by ring III of tobramycin and possibly simulate one
of its amino groups. We were pleased to find that the C2′′ (2-
aminoethyl-amino) ethyl and the C2′′ (3-aminopropyl-amino)
ethyl ether derivatives 5 and 6 of paromomycin (Figure 2)
showed in vitro activity against S. aureus comparable to that
of the parent antibiotic (see SAR below). However, the activity
was an order of magnitude less than that of tobramycin.
Although several other acyclic ethers were also prepared (see
below), we shifted the logic-based paradigm of aliphatic
N-aminoalkyl ether appendages at C2′′ to include N-arylalkyl
and N-heteroarylalkyl counterparts (compounds 7-9, for ex-
ample). This radical design concept led to the synthesis of a
series of highly active C2′′ ether tethered analogues (see SAR
below).
Paromomycin in particular, exhibits strong binding to the
A-site of 16S RNA by making effective contacts through amino
and hydroxyl groups while adopting a unique bioactive L-shaped
conformation, as seen in the crystal structure of the complex.16a
Thus, ring I is inserted into the A-site helix by forming a pseudo
base pair with two H-bonds to the Watson-Crick sites of A1408
and benefiting from a stacking interaction against G1491. In
the process, the A1492 and A1493 residues are sequestered in
the bulged out conformation, allowing them to make Watson-
Crick interactions with two base pairs in the minor groove. This
hallmark event of maintaining an ‘‘out” conformation for A1492
and A1493 in the aminoglycoside docked complex, forms the
molecular basis for codon misreading. The deoxystreptamine
(ring II) unit also makes contacts with different nucleotides,
which in conjunction with ring I, contributes to recognition,
binding, and stabilization. Rings III and IV, forming the lower
arm of the L-shaped structure, contribute to binding affinity and
charge interactions of phosphates, as well as correctly orienting
rings I and II in the bioactive conformation.
Chemistry
The readily available paromomycin derivative 1026 was
transformed first to the corresponding 5′′-OTBS ether and then
O-allylated at C2′′ in the presence of allyl iodide and KHMDS
to give 11 in good overall yield (Scheme 1). The selective
O-alkylation at C2′′ was ascertained by methanolysis with
TMSCl in MeOH and MS analysis of the resulting paromamine
and O-allylated methyl paromobiosaminide subunits. Benzoy-
lation followed by ozonolysis led to the selectively protected
benzoate ester 12. It should be noted that the O-benzoylation
of 11, containing four secondary hydroxyl groups, afforded a
product in which the C6 hydroxyl group remained free,
presumably due to steric reasons. A series of reductive amina-
tions led to the respective aminoethyl ethers 13a-z and 13aa-
ah (See Table 1). Cleavage of the benzoate esters in the presence
of MeONa in MeOH, followed by treatment with aqueous acetic
acid and hydrogenolysis, gave the corresponding C2′′ O-
substituted paromomycin analogues 5-9, 14a-z, and 14aa-
ae as their peracetate salts. The N-acetyl and N-benzoyl
derivatives 9a and 9b were also prepared from 13e (not shown,
see Table 2). To assess the importance of ring IV in the ether
analogues, we devised a mild method of oxidative cleavage of
These enormously informative insights into the molecular
basis of aminoglycoside interactions at the ribosomal level have
instigated renewed interest in the field.22 The charged anionic
nature of the rRNA subunits responsible for recognition led to
the logical derivatization of aminoglycosides with a variety of
pendant basic side chains. Indeed, in many cases, interesting in
vitro antibacterial activities were reported.22f With one excep-
tion,23 there are no X-ray structural data to delineate possible
beneficial interactions of such multidentate aminoalkyl append-
ages with the A-sites subunit.
Our strategy toward the design of new aminoglycoside
analogues was to take advantage of the structural information
available from X-ray complexes and to exploit the reactivity
pattern of paromomycin toward new sites for derivatization to
install functionality capable of making productive contacts.
Herein, we report the results of such efforts.

2354 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
Scheme 1. C2′′ Modification of Paromomycin by Reductive Aminationa
a Reagents and conditions: (a) TBSOTf, 2,4,6-collidine, CH2Cl2, 75%; (b) CH2dCHCH2I, KHMDS, THF, 70%; (c) BzCl, pyridine, DMAP, 95%; (d) (i)
O3, CH2Cl2, -78 °C; (ii) Me2S, -78 °C to RT, 80%; (e) amine, NaBH3CN, MeOH/AcOH (30:1), 90%; (f) MeONa/MeOH; (g) AcOH/H2O (4:1), 60 °C,
2 h; (h) Pd(OH)2/C, H2, AcOH/H2O (4:1).
the vicinal diol, followed by base-catalyzed elimination, to give
representative ring I, II, and III pseudotrisaccharides26 (Scheme
2). Thus, treatment of 11 with Pb(OAc)4 in pyridine resulted in
the cleavage of the 3′′′,4′′′-diol to give a dialdehyde, which
underwent â-elimination upon treatment with Et3N to give 15.
Conversion to the benzoate 16 and ozonolysis of the allyl ether
followed by reductive amination and debenzoylation gave the
products 17a-c. Final deprotection of the acetal as well as the
OTBS ether and hydrogenolysis of the N-Cbz groups gave the
intended ring IV truncated analogues 18a-c.
We next addressed the synthesis of neutral alkyl ethers at
C2′′ of paromomycin (Scheme 3). TBS protection of the primary
alcohol followed by alkylation of 10 with cinnamyl bromide in
the presence of KHMDS and Bu4NI at 0 °C gave 19 in 70%
yield. Alternatively, the same product could also be obtained
by performing a cross-metathesis reaction of 11 with styrene
in the presence of the Grubbs second generation catalyst27 in
75% yield. Cleavage of the benzylidene acetal as well as the
OTBS ether followed by catalytic hydrogenolysis gave 20.
Extended and truncated N-aryl ether analogues of paromomycin
were also prepared (Scheme 4). Thus, a Wittig reaction of 12
gave 21 as a mixture of isomeric olefins. Deprotection and
hydrogenation afforded the 5-phenylpentyl ether analogue 22.
Treatment of 12 with phenylmagnesium bromide or diphenyl
zinc gave 23 as a mixture of two alcohols. Acidic cleavage of
the acetal and TBS groups, followed by hydrogenolysis, gave
24 or 25, depending on the length of the reaction time.
Finally, we also deemed it necessary to prepare a 3-phenyl-
propyloxy ether to complete this series in which nitrogen,
carbon, or oxygen atoms were part of the ether chain (Scheme
5). Thus, reduction of 12 led to the alcohol 26, which was
deprotected to give 27. The alcohol 26 was also transformed
into 28 by standard alkylation with cinnamyl bromide and
afforded 29 upon global deprotection.
X-ray Structures of Complexes between the Aminogly-
cosides and A-Site RNA. The overall conformations of the
A-site complexed with 6, 7, and 8 are almost the same as those
of the previous RNA/aminoglycoside complexes.16,25 (Figure
3a-c). At both ends of the internal loop of the A-site, canonical
Watson-Crick pairs, G1405 ) C1496, G1494 ) C1407, and
C1409 ) G1491 are formed (Figure 3d,f,h). The U1406 residue
forms a bifurcated base pair with the U1495 residue (Figure
3e). The A1408 residue, which is conserved in bacteria, does
not form any base pair (Figure 3 g). All complexes between
oligonucleotides containing the A-site and paromomycin de-
rivatized at the 2′′ position present the same structural charac-
teristics as previously published.25 A cocrystal structure of 6
with the A-site oligonucleotides presented the same structural
characteristics seen in paromomycin complex.16a (Figure 3a).

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2355
Table 1. Side Chains in Compounds 13a-z and 13aa-ah
The paromamine moiety makes contact with the three conserved
adenines (A1408, A1492, and A1493). On the other hand, the
conformation of ring III is changed, and the rotation about the
linkage between rings II and III leads to a global rotation of
ring IV by 90°. The hydroxyl group O5′′ has rotated by 90°
and forms two H-bonds with the Hoogsteen sites of G1491.
Intrigued by the potent inhibitory activity of compounds 7 and
8, we obtained X-ray structures of their complexes (Figure 3b,c).
As previously observed,25 the â-D-ribofuranosyl ring was turned
by 40° with respect to the paromamine unit, which, together
with a change in the sugar pucker (C3′′ endo instead of C2′′
endo), results in a 90° change in the orientation of ring IV in
the new analogue 8. While the oxygen atom in the ring III still
forms an intermolecular H-bond with C2′NH2 of ring I, the
hydroxymethyl group at C5′′ forms an H-bond with O6 of
G1491 instead of N7, as in the case of paromomycin. A network
of additional H-bonds can be observed resulting in new
interactions. The new ether chain at C2′′ extends across the deep
major groove of the A-site and points into the solvent (Figure
3i). These unprecedented conformational changes and new
interactions as a result of the incorporation of a C2′′ ether chain
result in a compactly folded paromomycin structure with unique
binding sites. In the case of compound 8, the primary amine of
the aromatic ring forms two H-bonds with anionic phosphate
oxygen atoms (O2P of G1491 and O1P of U1490). In addition,
two water molecules bridge the N7′′ with the O2P of G1405.
Compound 8 makes 15 direct contacts with the A-site nucle-
otides and three indirect contacts mediated by water molecules

2356 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Table 2. Activities of Paromomycin C2′′ Ether Analogues
Hanessian et al.

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2357
Scheme 2. C2′′ Modification of Paromomycin without Ring IVa
a Reagents and conditions: (a) Pb(OAc)4, pyridine; (b) Et3N, THF, 30%, two steps; (c) BzCl, pyridine, DMAP, 95%; (d) (i) O3, CH2Cl2, -78 °C; (ii)
Me2S, -78 °C to RT, 80%; (e) amine, NaBH3CN, MeOH/AcOH (30:1), 90%; (f) MeONa/MeOH; (g) AcOH/H2O (4:1), 60 °C, 2 h; (h) Pd(OH)2/C, H2,
AcOH/H2O (4:1).
Scheme 3. C2′′ Modification of Paromomycin with a Carbon
paromomycin derivatives was discovered leading to potent in
vitro and in vivo (mouse) activity.
Side Chaina
Biological Results and Discussion
All compounds were tested for activity against E. coli
(ATCC#25922) and S. aureus (ATCC#13709). The minimum
inhibitory concentrations are shown in Tables 2-4. As shown
in Table 2, the SAR series of 2′′-ethylamino-substituted paro-
momycin analogues encompasses a variety of lipophilic and
lipophobic functionality, as well as aliphatic and aromatic
moieties. The steric space around the C2′′-position has been
explored using relatively small and large bulky substituents at
the extremity of the side chain. With few exceptions, all of these
modifications at the C2′′-position were well tolerated against
S. aureus (ATCC#13079), while the smaller, aliphatic, and
polar functionalities seemed to fare better against E. coli
(ATCC#25922). In the E. coli series, this is further confirmed
by the reduced activity of the nonpolar carbon and oxygen
analogues shown in Table 4, which is consistent with the
presence of a relatively large pocket about the C2′′ side chain
in the A-site, which is unoccupied. Substitution at C2′′ orients
the III-IV rings in the A-site in a manner different from that
of unsubstituted paromomycin and places the side-chain sub-
stituents in the deep major groove of the A-site. In fact, even
quite large and bulky substituents (for example, 14w, 14x, 14z,
14aa, 14ac, and 14ad) displayed activity close to, or better than,
that of paromomycin against S. aureus. This fact allows one to
append functionality that could drastically alter the pharmaco-
kinetic properties of the aminoglycosides, potentially avoiding
enzymes that confer resistance and possibly alter the toxicity
profile. While these factors have yet to be ascertained, we had
surmised that the alternative mode of binding for the C2′′-
substituted analogues in paromomycin places ring IV in an
orientation that does not benefit from critical interactions within
the A-site. As can be seen in Table 3, removal of ring IV of the
C2′′-substituted analogues results in active compounds, while
the paromomycin analogue is totally inactive. Although the
activity of C2′′ ethers lacking ring IV is slightly reduced
compared to that of the parent analogue, the loss of ring IV is
a Reagents and conditions: (a) TBSOTf, 2,4,6-collidine, CH2Cl2, 75%;
(b) cinnamyl bromide, KHMDS, Bu4NI, THF, 70%; (c) Grubbs II, styrene,
CH2Cl2, reflux, 16 h, 75%; (d) AcOH/H2O (4:1), 60 °C, 2 h; (e) Pd(OH)2/
C, H2, AcOH/H2O (4:1), 70% for two steps.
(see Figure 3d-h), while paromomycin forms 12 direct contacts.
Compound 8 also has the lowest dissociation constant from the
16S RNA and the best MICs. By contrast, compounds 6 and 7
form, respectively, 11 and 12 direct contacts, while paromo-
mycin forms 12 direct contacts. Thus, the expected extension
of C2′′-aminoalkyl ether appendages toward ring III of tobra-
mycin was not realized. Instead, a new mode of binding of these

2358 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
Scheme 4. C2′′ Modification of Paromomycin with a Carbon Side Chaina
a Reagents and conditions: (a) cinnamyl triphenylphosphonium bromide, KHMDS, THF, 60%; (b) MeONa/MeOH, 80%; (c) AcOH/H2O (4:1), 60 °C, 2
h; (d) Pd(OH)2/C, H2, AcOH/H2O (4:1), 70% for two steps; (e) Ph2Zn, RT, 90% or PhMgBr, -40 °C, 70%.
Scheme 5. C2′′ Modification of Paromomycin with an Oxygen Side Chaina
a Reagents and conditions: (a) NaBH3CN, MeOH/AcOH (30:1), 80%; (b) cinnamyl bromide, KHMDS, THF, Bu4NI, 45%; (c) MeONa/MeOH, 80%; (d)
AcOH:H2O (4:1), 60 °C, 2 h; (e) Pd(OH)2/C, H2, AcOH/H2O (4:1), 70% for two steps.
not as detrimental. Thus, ring IV appears to be less important
for binding in the C2′′ ether series.
To determine whether our analogues would have in vivo
activity, compounds 7 and 9 where chosen to test in a mouse
septicemia protection assay. Mice were infected IP with S.
aureus and treated at 1 h and 3 h post-infection with drug
delivered subcutaneously. All nondrug-treated mice were mori-
bund within 12 h and were sacrificed within 18 h. As shown in

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2359
Figure 3. X-ray complexes of analogues 6-8 with the A-site. (a), (b), and (c): Molecular contacts of 6, 7, and 8, respectively, with A-site RNA.
The structures for 6 and 7 were previously published.25 Hydrogen bonds are represented by dashed lines and water molecules are represented by
the letter w. (d), (e), (f), (g), and (h): Interactions between 8 and the base present in the A-site. Water molecules are represented by red spheres.
(i): Stereoview of 7 (green) superimposed with paromomycin 1 (yellow).
Table 5, compound 7 fully protected mice at 1.2 mg/kg.
Compound 9 showed an ED90 at 0.25 mg/kg and fully protected
the mice at 0.50 mg/kg. Furthermore, compound 7 showed no
overt signs of toxicity after observation of the mice up to two
weeks and after performing a histopathological analysis of the
kidney (data not shown). Compound 9 did display some acute
toxicity at the highest dose (75 mg/kg × 2), indicating the
phenethyl analogue was slightly more toxic. It is interesting to
note that histopathological analysis of the control aminoglyco-
sides indicated kidney toxicity at a lower dose (37 mg/kg × 2).
Clearly, these preliminary promising tests warrant further
validation in more extensive animal studies.
The useful information that we have obtained from our X-ray
data has guided our SAR in several significant ways. While it
should be noted that the improvement in activity in animal
models correlates well with the improvement in MIC activity,
this is most likely due to more favorable pharmacokinetic
properties. However, these improvements, due to extension of
the C-2′′ side chain, are greatly aided by the structural
information we have obtained. In particular, the extent of

2360 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
Table 3. Activities of Paromomycin C2′′ Ether Analogues without Ring
IV
importance of the ring IV when a C-2′′ substituent is present.
This is also borne out experimentally in the SAR.
Conclusion
The remarkable potency of the C2′′-substituted ether ana-
logues and their apparent reduced toxicity, exemplified by
analogue 7, demonstrate the potential of this series in addressing
the issues observed with aminoglycosides in a clinical setting.
Moreover, the alternative mode of binding adopted by the C2′′-
substituted analogues allows a high tolerance for substitution
at the C2′′-position and for additional modification, including
removal of ring IV. Additional work to ascertain their suscep-
tibility to resistance conferring enzymes, efflux pumps, and
broader antibacterial activities are underway. The encouraging
mouse protection results augur well for more extensive in vivo
studies with different bacterial strains of clinical relevance.
MIC
(µg/mL)
MIC
(µg/mL)
compound
R
E. coli
3-6
>100
S. aureus
1-2
>100
1
-
-
1 without
ring IV
18a
NH2
6-12
6-12
12-25
6-12
18b
Experimental Section
18c
>100
12-25
In Vitro Studies. Minimum Inhibitory Concentrations (MICs,
Bacteria). The assays are carried out in 150 µL volume in duplicate
in 96-well, clear, flat-bottom plates. The bacterial suspension from
an overnight culture growth in appropriate medium is added to a
solution of test compound in 2.5% DMSO in water. Final bacterial
inoculum is approximately 102-103 CFU/well. The percentage
growth of the bacteria in test wells relative to that observed for a
control well containing no compound is determined by measuring
absorbance at 595 nm (A595) after 20-24 h at 37 °C. The MIC is
determined as a range of concentrations, where complete inhibition
of growth is observed at the higher concentration and bacterial cells
are viable at the lower concentration. Both ampicillin and tetracy-
cline are used as antibiotic positive controls in each screening assay
for E. coli (ATCC#25922) and S. aureus (ATCC#13709).
In Vivo Studies. Bacteria. Staphylococcus aureus was obtained
from American Type Culture Collection repository (Smith strain
ATCC#13079). The bacterium was passaged several times through
mice to achieve and maintain mouse virulence. To be virulent in
mice, the Staphylococcus aureus needed to be suspended in 10%
sterile hog gastric mucin (obtained from Sigma).
Table 4. Activities of Paromomycin C2′′ Ether Analogues with Carbon
or Oxygen Side Chains
Mice. Female Balb/c mice (16-18 g) were obtained from Charles
River Labs. The mice were allowed to acclimate for one week prior
to use. The mice, when challenged with bacteria, showed symptoms
within 12 h of infection and were moribund and subsequently
sacrificed by 18 h. All treated groups had 10 mice per group.
Compounds Tested and Controls. Amikacin, tobramycin, and
neomycin obtained from the U.S. Pharmacopia were used as positive
control drugs. They were reconstituted in water and were used at
concentrations of 37.5, 18.8, 9.4, 4.7, and 2.3 mg/kg. The control
compounds protected the mice at all concentrations tested. Com-
pounds 7 and 9 were dissolved in water and used at 75, 37.5, 18.8,
9.4, 4.7, 2.3, 1.17, 0.5, 0.25, and 0.125 mg/kg. The data are shown
in Table 5.
Mouse Septicemia Protection Assay. Mice were infected IP
(intraperitoneally) with 0.5 mL of 106 Staph. organisms suspended
in 10% mucin. Mice were treated at 1 h and 3 h post-infection
with either serially 2-fold diluted control drug or serially 2-fold
dilutions of drug delivered in the amount of 0.5 mL subcutaneously.
Group I was a titration of S. aureus from 109 through 106 to illustrate
virulence. Group II was the mucin only control and illustrates the
need for Staph./mucin suspension to produce virulence in the mouse.
Group III was the Amikacin-treated control group. Group IV was
the tubercidin-treated control group. Group V was the neomycin-
treated control group. Group VI comprised compound 7 and group
VII was the compound 9-treated group. Mice were observed over
a two week period.
Table 5. Compounds 7 and 9 in a Mouse Septicemia Protection Assaya
number of mice moribund/total number of mice
cmpd
(dosage (mg/Kg))
(0.13) (0.25) (0.50) (1.2) (2.3) (4.7) (9.4) (19) (38) (75)
7
9
7/10
5/10
6/10
1/10
3/10
0/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10
0/10 0/10 0/10 0/10 0/10 0/10 4/10
a Compounds were dosed at the indicated amounts at 1 h and 3 h post
S. aureus infection.
toleration of the C-2′′ side chain is quite impressive due to the
alternative mode of binding and suggests further points of
contact could be made in this pocket. Our X-ray studies have
shown that new contacts are possible within the A-site.
Furthermore, this new mode of binding predicts a reduced
Crystallizations, X-ray Experiments, and Structure Refine-
ments. Crystallization procedures are essentially the same as
published previously.16,25 The asymmetrical loop of the bacterial
A-site was inserted between Watson-Crick pairs in sequences
designed to fold as a double helix (See Figure S1 in Supporting

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2361
Information). Prior to crystallization, each paromomycin derivative
was dissolved at 4 mM in 50 mM sodium cacodylate buffer (pH
6.4), and 2 mM RNA solution containing 100 mM sodium
cacodylate (pH 6.4), 25 mM sodium chloride, and 5 mM magnesium
sulfate was annealed by heating at 85 °C for 2 min, followed by
slow cooling to 37 °C. Same volumes of RNA solution and
aminoglycoside solution were mixed at 37 °C, and then the mixture
was cooled slowly to room temperature (21-25 °C). Crystallizations
were performed by the hanging-drop vapor diffusion method at 37
°C in conditions containing 1-6% 2-methyl-2,4-pentanediol, 1-5%
glycerol, and 50 mM sodium cacodylate. X-ray experiments were
performed using synchrotron radiation at beamline ID14-4 of the
European Synchrotron Radiation Facility (ESRF; Grenoble, France).
Images were processed with the HKL200028 and Crystalclear
(Rigaku/MSC). The statistics of data collection and the crystal data
are summarized in Table S1 (See Supporting Information). Initial
phases were derived with the molecular replacement program
AMoRe29 using the coordinates of the crystal structure of the RNA/
paromomycin complex (PDB code: 1J7T).16a The atomic param-
eters of the structure were refined with the program CNS.30 The
parameters and topology files for the antibiotic molecules were
obtained on the Hetero-compound Information Center-Uppsala
server (University of Uppsala, Sweden).31 The statistics of structure
refinement are summarized in Table S1 (See Supporting Informa-
tion). All figures were drawn using The PyMOL Molecular
Graphics system (2002), DeLano Scientific, San Carlos, CA (http://
www.pymol.org).
later, some of the CH2Cl2 was evaporated to a smaller volume for
washing with 0.5 M HCl twice and NaHCO3. Drying with Na2SO4
and purification by silica gel chromatography (2% MeOH/CH2-
Cl2) gave the O5′′-TBS monoprotected compound (4.86 g, 75%);
Rf 0.6 (CHCl3/EtOAc/MeOH (20:5:3)); 1H NMR (300 MHz, CDCl3)
δ 7.60-7.10 (m, 30H), 5.60-3.00 (m, 41H), 2.20 (m, 1H), 1.30
(m, 1H), 0.83 (s, 9H), 0.01 (s, 6H); ESI-MS m/z calcd for
C76H93N5O24Si [M + H]+, 1487.60580; found, 1488.9; TOF-HRMS
found, 1488.60258. This O5′′-protected compound (2.10 g, 1.411
mmol) was codistilled with toluene twice, and the residue was
dissolved in dry THF (70 mL) in a flask covered with aluminum
foil. Allyl iodide (1.29 mL, 14.11 mmol) was added followed by
the dropwise addition of 0.5 M KHMDS solution in toluene (1.41
mL, 0.706 mmol). The mixture was stirred overnight at room
temperature, then 0.3 equiv of KHMDS was added, and 6 h later,
the reaction mixture was quenched with an aqueous solution of
satd. NH4Cl (2 mL) and water. THF was evaporated, the aqueous
layer was extracted with ethyl acetate (3 times), and the organic
layer was washed with a sodium thiosulfate solution and brine and
dried over Na2SO4. The solvent was evaporated to dryness to afford
a crude solid, which was purified by silica gel flash chromatography
(1.5% MeOH/CH2Cl2), providing the known allyl ether 1125 (1.468
1
g, 68%); Rf 0.7 (CHCl3/EtOAc/MeOH (20:5:3)); H NMR (300
MHz, CDCl3) δ 7.60-7.10 (m, 30H), 6.30-3.00 (m, 41H), 2.20
(m, 1H), 1.30 (m, 1H), 0.83 (s, 9H), 0.01 (s, 6H); ESI-MS m/z
calcd for C79H97N5O24Si [M + H]+, 1528.63710; found, 1528.8;
TOF-HRMS found, 1528.63473.
Chemistry. General. Solvents were distilled under positive
pressure of dry argon before use and dried by standard methods:
THF from Na/benzophenone and CH2Cl2 from CaCl2. All com-
mercially available reagents were used without further purification.
All reactions were performed under nitrogen atmosphere. NMR (1H,
13C) spectra were recorded on AMX-300, ARX-400, and AV-400
spectrometers. Low- and high-resolution mass spectra were recorded
using electrospray technique. Optical rotations were recorded in a
1 dm cell at ambient temperature. Analytical thin-layer chroma-
tography was performed on precoated silica gel plates. Visualization
was performed by ultraviolet light and/or by staining with ceric
ammonium molybdate. Flash column chromatography was per-
formed using (40-60 µm) silica gel at increased pressure.
4′,6′-O-Benzylidene-penta-N-benzyloxycarbonyl paromomy-
cin (10). Sodium carbonate (55.0 g, 0.523 mol) and CbzCl (20.00
mL, 0.139 mol) were added to paromomycin sulfate (30.00 g,
0.0271 mol) in water (500 mL). After 18 h under vigorous stirring,
the water was decanted and the white precipitate was washed with
water twice. A solution of triethylamine (97.00 mL, 0.697 mol) in
methanol (600 mL) was added, followed by CbzCl (25.00 mL,
0.174 mol). After 5 h, the solvents were evaporated. The resulting
penta-NCbz-paromomycin was purified by flash column chroma-
tography over silica gel (5% MeOH/CH2Cl2). The obtained white
solid was codistilled with pyridine (200 mL) three times and at
half of the volume of the third codistillation, toluene (200 mL)
was added, and the solvents were evaporated to dryness. Another
codistillation with toluene (300 mL) was done before heating the
flask at 60 °C under 10 mmHg vacuum for 12 h. Freshly distilled
benzaldehyde (400 mL) was added to the resulting white solid, and
sonication was done to bring it in solution. To the stirred mixture
was added 4 Å molecular sieves (15 g) and formic acid (50.00
mL, 0.530 mol). After stirring for 12 h at room temperature, the
mixture was slowly added to a stirred ice-cold solution of saturated
aqueous NaHCO3, extracted with ethyl acetate (3 times), and the
organic layer was washed with water and dried over Na2SO4. The
solvent was evaporated to dryness to afford a crude solid, which
was purified by flash column chromatography over silica gel (3%
MeOH/CH2Cl2) to obtain the known compound 10 (18.91 g, 63%).26
4′,6′-O-Benzylidene-2′′-O-allyl-5′′-O-t-butyldimethylsilyl-penta-
N-benzyloxycarbonyl paromomycin (11). Compound 10 (6.00 g,
4.367 mmol), dried by two codistillations with toluene, was
dissolved in CH2Cl2 (400 mL) and 2,4,6-collidine (1.15 mL, 8.735
mmol), followed by TBDMSOTf (0.50 mL, 2.184 mmol) added at
0 °C. After 18 h, 0.6 equiv of TBDMSOTf was added, and 6 h
4′,6′-Benzylidene-3′,3′′′,4′′′-tri-O-benzoyl-2′′-O-carbonylmethyl-
5′′-O-t-butyldimethylsilyl-penta-N-benzyloxycarbonyl Paromo-
mycin (12). A solution of 11 (5.30 g, 3.46 mmol) in dry pyridine
(100 mL) was treated with benzoyl chloride (1.14 mL, 9.812 mmol).
The reaction mixture was stirred at room temperature for 16 h.
Water (5 mL) was added, and the solvent was removed under
vacuum. The residue was dissolved in ethyl acetate, and the organic
layer was washed with 0.5 M HCl, satd. NaHCO3, and water, dried
over Na2SO4, and concentrated under vacuum. The crude product
was purified by silica gel flash chromatography (1:1 EtOAc/hexane)
to yield the O-benzoyl-protected derivative (5.65 g, quant.); Rf 0.6
1
(1:1 EtOAc/hexane); H NMR (300 MHz, CDCl3) δ 8.10-7.10
(m, 47H), 6.30-3.00 (m, 44H), 2.20 (m, 1H), 1.30 (m, 1H), 0.83
(s, 9H), 0.01 (s, 6H); ESI-MS m/z calcd for C100H109N5O27Si [M
+ H]+, 1840.71574; found, 1840.9; TOF-HRMS found, 1840.72060.
The compound described above (2.00 g, 1.086 mmol) in CH2Cl2
(60 mL) was cooled at -78 °C, and ozone was bubbled for 2 h,
after which excess ozone was removed by bubbling argon. The
mixture was treated with Me2S (0.80 mL, 13.816 mmol) and
warmed to room temperature, and the solvent was removed under
vacuum. The crude solid was purified by silica gel flash chroma-
tography (2:3 EtOAc/ hexane) to give the known aldehyde 1225
(1.627 g, 80%); Rf 0.4 (1:1 EtOAc/hexane); ESI-MS m/z calcd for
C99H107N5O28Si [M + H]+, 1842.7; found, 1842.9.
General Procedure for Reductive Amination and Benzoate
Deprotection: To a mixture of the aldehyde 12 (80.0 mg, 0.043
mmol) and appropriate amine (0.129 mmol) in dry MeOH (3 mL)
was added acetic acid (0.1 mL), followed by NaBH3CN (1.0 M in
THF, 60 µL, 0.060 mmol). A total of 18 h later, a TLC indicated
a complete disappearance of the aldehyde. A freshly prepared
solution of MeONa/MeOH was added dropwise until the pH
reached approximately 10-10.5, and the solution was stirred until
a MS analysis indicated that the reaction was finished. This benzoate
deprotection needs to be carefully monitored by MS analysis to
minimize the formation of a methyl carbamate at the N6′′′ position.
If this undesired product appears in the MS analysis, it is necessary
to add more MeOH to dilute the reaction mixture and, therefore,
lower the pH at a level where the benzoate esters are cleaved, but
with no appreciable amount of methyl carbamate formation at N6′′′.
When the reaction was finished, the solution was cooled down to
-78 °C and dry ice was added until the pH was neutral. Solvent
was removed under vacuum, and the residue was taken in EtOAc,
washed with water, and dried over Na2SO4. After removal of the

2362 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
solvent under vacuum, the solid was purified by silica gel flash
chromatography to obtain the desired amine 13a-z and 13aa-ah.
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C88H110N6O26Si [M +
H]+, 1695.7; found, 1695.9. Compound 13u: 85%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +14.6 (c 1.1,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C88H104F6N6O24Si [M + H]+, 1771.7; found, 1771.5. Compound
13v: 87%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +12.7 (c 0.9, MeOH); Rf 0.2 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C90H108N6O24Si [M + H]+, 1684.7; found,
1685.8. Compound 13w: 78%; silica gel flash chromatography
eluent, 3 to 5% MeOH/CH2Cl2; [R]D +17.3 (c 1.2, MeOH); Rf 0.1
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C84H102N6O25Si [M +
H]+, 1623.7; found, 1623.8. Compound 13x: 80%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +15.6 (c 1.0,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C88H108N6O24Si [M + H]+, 1661.7; found, 1661.6. Compound 13y:
80%; silica gel flash chromatography eluent, 3 to 5% MeOH/CH2-
Cl2; [R]D +5.7 (c 0.8, MeOH); Rf 0.1 (5% MeOH/CH2Cl2); ESI-
MS m/z calcd for C84H108N6O24Si [M + H]+, 1613.7; found, 1614.9.
Compound 13z: 73%; silica gel flash chromatography eluent, 3
to 5% MeOH/CH2Cl2; [R]D +13.7 (c 1.0, MeOH); Rf 0.2 (5%
MeOH/CH2Cl2); ESI-MS m/z calcd for C87H112N6O24Si [M + H]+,
1653.8; found, 1653.7. Compound 13aa: 91%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +14.3 (c 1.0,
MeOH); Rf 0.1 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C88H112N6O25Si [M + H]+, 1681.8; found, 1681.6. Compound
13ab: 72%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +16.9 (c 1.2, MeOH); Rf 0.1 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C84H110N6O24Si [M + H]+, 1615.7; found,
1615.8. Compound 13ac: 87%; silica gel flash chromatography
eluent, 3 to 4% MeOH/CH2Cl2; [R]D +12.2 (c 1.0, MeOH); Rf 0.3
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C105H144N6O24Si [M
+ H]+, 1902.0; found, 1902.2. Compound 13ad: 75%; silica gel
flash chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +11.8
(c 0.8, MeOH); Rf 0.1 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C92H107N7O26Si [M + H]+, 1754.7; found, 1755.7. Compound
13ae: 91%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +10.9 (c 0.8, MeOH); Rf 0.2 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C80H102N6O24Si [M + H]+, 1559.7; found,
1560.5. Compound 13af: 88%; silica gel flash chromatography
eluent, 3 to 4% MeOH/CH2Cl2; [R]D +15.2 (c 1.0, MeOH); Rf 0.3
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C95H118N6O24Si [M +
H]+, 1753.8; found, 1754.1. Compound 13ag: 85%; silica gel flash
chromatography eluent, 3% MeOH/CH2Cl2; [R]D +21.7 (c 1.1,
MeOH); Rf 0.4 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C94H130N6O24Si [M + H]+, 1755.9; found, 1756.3. Compound
13ah: 89%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +17.6 (c 0.4, MeOH); Rf 0.3 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C90H111N7O26Si [M + H]+, 1734.7; found,
1735.1.
Compounds 13. Compound 13a: purified only after AcOH
(80%) treatment to remove TBS and benzylidene (67%); silica gel
flash chromatography eluent, 5 to 7% MeOH/CH2Cl2; Rf 0.1 (10%
MeOH/CH2Cl2); ESI-MS m/z calcd for C75H91N7O26 [M + H]+,
1506.6; found, 1506.5. Compound 13b: purified only after AcOH
(80%) treatment to remove TBS and benzylidene (71%); silica gel
flash chromatography eluent, 5 to 7% MeOH/CH2Cl2; Rf 0.1 (10%
MeOH/CH2Cl2); ESI-MS m/z calcd for C76H93N7O26 [M + H]+,
1520.6; found, 1520.7. Compound 13c: 87%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +16.6 (c 1.1,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C85H104N6O24Si [M + H]+, 1621.7; found, 1622.1. Compound 13d:
81%; silica gel flash chromatography eluent, 3 to 5% MeOH/CH2-
Cl2; [R]D +13.8 (c 1.0, MeOH); Rf 0.1 (5% MeOH/CH2Cl2); ESI-
MS m/z calcd for C89H112N8O26Si [M + H]+, 1737.7; found, 1737.9.
Compound 13e: 82%; silica gel flash chromatography eluent, 3
to 5% MeOH/CH2Cl2; [R]D +10.9 (c 1.0, MeOH); Rf 0.2 (5%
MeOH/CH2Cl2); ESI-MS m/z calcd for C86H106N6O24Si [M + H]+,
1635.7; found, 1636.0. Compound 13f: purified only after AcOH
(80%) treatment to remove TBS and benzylidene; 64%; silica gel
flash chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +11.3
(c 0.8, MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C73H88N6O24 [M + H]+, 1433.6; found, 1433.4. Compound 13g:
85%; silica gel flash chromatography eluent, 3 to 5% MeOH/CH2-
Cl2; [R]D +8.9 (c 1.4, MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-
MS m/z calcd for C83H101N7O24Si [M + Na]+, 1630.7; found,
1630.8. Compound 13h: 78%; silica gel flash chromatography
eluent, 3 to 5% MeOH/CH2Cl2; [R]D +10.3 (c 1.1, MeOH); Rf 0.15
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C85H106N7O24Si [M +
H]+, 1636.7; found, 1636.8. Compound 13i: 79%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +15.6 (c 1.2,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C87H103N7O24Si [M + H]+, 1658.7; found, 1657.7. Compound 13j:
87%; silica gel flash chromatography eluent, 3 to 5% MeOH/CH2-
Cl2; [R]D +17.2 (c 1.3, MeOH); Rf 0.1 (5% MeOH/CH2Cl2); ESI-
MS m/z calcd for C89H112N8O26Si [M + H]+, 1737.8; found, 1738.2.
Compound 13k: 85%; silica gel flash chromatography eluent, 3
to 5% MeOH/CH2Cl2; [R]D +10.4 (c 0.8, MeOH); Rf 0.2 (5%
MeOH/CH2Cl2); ESI-MS m/z calcd for C83H101N7O24Si [M + H]+,
1608.7; found, 1608.8. Compound 13l: purified only after AcOH
(80%) treatment to remove TBS and benzylidene; 67%; silica gel
flash chromatography eluent, 6 to 8% MeOH/CH2Cl2; [R]D +7.3
(c 1.6, MeOH); Rf 0.2 (10% MeOH/CH2Cl2); ESI-MS m/z calcd
for C73H86N8O24 [M + H]+, 1459.6; found, 1459.7. Compound
13m: 82%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +21.3 (c 1.2, MeOH); Rf 0.2 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C84H102N6O24Si [M + H]+, 1607.7; found,
1608.9. Compound 13n: 78%; silica gel flash chromatography
eluent, 3 to 5% MeOH/CH2Cl2; [R]D +6.3 (c 0.7, MeOH); Rf 0.15
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C85H104N6O24Si [M +
H]+, 1621.7; found, 1621.8. Compound 13o: 90%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +14.7 (c 1.0,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C87H108N6O24Si [M + Na]+, 1671.7; found, 1671.9. Compound
13p: 86%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +12.8 (c 0.9, MeOH); Rf 0.2 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C88H110N6O24Si [M + Na]+, 1685.7; found,
1686.1. Compound 13q: 83%; silica gel flash chromatography
eluent, 3 to 5% MeOH/CH2Cl2; [R]D +8.4 (c 0.8, MeOH); Rf 0.2
(5% MeOH/CH2Cl2); ESI-MS m/z calcd for C87H108N6O25Si [M +
H]+, 1665.7; found, 1665.6. Compound 13r: 94%; silica gel flash
chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D +13.8 (c 0.8,
MeOH); Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for
C87H105F3N6O24Si [M + H]+, 1703.7; found, 1703.5. Compound
13s: 81%; silica gel flash chromatography eluent, 3 to 5% MeOH/
CH2Cl2; [R]D +11.7 (c 0.8, MeOH); Rf 0.15 (5% MeOH/CH2Cl2);
ESI-MS m/z calcd for C92H110N6O24Si [M + H]+, 1711.7; found,
1711.7. Compound 13t: 84%; silica gel flash chromatography
eluent, 3 to 5% MeOH/CH2Cl2; [R]D +14.9 (c 1.0, MeOH); Rf 0.15
General Procedure for Final Deprotection: The appropriate
substrate was dissolved in 80% aqueous acetic acid (3 mL), and
the solution was heated at 60 °C for 2-5 h or until a MS analysis
showed total conversion of the starting material into the benzylidene
as well as TBS deprotected product. The solution was cooled down
to room temperature, a catalytic amount of 20% palladium
hydroxide on carbon was added, and the suspension was stirred at
room temperature under an atmosphere of hydrogen (hydrogen
balloon) until the conversion of the starting material into the product
was completed as indicated by MS analysis. The mixture was
filtered through a layer of Celite on cotton, concentrated under
vacuum, washed with CH2Cl2 twice, and lyophilized to afford
compounds 5-9, 9a,b, 14a-z, and 14aa-ae as per acetate salts.
Compounds 5-9. Compound 5: 75%; [R]D +8.8 (c 0.6, H2O);
1H NMR (400 MHz, D2O) δ 5.75 (m, 1H), 5.44 (m, 1H), 5.20 (m,
1H), 4.30-4.00 (m, 6H), 3.85-3.50 (m, 12H), 3.40-3.15 (m, 6H),
3.00-2.55 (m, 6H) 2.31 (m, 1H), 1.91 (s, 21H), 1.63 (m, 1H); 13
C
NMR (125 MHz, D2O) δ 181.9, 109.8, 96.7, 96.3, 86.2, 81.5, 81.0,
75.3, 73.8, 73.3, 70.9, 70.4, 69.7, 68.4, 67.9, 61.2, 60.6, 60.4, 54.7,
51.6, 50.7, 49.9, 41.4, 39.4, 31.9, 32.7, 28.5, 28.1, 23.7; ESI-MS
m/z calcd for C27H55N7O14 [M + H]+, 701.38852; found, 702.4;
TOF-HRMS found, 702.38698. Compound 6: 80%; [R]D +5.7 (c

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2363
1
0.4, H2O); H NMR (400 MHz, D2O) δ 5.72 (m, 1H), 5.44 (m,
70.7, 69.4, 69.1, 67.9, 67.5, 60.5, 60.1, 54.1, 51.1, 50.3, 50.2, 49.1,
48.9, 46.1, 44.2, 44.1, 40.6, 30.3, 28.6, 22.7, 22.1, 21.4, 18.2; ESI-
MS m/z calcd for C31H61N7O14 [M + H]+, 756.43547; found, 756.7;
TOF-HRMS found, 756.43313. Compound 14d: 75%; [R]D +6.8
1H), 5.21 (m, 1H), 4.59 (m, 1H), 4.20-4.00 (m, 5H), 3.95-3.50
(m, 12H), 3.45-2.7 (m, 14H), 2.26 (m, 1H), 1.87 (s, 21H), 1.59
(m, 1H); 13C NMR (125 MHz, D2O) δ 182.2, 108.9, 96.8, 96.0,
86.0, 81.8, 79.8, 74.5, 74.3, 71.3, 71.2, 70.1, 68.6, 68.2, 67.5, 61.0,
54.8, 51.8, 51.1, 50.3, 49.8, 48.8, 45.5, 43.7, 41.1, 37.3, 31.1, 27.4,
24.5, 24.0; ESI-MS m/z calcd for C28H57N7O14 [M + H]+,
716.40417; found, 716.4; TOF-HRMS found, 716.40380. Com-
pound 7: 80%; [R]D +5.4 (c 0.6, H2O); 1H NMR (400 MHz, D2O)
δ 8.40-8.32 (m, 2H), 7.70-7.30 (m, 2H), 5.71 (m, 1H), 5.38 (m,
1H), 5.16 (m, 1H), 4.55 (m, 1H), 4.20-4.00 (m, 7H), 3.95-3.50
(m, 12H), 3.45-3.15 (m, 8H), 2.32 (m, 1H), 1.81 (s, 21H), 1.60
(m, 1H); 13C NMR (125 MHz, D2O) δ 181.4, 150.5, 140.0, 132.8,
129.8, 125.5, 109.0, 96.6, 95.7, 85.7, 81.4, 78.5, 74.3, 73.7, 71.1,
70.3, 69.9, 69.3, 68.5, 68.1, 67.5, 66.3, 61.0, 60.1, 54.6, 51.6, 50.8,
49.6, 46.6, 43.5, 41.1, 29.7, 23.5; ESI-MS m/z calcd for C31H55N7O14
[M + H]+, 750.38852; found, 750.4; TOF-HRMS found, 750.38473.
Compound 8: 70%; [R]D +10.6 (c 0.6, H2O); 1H NMR (400 MHz,
D2O) δ 7.82 (s, 1H), 7.66 (d, 5 Hz, 1H), 6.76 (d, 5 Hz, 1H), 5.69
(m, 1H), 5.32 (m, 1H), 5.12 (m, 1H), 4.50 (m, 1H), 4.14-4.04 (m,
5H), 3.85-3.50 (m, 12H), 3.13-3.17 (m, 10H), 2.31 (m, 1H), 1.91
(s, 21H), 1.65 (m, 1H); 13C NMR (125 MHz, D2O) δ 180.1, 156.7,
143.5, 142.1, 116.2, 113.0, 108.5, 96.2, 95.1, 85.1, 81.0, 80.9, 77.2,
74.1, 73.8, 72.9, 70.7, 69.4, 69.1, 68.0, 67.5, 66.8, 66.0, 60.5, 59.5,
54.1, 51.1, 49.0, 47.9, 46.7, 40.6, 28.6, 22.6; ESI-MS m/z calcd
for C31H56N8O14 [M + H]+, 765.39942; found, 765.7; TOF-HRMS
m/z calcd for C31H56N8O14 [M + Na]+, 787.38137; found,
787.38206. Compound 9: 80%; [R]D +8.3 (c 0.5, H2O); 1H NMR
(400 MHz, D2O) δ 7.22 (m, 5H), 5.68 (m, 1H), 5.32 (m, 1H), 5.11
(m, 1H), 4.52 (m, 1H), 4.15-4.05 (m, 5H), 3.90-3.54 (m, 12H),
3.35-3.17 (m, 10H), 2.71 (m, 2H), 2.29 (m, 1H), 1.93 (s, 18H),
1.67 (m, 1H); 13C NMR (125 MHz, D2O) δ 180.9, 136.6, 129.4
(2C), 129.1 (2C), 127.8, 108.5, 96.2, 95.1, 85.2, 80.9, 80.8, 77.6,
74.0, 73.7, 73.5, 73.0, 70.7, 69.8, 69.4, 68.0, 67.6, 65.7, 60.5, 59.5,
54.1, 51.1, 49.0, 48.7, 47.2, 46.7, 40.6, 29.0, 23.2; ESI-MS m/z
calcd for C33H58N6O14 [M + H]+, 762.4011; found, 763.4; TOF-
HRMS found, 763.40657. Compound 9a: 76%; [R]D +9.4 (c 0.6,
1
(c 0.4, H2O); H NMR (400 MHz, D2O) δ 8.00-7.70 (m, 2H),
7.60-7.40 (m, 2H), 5.70 (m, 1H), 5.33 (m, 1H), 5.11(m, 1H), 4.50
(m, 1H), 4.20-4.00 (m, 5H), 3.85-3.50 (m, 14H), 3.40-3.15 (m,
6H), 2.37 (m, 1H), 1.79 (s, 21H), 1.70 (m, 1H); 13C NMR (125
MHz, D2O) δ 181.4, 132.3, 131.6, 129.5, 128.7, 126.8, 108.8, 96.2,
95.3, 85.3, 81.6, 81.0, 78.0, 74.1, 73.1, 71.5, 70.7, 69.6, 69.3, 68.0,
67.7, 60.7, 60.3, 54.2, 51.5, 51.1, 50.3, 49.2, 43.0, 40.7, 29.2, 23.5;
ESI-MS m/z calcd for C30H53N7O14 [M + H]+, 736.37287; found,
736.5; TOF-HRMS found, 736.37359. Compound 14e: 70%; [R]D
1
+7.6 (c 0.4, H2O); H NMR (400 MHz, D2O) δ 8.41-8.42 (m,
2H), 7.92 (d, J ) 4.4 Hz, 1H), 7.52 (m, 1H), 5.69 (m, 1H), 5.33
(m, 1H), 5.13 (m, 1H), 4.50 (m, 1H), 4.20-4.00 (m, 7H), 3.90-
3.52 (m, 12H), 3.41-3.02 (m, 10H), 2.33 (m, 1H), 1.99 (m, 1H)
1.84 (s, 21H); 13C NMR (125 MHz, D2O) δ 179.7, 146.9, 141.8,
134.5, 130.1, 126.2, 109.0, 96.7, 95.6, 85.6, 81.3, 77.7, 74.6, 74.3,
73.4, 71.2, 70.4, 69.9, 69.5, 68.4, 68.0, 67.4, 66.2, 61.0, 60.0, 54.5,
51.6, 50.7, 49.5, 48.4, 47.9, 41.1, 29.4, 22.5; ESI-MS m/z calcd
for C32H57N7O14 [M + H]+, 764.40417; found, 764.7; TOF-HRMS
found, 764.40547. Compound 14f: 80%; [R]D +7.5 (c 0.2, H2O);
1H NMR (400 MHz, D2O) δ 8.42 (m, 1H), 7.85 (m, 2H), 7.41 (m,
3H), 5.72 (m, 1H), 5.38 (m, 1H), 5.17 (m, 1H), 4.55 (m, 1H), 4.20-
4.00 (m, 5H), 3.95-3.50 (m, 12H), 3.45-3.15 (m, 8H), 2.32 (m,
1H), 1.81 (s, 21H), 1.65-1.40 (m, 1H); 13C NMR (125 MHz, D2O)
δ 181.8, 143.4, 140.5, 138.4, 130.2, 129.6, 127.3, 126.9, 122.5,
114.6, 109.1, 96.5, 95.6, 85.1, 81.8, 78.5, 75.6, 74.4, 72.8, 70.1,
69.9, 69.6, 68.3, 67.5, 60.9, 60.4, 54.5, 52.6, 51.5, 50.4, 49.5, 41.0,
29.2, 28.0, 23.8; ESI-MS m/z calcd for C34H55N7O14 [M + H]+,
786.38852; found, 786.5; TOF-HRMS found, 786.38421. Com-
1
pound 14g: 75%; [R]D +5.6 (c 0.3, H2O); H NMR (400 MHz,
D2O) δ 7.91 (m, 1H), 7.45 (m, 1H), 6.65 (m, 1H), 5.67 (m, 1H),
5.32 (m, 1H), 5.10 (m, 1H), 4.51 (m, 1H), 4.15-4.00 (m, 5H),
3.76-3.50 (m, 12H), 3.41-3.16 (m, 10H), 2.32 (m, 1H), 1.81 (s,
21H), 1.65-1.50 (m, 1H); 13C NMR (125 MHz, D2O) δ 180.8,
157.6, 116.31, 142.7, 136.2, 112.3, 108.5, 96.2, 95.1, 85.2, 82.1,
81.1, 80.8, 77.5, 74.0, 73.8, 73.0, 70.7, 69.4, 69.2, 67.9, 67.6, 65.5,
60.5, 59.5, 54.1, 51.1, 50.3, 49.0, 46.6, 40.6, 28.8, 23.2; ESI-MS
m/z calcd for C31H56N8O14 [M + H]+, 765.39942; found, 765.6;
TOF-HRMS found, 765.39871. Compound 14h: 80%; [R]D +9.7
(c 0.5, H2O); 1H NMR (400 MHz, D2O) δ 7.78 (m, 1H), 7.31 (m,
1H), 6.94 (m, 1H), 6.79 (m, 1H), 5.72 (m, 1H), 5.34 (m, 1H), 5.10
(m, 1H), 4.51 (m, 1H), 4.21-4.06 (m, 5H), 3.90-3.46 (m, 14H),
1
H2O); H NMR (400 MHz, D2O) δ 7.25 (m, 5H), 5.66 (m, 1H),
5.33 (m, 1H), 5.11 (m, 1H), 4.55 (m, 1H), 414.20-4.05 (m, 5H),
3.85-3.45 (m, 12H), 3.37-3.22 (m, 8H), 2.95 (m, 2H), 2.31 (m,
1H), 1.99-1.88 (s, 18H), 1.69 (m, 1H); ESI-MS m/z calcd for
C35H60N6O15 [M + H]+, 805.41949; found, 805.6; TOF-HRMS
found, 805.41607. Compound 9b: 70%; [R]D +8.7 (c 0.5, H2O);
1H NMR (400 MHz, D2O) δ 7.79 (m, 2H), 7.46-7.35 (m, 3H),
7.24 (m, 5H), 5.69 (m, 1H), 5.31 (m, 1H), 5.12 (m, 1H), 4.54 (m,
1H), 4.15-4.00 (m, 5H), 3.85-3.50 (m, 12H), 3.35-3.21 (m, 8H),
2.91 (m, 2H), 2.30 (m, 1H), 1.91 (s, 15H), 1.67 (m, 1H); ESI-MS
m/z calcd for C35H60N6O15 [M + H]+, 867.43514; found, 867.8;
TOF-HRMS found, 867.43400.
3.39-3.22 (m, 6H), 2.37 (m, 1H), 1.81 (s, 21H), 1.68 (m, 1H); 13
C
NMR (125 MHz, D2O) δ 180.9, 154.1, 144.2, 136.3, 129.6, 113.5,
109.1, 96.5, 95.5, 85.6, 81.7, 81.3, 77.6, 74.6, 74.1, 73.3, 71.1, 69.9,
69.5, 68.4, 68.3, 68.1, 61.0, 60.4, 54.5, 51.6, 50.6, 49.5, 42.4, 41.1,
29.1, 23.3; ESI-MS m/z calcd for C30H53N7O14 [M + H]+,
736.37287; found, 736.5; TOF-HRMS found, 736.37085. Com-
Compounds 14. Compound 14a: 78%; [R]D +10.6 (c 0.7, H2O);
1H NMR (400 MHz, D2O) δ 5.78 (m, 1H), 5.46 (m, 1H), 5.26 (m,
1H), 4.30-4.00 (m, 6H), 3.95-3.50 (m, 14H), 3.45-3.00 (m, 6H),
2.35 (m, 1H), 1.91 (s, 18H), 1.71 (m, 1H); 13C NMR (125 MHz,
D2O) δ 181.7, 110.2, 97.3, 95.7, 86.2, 82.3, 77.8, 76.0, 74.7, 73.9,
72.6, 70.8, 69.4, 69.3, 68.2, 67.6, 61.4, 60.5, 58.8, 54.9, 52.6, 52.1,
49.3, 48.1, 42.3, 27.6, 23.4; ESI-MS m/z calcd for C25H50N6O14
[M + H]+, 659.34633; found, 659.4; TOF-HRMS found, 659.34410.
1
pound 14i: 75%; [R]D +8.6 (c 0.6, H2O); H NMR (400 MHz,
D2O) δ 7.80-7.40 (m, 4H), 5.81 (m, 1H), 5.44 (m, 1H), 5.24 (m,
1H), 4.35-4.10 (m, 6H), 3.95-3.50 (m, 14H), 3.45-3.15 (m, 8H),
2.42 (m, 1H), 1.91 (s, 21H), 1.61 (m, 1H); ESI-MS m/z calcd for
C33H56N8O14 [M + H]+, 789.39942; found, 789.5; TOF-HRMS
found, 789.39932. Compound 14j: 80%; [R]D +6.7 (c 0.5, H2O);
1H NMR (400 MHz, D2O) δ 7.22-6.91 (m, 5H), 5.65 (m, 1H),
5.25 (m, 1H), 5.16 (m, 1H), 4.41 (m, 1H), 4.25-4.05 (m, 5H),
3.91-3.55 (m, 12H), 3.44-3.20 (m, 8H), 2.33 (m, 1H), 1.80 (s,
18H), 1.72 (m, 1H); 13C NMR (125 MHz, D2O) δ 180.9, 144.7,
129.1 (2C), 119.6, 116.3 (2C), 109.5, 95.7, 94.8, 84.1, 80.7, 78.1,
74.5, 74.6, 73.2, 72.8, 72.2, 69.8, 69.5, 68.7, 68.6, 67.2, 66.8, 59.8,
59.5, 53.5, 50.4, 49.5, 48.4, 40.0, 28.8, 22.8; ESI-MS m/z calcd
for C31H54N6O14 [M + H]+, 735.37763; found, 735.5; TOF-HRMS
found, 735.37748. Compound 14k: 85%; [R]D +8.5 (c 0.4, H2O);
1H NMR (400 MHz, D2O) δ 7.33 (m, 5H), 5.67 (m, 1H), 5.32 (m,
1H), 5.08 (m, 1H), 4.48 (m, 1H), 4.16-4.01 (m, 5H), 3.89-3.48
(m, 14H), 3.36-3.16 (m, 8H), 2.31 (m, 1H), 1.91 (s, 18H), 1.65
(m, 1H); 13C NMR (125 MHz, D2O) δ 181.5, 131.2, 130.7 (2C),
130.1 (2C), 129.6, 108.9, 96.6, 95.6, 85.6, 81.6, 78.1, 74.5, 74.3,
1
Compound 14b: 70%; [R]D +7.8 (c 1.0, H2O); H NMR (400
MHz, D2O) δ 5.66 (m, 1H), 5.30 (m, 1H), 5.11 (m, 1H), 4.46 (m,
1H), 4.20-4.00 (m, 5H), 3.95-3.50 (m, 14H), 3.40-2.95 (m, 11H),
2.37 (m, 1H), 2.1-1.9 (m, 4H) 1.79 (s, 21H), 1.70 (m, 1H); 13C
NMR (125 MHz, D2O) δ 181.0, 108.9, 96.6, 95.7, 90.9, 85.5, 81.5,
77.7, 74.5, 74.3, 73.3, 71.1, 69.9, 69.5, 68.4, 68.3, 68.0, 61.0, 54.5,
52.4, 51.5, 50.6, 49.4, 44.7, 44.2, 41.0, 40.1, 34.5, 28.9, 23.3 20.9,
20.2; ESI-MS m/z calcd for C30H59N7O14 [M + H]+, 741.41982;
found, 742.7; TOF-HRMS found, 742.41783. Compound 14c:
1
75%; [R]D +12.4 (c 1.1, H2O); H NMR (400 MHz, D2O) δ 5.67
(m, 1H), 5.32 (m, 1H), 5.25 (m, 1H), 4.48 (m, 1H), 4.20-4.00 (m,
5H), 3.95-3.30 (m, 14H), 3.30-2.75 (m, 13H), 2.21 (m, 1H), 1.81
(s, 21H), 1.72 (m, 1H), 1.63-1.45 (m, 4H); 13C NMR (125 MHz,
D2O) δ 180.3, 108.5, 96.2, 95.2, 85.0, 81.1, 80.8, 77.2, 74.1, 72.9,

2364 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
73.5, 71.2, 69.9, 69.6, 68.4, 68.0, 66.2, 60.9, 60.1, 54.6, 51.8, 51.6,
50.7, 49.5, 47.1, 41.1, 40.1, 29.5, 23.6; ESI-MS m/z calcd for
C32H56N6O14 [M + H]+, 749.39328; found, 749.6; TOF-HRMS
found, 749.39319. Compound 14l: 90%; [R]D +11.4 (c 0.4, H2O);
1H NMR (400 MHz, D2O) δ 7.27 (m, 5H), 5.74 (m, 1H), 5.36 (m,
1H), 5.27 (m, 1H), 4.29 (m, 1H), 4.21-4.07 (m, 5H), 3.90-3.14
(m, 22H), 2.70-2.61 (m, 2H), 2.32 (m, 1H) 2.21 (m, 2H), 1.89 (s,
18H), 1.61 (m, 1H); 13C NMR (125 MHz, D2O) δ 182.3, 132.8,
129.5 (2C), 129.3 (2C), 126.9, 110.5, 97.6, 97.0, 85.6, 82.0, 81.8,
76.0, 74.5, 74.2, 74.0, 71.3, 71.1, 70.2, 69.1, 68.4, 61.7, 61.1, 55.2,
52.0, 51.2, 50.1, 41.3, 39.7, 33.2, 32.6, 30.7, 29.7, 28.7, 24.1; ESI-
MS m/z calcd for C34H60N6O14 [M + H]+, 777.42458; found, 777.6;
TOF-HRMS found, 777.42090. Compound 14m: 90%; [R]D +9.6
(c 0.5, H2O); 1H NMR (400 MHz, D2O) δ 7.22 (m, 5H), 5.55 (m,
1H), 5.27 (m, 1H), 5.14 (m, 1H), 4.45 (m, 1H), 4.25-4.08 (m,
5H), 3.81-2.94 (m, 22H), 2.61 (m, 2H), 2.31-2.15 (m, 3H), 1.79
(s, 18H), 1.4-1.65 (m, 3H); 13C NMR (125 MHz, D2O) δ 181.8,
143.2, 128.9 (4C), 126.3, 110.1, 96.9, 96.3, 85.2, 81.5, 81.1, 75.4,
73.9, 73.7, 73.6, 70.7, 70.3, 69.7, 68.4, 67.8, 61.2, 60.6, 60.4, 54.7,
51.4, 50.7, 49.6, 40.8, 39.4, 34.9, 31.8, 32.7, 28.3, 28.1, 23.6; ESI-
MS m/z calcd for C35H62N6O14 [M + H]+, 791.44023; found, 791.7;
TOF-HRMS found, 791.44394. Compound 14n: 90%; [R]D +5.4
182.1, 136.6, 131.7, 130.9, 130.2, 129.7, 129.3 128.7, 128.2, 127.8,
124,7, 109.6, 97.1, 96.3, 84.3, 81.7, 81.2, 78.2, 74.3, 74.0, 73.6,
73.2, 71.2, 70.4, 69.8, 68.5, 68.0, 66.2, 60.7, 60.1, 54.6, 51.7, 49.5,
49.1, 48.0, 46.9, 41.4, 31.3, 23.7; ESI-MS m/z calcd for C37H60N6O14
[M + H]+, 813.42458; found, 813.4; TOF-HRMS found, 813.42068.
Compound 14t: 85%; [R]D +7.3 (c 0.3, H2O); 1H NMR (400 MHz,
D2O) δ 6.95 (m, 1H), 6.15-6.05 (m, 3H), 5.67 (m, 1H), 5.24 (m,
1H), 5.13 (m, 1H), 4.41 (m, 1H), 414.28-4.07 (m, 5H), 3.90-
3.55 (m, 12H), 3.46-3.11 (m, 8H), 2.34 (m, 1H), 1.79 (s, 18H),
1.67 (m, 1H); 13C NMR (125 MHz, D2O) δ 181.7, 156.5, 149.2,
130.8, 129.1, 109.0, 107.5, 105.3, 96.3, 95.6, 85.6, 81.0, 79.1, 73.8,
73.5, 73.2, 70.6, 69.5, 69.2, 68.7, 68.0, 67.9, 67.6, 60.5, 60.3, 54.3,
51.2, 50.4, 49.3, 49.2, 40.6, 29.3, 23.5; ESI-MS m/z calcd for
C31H54N6O15 [M + H]+, 751.37254; found, 751.6; TOF-HRMS
found, 751.36768. Compound 14u: 90%; [R]D +12.6 (c 0.8, H2O);
1H NMR (400 MHz, D2O) δ 7.22-7.09 (m, 4H), 5.66 (m, 1H),
5.25 (m, 1H), 5.17 (m, 1H), 4.42 (m, 1H), 4.29-4.06 (m, 5H),
3.88-3.55 (m, 12H), 3.44-3.19 (m, 8H), 2.75-2.70 (m, 3H), 2.33
(m, 1H), 2.00-1.84 (m, 4H), 1.78 (s, 18H), 1.70 (m, 1H); 13C NMR
(125 MHz, D2O) δ 182.1, 139.1, 132.0, 130.7, 129.8, 129.1, 127.3,
110.3, 98.1, 97.5, 85.4, 82.2, 82.0, 76.0, 74.9, 74.2, 74.1, 72.7, 71.6,
71.4, 70.3, 69.5, 68.6, 67.3, 61.2, 61.1, 55.4, 52.2, 51.2, 50.2, 49.8,
41.3, 32.9, 28.8, 28.1, 24.2, 18.6; ESI-MS m/z calcd for C35H60N6O14
[M + H]+, 789.42458; found, 789.5; TOF-HRMS found, 789.42387.
Compound 14v: 85%; [R]D +4.9 (c 0.3, H2O); 1H NMR (400 MHz,
D2O) δ 5.68 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H), 4.42 (m, 1H),
4.28-4.07 (m, 5H), 3.88-3.59 (m, 12H), 3.47-3.19 (m, 8H), 2.70
(m, 1H), 2.34 (m, 1H), 2.1-0.8 (m, 39H); 13C NMR (125 MHz,
D2O) δ 182.2, 110.2, 98.5, 98.0, 85.2, 82.5, 82.0, 76.0, 75.3, 74.2,
74.0, 72.0, 71.8, 71.6, 70.4, 69.9, 68.8, 61.2, 55.5, 52.4, 52.2, 51.2,
50.4, 50.0, 41.4, 33.3, 31.1 (2C), 25.1, 24.6, 24.2, 24.1 (2C); ESI-
MS m/z calcd for C31H60N6O14 [M + H]+, 741.41458; found, 741.5;
TOF-HRMS found, 741.42466. Compound 14w: 80%; [R]D +8.5
(c 0.6, H2O); 1H NMR (400 MHz, D2O) δ 5.66 (m, 1H), 5.26 (m,
1H), 5.18 (m, 1H), 4.41 (m, 1H), 4.28-4.10 (m, 5H), 3.88-3.54
(m, 12H), 3.47-3.20 (m, 8H), 2.82 (m, 2H), 2.36-1.02 (m, 43H);
13C NMR (125 MHz, D2O) δ 180.9, 108.5, 96.2, 95.1, 85.2, 80.9,
80.8, 77.6, 74.0, 73.7, 73.5, 73.0, 70.7, 69.8, 69.4, 68.0, 67.6, 65.7,
60.5, 54.1, 51.1, 49.0, 48.7, 47.2, 46.7, 42.4, 40.6, 35.8, 35.1, 32.6,
30.1, 29,7 29.0, 28.5, 27.9, 23.2; ESI-MS m/z calcd for C34H64N6O14
[M + H]+, 781.45588; found, 781.5; TOF-HRMS found, 781.45282.
Compound 14x: 80%; [R]D +7.8 (c 0.5, H2O); 1H NMR (400 MHz,
D2O) δ 5.66 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H), 4.42 (m, 1H),
4.31-4.04 (m, 5H), 3.88-3.57 (m, 12H), 3.46-3.18 (m, 8H), 2.34
(m, 1H), 2.22 (m, 3H), 1.79 (s, 18H), 1.70-1.66 (m, 4H), 1.57 (m,
6H), 1.44 (m, 3H); ESI-MS m/z calcd for C35H64N6O15 [M + H]+,
809.45079; found, 809.4; TOF-HRMS found, 809.44818. Com-
1
(c 0.2, H2O); H NMR (400 MHz, D2O) δ 7.13 (s, 2H), 6.85 (m,
2H), 5.66 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H), 4.42 (m, 1H),
414.28-4.08 (m, 5H), 3.88-3.56 (m, 15H), 3.46-3.20 (m, 10H),
2.81 (m, 2H), 2.34 (m, 1H), 1.79 (s, 18H), 1.72 (m, 1H); 13C NMR
(125 MHz, D2O) δ 182.2, 158.7, 130.9 (2C), 115.2 (2C), 110.5,
97.5, 96.8, 85.5, 82.0, 81.8, 75.9, 74.4, 74.1, 72.5, 71.2, 70.9, 70.2,
69.0, 68.3, 66.3, 61.1, 61.0, 56.1, 55.9, 55.2, 52.0, 51.2, 50.3, 41.5,
32.6, 32.1, 24.1; ESI-MS m/z calcd for C34H60N6O15 [M + H]+,
793.41949; found, 793.7; TOF-HRMS found, 793.41776. Com-
1
pound 14o: 85%; [R]D +6.5 (c 0.2, H2O); H NMR (400 MHz,
D2O) δ 7.57 (s, 2H), 7.35 (m, 2H), 5.66 (m, 1H), 5.25 (m, 1H),
5.16 (m, 1H), 4.41 (m, 1H), 414.29-4.06 (m, 5H), 3.87-3.56 (m,
12H), 3.47-3.15 (m, 10H), 2.94 (m, 2H), 2.33 (m, 1H), 1.79 (s,
18H), 1.68 (m, 1H); 13C NMR (125 MHz, D2O) δ 182.2, 141.7,
130.1, 126.5, 110.2, 98.4, 97.8, 85.2, 82.4, 82.0, 76.0, 75.2, 74.1,
74.0, 72.4, 71.9, 71.5, 70.3, 69.8, 68.7, 67.2, 61.2, 55.5, 59.8, 52.3,
51.2, 50.3, 41.3, 41.0, 36.7, 33.4 24.1; ESI-MS m/z calcd for
C34H57F3N6O14 [M + H]+, 831.39631; found, 831.5; TOF-HRMS
found, 831.39758. Compound 14p: 85%; [R]D +10.8 (c 0.6, H2O);
1H NMR (400 MHz, D2O) δ 7.57 (m, 2H), 7.42 (m, 2H), 7.31 (m,
5H), 5.69 (m, 1H), 5.32 (m, 1H), 5.12 (m, 1H), 4.5 (m, 1H),
414.14-4.04 (m, 5H), 3.85-3.50 (m, 12H), 3.30-3.17 (m, 10H),
2.78 (m, 2H), 2.31 (m, 1H), 1.91 (s, 18H), 1.65 (m, 1H); 13C NMR
(125 MHz, D2O) δ 181.2, 138.4, 136.1, 132.7 (2C), 130.9 (2C),
130.0 (2C), 128.4, 127.1 (2C), 126.3, 109.1, 97.6, 95.4, 87.1, 81.5,
80.6, 78.1, 74.8, 74.1, 73.9, 73.4, 71.2, 70.1, 69.4, 68.6, 67.9, 65.7,
60.7, 59.3, 54.7, 53.2, 50.8, 49.4, 48.0, 46.9, 41.5, 29.3, 23.8; ESI-
MS m/z calcd for C39H62N6O14 [M + H]+, 839.44023; found, 839.5;
TOF-HRMS found, 839.43844. Compound 14q: 90%; [R]D +11.4
(c 0.7, H2O); 1H NMR (400 MHz, D2O) δ 6.40 (m, 3H), 5.67 (m,
1H), 5.24 (m, 1H), 5.15 (m, 1H), 4.41 (m, 1H), 4.28-4.07 (m,
5H), 3.90-3.55 (m, 18H), 3.46-3.11 (m, 10H), 2.82 (m, 2H), 2.34
(m, 1H), 1.79 (s, 18H), 1.67 (m, 1H); 13C NMR (125 MHz, D2O)
δ 181.0, 160.0 (2C), 138.9, 109.4, 106.8 (2C), 98.6, 96.1, 95.3,
84.3, 80.6, 80.2, 74.5, 73.0, 72.8, 71.9, 71.4, 69.9, 69.4, 68.9, 67.6,
67.0, 61.3, 59.8, 59.6, 55.0, 53.8, 50.6, 49.9, 48.7, 40.0, 32.4, 30.8,
22.8; ESI-MS m/z calcd for C35H62N6O16 [M + H]+, 823.43006;
found, 823.5; TOF-HRMS found, 823.42847. Compound 14r:
1
pound 14y: 80%; [R]D +13.6 (c 0.8, H2O); H NMR (400 MHz,
D2O) δ 5.66 (m, 1H), 5.26 (m, 1H), 5.18 (m, 1H), 4.41 (m, 1H),
4.28-4.10 (m, 5H), 3.88-3.54 (m, 12H), 3.47-3.20 (m, 8H), 2.88
(m, 2H), 2.33 (m, 1H), 1.80 (s, 18H), 1.71 (m, 1H), 1.42 (m, 2H),
0.80 (m, 9H); 13C NMR (125 MHz, D2O) δ 182.7, 109.6, 96.9,
94.3, 84.8, 81.3, 77.5, 75.2, 74.5, 73.7, 71.9, 70.8, 69.6, 69.2, 68.4,
67.8, 61.4 60.3, 59.9, 54.8, 51.4, 50.5, 49.6, 47.5, 46.5, 40.2, 34.3
(3C), 31.6, 29.3 26.1, 23.9; ESI-MS m/z calcd for C31H62N6O14
[M + H]+, 743.44023; found, 743.4; TOF-HRMS found, 743.43885.
Compound 14z: 80%; [R]D +7.4 (c 0.4, H2O); 1H NMR (400 MHz,
D2O) δ 5.66 (m, 1H), 5.26 (m, 1H), 5.18 (m, 1H), 4.41 (m, 1H),
4.28-4.10 (m, 5H), 3.88-3.54 (m, 12H), 3.47-3.20 (m, 8H), 2.31
(m, 1H), 2.01-0.65 (m, 66H); ESI-MS m/z calcd for C52H96N6O14
[M + H]+, 1029.70628; found, 1029.7; TOF-HRMS found,
1
90%; [R]D +5.6 (c 0.3, H2O); H NMR (400 MHz, D2O) δ 7.72
1
(m, 1H), 7.65 (m, 2H), 5.66 (m, 1H), 5.26 (m, 1H), 5.17 (m, 1H),
4.42 (m, 1H), 414.28-4.08 (m, 5H), 3.88-3.56 (m, 12H), 3.46-
3.20 (m, 10H), 2.94 (m, 2H), 2.34 (m, 1H), 1.79 (s, 24H), 1.72 (m,
1H); ESI-MS m/z calcd for C35H56F6N6O14 [M + H]+, 899.38369;
found, 899.4; TOF-HRMS found, 899.38216. Compound 14s:
1029.70511. Compound 14aa: 75%; [R]D +8.4 (c 0.5, H2O); H
NMR (400 MHz, D2O) δ 8.61 (d, J ) 3.5 Hz, 2H), 8.22 (d, J )
3.5 Hz, 2H), 7.77 (t, J ) 7 Hz, 2H), 5.66 (m, 1H), 5.30 (m, 1H),
5.11 (m, 1H), 4.46 (m, 1H), 4.20-4.00 (m, 5H), 3.95-3.50 (m,
12H), 3.40-2.95 (m, 12H), 2.37 (m, 1H), 1.79 (s, 18H), 1.70 (m,
1H); 13C NMR (125 MHz, D2O) δ 181.1, 165.4 (2C), 134.8 (2C),
129.7 (2C), 128.4 (2C), 126.4 (2C), 123.6 (2C), 110,7, 96.8, 96.1,
85.0, 82.3, 78.1, 76.1, 74.6, 73.2, 71.3, 70.8, 70.1, 69.7, 68.8, 68.1,
67.6, 61.8, 61.1, 56.7 54.6, 52.8, 51.7, 51.4, 50.8, 49.5, 41.6, 28.9,
23.4; ESI-MS m/z calcd for C39H61N7O16 [M + H]+, 882.40965;
1
80%; [R]D +9.2 (c 0.5, H2O); H NMR (400 MHz, D2O) δ 7.82
(m, 3H), 7.66 (m, 1H), 7.47 m, (2H), 7.34 (m, 1H), 5.68 (m, 1H),
5.32 (m, 1H), 5.11 (m, 1H), 4.52 (m, 1H), 4.15-4.05 (m, 5H),
3.90-3.54 (m, 12H), 3.35-3.17 (m, 10H), 2.81 (m, 2H), 2.29 (m,
1H), 1.93 (s, 18H), 1.67 (m, 1H); 13C NMR (125 MHz, D2O) δ

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2365
found, 882.5; TOF-HRMS found, 882.40762. Compound 14ab:
80%; [R]D +7.3 (c 0.6, H2O); H NMR (400 MHz, D2O) δ 5.76
ozone was bubbled for 2 h, after which excess ozone was removed
by bubbling argon. The mixture was treated with Me2S (0.130 mL,
1.77 mmol) and warmed to room temperature and the solvent was
removed under vacuum. The crude solid was purified by silica gel
flash chromatography (2:3 EtOAc/hexane) to give the corresponding
aldehyde (200 mg, 80%): Rf 0.25 (1:1 EtOAc/hexane); ESI-MS
m/z calcd for C77H83N3O21Si [M + H]+, 1414.5; found, 1414.7.
To a mixture of the above aldehyde (70 mg, 0.050 mmol) and
appropriate amine (0.15 mmol) in dry MeOH (3 mL) was added
acetic acid (0.1 mL), followed by NaBH3CN (1.0 M in THF, 148
µL, 0.148 mmol). A TLC indicated a complete disparition of the
aldehyde after 18 h. A freshly prepared solution of MeONa/MeOH
was added dropwise until the pH reached approximately 10-10.5.
When the reaction was complete, as indicated by a MS analysis,
the solution was cooled down to -78 °C and dry ice was added
until neutral pH. Solvent was removed under vacuum, and the
residue was taken up in EtOAc, washed with water, and dried over
Na2SO4. After removal of the solvent under vacuum, the solid was
purified by silica gel flash chromatography to obtain the desired
amine 17a-c. Compound 17a: 64%; silica gel flash chromatog-
raphy eluent, 3 to 5% MeOH/CH2Cl2; [R]D +10.3 (c 0.5, MeOH);
Rf 0.2 (5% MeOH/CH2Cl2); ESI-MS m/z calcd for C64H82N4O17Si
[M + H]+, 1207.6; found, 1208.2. Compound 17b: 77%; silica
gel flash chromatography eluent, 3 to 5% MeOH/CH2Cl2; [R]D
+14.8 (c 1.0, MeOH); Rf 0.15 (5% MeOH/CH2Cl2); ESI-MS m/z
calcd for C67H87N5O19Si [M + H]+, 1294.6; found 1294.9.
Compound 17c: 82%; silica gel flash chromatography eluent, 3
to 5% MeOH/CH2Cl2; [R]D +11.7 (c 0.9, MeOH); Rf 0.2 (5%
MeOH/CH2Cl2); ESI-MS m/z calcd for C64H82N4O17Si [M + H]+,
1207.6; found, 1207.9.
1
(m, 1H), 5.46 (m, 1H), 5.26 (m, 1H), 4.62 (m, 1H), 4.41-4.04 (m,
5H), 3.90-3.50 (m, 14H), 3.45-3.20 (m, 6H), 2.9 (s, 6H), 2.33
(m, 1H), 1.88 (s, 18H), 1.70 (m, 1H); 13C NMR (125 MHz, D2O)
δ 182.0, 108.8, 96.7, 95.6, 85.8, 81.4, 81.2, 78.9, 74.3, 74.2, 73.9,
71.2, 69.9, 69.8, 68.5, 68.0, 64.9, 60.9, 59.9, 57.5, 54.7, 51.7, 50.9,
49.6, 43.6 (2C), 41.1, 30.2, 23.9; ESI-MS m/z calcd for C27H54N6O14
[M + H]+, 687.37763; found, 687.4; TOF-HRMS found, 687.37907.
1
Compound 14ac: 90%; [R]D +11.6 (c 0.8, H2O); H NMR (400
MHz, D2O) δ 7.31-7.23 (m, 10H), 5.74 (m, 1H), 5.36 (m, 1H),
5.27 (m, 1H), 4.29 (m, 1H), 4.21-4.07 (m, 5H), 3.90-3.14 (m,
24H), 2.78-2.69 (m, 4H), 2.35-2.23 (m, 3H), 1.89 (s, 18H), 1.61
(m, 1H); ESI-MS m/z calcd for C42H70N6O14 [M + H]+, 883.50283;
found, 883.8; TOF-HRMS found, 883.49692. Compound 14ad:
1
80%; [R]D +8.3 (c 0.5, H2O); H NMR (400 MHz, D2O) δ 5.69
(m, 1H), 5.33 (m, 1H), 5.13 (m, 1H), 4.50 (m, 1H), 4.20-4.00 (m,
5H), 3.90-3.52 (m, 12H), 3.41-3.02 (m, 8H), 2.72 (m, 4H), 2.33
(m, 1H), 1.84 (s, 18H), 1.62 (m, 1H), 1.53 (m, 4H), 1.29 (m, 20H)
0.88 (m, 6H); 13C NMR (125 MHz, D2O) δ 182.1, 110.3, 98.3,
97.6, 85.4, 82.9, 82.2, 76.3, 75.8, 74.1, 72.1, 71.9, 71.6, 70.7, 69.9,
68.5, 67.8, 62.1, 61.7, 60.8, 56.5, 54.6, 51.5, 50.8 (2C), 50.5, 41.6,
33.7 (2C), 32.4 (2C), 30.8 (2C), 29.7 (2C), 29.1, 28.6 (2C), 24.2,
21.3 (2C), 16.4 (2C); ESI-MS m/z calcd for C41H82N6O14 [M +
H]+, 883.59673; found, 883.9; TOF-HRMS found, 883.59434.
1
Compound 14ae: 85%; [R]D +14.5 (c 0.7, H2O); H NMR (400
MHz, D2O) δ 5.70 (m, 1H), 5.35 (m, 1H), 5.12 (m, 1H), 4.49 (m,
1H), 4.30-4.00 (m, 5H), 3.95-3.40 (m, 14H), 3.45-3.05 (m, 10H),
2.68 (m, 4H), 2.26 (m, 1H), 1.87 (s, 21H), 1.62 (m, 1H); 13C NMR
(125 MHz, D2O) δ 181.6, 108.9, 96.6, 95.9, 87.5, 81.6, 78.5, 74.6,
74.5, 73.7, 71.2, 70.0, 69.8, 68.5, 68.1, 68.0, 61.0, 60.6, 57.2, 54.7,
51.8, 51.1, 50.8, 50.2 (2C), 49.7, 43.6 (2C), 41.1, 31.1, 23.6; ESI-
MS m/z calcd for C29H58N7O14 [M + H]+, 728.40417; found, 728.3;
TOF-HRMS found, 728.40202.
Compounds 18a-c: The appropriate substrate 17a-c was
dissolved in 80% aqueous acetic acid (3 mL), and the solution was
heated at 60 °C for 2-5 h or until a MS analysis showed total
conversion of the starting material into the benzylidene as well as
TBS-deprotected product. The solution was cooled down to room
temperature, a catalytic amount of 20% palladium hydroxide on
carbon was added, and the suspension was stirred at room
temperature under an atmosphere of hydrogen (hydrogen balloon)
until the conversion of the starting material into the product was
completed, as indicated by MS analysis. The mixture was filtered
through a layer of Celite on cotton, concentrated under vacuum,
washed with CH2Cl2 twice, and lyophilized to afford compounds
18a-c as fluffy white solids. Compound 18a: 75%; [R]D +6.9 (c
Compound 15: To 11 (1.00 g, 0,654 mmol) in solution in
pyridine (20 mL) was added Pb(OAc)4 (725 mg, 1.635 mmol). After
stirring overnight, oxalic acid (295 mg, 3,271 mmol) was added
and the solvent was evaporated. The resulting yellow solid was
taken up in EtOAc and the solids were filtered. The filtrate was
washed with water and dried over Na2SO4, the solvent was removed
under vacuum, and the resulting yellow oil was purified by silica
gel chromatography (2% MeOH in CH2Cl2). The resulting yellow
solid was dissolved in THF (20 mL) and Et3N (5 mL) was added.
After stirring for 36 h, the solvent was removed under vacuum
and the oil was purified by silica gel chromatography (3% MeOH
in CH2Cl2) to get 15 (220 mg, 30%) as a white solid. [R]D +18.82
1
0.5, H2O); H NMR (400 MHz, D2O) δ 5.67 (m, 1H), 5.34 (m,
1H), 4.35 (m, 1H), 4.22 (m, 1H), 3.99-3.12 (m, 16H), 2.81 (m,
2H), 2.21 (m, 1H), 1.82 (s, 12H), 1.53 (m, 1H); 13C NMR (125
MHz, D2O) δ 182.6, 108.5, 96.4, 85.7, 83.3, 82.6, 73.9, 73.8, 73.5,
69.7, 69.6, 68.9, 66.6, 60.9, 60.6, 54.5, 50.8, 49.4, 39.8, 28.7, 23.5;
ESI-MS m/z calcd for C19H38N4O11 [M + H]+, 499.26153; found,
499.4; TOF-HRMS found, 499.26104. Compound 18b: 85%; [R]D
1
(c 1.0, CHCl3); Rf 0.6 (CHCl3/EtOAc/MeOH (20:5:3)); H NMR
(300 MHz, CDCl3) δ 7.60-7.10 (m, 20H), 6.30-3.00 (m, 30H),
2.27 (m, 1H), 1.33 (m, 1H), 0.85 (s, 9H), 0.03 (s, 6H); ESI-MS
m/z calcd for C57H73N3O17Si [M + H]+, 1100.47875; found, 1100.6;
TOF-HRMS found, 1100.47681.
1
+10.8 (c 1.0, H2O); H NMR (400 MHz, D2O) δ 5.66 (m, 1H),
5.34 (m, 1H), 4.35 (m, 1H), 4.24 (m, 1H), 3.99-2.97 (m, 20H),
2.49 (m, 2H), 2.21 (m, 1H), 2.03 (m, 2H), 1.82 (s, 15H), 1.54 (m,
1H); 13C NMR (125 MHz, D2O) δ 182.3, 108.6, 96.8, 86.1, 83.7,
82.8, 80.3, 74.3, 74.1, 73.6, 70.0, 69.2, 66.0, 61.2, 61.0, 54.9, 51.1,
49.8, 48.2, 45.3, 37.3, 31.5, 24.5, 24.1; ESI-MS m/z calcd for
C22H45N5O11 [M + H]+, 556.31938; found, 556.4; TOF-HRMS
found, 556.31849. Compound 18c: 85%; [R]D +7.8 (c 0.9, H2O);
1H NMR (400 MHz, D2O) δ 7.36-7.25 (m, 5H), 5.72 (m, 1H),
5.33 (m, 1H), 4.37 (m, 1H), 4.26 (m, 1H), 3.98-2.95 (m, 20H),
2.73 (m, 2H), 2.21 (m, 1H), 1.87 (s, 12H), 1.71 (m, 1H); 13C NMR
(125 MHz, D2O) δ 181.4, 132.3, 131.6, 129.5, 129.2, 128.7, 127.1,
126.8, 108.8, 96.2, 95.3, 85.3, 81.6, 78.0, 74.1, 73.1, 70.7, 69.6,
69.3, 68.0, 67.7, 60.7, 60.3, 54.2, 50.3, 49.2, 43.0, 40.7, 29.2, 23.5;
ESI-MS m/z calcd for C27H46N4O11 [M + H]+, 602.3163; found,
603.32413; TOF-HRMS found, 603.32322.
Compound 16: A solution of 15 (200 mg, 0.182 mmol) in dry
pyridine (10 mL) containing DMAP (2 mg) was treated with
benzoyl chloride (0.21 mL, 1.812 mmol). The reaction mixture was
stirred at room temperature for 16 h, water (1 mL) was added, and
the solvent was removed under vacuum. The residue was dissolved
in ethyl acetate, and the organic layer was washed with 0.5 M HCl,
satd. NaHCO3, and water, dried over Na2SO4, and concentrated
under vacuum. The crude product was purified by silica gel flash
chromatography (1:1 EtOAc/hexane) to yield the O-benzoyl-
protected derivative 16 (240 mg, quant.): [R]D +21.6 (c 0.7,
1
CHCl3); Rf 0.5 (1:1 EtOAc/hexane); H NMR (300 MHz, CDCl3)
δ 8.15-7.10 (m, 35H), 6.30-3.00 (m, 33H), 2.23 (m, 1H), 1.37
(m, 1H), 0.89 (s, 9H), 0.03 (s, 6H); 13C NMR (75 MHz, CDCl3) δ
160.3, 158.6, 142.4, 139.4, 138.2, 137.5, 134.1, 131.6, 130.5, 129.4,
129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.5, 128.3, 128.2, 128.1,
128.0, 126.7, 99.4, 84.5, 77.6, 71.8, 68.7, 67.9, 67.6, 67.2, 29.5;
ESI-MS m/z calcd for C78H85N3O20Si [M + H]+, 1412.55439;
found, 1412.9; TOF-HRMS found. 1412.55173.
4′,6′-O-Benzylidene-2′′-O-cinnamyl-5′′-O-t-butyldimethylsilyl-
penta-N-benzyloxycarbonyl Paromomycin (19). Procedure A:
The alcohol 10 (6.00 g, 4.367 mmol) dried by two codistillations
with toluene was dissolved in CH2Cl2 (400 mL) and 2,4,6-collidine
(1.15 mL, 8.735 mmol) followed by TBDMSOTf (0.50 mL, 2.184
Compounds 17a-c: The O-benzoyl-protected derivative 16 (250
mg, 0.177 mmol) in CH2Cl2 (15 mL) was cooled at -78 °C and

2366 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
mmol) were added at 0 °C. After 18 h, 0.6 equiv of TBDMSOTf
was added, and 6 h later, some of the CH2Cl2 was evaporated to a
smaller volume for washing with HCl (0.5 M) twice and H2O.
Drying with Na2SO4 and purification by silica gel chromatography
(2% MeOH/CH2Cl2) gave the O5′′-TBS monoprotected compound
(4.861 g, 75%): [R]D +41.8 (c 0.9, CHCl3); Rf 0.6 (CHCl3/EtOAc/
vacuum, and the residue was taken up in EtOAc, washed with water,
and dried over Na2SO4. After removal of the solvent under vacuum,
the solid was purified by silica gel flash chromatography to obtain
21 (25 mg, 82%): [R]D +21.8 (c 0.9, CHCl3); Rf 0.7 (CHCl3/EtOAc/
1
MeOH (20:5:3); H NMR (300 MHz, CDCl3) δ 7.60-7.10 (m,
35H), 6.57-3.00 (m, 47H), 2.25 (m, 1H), 1.41 (m, 1H), 0.91 (s,
9H), 0.02 (s, 6H); ESI-MS m/z calcd for C87H103N5O24Si [M +
Na]+, 1652.66600; found, 1653.9; TOF-HRMS found, 1652.66981.
2′′-O-(5-Phenylpentyl)-paromomycin (22). The ether 21 was
dissolved in 80% aqueous acetic acid (3 mL), and the solution was
heated at 60 °C until a MS analysis showed total conversion of the
starting material into the benzylidene as well as TBS-deprotected
product (5 h). The solution was cooled down to room temperature,
a catalytic amount of 20% palladium hydroxide on carbon was
added, and the suspension was stirred at room temperature under
an atmosphere of hydrogen (hydrogen balloon) until the conversion
of the starting material into the product was completed, as indicated
by MS analysis. The mixture was filtered through a layer of Celite
on cotton, concentrated under vacuum, washed with CH2Cl2 twice,
and lyophilized to afford 22 as a fluffy white solid (11 mg, 70%):
[R]D +10.5 (c 0.6, H2O); 1H NMR (400 MHz, D2O) δ 7.36-7.26
(m, 5H), 5.69 (m, 1H), 5.43 (m, 1H), 5.12 (m, 1H), 4.39 (m, 1H),
4.18-4.12 (m, 5H), 3.93-3.04 (m, 18H), 2.63 (m, 2H), 2.22 (m,
1H), 1.90 (s, 15H), 1.71 (m, 1H) 1.64 (m, 4H) 1.35 (m, 2H); 13C
NMR (125 MHz, D2O) δ 182.6, 141.0, 129.7 (2C), 129.1 (2C),
125.9, 107.6, 97.7, 96.3, 84.9, 82.3, 81.2, 78.4, 74.5, 74.3, 72.1,
71.2, 70.4, 69.8, 68.5, 68.0, 62.7, 61.9, 60.1, 54.1, 50.8, 49.7, 49.2,
40.6, 37.3, 33.4, 32.5, 28.7, 26.4, 23.9; ESI-MS m/z calcd for
C34H59N5O14 [M + H]+, 762.41368; found, 762.4; TOF-HRMS
found, 762.41423.
1
MeOH (20:5:3)); H NMR (300 MHz, CDCl3) δ 7.60-7.10 (m,
30H), 5.60-3.00 (m, 41H), 2.20 (m, 1H), 1.30 (m, 1H), 0.83 (s,
9H), 0.01 (s, 6H); ESI-MS m/z calcd for C76H93N5O24Si [M + H]+,
1487.60580; found, 1488.9; TOF-HRMS found, 1488.60258. This
O5′′-TBS monoprotected compound (200 mg, 0.134 mmol) was
dissolved in dry THF (10 mL). A catalytic amount of Bu4NI and
cinnamyl bromide (66 mg, 0.269 mmol) were added followed by
the dropwise addition of 0.5 M KHMDS solution in toluene (0.350
mL, 0.175 mmol). The mixture was stirred overnight at room
temperature, then 0.3 equiv of KHMDS was added, and 6 h later,
the reaction mixture was quenched with an aqueous solution of
satd. NH4Cl (2 mL) and water. THF was evaporated and the
aqueous layer was extracted with EtOAc. The organic layer was
washed with a sodium thiosulfate solution and brine and dried over
Na2SO4. The solvent was evaporated to dryness to afford a crude
solid, which was purified by silica gel flash chromatography (1 to
2% MeOH in CH2Cl2), providing the corresponding cinnamyl ether
19 (152 mg, 70%): [R]D +16.4 (c 1.0, CHCl3); Rf 0.7 (CHCl3/
EtOAc/MeOH (20:5:3); 1H NMR (300 MHz, CDCl3) δ 7.65-7.12
(m, 35H), 6.54-3.05 (m, 45H), 2.24 (m, 1H), 1.36 (m, 1H), 0.84
(s, 9H), 0.01 (s, 6H); ESI-MS m/z calcd for C85H101N5O24Si [M +
H]+, 1604.66840; found, 1605.1; TOF-HRMS found, 1604.67026.
Procedure B: To 11 (200 mg, 0.131 mmol) in solution in CH2-
Cl2 (0.01M) was added the Grubbs second generation catalyst (11
mg, 0.013 mmol), and after 16 h, the solution was heated to reflux
for an additional 24 h. The solvent was removed under vacuum
and the resulting brown solid was purified by silica gel chroma-
tography (3% MeOH in CH2Cl2) to give the above described 19
(160 mg, 75%) as a white solid.
2′′-O-(3-Phenylpropyl)-paromomycin (20). Compound 19 (100
mg, 0.062 mmol) was dissolved in 80% aqueous acetic acid (3 mL),
and the solution was heated at 60 °C until a MS analysis showed
total conversion of the starting material into the benzylidene as
well as TBS-deprotected product (5 h). The solution was cooled
down to room temperature, a catalytic amount of 20% palladium
hydroxide on carbon was added, and the suspension was stirred at
room temperature under an atmosphere of hydrogen (hydrogen
balloon) until the conversion of the starting material into the product
was completed, as indicated by MS analysis. The mixture was
filtered through a layer of Celite on cotton, concentrated under
vacuum, washed with CH2Cl2 twice, and lyophilized to afford 20
as a fluffy white solid (32 mg, 70%): [R]D +10.5 (c 0.6, H2O); 1H
NMR (400 MHz, D2O) δ 7.28-7.19 (m, 5H), 5.67 (m, 1H), 5.32
(m, 1H), 5.13 (m, 1H), 4.47 (m, 1H), 414.18-4.11 (m, 5H), 3.86-
3.51 (m, 12H), 3.40-3.19 (m, 6H), 2.61-2.36 (m, 4H), 2.31 (m,
1H), 1.84 (s, 15H), 1.71 (m, 1H); 13C NMR (125 MHz, D2O) δ
181.9, 142.3, 129.1 (2C), 128.9 (2C), 126.5, 108.7, 96.2, 95.5, 85.4,
81.8, 80.9, 78.0, 74.3, 74.1, 73.1, 71.8, 70.7, 69.9, 69.6, 69.3, 68.0
67.9, 67.7, 60.4, 54.2, 51.3, 50.3, 49.2, 40.7, 31.7, 30.7, 23.8; ESI-
MS m/z calcd for C32H55N5O14 [M + H]+, 734.38238; found, 734.5;
TOF-HRMS found, 734.38213.
Compound 23. Procedure A: A solution of 12 (100 mg, 0.055
mmol) in THF (5 mL) was cooled down to -40 °C and PhMgBr
(1 M in THF, 81 µL, 0.081 mmol) was slowly added. After 1 h, a
few drops of satd. NH4Cl was added, the solvent was evaporated,
the resulting solid was taken up in AcOEt, washed with water, and
dried over Na2SO4, and the solvent was evaporated. Procedure B:
Alternatively, Ph2Zn (24 mg, 0.109 mmol) was added to a solution
of 12 (100 mg, 0.055 mmol) in THF (5 mL) at room temperature
and NH4Cl was added after 30 min. This later procedure was used
without affecting any benzoate ester protecting group.
To the combined alcohol dissolved in MeOH was added a freshly
prepared solution of MeONa/MeOH until the pH reached ap-
proximately 10-10.5. When the reaction was finished, as indicated
by a MS analysis, the solution was cooled down to -78 °C and
dry ice was added until neutral pH. Solvent was removed under
vacuum, and the residue was taken up in EtOAc, washed with water,
and dried over Na2SO4. After removal of the solvent under vacuum,
the resulting solid was purified by silica gel flash chromatography
(2% MeOH in CH2Cl2) to obtain 23 (108 mg, 65%): [R]D +16.2
1
(c 0.7, CHCl3); Rf 0.6 (CHCl3/EtOAc/MeOH (20:5:3)); H NMR
(300 MHz, CDCl3) δ 7.60-7.10 (m, 35H), 5.65-3.12 (m, 45H),
2.21 (m, 1H), 1.35 (m, 1H), 0.91 (s, 9H), 0.03 (s, 6H); ESI-MS
m/z calcd for C84H101N5O25Si [M + Na]+, 1630.64526; found,
1632.2; TOF-HRMS found, 1630.64331.
2′′-O-(2-Phenylethyl)-paromomycin (25) and Hydroxyl Ana-
logue (24). Compound 23 (150 mg, 0.093 mmol) was dissolved in
80% aqueous acetic acid (3 mL), and the solution was heated at
60 °C until a MS analysis showed total conversion of the starting
material into the benzylidene as well as TBS-deprotected product
(5 h). The solvent was removed and replaced by 3 mL of MeOH/
water (1:1), a catalytic amount of 20% palladium hydroxide on
carbon was added, and the suspension was stirred at room
temperature under an atmosphere of hydrogen (hydrogen balloon)
until the conversion of the starting material into the product was
completed, as indicated by MS analysis. After 6 h, half of the
mixture (1.5 mL) was filtered through a layer of Celite on cotton,
concentrated under vacuum, washed with CH2Cl2 twice, and
lyophilized to afford a fluffy white solid 24 (21 mg, 70%). To the
remaining half AcOH (1.5 mL) was added and the hydrogenation
was pursued for an additional 18 h to provide 25 (24 mg, 80%).
Compounds 24 and 25. Compound 24: [R]D +9.2 (c 0.5, H2O);
Compound 21: To cinnamyl triphenylphosphonium bromide
(31 mg, 0.081 mmol) in solution in THF (1 mL) was added
KHMDS (0.5 M in toluene, 0.17 mL, 0.085 mmol), and the resulting
orange solution was added to a solution of 12 (75 mg, 0.041 mmol)
in THF (3 mL) at -40 °C. After 1 h, the solution was warmed to
0 °C for 2 h. NH4Cl was added, the solvent was removed under
vacuum, and the resulting yellow solid was taken up in EtOAc,
washed with water, and dried over Na2SO4. The solvent was
removed under vacuum to give the olefin that was used directly in
the next step. To this olefin dissolved in MeOH was added a freshly
prepared solution of MeONa/MeOH until the pH reached ap-
proximately 10-10.5. When the reaction was finished as indicated
by a MS analysis, the solution was cooled down to -78 °C and
dry ice was added until neutral pH. Solvent was removed under

Aminoglycoside Antibiotics
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2367
1H NMR (400 MHz, D2O) δ 7.43 (m, 5H), 5.83 (m, 1H), 5.44 (m,
1H), 5.14 (m, 1H), 4.56 (m, 1H), 4.42 (m, 1H), 4.24-4.14 (m,
4H), 3.92-3.62 (m, 23H), 3.51-3.31 (m, 6H), 3.12 (m, 1H), 2.43
(m, 1H), 1.82 (m, 1H); 13C NMR (125 MHz, D2O) δ 141.0, 129.5
(2C), 129.1, 127.2, 109.0, 96.6, 96.0, 85.6, 82.5, 81.7, 78.0, 74.5,
73.9, 73.4, 72.9, 71.0, 69.6, 68.2, 68.0, 61.0, 59.8, 58.3, 54.6, 51.7,
51.5, 50.5, 49.6, 41.1, 29.3; ESI-MS m/z calcd for C31H53N5O15
[M + H]+, 736.36164; found, 736.7; TOF-HRMS found, 736.36153.
50.2, 41.2, 29.3, 24.6; ESI-MS m/z calcd for C25H49N5O15,
660.33034; found, 660.5; TOF-HRMS found, 660.32818.
Compound 28. Alcohol 26 (75 mg, 0.0431 mmol) was dissolved
in dry THF (5 mL). A catalytic amount of Bu4NI and cinnamyl
bromide (17 mg, 0.085 mmol) were added followed by the dropwise
addition of 0.5 M KHMDS solution in toluene (0.11 mL, 0.055
mmol). The mixture was stirred overnight at room temperature,
then 0.3 equiv of KHMDS was added, and 6 h later, the reaction
mixture was quenched with a few drops of an aqueous solution of
satd. NH4Cl and water. THF was evaporated, the aqueous layer
was extracted with ethyl acetate (2 times), and the organic layer
was washed with a sodium thiosulfate solution and brine and dried
over Na2SO4. The solvent was evaporated to dryness to afford a
crude solid that was purified by silica gel flash chromatography
(2:3 EtOAc/hexane), providing the corresponding cinnamyl ether
(30 mg, 35%): [R]D +21.5 (c 1.0, CHCl3); Rf 0.5 (AcOEt/hexane
1:1); ESI-MS m/z calcd for C108H117N5O28Si [M + H]+, 1960.8;
found, 1960.9. To this cinnamyl ether was added a freshly prepared
solution of MeONa/MeOH until the pH reached approximately 10-
10.5, and the solution was stirred until a MS analysis indicated
that the reaction was finished. This benzoate deprotection needs to
be carefully monitored by MS analysis to control the formation of
a methyl carbamate at the N6′′′ position. If this undesired product
appears in the MS analysis, it is necessary to add more MeOH to
dilute the reaction mixture and, therefore, lower the pH at a level
where the benzoate ester is cleaved but with no appreciable amount
of methyl carbamate formation at N6′′′. When the reaction was
finished, the solution was cooled down to -78 °C and dry ice was
added until neutral pH. Solvent was removed under vacuum, and
the residue was taken up in EtOAc, washed with water, and dried
over Na2SO4. After removal of the solvent under vacuum, the solid
was purified by silica gel flash chromatography (3% MeOH in CH2-
Cl2) to obtain 28 (19 mg, 80%): [R]D +11.2 (c 0.8, CHCl3); Rf 0.4
1
Compound 25: [R]D +10.3 (c 1.0, H2O); H NMR (400 MHz,
D2O) δ 7.41 (m, 5H), 5.80 (m, 1H), 5.32 (m, 1H), 5.08 (m, 1H),
4.68 (m, 1H), 4.42-4.12 (m, 5H), 4.03 (m, 1H), 3.90-3.57 (m,
11H), 3.47-3.30 (m, 6H), 2.94 (m, 2H), 2.40 (m, 1H), 1.90 (s,
15H), 1.82 (m, 1H); 13C NMR (125 MHz, D2O) δ 182.0, 139.8,
129.9 (2C), 129.5 (2C), 127.3, 109.0, 96.6, 95.8, 85.9, 82.2, 81.2,
78.5, 74.8, 73.5, 72.9, 72.1, 71.0, 69.7, 68.2, 68.0, 61.0, 59.6, 55.3,
54.6, 51.5, 50.6, 49.6, 41.1, 36.0, 29.8, 23.8; ESI-MS m/z calcd
for C31H53N5O14 [M + H]+, 720.36673; found, 720.7; TOF-HRMS
found, 720.36462.
4′,6′-Benzylidene-3′,3′′′,4′′′-tri-O-benzoyl-5′′-O-t-butyldimeth-
ylsilyl-2′′-O-(2-hydroethyl)-penta-N-benzyloxycarbonyl Paro-
momycin (26). To a mixture of the aldehyde 12 (240.0 mg, 0.130
mmol) in dry MeOH (10 mL) was added acetic acid (0.1 mL)
followed by NaBH3CN (1.0 M in THF, 180 µL, 0.180 mmol). A
TLC indicated a complete disparition of the aldehyde after 18 h.
After removal of the solvent under vacuum, the solid was purified
by silica gel flash chromatography (2:3 EtOAc/hexane) to obtain
26 (190 mg, 80%): [R]D +23.7 (c 1.0, CHCl3); Rf 0.3 (1:1 EtOAc/
hexane); ESI-MS m/z calcd for C99H109N5O28Si [M + Na]+,
1866.69260; found, 1868.0; TOF-HRMS m/z calcd for C99H109N5O28-
Si [M + H]+, 1844.71066; found, 1844.71460.
2′′-O-(2-Hydroxyethyl)-paromomycin (27). To the perester 26
(75 mg, 0.043 mmol) was added a freshly prepared solution of
MeONa/MeOH dropwise until the pH reached approximately 10-
10.5, and the solution was stirred until a MS analysis indicated
that the reaction was finished. This benzoate deprotection needs to
be carefully monitored by MS analysis to control the formation of
a methyl carbamate at the N6′′′ position. If this undesired product
appears in the MS analysis, it is necessary to add more MeOH to
dilute the reaction mixture and, therefore, lower the pH at a level
where the benzoate esters are cleaved but with no appreciable
amount of methyl carbamate formation at N6′′′. When the reaction
was finished, the solution was cooled down to -78 °C and dry ice
was added until a neutral pH. Solvent was removed under vacuum,
and the residue was taken up in EtOAc, washed with water, and
dried over Na2SO4. After removal of the solvent under vacuum,
the solid was purified by silica gel flash chromatography (3%
MeOH in CH2Cl2) to obtain the desired deprotected polyol (58 mg,
80%): [R]D +13.2 (c 0.7, CHCl3); Rf 0.4 (CHCl3/EtOAc/MeOH
1
(CHCl3/EtOAc/MeOH (20:5:3)); H NMR (300 MHz, CDCl3) δ
7.60-7.10 (m, 35H), 6.52-3.05 (m, 49H), 2.23 (m, 1H), 1.33 (m,
1H), 0.86 (s, 9H), 0.01 (s, 6H); ESI-MS m/z calcd for C87H105N5O25-
Si [M + Na]+, 1670.67656; found, 1670.7; TOF-HRMS found,
1670.67734.
2′′-O-(3-Phenylpropoxyethyl)-paromomycin (29). The ether 28
was dissolved in 80% aqueous acetic acid (3 mL), and the solution
was heated at 60 °C until a MS analysis showed total conversion
of the starting material into the benzylidene as well as TBS-
deprotected product (2 h): ESI-MS m/z calcd for C74H87N5O25 [M
+ H]+, 1446.6; found, 1446.4. The solution was cooled down to
room temperature, a catalytic amount of 20% palladium hydroxide
on carbon was added, and the suspension was stirred at room
temperature under an atmosphere of hydrogen (hydrogen balloon)
until the conversion of the starting material into the product was
completed, as indicated by MS analysis. The mixture was filtered
through a layer of Celite on cotton, concentrated under vacuum,
washed with CH2Cl2 twice, and lyophilized to afford the over-
reduced solids 29 (5 mg, 60%): [R]D +6.8 (c 0.4, H2O); 1H NMR
(400 MHz, D2O) δ 7.38, (m, 5H), 5.68 (m, 1H), 5.26 (m, 1H),
5.17 (m, 1H), 4.42 (m, 1H), 414.28-4.07 (m, 5H), 3.88-3.59 (m,
16H), 3.47-3.19 (m, 10H), 2.34 (m, 1H), 1.82 (s, 15H), 1.47 (m,
1H); 13C NMR (125 MHz, D2O) δ 182.6, 140.7, 129.3 (2C), 129.1
(2C), 127.3, 109.0, 96.7, 95.3, 85.7, 81.5, 80.6, 77.7, 74.1, 73.8,
73.0, 71.4, 70.2, 69.7, 69.3, 69.1, 67.8 67.4, 67.1, 63.4, 61.5, 60.2,
53.9, 51.3, 49.8, 49.1, 40.5, 31.6, 29.2, 23.4; ESI-MS m/z calcd
for C34H59N5O15 [M + H]+, 778.40858; found, 778.6; TOF-HRMS
found, 778.40746.
1
(20:5:3)); H NMR (300 MHz, CDCl3) δ 7.60-7.10 (m, 30H),
5.65-3.00 (m, 45H), 2.26 (m, 1H), 1.41 (m, 1H), 0.85 (s, 9H),
0.01 (s, 6H); ESI-MS m/z calcd for C78H97N5O25Si [M + Na]+,
1554.6; found, 1656.0. The above polyol was dissolved in 80%
aqueous acetic acid (3 mL), and the solution was heated at 60 °C
until a MS analysis showed total conversion of the starting material
into the benzylidene as well as TBS-deprotected product (2 h): ESI-
MS m/z calcd for C65H79N5O25 [M + Na]+, 1352.5; found, 1352.7.
The solution was cooled down to room temperature, and a catalytic
amount of 20% palladium hydroxide on carbon was added and the
suspension was stirred at room temperature under an atmosphere
of hydrogen (hydrogen balloon) until the conversion of the starting
material into the product was completed, as indicated by MS
analysis. The mixture was filtered through a layer of Celite on
cotton, concentrated under vacuum, washed with CH2Cl2 twice, and
lyophilized to afford 27 as a fluffy white solid in 75% yield: [R]D
Supporting Information Available: Crystal data, statistics of
data collection, and structure refinement (Table S1); secondary
structures of the bacterial 16S ribosomal A-site (Figure S1); LCMS
retention times and HRMS analysis of paromomycin analogues
(Table S2); and LCMS retention times and HRMS analysis of
paromomycin analogues without ring IV (Table S3). This material
is available free of charge via the Internet at http://pubs.acs.org.
1
+11.4 (c 0.8, H2O); H NMR (400 MHz, D2O) δ 5.76 (m, 1H),
5.47 (m, 1H), 5.26 (m, 1H), 4.58 (m, 1H), 4.40 (m, 1H), 4.29-
4.20 (m, 4H), 3.90-3.26 (m, 16H), 3.11-3.05 (m, 4H), 2.38 (m,
1H), 1.88 (s, 15H), 1.72 (m, 1H); 13C NMR (125 MHz, D2O) δ
182.2, 108.9, 96.8, 96.3, 85.8, 82.5, 81.8, 79.7, 75.3, 74.3, 73.8,
72.7, 71.2, 70.0, 68.7, 68.5, 68.0, 61.4, 61.3, 58.9, 54.8, 51.8, 50.8,

2368 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Hanessian et al.
4, 1078-1088. (e) Franc¸ois, B.; Russell, R. J.; Murray, J. B.; Aboul-
ela, F.; Masquida, B.; Vicens, Q.; Westhof, E. Crystal structures of
complexes between aminoglycosides and decoding A site oligo-
nucleotides: Role of the number of rings and positive charges in the
specific binding leading to miscoding. Nucleic Acids Res. 2005, 33,
5677-5690. (f) Kondo, J.; Franc¸ois, B.; Russell, R. J.; Murray, J.
B.; Westhof, E. Crystal structure of the bacterial ribosomal decoding
site complexed with amikacin containing the γ-amino-R-hydroxy-
butyryl (haba) group. Biochimie 2006, 88, 1027-1031.
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