Surprising alteration of antibacterial activity of 5″-modified neomycin against resistant bacteria

Article abstract of DOI:10.1021/jm800997s

A facile synthetic protocol for the production of neomycin B derivatives with various modifications at the 5″ position has been developed. The structural activity relationship (SAR) against aminoglycoside resistant bacteria equipped with various aminoglycoside-modifying enzymes (AMEs) was investigated. Enzymatic and molecular modeling studies reveal that the superb substrate promiscuity of AMEs allows the resistant bacteria to cope with diverse structural modifications despite the observation that several derivatives show enhanced antibacterial activity compared to the parent neomycin. Surprisingly, when testing synthetic neomycin derivatives against other human pathogens, two leads exhibit prominent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) that are known to exert a high level of resistance against clinically used aminoglycosides. These findings can be extremely useful in developing new aminoglycoside antibiotics against resistant bacteria. Our result also suggests that new biological and antimicrobial activities can be obtained by chemical modifications of old drugs.

Full text of DOI:10.1021/jm800997s

J. Med. Chem. 2008, 51, 7563–7573
7563
Surprising Alteration of Antibacterial Activity of 5′′-Modified Neomycin against Resistant
Bacteria
Jianjun Zhang,† Fang-I. Chiang,† Long Wu,‡ Przemyslaw Greg Czyryca,§ Ding Li,‡ and Cheng-Wei Tom Chang*,†
Department of Chemistry and Biochemistry, Utah State UniVersity, 0300 Old Main Hill, Logan, Utah 84322-0300, Department of Biology and
Chemistry, City UniVersity of Hong Kong, 83 Tat Chee AVenue, Kowloon, Hong Kong, P. R. China, and Allosterix Pharmaceuticals, LLC,
126 South, 100 East, ProVidence, Utah 84332
ReceiVed August 6, 2008
A facile synthetic protocol for the production of neomycin B derivatives with various modifications at the
5′′ position has been developed. The structural activity relationship (SAR) against aminoglycoside resistant
bacteria equipped with various aminoglycoside-modifying enzymes (AMEs) was investigated. Enzymatic
and molecular modeling studies reveal that the superb substrate promiscuity of AMEs allows the resistant
bacteria to cope with diverse structural modifications despite the observation that several derivatives show
enhanced antibacterial activity compared to the parent neomycin. Surprisingly, when testing synthetic
neomycin derivatives against other human pathogens, two leads exhibit prominent activity against both
methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) that are
known to exert a high level of resistance against clinically used aminoglycosides. These findings can be
extremely useful in developing new aminoglycoside antibiotics against resistant bacteria. Our result also
suggests that new biological and antimicrobial activities can be obtained by chemical modifications of old
drugs.
Introduction
can be carried out in large quantity, appear to be a more practical
approach for generating clinically useful novel aminoglycosides.
Thus, we began to ponder the possibility of developing diversity-
oriented synthesis of aminoglycoside derivatives via the use of
carbamate type protecting groups.
Chemical modification of aminoglycosides has been an
effective way of reviving the antibacterial activity of aminogly-
cosides against resistant bacteria, especially against those that
are equipped with aminoglycoside-modifying enzymes (AM-
Esa).1-3 For example, attachment of (S)-4-amino-2-hydroxybu-
tanoyl (AHB) and (S)-3-amino-2-hydroxypropanoyl groups at
the N-1 position has led to the production of clinically useful
aminoglycosides, amikacin and isepamicin, respectively (Figure
1). In general, protection of amino groups as carbamates or
transformation of amino groups into azido groups on aminogly-
cosides are the most commonly employed strategies for
introducing desired structural motifs regiospecifically.
Our group has been working on modifications of aminogly-
coside antibiotics via the amine/azide transformation approach.4,5
The polyazido intermediates employed in the synthesis can be
conveniently manipulated in organic media using traditional
synthetic methods, which is ideal for constructing a library of
aminoglycosides for identifying leads. However, the safety
concern of handling azido compounds, the higher cost involved
in the synthesis of azidoaminoglycosides, and the reduction of
azido groups impose challenges in scale-up production of the
leads for subsequent animal studies and clinical trials. By
comparison, direct modification of commercially available
aminoglycosides using carbamate type protecting groups, which
Design and Synthesis of 5′′-Modified Neomycin B. Re-
cently, several examples of 5′′-modified neomycin class of
aminoglycosides have been reported including conformational
constrained aminoglycosides with intramolecular linkage be-
tween N-2′ and C-5′′6,7 and between N-3 and N-6′,8 the dimer
of neomycin with a spacing linkage via C-5′′,9 neomycin with
a 2,3-diaminopropanoyl group at the 5′′ position,10 and orthogo-
nal divergent synthesis of neomycin derivatives with modifica-
tions at various amine functionalities.11 Although the constructed
conformationally constrained aminoglycoside manifested a much
lower turn-over rate toward the AME of interest, the antibacterial
activity also decreased significantly.6 The design of orthogonal
divergent synthesis appears to have the potential of being
employed for diversity-oriented synthesis. Nevertheless, the
reported synthesis did not include the deprotection of carbamate
protecting groups and the production of neomycin derivatives
that can be assayed for their antibacterial activity.
In addition, it has also been suggested that aminoglycosides
bearing deoxygenation at the 3′ position, such as in the case of
tobramycin, display relatively higher cytotoxicity than those that
still contain 3′-OH.12 Neomycin B, which contains 3′-OH, exerts
broad spectrum antibacterial activity and can be available in a
large quantity at a relatively low cost. Thus, we reason that
derivatizing neomycin with 5′′-modifications could be valuable
in investigating the structure-activity relationship (SAR) against
resistant bacteria, the efficacy of avoiding enzyme-catalyzed
modifications from aminoglycoside phosphotransferases (APH)
that target 3′-OH or, perhaps, aminoglycoside nucleotidyltrans-
ferases(ANT)thattarget4′-OH,andeventhestructure-cytotoxicity
relationship. Neomycin derivatives bearing carbohydrate moi-
eties at the 5′′-OH have also been shown to have good activity
against a panel of bacteria.13,14
* To whom correspondence should be addressed. E-mail: tom.chang@
usu.edu. Phone: (435) 797-3545. Fax: 435-797-3390.
†
Utah State University.
City University of Hong Kong.
Allosterix Pharmaceuticals, LLC.
‡
§
a Abbreviations: SAR, structural activity relationship; AME, aminogly-
coside-modifying enzyme; MRSA, methicillin-resistant Staphylococcus
aureus; VRE, vancomycin-resistant enterococci; AHB, (S)-4-amino-2-
hydroxybutanoyl; APH, aminoglycoside phosphotransferases; ANT, ami-
noglycoside nucleotidyltransferases; Cbz or Z, carbobenzyloxy; AAC,
aminoglycoside acetyltransferase; MIC, minimum inhibitory concentration;
rRNA, ribosomal RNA; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride; HOBt, 1-hydroxybenzotriazole.
10.1021/jm800997s CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

7564 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Zhang et al.
Figure 1. Structures of neomycin and kanamycin class aminoglycosides.
We prefer to employ the carbobenzyloxy (Cbz or Z) group
as the protecting group of amines on neomycin since it can
be tracelessly removed using hydrogenolysis. The synthetic
strategy should be simple, which is suitable in producing a
library of such derivatives for expedient screening and
subsequent gram-scale synthesis of the leads. From our
molecular modeling studies and SAR of others,13,14 we have
also noticed that diverse functional groups can be attached
at C-5′′ without significantly obstructing the antibacterial
activity. Finally, 5′′-OH is the only primary hydroxyl group
on neomycin B which can be regioselectively converted to
azido group and serve as the site of diversity-oriented
modifications via “Click” chemistry or amide linkage. Our
initial design was to randomly introduce structural moieties
containing alkyl, aryl, cationic, and anionic substitutents for
probing the SAR. The diversity-oriented synthesis started
from a known Cbz-protected neomycin B derivative, 1,8
followed by regioselective substitution of 5′′-OH with azide
(Scheme 1). The 5′′-azido group can undergo 1,3-dipolar
cyclization with alkynes (class I design).15 Alternatively, the
5′′-azido group of 1 can be reduced to an amino group and
coupled with various carboxylic acids or amino acids (class
II design). Global deprotection of the Cbz groups of these
derivatives from both routes provided 5′′-modified neomycin
derivatives ready for antibacterial assay. The yields and
structural designs are summarized in Tables 1 and 2. With
the goal to expand the structural diversity, a dimeric
neomycin derivative, 33, can be prepared by reacting the 5′′-
NH2 group with succinic anhydride providing 32 and then
coupled to another molecule of 3 (Scheme 2).
Scheme 1. Synthesis of Neomycin Derivatives
Antibacterial Activity of 5′′-Modified Neomycin B. The
synthesized aminoglycosides were assayed against susceptible
and resistant bacterial strains using neomycin B as the control.
Aminoglycoside susceptible Escherichia coli (ATCC 25922) and
Staphylococcus aureus (ATCC 25923) were used as standard
reference strains. E. coli (pTZ19U-3) and E. coli (pSF815) are
laboratory resistant strains using E. coli (TG1) as the host. The
first one is equipped with the pTZ19U-3 plasmid encoded for
APH(3′)-I, which catalyzes phosphorylation at the 3′-OH of both
neomycin and kanamycin classes of aminoglycosides. The
second one is equipped with the pSF815 plasmid encoded for
a bifunctional enzyme, AAC(6′)/APH(2′′), which catalyzes
acetylation of the amino group at 6′-NH2 and phosphorylation
of the hydroxyl group at 2′′-OH. These enzymes are among
the most prevalent modes of resistance found in aminoglycoside
resistant strains. The minimum inhibitory concentrations (MICs)
are summarized in Table 3.
From the MIC results, it appears that the 5′′ position can
tolerate the incorporation of diverse structural components,
which is consistent with previous literature results.13,14 Deriva-
tives with a 1,2,3-triazole linkage manifested modest antibacte-
rial activities comparable to neomycin B with 4e and 4g as the
most active derivatives. Most of the derivatives bearing amide-
based linkages were less active than the parent neomycin B.
Nevertheless, derivatives incorporated with long linear alkyl
chains (5b and 5c) showed better antibacterial activity than
neomycin B, and the one with a shorter linear alkyl chain (5a),
even against resistant strains. Among the derivatives attached
with amino acids, glycine (5f), alanine (5g and 5m), and proline
(5h and 5o) manifested similar levels of antibacterial activity
with no particular difference between D and L amino acids. On
average, it appears that these 5′′-modified neomycin derivatives

Antibacterial ActiVity of 5′′-Modified Neomycin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7565
Table 1. Yields and Structural Designs of Class I 5′′-Modified Neomycin Derivatives
compounds
alkynes
yield (%)
source of alkynes
ref 35
commercially available
commercially available
prepared in this work
commercially available
commercially available
ref 36
4a
4b
4c
4d
4e
4f
4g
4h
4i
N-propargyl-5-acetamidomethyl-2-oxazolidinone (6)
1-octyne (7)
N,N-dimethyl propargylamine (8)
N-propargyl-2-phenyl-4-quinolinecarboxamide (9)
N-dimethyl propargylamine (10)
86
69
66
69
61
68
77
40
49
2-ethynylpyridine (11)
N-carbobenzyloxypropargylamine (12)
N-propargylisonicotinamide (13)
N-carbobenzyloxy-L-proline N′- propargylamine (14)
prepared in this work
prepared in this work
Table 2. Yields and Structural Designs of Class II 5′′-Modified Neomycin Derivatives
source of carboxylic
acids or amino acids
carboxylic acids or amino acids
compounds
yield (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
heptanoic acid (15)
palmitic acid (16)-
stearic acid (17)-
2-phenyl-4-quinolinecarboxylic acid (18)
(S)-Z-4-amino-2-benzyloxybutyric acid (19)
Z-Gly (20)
Z-L-Ala (21)
Z- L-Pro (22)
Z- L-Trp (23)
Z- L-Ser (24)
Z- L-Lys (25)
succinic anhydride (26)
Z-D-Ala (27)
Z- D-Ser (28)
Z- D-Pro (29)
Z- D-Lys (30)
Z-Gly-Gly (31)
88
63
54
30
90
27
36
50
67
52
61
85
69
57
53
25
42
commercially available
commercially available
commercially available
commercially available
ref 37
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
commercially available
Scheme 2. Synthesis of Neomycin Dimer
against resistant bacteria is of particular interest, which prompts
further enzymatic and molecular modeling investigations.
Enzymatic and Antibacterial Studies of 5′′-Modified
Neomycin B against APH(3′)-IIIa. Although no significant
activity against bacteria equipped with APH(3′)-I was observed
for most of the derivatives, several derivatives did appear to
have improved or similar activities as compared to the parent
neomycin B. Thus, effort was devoted to the investigation of
the relationship between enzyme kinetic studies and the whole-
cell based assay. Our initial strategy is to introduce structural
variants at the 5′′-position, which is close to the 3′-OH targeted
by many APH(3′). There are as many as seven isoforms of
APH(3′) (I-VII) which have been identified with various
substrate specificity among gram-negative and gram-positive
bacteria.16-19 We selected APH(3′)-IIIa for enzymatic study due
to consideration of its availability and clinical significance. For
example, studies of the epidemiology of AMEs have revealed
the prevalence of the APH(3′)-IIIa in Methicillin-resistant
Staphylococcus aureus (MRSA), Enterococcus faecalis, and
Enterococcus faecium.20-23
are more active against G+ bacteria (S. aureus) than G-
bacteria (E. coli). For example, there is only a 4-fold MIC
difference for neomycin B between G+ and G- bacteria while,
in most of the synthetic derivatives, the differences are in the
range of 4 to 16-fold.
As expected, compound 5l incorporated with a negatively
charged carboxylate group showed much lower antibacterial
activity as compared to other derivatives. With the exception
of 5b and 5c, all the derivatives are inactive against the resistant
strain equipped with APH(3′)-I while retaining moderate activi-
ties against the susceptible control strain. This finding implies
that APH(3′)-I has much broader substrate promiscuity than the
RNA binding site for 5′′-modified neomycin derivatives, which
again, exemplifies the challenges in developing aminoglycosides
against drug-resistant bacteria. The activities of 5b and 5c
The enzyme APH(3′)-IIIa was expressed and purified ac-
cording to the literature procedure.24 Enzyme kinetic studies
for 5′′-modified neomycin derivatives, kanamycin A, amikacin,
and neomycin B were performed as reported.25 The laboratory
resistant strain was generated using E. coli (TG1) as the host
harboring pET28a plasmid encoded for APH(3′)-IIIa. Neomycin
B, kanamycin B (Kan B), and amikacin were used as controls
in the antibacterial assay. The results from enzymatic studies
and the MIC are summarized in Table 4.
By comparison of the kinetic parameters from the com-
mercially available aminoglycosides (kanamycin A, amikacin,
and neomycin B), the presence of the AHB group increases
KM but lowers Vmax and kcat indicating that amikacin has a lower
binding affinity and turnover rate toward APH(3′)-IIIa. Interest-
ingly, most of the 5′′-modified neomycin derivatives manifest

7566 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Table 3. MIC of the 5′′-Modified Neomycin Derivativesa
Zhang et al.
compounds
E. colib
S. aureusc
E. coli (TG1)d
E. coli (pSF815)e
E. coli (pTZ19U-3)f
neomycin B
4
32
32
8
32
8
8-16
8-16
16-32
16
1
4-8
4
2
8
1-2
2
1-2
4
2
4
2
4
4-8
1-2
1
0.5
0.5-1
2
1
2
8
1
4-8
16
8-16
8-16
8-16
16
16
16
16
16
4-8
32
16
16
32
16
16
16
32
16
16-32
16
32
16
16-32
8-16
16
16-32
16
16
32
64
16-32
16
16
64
16
16-32
g2000
g2000
500
g2000
500-1000
g2000
g2000
1000
g2000
g2000
g2000
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
33
16-32
4
8
2-4
2-4
8-16
16
8-16
8-16
16
8-16
8-16
16
32
16
8-16
8
16
16-32
8-16
4-8
8-16
8-16
16
8-16
16-32
16
8
g2000
g2000
500-1000
g2000
g2000
g2000
g2000
125-250
g2000
g2000
g2000
g2000
g2000
125-250
500
8
16-32
64
16-32
8-16
8-16
32
1
0.5-1
1-2
2-4
2-4
16-32
32
a Unit: µg/mL. b Escherichia coli (ATCC 25922), c Staphylococcus aureus (ATCC 25923). d E. coli (TG1) (aminoglycoside susceptible strain). e E. coli
(TG1) (pSF815 plasmid encoded for (AAC(6′)/APH(2′′)). f E. coli (TG1) (pTZ19U-3 plasmid encoded for APH(3′)-I).
Table 4. Kinetic Parameters and MIC of Aminoglycosides for APH(3′)-IIIa
MIC against bacterium
KM (µM)
Vmax (µmol/mg/min)
kcat (S-1
)
kcat/KM/104 (S-1M-1
)
MIC ratiob
compound
with APH(3′)-IIIaa
kanamycin A
amikacin
neomycin B
8.2 ( 0.7
57.4 ( 5.7
6.0 ( 0.8
160 ( 15
127 ( 13
120 ( 14
410 ( 38
100 ( 9
150 ( 16
111 ( 12
171 ( 18
175 ( 18
48 ( 6
6.96 ( 0.12
2.75 ( 0.13
3.22 ( 0.17
0.41 ( 0.03
0.75 ( 0.04
0.65 ( 0.05
0.30 ( 0.03
0.72 ( 0.07
0.54 ( 0.05
0.68 ( 0.04
0.41 ( 0.04
0.40 ( 0.04
0.95 ( 0.06
0.85 ( 0.08
0.84 ( 0.05
0.38 ( 0.04
0.93 ( 0.07
0.88 ( 0.06
0.90 ( 0.09
0.63 ( 0.07
0.57 ( 0.06
1.01 ( 0.09
0.79 ( 0.08
0.86 ( 0.09
0.82 ( 0.09
0.93 ( 0.10
0.72 ( 0.07
0.70 ( 0.07
<0.1
3.48 ( 0.06
1.38 ( 0.07
1.61 ( 0.09
0.21 ( 0.02
0.38 ( 0.02
0.33 ( 0.03
0.15 ( 0.02
0.36 ( 0.04
0.27 ( 0.03
0.34 ( 0.02
0.21 ( 0.02
0.20 ( 0.02
0.48 ( 0.05
0.42 ( 0.05
0.42 ( 0.03
0.19 ( 0.02
0.47 ( 0.05
0.44 ( 0.03
0.45 ( 0.05
0.32 ( 0.03
0.29 ( 0.03
0.51 ( 0.05
0.40 ( 0.03
0.43 ( 0.04
0.41 ( 0.04
0.47 ( 0.05
0.36 ( 0.04
0.35 ( 0.04
<0.05
42.4
2.40
26.8
0.13
0.30
0.28
0.037
0.36
0.18
0.3
0.12
0.14
1.0
0.72
0.7
125 (KanB)
1
64
32 (Kan B)
1
8
8
4
8
2
8
8
4
8
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
33
125
32-64
64-125
16-32
125
125
64
64-125
64-125
64
1-2
2
32-64
125
32
64
64
64
32-64
32-64
250
8
58 ( 4
0.5
1-2
4
8
2
4
4
4
4
4
8
8
4
8
8
8
60 ( 7
400 ( 35
50 ( 4
0.048
0.94
0.6
0.75
0.4
75 ( 8
60 ( 5
80 ( 9
322 ( 31
42 ( 4
0.1
1.2
78 ( 8
0.51
0.81
0.65
0.82
0.51
0.47
<0.01
53 ( 6
63 ( 7
64-125
64
64
64-125
64-125
57 ( 6
70 ( 8
75 ( 8
>500
a Unit: µg/mL. b MIC ratio ) (MIC against E. coli (TG1))/(MIC against E. coli (TG1) with APH(3′)-IIIa).
much higher KM and lower Vmax and kcat implying that these
derivatives can better evade the enzyme-catalyzed modification
as well. The high KM indicates that these 5′′-modified neomycin
derivatives are poor substrates for APH(3′)-IIIa. The low kcat
suggests that the added functionalities at the 5′′ position could
disrupt the rate of phosphorylation at the 3′-OH even in the
case of 5f where a relatively smaller glycine is incorporated.
From the kinetic data, no obvious correlation between the kcat/
KM and MIC can be deduced. For example, 4a has a relatively
low kcat/KM but still exerts no significant activity in the whole-
cell based antibacterial assay against the resistant strain. Several
possible reasons can account for these observations: (1) binding
of the 5′′-modified neomycin derivatives to the RNA targeted
site could have been disrupted by the structural modifications

Antibacterial ActiVity of 5′′-Modified Neomycin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7567
Figure 2. (a) APH(3′)-IIIa with 5f, (b) APH(3′)-IIIa with 4d, (c) RNA with 5f, and (d) RNA with 4d.
leading to lower antibacterial activity; (2) APH(3′)-IIIa can still
inactivate the 5′′-modified neomycin derivatives. Although
inactivation occurs at a much slower rate, the higher concentra-
tion of APH(3′)-IIIa as compared to the targeted rRNA site
renders the lower kcat/KM values insignificant for most of the
neomycin derivatives in regaining their antibacterial activity;
and (3) the uptake of the 5′′-modified neomycin derivatives by
bacteria could have also been hampered by the structural
modifications.
of these modified aminoglycosides. Therefore, 5b and 5c may
exert their antibacterial activity not by binding to the rRNA
which occurs within bacteria but by a different mode of
antibacterial action.
Compounds 5f and 4d exhibit better activity against resistant
strains than does neomycin without suffering significant activity
loss against susceptible strains. Since protected glycine can be
readily prepared in large-scale and, in particular, 5f has excellent
activity against S. aureus, we decided to treat compound 5f as
a lead for further chemical modifications to be discussed later.
Finally, despite having a very low kcat/KM value, the dimer, 33,
displayed only modest activities against susceptible strains and
low activity against resistant strains. Once again, this result
implies the broader substrate promiscuity of APH(3′)-IIIa than
the binding pocket of the targeted rRNA site. Overall, our results
from enzymatic studies suggest that a low kcat/KM value may
not be applicable to predict the antibacterial activity of ami-
noglycoside constructs against resistant bacteria equipped with
AMEs.
Molecular Modeling of 5′′-Modified Neomycin B. The
bactericidal activity of neomycin and kanamycin arises from
the binding of aminoglycosides to the A-site decoding region
of 16S rRNA. To examine whether the targeted site of rRNA
can accommodate the added functional groups at the 5′′ position,
molecular modeling27 was carried out using the reported X-ray
structures of APH(3′)-IIIa and neomycin-bound rRNA as the
templates.28,29 Two particularly active compounds, 5f and 4d,
representative of classes I and II, were selected for the molecular
modeling analysis (Figure 2). While the part of the APH (3′)-
IIIa enzyme responsible for the catalysis and the tight binding
of the neamine portion of the molecule (rings I and II) is shaped
in the form of a deep cleft, other fragments of the aminogly-
coside molecule are bound to the rather sterically unhindered
surface of the enzyme. This allows for the enzyme’s unique
substrate promiscuity, which is facilitated also by the substrate’s
conformational flexibility. Because of the steric consideration,
To better compare the changes of antibacterial efficiency, we
calculated the MIC ratio by dividing the MIC against susceptible
strain with the MIC against resistant one (right column, Table
4). Amikacin, which has excellent activity against the resistant
strain, has an MIC ratio of 1, which means there is no loss of
activity against the resistant strain. Several interesting findings
were noted. While neomycin and most of the neomycin
derivatives have an MIC ratio ranging from 4 to 8, two
compounds, 5b and 5c, have ratios of 0.5 and 1, respectively.
The kinetic studies also suggested that these derivatives should
be “poor substrates” for the enzyme. Thus, these results point
to an interesting question: why are these two compounds still
active against resistant strains while other derivatives with
similar or lower kcat/KM values are inactive or less active?
Two possible reasons could account for the observed activity
of 5b and 5c having similar antibacterial efficiency as compared
to amikacin and superior efficiency as compared to neomycin.
First: the structural modifications at the N-5′′ of 5b and 5c may
have similar effects as the AHB group at the N-1 of amikacin.
From the X-ray crystallography study,26 it has been demon-
strated that the AHB side chain can be accommodated by the
binding site of rRNA while rendering amikacin a “poor
substrate” for APH(3′)-IIIa. Second: these two derivatives may
have different modes of antibacterial action. We favor the second
postulate. Since the enzyme-catalyzed modifications occur
within bacteria, neomycin derivatives with similar kcat/KM values
should still undergo phosphorylation leading to the inactivation

7568 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Zhang et al.
threat to the public health.32 Enterococcus faecalis (ATCC51299),
which is one of the vancomycin-resistant enterococci (VRE),
contains VanB, ant(6)-I, and aac(6′)-aph(2′′) resistance genes
and exerts high level of resistance to aminoglycosides and
vancomycin.33 E. faecalis (ATCC 29212), which is susceptible
to vancomycin but manifests moderate resistance to aminogly-
cosides, is used as comparison.
The results from the antibacterial assay are summarized in
Table 5. As expected, the derivatives with relatively smaller
structural modifications (5f and 5q) display similar antibacterial
profiles as neomycin suggesting unaltered modes of bactericidal
and resistant mechanisms. 5f and 5q are more active against P.
aeruginosa as compared to neomycin but are less active as
compared to amikacin and gentamicin. Overall, 5f is slightly
more active than neomycin and implies that the structural
modification with the glycidyl group is beneficial. Nonetheless,
such a modification is insufficient in reviving the antibacterial
activity against aminoglycoside resistant bacteria.
Intriguing results were obtained from the screening of 5b and
5c that have drastic differences in their antibacterial activity
profiles from other aminoglycosides. In general, 5b and 5c are
also more active against G+ bacteria than G- bacteria as
indicated in the MIC values. However, 5b and 5c exhibit
prominent activities against E. faecalis (ATCC29212), while
other aminoglycosides, including clinically used amikacin and
gentamicin, are much less active. More impressively, 5b and
5c show excellent activities against VRE as compared to
amikacin, gentamicin, and even vancomycin. As mentioned
previously, enterococci have intrinsic resistance against ami-
noglycosides. This result strongly supports our speculation that
5b and 5c have different modes of antibacterial action. We have
also introduced similar structural modifications on the kana-
mycin class of aminoglycosides.34 However, from the prelimi-
nary screening, such modifications are ineffective in reviving
the activity of these kanamycin analogues against aminoglyco-
side resistant bacteria. Thus, to our knowledge, the modifications
on 5b and 5c are one of the very few examples of aminogly-
coside derivatives with clinically useful activity against MRSA,
VRE, and other resistant strains.
Figure 3. Structures of 5f and 5q.
the 5′′-side chain of 4d is pointed away from the enzyme
implying poor recognition and turnover from APH(3′)-IIIa,
which is supported by the high KM and low kcat/KM.
Both 5f and 4d can also bind to the targeted rRNA site
although the 5′′-side chain appears to have significant flexibility
by adopting different conformations in both cases. In contrast
to the binding of neomycin derivatives to APH(3′)-IIIa, all four
rings from the neomycin scaffold interact with the RNA
suggesting a lesser tolerance to structural modifications. It is
expected that the larger 5′′-side chain of 4d will lower the
binding affinity thus accounting for the lower antibacterial
activity. Nevertheless, as mentioned previously, the superior
substrate promiscuity and higher concentration of APH(3′) will
render most of the structural modifications at rings III and IV
less effective. Unfortunately, the 5′′-side chain of 5b and 5c
exhibit too much freedom to be properly predicted by molecular
modeling.
Although scoring was attempted, the scores obtained do not
correlate well with the experimental MICs. We have noticed
earlier that theoretical scores are better correlated with experi-
ment for less active compounds.4 The correlation deteriorates
for more active compounds. This suggests that the predictions
are accurate when the ligand-macromolecule fit is the limiting
factor. When it is not, other factors, such as solubility, transport,
or degradation of the aminoglycosides, cannot be neglected
when constructing a quantitative, linear SAR.
Antibacterial Assay of Selected Derivatives against
Other Pathogens. As mentioned previously, 5′′-modified neo-
mycin derivatives seem to be more active against G+ bacteria
(S. aureus) than G- bacteria (E. coli), and 5f appears to be
one of the most prominent leads having a similar MIC ratio to
amikacin. We decided to further derivatize 5f by preparing 5q
by extending one glycidal unit (Figure 3). In addition, we also
intend to explore the spectrum of the antibacterial activity of
5b and 5c against other clinically significant pathogens including
Klebsiella pneumoniae (Gram negative), Methicillin-resistant
S. aureus (MRSA), Pseudomonas aeruginosa (Gram negative),
and Enterococcus faecalis (Gram positive).
K. pneumoniae (ATCC 700603) is a clinical isolate that is
resistant to ceftazidime, other ꢀ-lactams, and several aminogly-
cosides (ANT(2′′).30 K. pneumoniae (ATCC 13883) is resistant
to ampicillin but susceptible to aminoglycosides. P. aeruginosa
(ATCC 27853) that expresses APH(3′)-IIb manifests modest
resistance toward aminoglycosides.31 Methicillin-resistant S.
aureus (ATCC 33591) (MRSA) is the leading cause of bacterial
infections, and many MRSA strains harbor genes encoded for
APH(3′), ANT(4′), and AAC(6′)/APH(2′′), which render the
bacteria resistant to various aminoglycosides.20 It has been
reported that microorganisms like enterococci and other anaer-
obes are intrinsically resistant to aminoglycosides due to the
dull uptake of aminoglycosides as a result of the deficiency in
their membrane-associated electron transport systems.19 Like
MRSA, vancomycin-resistant enterococci (VRE) that cause
various types of nosocomial infections also represent a great
Discussion and Conclusion
We have developed a facile and cost-effective method for
constructing neomycin derivatives. Although the original strat-
egy was to avoid the inactivation from APH(3′), unexpected
and useful outcomes were obtained. First of all, structural
modifications, which can be accommodated by the binding site
of rRNA, can be readily accommodated by AMEs as well.
Results from our enzymatic studies show that low kcat/KM values
for the structurally modified aminoglycosides may not be
applicable to suggest that these aminoglycosides could avoid
the enzyme-catalyzed inactivation and regain their antibacterial
activity. Molecular modeling results indicate that AMEs rec-
ognition on the neamine ring is sufficient for enzymes to
inactivate aminoglycoside derivatives bearing diverse modifica-
tions at rings III and IV of neomycin or ring III of kanamycin.
Structural modifications targeting neamine, such as the attach-
ment of the AHB group at N-1 and 3′,4′-dideoxygenation,
remain to be the few effective approaches. Although several
examples from our work show enhanced activity is possible, it
could be challenging to reproduce the effectiveness of the N-1
AHB group on kanamycin or ribostamycin. In summary, it may
not be easy to develop structurally modified aminoglycosides
active against resistant bacteria while maintaining the same
mode of antibacterial action of traditional aminoglycosides.

Antibacterial ActiVity of 5′′-Modified Neomycin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7569
Table 5. MIC of the 5′′-Modified Neomycin Derivatives against Other Strains of Bacteriaa
d
entry
compound
5b
5c
5f
5q
neomycin B
amikacin
gentamicin
vancomycin
K. pneumoniaeb
K. pneumoniaec
S. aureus
P. aeruginosae
E. faecalisf
E. faecalisg
1
2
3
4
5
6
7
8
8-16
8-16
4
4
4
1
16-32
32
8
8
16-32
0.5
8
2-4
4-8
16-32
32
4
2-4
4-8
4-8
8-16
8-16
16-32
64
0.5-1
0.5-1
ND
8-16
g250
g250
g250
g250
g250
125
32-64
64-125
64-125
32-64
8-16
ND
125
8-16
2-4
ND
1
ND
ND
a Unit, µg/mL; ND, not determined. b Klebsiella pneumoniae (ATCC 13883). c K. pneumoniae (ATCC 700603). d Staphylococcus aureus (ATCC 33591)
(MRSA). e Pseudomonas aeruginosa (ATCC 27853). f Enterococcus faecalis (ATCC 29212). g E. faecalis (ATCC51299) (VRE).
and propargylamine (0.49 g, 8.83 mmol) in DMF (30 mL), Et3N
(2.3 mL), EDC (2.31 g, 12.0 mmol), and HOBt (1.63 g, 12.0 mmol)
were added. The reaction mixture was stirred at room temperature
for 2 days. After completion of the reaction, the reaction mixture
was concentrated and then diluted with EtOAc. The organic solution
was washed with 1 N HCl, H2O, NaHCO3, and brine and then dried
over Na2SO4. After removal of solvent and recrystallization from
ether, the desired product was obtained as a crystal (1.69 g, 5.90
mmol, 74%). 1H NMR (CDCl3, 400 MHz) δ 8.17 (d, J ) 9.5 Hz,
1H), 8.1 (m, 2H), 7.81 (s, 1H), 7.72 (td, J ) 9.2 Hz, J ) 1.1 Hz,
1H), 7.4-7.5 (m, 5H), 6.54 (s, 1H), 4.36 (dd, J ) 5.9 Hz, J ) 2.9
Hz, 2H), 2.35 (t, J ) 2.9 Hz, 1H). 13C (CDCl3, 100 MHz) δ 166.6,
156.1, 148.0, 141.1, 138.1, 129.8, 129.5, 129.3, 128.4 (2 carbons),
127.0, 126.9 (2 carbons), 124.3, 122.7, 116.0, 78.3, 72.0, 29.3. ESI/
APCI calcd for C19H15N2O ([M + H]+) m/e 287.1179; found m/e
287.1176.
Nonetheless, our results also reveal another strategy to combat
the problem of resistance. It is possible to revive the antibacterial
activity of aminoglycoside using structural modifications that
can alter the original mode of action. For example MRSA and
VRE are known to exert a high level resistance to aminogly-
cosides. The discovery of two leads active against both MRSA
and VRE is particularly significant since the transfer of
resistance from enterococci to opportunistic S. aureus has been
recognized as one of the stringent threats to public health.33
Ongoing efforts have been devoted into the investigation of
possible antibacterial modes of action for 5b and 5c and further
structural optimization from these two leads.
Experimental Section
General Procedure for Coupling of Compound 5 with
Carboxylic Acids. To a solution of compound 3 (0.20 g, 0.14
mmol) and carboxylic acids (0.28 mmol) in DMF (10 mL) and
Et3N (0.04 mL, 0.28 mmol), HOBt (0.030 g, 0.21 mmol) and EDC
(0.040 g, 0.21 mmol) were added. The reaction mixture was stirred
at room temperature overnight. After completion of the reaction,
the reaction mixture was concentrated and diluted with EtOAc. The
organic solution was washed with water, saturated NaHCO3(aq),
and brine and dried over anhydrous Na2SO4. After removal of the
solvent followed by a fast gradient column chromatography (eluted
from hexane/EtOAc ) 1/1 to EtOAc/MeOH ) 9/1), the product
N-Propargylisonicotinamide (13). The product was synthesized
similarly as compound 9 except the purification was accomplished
using gradient column chromatography (eluted from hexane/EtOAc
1
) 60/40 to hexane/EtOAc ) 0/100, 92%). H NMR (CDCl3, 400
MHz) δ 8.71 (dd, J ) 4.5 Hz, J ) 1.7 Hz, 2H), 7.61 (dd, J ) 4.5
Hz, J ) 1.6 Hz, 2H), 6.81 (s, 1H), 4.23 (dd, J ) 5.3 Hz, J ) 2.6
Hz, 2H), 2.27 (dd, J ) 2.4 Hz, J ) 1.1 Hz, 1H); 13C NMR (CDCl3,
100 MHz) δ 165.5, 150.8 (2 carbons), 136.0, 121.2 (2 carbons),
79.0, 72.5, 30.1. ESI/APCI calcd for C9H9N2O ([M + H]+) m/e
161.0709; found m/e 161.0713.
1
N-Propargylcarbobenzyloxy-L-prolinamide (14). The product
was synthesized similarly as compound 9 except the purification
was accomplished using gradient column chromatography (eluted
from hexane/EtOAc ) 90/10 to hexane/EtOAc ) 30/70, 74%). 1H
NMR (CDCl3, 400 MHz) δ 7.3 (m, 5H), 5.1 (m, 2H), 4.26 (s, 1H),
3.90 (d, J ) 9.7 Hz, 2H), 3.4 (m, 2H), 1.8-2.2 (m, 6H). 13C NMR
(CDCl3, 100 MHz) δ 172.4, 156.1, 136.6, 128.7 (2 carbons), 128.2,
128.0 (2 carbons), 79.9, 71.6, 60.8, 47.7, 31.3, 29.2, 28.9, 24.7.
ESI/APCI calcd for C16H19N2O3+ ([M + H]+) m/e 287.1390; found
m/e 287.1401.
was usually obtained as a solid, which was characterized with H
and/or 13C NMR and subjected to hydrogenation without further
purification.
General Procedure for the 1,3-Dipolar Cycloaddition. A
solution of compound 2 (0.028 mmol), alkyne (0.05 mmol),
Cu(OAc)2 (0.05 mmol), and sodium ascorbate (0.05 mmol) in a
mixed solution of MeOH (1.7 mL), THF (0.46 mL), and water (0.3
mL) was sonicated at ambient temperature for 14 min (7 min, 2
times). After completion of the reaction, the reaction mixture was
diluted with CH2Cl2 then filtered through Celite. After removal of
the solvent followed by a fast gradient column chromatography
purification (CH2Cl2/MeOH ) 100:0 to 60:40), the product was
typically obtained as a solid, which was characterized with 1H and/
or 13C NMR and subjected to hydrogenation without further
purification.
General Procedure for Hydrogenation and Purification. The
solids from 1,3-dipolar cycloaddition or acid/amine coupling
reaction (0.1-0.2 mmol) were dissolved in degassed MeOH (9 mL)
followed by the addition of 1 mL of HOAc/H2O (1/4 ratio) solution.
A catalytic amount of Pd(OH)2/C powder was added, and the system
was well sealed and further degassed. The system was stirred under
atmospheric H2 at room temperature for 10 h. The reaction mixture
was then quenched by filtering through Celite and the residue was
washed with H2O and the combined solutions were concentrated.
The crude product was purified with Amberlite CG50 (NH4+) eluted
with a gradient of NH4OH solution (0%-20%). After collection
of the desired fractions and removal of solvent, the product was
redissolved in water and loaded onto an ion-exchange column
packed with Dowex 1 × 8-200 (Cl- form) and eluted with water.
After removal of solvent, the product was obtained as a white solid.
N-Propargyl-2-phenyl-4-quinolinecarboxamide (9). To a solu-
tion of 2-phenyl-4-quinolinecarboxylic acid (2.09 g, 8.02 mmol)
Compound 2. A solution of compound 1 (1.50 g, 0.89 mmol)
and NaN3 (0.12 g, 1.78 mmol) in DMF was stirred at 80 °C
overnight. TLC showed the completion of the reaction (Rf ) 0.50
eluted with EtOAc/MeOH ) 97/3). The reaction mixture was
filtered through Celite, and the residue was washed with EtOAc.
After the removal of the solvent followed by purification with
gradient column chromatography (CH2Cl2/MeOH ) 100/0 to 90/
1
10), the product was obtained as a white solid (0.81 g, 63%). H
NMR (CD3COCD3, 400 MHz) δ 7.2-7.4 (m, 30H), 6.56 (m, 2H),
6.36 (d, J ) 9.6 Hz, 1H), 4.8-5.3 (m, 18H), 4.5 (m, 4H), 3.8-4.1
(m, 6H), 3.3-3.9 (m, 20H). 13C NMR (CD3COCD3, 100 MHz) δ
157.6 (2 carbons), 157.4, 156.8, 156.5 (2 carbons), 137.4-137.7
(6 carbons), 127.7-128.7 (30 carbons), 110.0, 100.3, 99.1, 86.6,
80.5, 79.4, 74.8, 74.4, 73.1, 72.6, 72.3, 71.7, 70.3, 67.9, 66.0-67.9
(8 carbons), 56.6, 53.2, 51.9, 51.3, 42.6, 41.3, 34.8. ESI/APCI calcd
for C71H81N9O24Na ([M + Na]+) m/e 1466.5292; found m/e
1466.5263.
Compound 3. To a solution of compound 2 (1.98 g, 1.37 mmol)
in THF (20 mL) and several drops of water, PMe3 in THF (8.2
mL, 8.2 mmol) was added. The reaction mixture was stirred at 50
°C for an hour, and TLC showed the completion of the reaction
(Rf ) 0.02 eluted with EtOAc/MeOH ) 97/3). After the removal

7570 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Zhang et al.
of the solvent followed by purification with gradient column
82.0, 79.7, 77.0, 76.9, 75.2, 72.9, 71.0, 70.5, 69.3, 69.0, 67.9, 67.6,
53.8, 52.0, 51.0, 50.4, 48.9, 45.1, 40.7, 40.4, 30.5, 28.3, 21.7, 21.6.
ESI/APCI calcd for C30H57N10O12 ([M + H]+) m/e 749.4152; found
m/e 749.4165.
chromatography (CH2Cl2/MeOH ) 100/0 to 70/30), the product
1
was obtained as a white solid (1.80 g, 93%). H NMR (CD3OD,
400 MHz) δ 7.3-7.4 (m, 30H), 5.1 (m, 10H), 3.3-3.9 (m, 37H).
13C NMR (CD3OD, 100 MHz) δ 157.6-158.0 (6 carbons),
136.9-137.1 (6 carbons), 127.7-128.4 (30 carbons), 110.9, 98.8,
98.1, 86.6, 79.4, 78.7, 74.8, 74.0, 73.3, 71.9, 71.8, 71.1, 70.3, 67.8,
56.2, 52.9, 51.7, 50.8, 43.4, 42.0, 41.1, 39.0, 33.6, 30.4, 29.6, 28.9,
24.4, 23.7, 22.8. ESI/APCI calcd for C71H84N7O24 ([M + H]+) m/e
1418.5568; found m/e 1418.5536.
Compound 4g. 1H NMR (D2O, 400 MHz) δ 8.19 (s, 1H), 6.04
(d, J ) 3.9 Hz, 1H), 5.36 (d, J ) 3.1 Hz, 1H), 5.26 (d, J ) 1.6 Hz,
1H), 4.53 (t, J ) 5.2 Hz, 1H), 4.39 (m, 1H), 4.26 (m, 3H), 4.0-4.1
(m, 2H), 3.9-4.0 (m, 4H), 3.7-3.8 (m, 2H), 3.2-3.5 (m, 10H),
2.42 (m, 1H), 1.97 (m, 1H). 13C NMR (D2O, 100 MHz) δ 140.3,
126.1, 110.2, 95.5, 95.1, 85.0, 80.0, 76.8, 75.0, 73.0, 72.5, 70.6,
70.4, 70.0, 68.2, 67.8, 67.5, 53.4, 52.7, 51.0, 49.9, 48.6, 40.7, 40.3,
34.2, 28.1. ESI/APCI calcd for C26H50N10O12 ([M + Na]+) m/e
717.3502; found m/e 717.3525.
Compound 4a. 1H NMR (D2O, 400 MHz) δ 8.08 (s, 1H), 5.95
(d, J ) 3.4 Hz, 1H), 5.34 (s, 1H), 5.24 (s, 1H), 4. 3 (t, J ) 5.0 Hz,
2H), 4.14 (s, 2H), 4.06 (t, J ) 9.7 Hz, 1H), 3.8-4.0 (m, 6H), 3.8
(m, 2H), 3.6-3.8 (m, 3H), 3.4-3.5 (m, 4H), 3.2-3.4 (m, 11H),
2.3 (m, 1H), 2.0 (m, 1H), 1.82 (s, 3H). 13C NMR (D2O, 100 MHz)
δ 174.8, 159.4, 142.8, 125.5, 110.1, 95.5, 95.0, 84.9, 79.9, 76.8,
75.3, 73.5, 73.1, 72.4, 70.6, 70.4, 69.9, 68.2, 67.8, 67.5, 53.5, 52.2,
51.0, 49.9, 48.7, 47.0, 41.9, 40.7, 40.2, 38.6, 28.3, 22.0. ESI/APCI
calcd for C32H58N11O15 ([M + H]+) m/e 836.4108; found m/e
836.4107.
Compound 4h. 1H NMR (D2O, 400 MHz) δ 7.96 (s, 1H), 5.90
(d, J ) 3.8 Hz, 1H), 5.31 (d, J ) 3.3 Hz, 1H), 5.24 (d, J ) 1.6 Hz,
1H), 4.48 (m, 2H), 4.39 (s, 2H), 4.26 (t, J ) 4.6 Hz, 1H), 4.14 (t,
J ) 3.1 Hz, 1H), 3.8-3.9 (m, 5H), 3.74 (m, 1H), 3.6 (dd, J ) 9.0
Hz, J ) 7.0 Hz, 1H), 3.51 (m, 1H), 3.2-3.4 (m, 12H), 2.96 (td, J
) 12.9 Hz, J ) 3.0 Hz, 2H), 2.6 (m, 1H), 23 (dt, J ) 8.6 Hz, J )
4.2 Hz, 1H), 2.0 (m, 2H), 1.7-1.8 (m, 3H). 13C NMR (D2O, 100
MHz) δ 176.5, 145.2, 124.6, 110.2, 95.6, 95.2, 85.2, 79.9, 76.9,
76.8, 73.0, 72.8, 70.8, 70.4, 69.5, 68.6, 67.8, 67.5, 53.7, 52.3, 51.0,
50.1, 48.7, 43.3 (2 carbons), 40.7, 40.3, 39.8, 34.6, 29.0, 25.2 (2
carbons). ESI/APCI calcd for C32H59N11O13Na ([M + Na]+) m/e
828.4186; found m/e 828.4170.
Compound 4b. 1H NMR (D2O, 400 MHz) δ 7.80 (s, 1H), 5.78
(d, J ) 3.7 Hz, 1H), 5.30 (d, J ) 3.8 Hz, 1H), 5.21 (m, 1H), 4.5
(m, 2H), 4.2 (m, 1H), 3.9 (m, 1H), 3.7-3.9 (m, 6H), 3.5 (m, 2H),
3.3 (m, 5H), 3.2 (m, 3H), 2.62 (t, J ) 7.4 Hz, 2H), 2.22 (m, 1H),
1.97 (m, 1H), 1.6 (m, 2H), 1.1-1.2 (m, 8H), 0.75 (m, 3H). 13C
NMR (D2O, 100 MHz) δ 149.4, 123.9, 109.9, 95.9, 95.4, 85.4,
79.9, 78.0, 77.2, 73.2, 73.0, 70.9, 69.3, 68.9, 67.8, 67.5, 53.8, 51.7,
51.0, 50.3, 48.8, 46.8, 40.7, 40.3, 30.8, 30.4, 28.8, 27.9, 24.5, 22.1,
13.5. ESI/APCI calcd for C31H60N9O12 ([M + H]+) m/e 750.4356;
found m/e 750.4333.
1
Compound 4i. H NMR (D2O, 400 MHz) δ 8.01 (s, 1H), 5.98
(d, J ) 4.0 Hz, 1H), 5.34 (d, J ) 3.5 Hz, 1H), 5.24 (s, 1H), 4.4-4.5
(m, 4H), 4.31 (t, J ) 2.9 Hz, 1H), 4.26 (t, J ) 5.4 Hz, 1H), 4.14
(t, J ) 3.0 Hz, 1H), 3.97 (t, J ) 9.0 Hz, 1H), 3.95 (dd, J ) 10.5
Hz, J ) 9.0 Hz, 1H), 3.9 (m, 3H), 3.8 (s, 1H), 3.64 (t, J ) 10 Hz,
1H), 3.2-3.5 (m, 13H), 2.4 (m, 2H), 2.0 (m, 3H), 1.84 (dd, J )
14.0 Hz, J ) 12.0 Hz, 1H). 13C NMR (D2O, 100 MHz) δ 169.8,
144.6, 124.7, 110.1, 95.7, 95.1, 84.9, 80.0, 77.0, 75.1, 73.1, 72.5,
70.6, 70.4, 69.8, 68.2, 67.8, 67.5, 59.9, 53.4, 52.3, 50.9, 49.8, 48.6,
46.7, 40.7, 40.2, 34.8, 29.8, 28.1, 24.0. ESI/APCI calcd for
C31H58N11O13 ([M + H]+) m/e 792.4210; found m/e 792.4236.
Compound 4c. 1H NMR (D2O, 400 MHz) δ 8.39 (s, 1H), 5.97
(d, J ) 3.6 Hz, 1H), 5.40 (d, J ) 2.9 Hz, 1H), 5.33 (s, 1H), 4.9
(dd, J ) 14 Hz, J ) 1.0 Hz, 1H), 4.81 (dd, J ) 15 Hz, J ) 7.4 Hz,
1H), 4.61 (t, J ) 5.7 Hz, 1H), 4.5 (m, 4H), 4.5 (s, 2H), 4.35 (t, J
) 5.3 Hz, 1H), 4.22 (t, J ) 3.0 Hz, 1H), 4.12 (dd, J ) 7.6 Hz, J
) 2.9 Hz, 1H), 4.0 (m, 1H), 3.98 (t, J ) 9.2 Hz, 2H), 3.8-3.9 (m,
3H), 3.7 (m, 2H), 3.6 (m, 1H), 3.4-3.5 (m, 6H), 3.2-3.3 (m, 3H),
2.3 (m, 1H), 1.7 (m, 1H). 13C NMR (D2O, 100 MHz) δ 137.2,
128.4, 110.4, 95.7, 95.4, 85.3, 79.8, 77.0, 76.9, 73.1, 73.0, 71.0,
70.6, 69.5, 69.0, 67.9, 67.6, 53.9, 52.9, 51.3, 51.1, 50.4, 49.0, 42.5
(2 carbons), 40.8, 40.5, 30.1. ESI/APCI calcd for C28H55N10O12
([M + H]+) m/e 723.3995; found m/e 723.4022.
1
Compound 5a. H NMR (D2O, 400 MHz) δ 5.80 (d, J ) 3.8
Hz, 1H), 5.30 (d, J ) 10.7 Hz, 1H), 5.19 (d, J ) 1.3 Hz, 1H), 4.33
(t, J ) 4.9 Hz, 1H), 4.22 (t, J ) 5.0 Hz, 1H), 4.16 (t, J ) 2.9 Hz,
1H), 3.8-3.9 (m, 4H), 3.7 (m, 1H), 3.65 (t, J ) 9.6 Hz, 1H), 3.55
(dd, J ) 12.8 Hz, J ) 3.8 Hz, 1H), 3.5 (m, 1H), 3.2-3.4 (m, 10H),
2.61 (m, 1H), 2.17 (t, J ) 7.2 Hz, 2H), 1.5 (m, 1H), 1.2 (m, 2H),
1.2 (m, 8 H), 0.74 (t, J ) 6.5 Hz, 3H); 13C NMR (D2O, 100 MHz)
δ 178.0, 109.3, 95.9, 95.2, 85.2, 80.8, 77.5, 76.5, 73.6, 72.7, 70.7,
70.3, 69.3, 68.5, 67.8, 67.5, 53.6, 51.0, 49.9, 48.8, 41.5, 40.7, 40.3,
36.1, 30.9, 27.8, 28.2, 25.6, 22.1, 13.5. ESI/APCI calcd for
C30H60N7O13 ([M + H]+) m/e 726.4244; found m/e 726.4226.
Compound 4d. 1H NMR (D2O, 400 MHz) δ 8.14 (s, 1H), 7.88
(t, J ) 8.6 Hz, 2H), 7.73 (m, 3H), 7.68 (t, J ) 8.2 Hz, 1H), 7.49
(t, J ) 8.1 Hz, 1H), 7.4 (m, 3H), 5.94 (d, J ) 3.9 Hz, 1H), 5.32 (d,
J ) 3.5 Hz, 1H), 5.21 (d, J ) 1.5 Hz, 1H), 4.5 (m, 2H), 4.2 (m,
2H), 4.1 (m, 1H), 3.8-3.9 (m, 6H), 3.7 (m, 1H), 3.6 (m, 1H), 3.5
(m, 2H), 3.2-3.4 (m, 9H), 2.31 (dt, J ) 8.4 Hz, J ) 4.1 Hz, 1H),
1.73 (m, 1H). 13C NMR (D2O, 100 MHz) δ 169.7, 157.7, 147.5,
144.8, 142.2, 137.9, 131.3, 130.4, 129.2 (2 carbons), 128.3, 128.1,
127.9 (2 carbons), 125.0, 124.8, 123.0, 118.2, 110.0, 95.7, 95.2,
85.0, 79.9, 76.9, 76.2, 73.1, 72.6, 70.7, 70.3, 69.7, 68.4, 67.8, 67.5,
53.6, 52.2, 50.9, 50.0, 48.7, 40.7, 40.2, 35.1, 28.9. ESI/APCI calcd
for C42H59N11O13 ([M + H]+) m/e 948.4186; found m/e 948.4208.
Compound 4e. 1H NMR (D2O, 400 MHz) δ 8.22 (s, 1H), 5.85
(d, J ) 3.5 Hz, 1H), 5.30 (d, J ) 2.9 Hz, 1H), 5.24 (d, J ) 1.5 Hz,
1H), 4.8 (dd, J ) 15 Hz, J ) 2.8 Hz, 2H), 4.52 (t, J ) 5.9 Hz,
1H), 4.5 (m, 1H), 4.32 (s, 2H), 4.27 (t, J ) 5.1 Hz, 1H), 4.14 (t, J
) 3.0 Hz, 1H), 4.05 (dd, J ) 4.6 Hz, J ) 2.9 Hz, 1H), 3.96 (m,
1H), 3.85 (t, J ) 9.3 Hz, 2H), 3.8 (m, 1H), 3.6 (m, 1H), 3.51 (s,
1H), 3.3-3.4 (m, 5H), 3.2 (m, 4H), 2.66 (m, 3H), 2.20 (m, 1H),
1.57 (m, 1H). 13C NMR (D2O, 100 MHz) δ 138.5, 127.1, 110.3,
95.6, 95.4, 85.4, 79.7, 78.0, 76.8, 73.0, 72.9, 71.0, 70.5, 69.3, 69.2,
67.9, 67.6, 53.9, 52.7, 51.0, 50.4, 48.9, 42.7, 40.7, 40.4, 32.3, 31.2.
ESI/APCI calcd for C27H52N10O12Na ([M + Na]+) m/e 731.3658;
found m/e 731.3693.
1
Compound 5b. H NMR (D2O, 400 MHz) δ 5.72 (d, J ) 3.6
Hz, 1H), 5.28 (d, J ) 4.1 Hz, 1H), 5.18 (d, J ) 1.4 Hz, 1H), 4.31
(t, J ) 3.6 Hz, 1H), 4.22 (t, J ) 4.0 Hz, 1H), 4.17 (t, J ) 4.9 Hz,
2H), 4.12 (t, J ) 3.0 Hz, 1H), 3.95 (m, 1H), 3.7-3.8 (m, 3H),
3.4-3.6 (m, 3H), 3.3-3.4 (m, 6H), 3.2 (m, 3H), 2.2 (m, 3H), 1.6
(m, 1H), 1.48 (m, 2H), 1.2 (m, 26H), 0.74 (m, 3H). 13C NMR (D2O,
100 MHz) δ 178.1, 109.4, 96.0, 95.5, 85.4, 80.6, 77.5, 73.6, 73.0,
70.9, 70.3, 69.4, 69.1, 67.8, 67.6, 53.9, 51.0, 50.2, 49.0, 41.2, 40.7,
40.3, 36.1, 31.4, 28.5-29.0 (12 carbons), 25.6, 22.2, 13.6. ESI/
APCI calcd for C39H78N7O13 ([M + H]+) m/e 852.5652; found m/e
852.5633.
1
Compound 5c. H NMR (D2O, 400 MHz) δ 5.75 (d, J ) 3.3
Hz, 1H), 5.31 (s, 1H), 5.17 (s, 1H), 4.35 (t, J ) 4.6 Hz, 1H),
4.1-4.2 (m, 4H), 3.95 (m, 1H), 3.8-3.9 (m, 3H), 3.62 (t, J ) 8.8
Hz, 1H), 3.5 (m, 3H), 3.1-3.4 (m, 8H), 2.2 (m, 3H), 1.6 (m, 1H),
1.49 (m, 2H), 1.16 (m, 30H), 0.76 (m, 3H). 13C NMR (D2O, 100
MHz) δ 177.6, 109.4, 96.0, 95.6, 85.1, 80.3, 77.6, 73.4, 72.9, 71.1,
70.3, 69.4, 67.9, 67.7, 54.0, 51.1, 50.3, 49.0, 41.5, 40.7, 40.4, 39.0,
36.2, 31.7, 28.9-29.9 (14 carbons), 25.8, 22.5, 13.9. ESI/APCI
calcd for C41H82N7O13 ([M + H]+) m/e 880.5965; found m/e
880.5959.
1
Compound 4f. H NMR (D2O, 400 MHz) δ 8.18 (s, 1H), 5.90
(d, J ) 3.2 Hz, 1H), 5.31 (s, 1H), 5.21 (s, 1H), 4.53 (m, 1H), 4.46
(d, J ) 8.9 Hz, 2H), 4.27 (m, 1H), 4.1 (m, 2H), 3.8-3.9 (m, 5H),
3.1-3.6 (m, 14H), 2.20 (m, 2H), 1.9 (m, 3H), 1.6 (m, 3H). 13C
NMR (D2O, 100 MHz) δ 144.4, 124.9, 110.4, 95.6, 95.3, 85.4,
1
Compound 5d. H NMR (D2O, 400 MHz) δ 8.13 (t, J ) 8.8
Hz, 2H), 7.98 (dd, J ) 8.0 Hz, J ) 2.0 Hz, 1H), 7.95 (s, 1H), 7.87
(t, J ) 8.4 Hz, 1H), 7.70 (t, J ) 7.2 Hz, 1H), 7.6 (m, 3H), 7.4 (m,

Antibacterial ActiVity of 5′′-Modified Neomycin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7571
1H), 5.71 (m, 1H), 5.36 (d, J ) 3.7 Hz, 1H), 5.2 (m, 1H), 4.55 (t,
J ) 5.3 Hz, 1H), 4.3 (m, 3H), 4.2 (m, 1H), 4.1 (m, 1H), 4.0 (m,
2H), 3.7-3.9 (m, 3H), 3.6 (m, 3H), 3.5 (m, 2H), 3.3-3.4 (m, 2H),
3.1-3.3 (m, 2H), 3.0-3.1 (m, 2H), 2.8 (m, 1H), 2.22 (m, 1H), 1.6
(m, 1H). 13C NMR (D2O, 100 MHz) δ 170.6, 158.1, 147.0, 142.8,
137.0, 131.6, 130.6, 129.5 (2 carbons), 128.6, 128.4, 128.0 (2
carbons), 124.9, 123.0, 118.7, 109.8, 95.8, 95.5 (2 carbons), 85.3,
80.3, 77.8, 73.6, 73.2, 70.8, 70.4, 69.2, 67.6, 67.2, 66.0, 53.9, 51.1,
50.3, 49.0, 41.5, 40.4, 40.2, 32.0. ESI/APCI calcd for C41H82N7O13
([M + H]+) m/e 845.4040; found m/e 845.4021.
(t, J ) 5.3 Hz, 1H), 4.26 (t, J ) 3.7 Hz, 1H), 4.1-4.2 (m, 3H),
3.9-4.0 (m, 2H), 3.8-3.9 (m, 3H), 3.7 (m, 1H), 3.6-3.7 (m, 2H),
3.2-3.5 (m, 11H), 2.9 (t, J ) 7.7 Hz, 1H), 2.2 (m, 1H), 1.8 (m,
2H), 1.6 (m, 3H), 1.38 (m, 2H). 13C NMR (D2O, 100 MHz) δ 170.9,
109.0, 96.0, 95.9, 84.8, 80.9, 77.1, 73.2, 72.8, 70.8, 70.2, 69.5, 69.1,
67.8, 67.7, 53.9, 53.3, 51.0, 50.2, 49.0, 42.0, 40.8, 40.3, 39.2 (2
carbons), 30.8, 30.0, 26.6, 21.7. MALDI calcd for C29H59N9O13Na
([M + Na]+) m/e 764.4125; found m/e 764.4163.
Compound 5l. This compound was prepared by subjecting
compound 32 to hydrogenation and sequential purification. 1H NMR
(D2O, 300 MHz) δ 5.85 (d, J ) 4.2 Hz, 1H), 5.31 (d, J ) 4.1 Hz,
1H), 5.20 (d, J ) 1.4 Hz, 1H), 4.35 (t, J ) 4.8 Hz, 1H), 4.1-4.2
(m, 4H), 3.8-4.0 (m, 4H), 3.7 (m, 2H), 3.2-3.6 (m, 11H), 2.4-2.6
(m, 5H), 1.90 (m, 1H). 13C NMR (D2O, 100 MHz) δ 177.4, 175.6,
84.8, 80.8, 77.2, 75.3, 75.1, 73.5, 72.2 (2 carbons), 70.4, 70.2, 69.8
(2 carbons), 68.1, 67.7, 67.4, 53.3, 50.8, 49.7, 48.6, 41.0, 40.6,
40.1, 30.4, 29.5, 28.0. MALDI calcd for C21H57N7O15Na ([M +
Na]+) m/e 736.3335; found m/e 736.3331.
Compound 5e. 1H NMR (D2O, 400 MHz) δ 5.80 (s, 1H), 5.31
(d, J ) 3.3 Hz, 1H), 5.22 (s, 1H), 4.40 (t, J ) 5.2 Hz, 1H), 4.2-4.3
(m, 6H), 4.13 (t, J ) 3.1 Hz, 1H), 3.8-3.9 (m, 5H), 3.74 (s, 1H),
3.65 (t, J ) 9.7 Hz, 1H), 3.5 (m, 4H), 3.2-3.4 (m, 4H), 3.07 (t, J
) 7.2 Hz, 2H), 2.3 (m, 1H), 2.1 (m, 1H), 1.9 (m, 1H), 1.7 (m, 1H).
13C (D2O, 100 MHz) δ 176.1, 109.5, 95.8, 95.3, 85.0, 80.4, 77.4,
73.5, 72.7, 70.8, 70.3, 69.7, 69.5, 69.0, 67.9, 67.6, 53.8, 51.0, 50.2,
48.9, 41.3, 40.7, 40.3, 36.9, 31.2, 30.1, 30.0. ESI/APCI calcd for
C27H54N8O14Na ([M + Na]+) m/e 737.3652; found m/e 737.3682.
1
Compound 5m. H NMR (D2O, 300 MHz) δ 5.87 (d, J ) 7.9
1
Compound 5f. H NMR (D2O, 400 MHz) δ 5.88 (d, J ) 3.9
Hz, 1H), 5.34 (d, J ) 3.8 Hz, 1H), 5.20 (s, 1H), 4.40 (t, J ) 5.5
Hz, 1H), 3.8-4.3 (m, 10H), 3.7 (m, 2H), 3.2-3.7 (m, 10H), 2.41
(dt, J ) 8.6 Hz, J ) 4.1 Hz, 1H), 1.90 (m, 1H), 1.46 (d, J ) 6.9
Hz, 3H). 13C NMR (D2O, 100 MHz) δ 171.5, 108.8, 95.7, 95.2,
84.5, 80.9, 76.8, 75.2, 73.4, 72.2, 70.5, 70.2, 69.8, 68.2, 67.7, 67.5,
53.3, 50.9, 49.7, 49.2, 48.6, 40.6, 40.2, 28.0, 16.9. MALDI calcd
for C26H52N8O13Na ([M + Na]+) m/e 707.3546; found m/e
707.3518.
Hz, 1H), 5.32 (d, J ) 3.5 Hz, 1H), 5.21 (d, J ) 1.5 Hz, 1H), 4.35
(t, J ) 5.4 Hz, 1H), 4.26 (t, J ) 5.3 Hz, 1H), 4.22 (d, J ) 3.7 Hz,
1H), 4.1 (m, 2H), 4.03 (t, J ) 9.0 Hz, 1H), 3.9 (m, 2H), 3.78 (s,
1H), 3.74 (m, 2H), 3.71 (t, J ) 9.4 Hz, 1H), 3.6 (m, 1H), 3.2-3.5
(m, 11H), 2.35 (m, 1H), 1.80 (dd, J ) 24.0 Hz, J ) 14.6 Hz, 1H).
13C (D2O, 100 MHz) δ 170.0, 109.2, 95.9, 95.6, 84.8, 80.6, 78.3,
77.0, 73.3, 72.8, 70.8, 70.3, 69.5, 69.1, 67.9, 67.6, 53.8, 51.0, 50.3,
49.0, 41.8, 40.7 (2 carbons), 40.3, 30.0. ESI/APCI calcd for
C25H51N8O13 ([M + H]+) m/e 671.3570; found m/e 671.3591.
1
Compound 5n. H NMR (D2O, 300 MHz) δ 5.69 (d, J ) 3.8
Hz, 1H), 5.28 (d, J ) 5.0 Hz, 1H), 5.20 (s, 1H), 4.33 (t, J ) 5.1
Hz, 1H), 4.1-4.2 (m, 5H), 3.6-3.8 (m, 9H), 3.1-3.5 (m, 10H),
2.19 (dt, J ) 11.0 Hz, J ) 4.5 Hz, 1H), 1.55 (m, 1H). 13C NMR
(D2O, 100 MHz) δ 170.2, 109.0, 95.9, 84.8, 80.4, 79.1, 77.0, 73.2,
72.9, 70.9, 70.2, 69.4, 69.2, 67.8, 67.6, 61.1, 54.9, 53.9, 52.0, 50.9,
50.3, 49.0, 41.5, 40.6, 40.3, 30.7. MALDI calcd for C26H52N8O14Na
([M + Na]+) m/e 723.3495; found m/e 723.3494.
1
Compound 5g. H NMR (D2O, 400 MHz) δ 5.88 (d, J ) 3.9
Hz, 1H), 5.34 (d, J ) 3.8 Hz, 1H), 5.22 (s, 1H), 4.31 (t, J ) 5.3
Hz, 1H), 4.26 (t, J ) 2.0 Hz, 1H), 4.18 (t, J ) 4.9 Hz, 2H), 4.13
(t, J ) 3.0 Hz, 1H), 4.0-4.1 (m, 2H), 3.9-4.0 (m, 3H), 3.74 (dd,
J ) 3.1 Hz, J ) 1.5 Hz, 1H), 7.72 (d, J ) 9.7 Hz, 1H), 3.2-3.6
(m, 11H), 2.36 (dt, J ) 8.5 Hz, J ) 4.2 Hz, 1H), 1.81 (m, 1H),
1.45 (d, J ) 7.1 Hz, 3H). 13C NMR (D2O, 100 MHz) δ 171.6,
108.8, 95.9, 95.2, 84.6, 80.9, 77.0, 73.4, 72.4, 70.6, 70.3, 69.8, 68.6,
67.8, 67.7, 53.5, 51.0, 50.0, 49.3, 48.8, 41.7, 40.8, 40.2, 27.0, 16.9.
ESI/APCI calcd for C25H53N8O13 ([M + H]+) m/e 685.3727; found
m/e 685.3735.
1
Compound 5o. H NMR (D2O, 400 MHz) δ 5.86 (d, J ) 3.9
Hz, 1H), 5.33 (d, J ) 3.6 Hz, 1H), 5.22 (s, 1H), 4.3-4.4 (m, 2H),
4.3 (m, 1H), 4.1-4.2 (m, 4H), 3.9-4.0 (m, 3H), 3.2-4.0 (m, 14H),
2.4 (M, 2H), 2.0 (m, 4H), 1.9 (m, 1H). 13C NMR (D2O, 100 MHz)
δ 170.4, 109.1, 95.8, 95.3, 84.7, 80.9, 77.0, 75.3, 73.5, 72.4, 70.7,
70.3, 70.0, 68.3, 67.8, 67.6, 60.0, 53.4, 51.0, 49.8, 48.7, 46.8, 41.8,
40.7, 40.4, 30.2, 28.1, 24.1. ESI/APCI calcd for C28H55N8O13 ([M
+ H]+) m/e 711.3883; found m/e 711.3873.
1
Compound 5h. H NMR (D2O, 400 MHz) δ 5.91 (d, J ) 4.0
Hz, 1H), 5.35 (d, J ) 3.9 Hz, 1H), 5.21 (d, J ) 1.7 Hz, 1H), 4.30
(t, J ) 9.2 Hz, 1H), 4.31 (d, J ) 10.8 Hz, 1H), 4.3 (m, 1H), 4.1-4.2
(m, 2H), 3.9-4.0 (m, 3H), 3.7 (m, 2H), 3.3-3.6 (m, 15H), 2.4 (m,
2H), 2.0 (m, 3H), 1.86 (m, 1H). 13C NMR (D2O, 100 MHz) δ 170.3,
108.8, 95.9, 95.2, 84.5, 81.0, 77.1, 75.2, 73.4, 72.3, 70.5, 70.2, 70.0,
68.4, 67.8, 67.7, 60.1, 53.3, 50.9, 49.8, 48.7, 46.6, 41.8, 40.7, 40.2,
29.9, 28.0, 24.1. ESI/APCI calcd for C28H54N8O13Na ([M + Na]+)
m/e 733.3703; found m/e 733.3725.
1
Compound 5p. H NMR (D2O, 400 MHz) δ 5.90 (d, J ) 4.0
Hz, 1H), 5.34 (d, J ) 3.4 Hz, 1H), 5.21 (d, J ) 1.5 Hz, 1H), 4.39
(t, J ) 6.5 Hz, 1H), 4.26 (m, 1H), 4.1-4.3 (m, 4H), 3.9-4.0 (m,
4H), 3.7 (m, 2H), 3.2-3.6 (m, 11H), 2.91 (t, J ) 7.6 Hz, 2H),
2.41 (dt, J ) 8.4 Hz, J ) 4.0 Hz, 1H), 1.8-2.0 (m, 3H), 1.6-1.7
(m, 2H), 1.4 (m, 2H). 13C NMR (D2O, 100 MHz) δ 170.6, 109.1,
95.6, 95.3, 84.6, 80.8, 76.6, 75.2, 73.3, 72.4, 70.7, 70.4, 69.9, 68.4,
67.8, 67.6, 53.4, 53.3, 51.0, 49.9, 48.7, 41.7, 40.8, 40.3, 39.2, 30.7,
28.1, 26.6, 21.7. ESI/APCI Calcd for C29H59N9O13Na ([M + Na]+)
m/e 764.4125; found m/e 764.4116.
1
Compound 5i. H NMR (D2O, 400 MHz) δ 7.55 (d, J ) 7.9
Hz, 1H), 7.44 (d, J ) 8.3 Hz, 1H), 7.26 (s, 1H), 7.17 (t, J ) 7.1
Hz, 1H), 7.10 (t, J ) 7.8 Hz, 1H), 5.66 (d, J ) 3.7 Hz, 1H), 5.19
(d, J ) 3.4 Hz, 1H), 4.94 (s, 1H), 4.26 (t, J ) 6.9 Hz, 2H), 4.1 (m,
2H), 3.6-4.0 (m, 8H), 3.1-3.5 (m, 12H), 2.22 (dt, J ) 8.8 Hz, J
) 4.6 Hz, 1H), 1.6 (m, 1H), 1.2 (m, 1H). 13C NMR (D2O, 100
MHz) δ 170.7, 136.3, 126.8, 125.3, 122.5, 119.8, 118.4, 112.4,
108.5, 106.7, 96.0, 95.9, 84.7, 80.6, 78.0, 76.7, 70.9, 71.3, 70.0,
69.4, 69.3, 67.8, 67.5, 53.9, 53.8, 51.2, 50.8, 50.3, 49.1, 41.2, 40.7,
40.3, 30.2, 27.1. ESI/APCI calcd for C34H58N9O13 ([M + H]+) m/e
800.4149; found m/e 800.4139.
Compound 33. To a solution of compound 3 (0.20 g, 0.14 mmol)
in anhydrous DMF (5 mL), succinic anhydride (0.02 g, 0.20 mmol)
was added. After being stirred at room temperature overnight, the
reaction mixture was concentrated. After removal of the solvent
followed by fast gradient column chromatography (eluted from
CH2Cl2/MeOH ) 100/0 to 80/20), the product, 32 was obtained as
a solid and subjected to the next step without further purification.
The acid crude product was redissolve in DMF (8 mL) and added
with compound 3 (0.20 g, 0.14 mmol), Et3N (0.04 mL, 0.28 mmol),
HOBt (0.030 g, 0.21 mmol), and EDC (0.040 g, 0.21 mmol). After
being stirred overnight at room temperature, the reaction mixture
was concentrated and diluted with EtOAc. The organic solution
was washed with saturated NaHCO3(aq), water, and brine and dried
over anhydrous Na2SO4. After removal of the solvent followed by
a fast gradient column chromatography (eluted from CH2Cl2/MeOH
) 100/0 to 80/20), the product was obtained as a solid subjected
to hydrogenation without further purification. 1H NMR (D2O, 400
MHz) δ 5.94 (d, J ) 3.8 Hz, 2H), 5.37 (d, J ) 3.5 Hz, 2H), 5.22
1
Compound 5j. H NMR (D2O, 400 MHz) δ 5.95 (d, J ) 3.9
Hz, 1H), 5.35 (d, J ) 3.1 Hz, 1H), 4.38 (t, J ) 5.4 Hz, 1H), 4.26
(t, J ) 3.2 Hz, 2H), 4.1-4.2 (m, 4H), 3.9-4.0 (m, 5H), 3.6-3.8
(m, 3H), 3.2-3.5 (m, 11H), 2.42 (dt, J ) 8.5 Hz, J ) 4.3 Hz, 1H),
1.86 (q, J ) 12.6 Hz, 1H). 13C NMR (D2O, 100 MHz) δ 168.8,
109.3, 95.6, 95.2, 84.6, 80.3, 76.7, 75.1, 73.3, 72.4, 70.6, 70.3, 69.9,
68.3, 67.8, 67.6, 60.5, 54.5, 53.3, 51.0, 49.9, 48.7, 41.8, 40.7, 40.2,
28.1. ESI/APCI calcd for C26H53N8O14 ([M + H]+) m/e 701.3676;
found m/e 701.3695.
1
Compound 5k. H NMR (D2O, 400 MHz) δ 5.75 (d, J ) 3.5
Hz, 1H), 5.32 (d, J ) 4.1 Hz, 1H), 5.21 (d, J ) 1.5 Hz, 1H), 4.30

7572 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Zhang et al.
the HIV-1 Rev response element. Biochemistry 2003, 42, 11391–
(s, 2H), 4.40 (t, J ) 5.2 Hz, 2H), 4.2-4.3 (m, 8H), 3.9 (m, 6H),
3.8 (m, 6H), 3.2-3.6 (m, 22H), 2.5 (s, 4H), 2.43 (dt, J ) 8.8 Hz,
J ) 4.5 Hz, 2H), 1.87 (m, 2H). 13C NMR (D2O, 100 MHz) δ 175.5
(2 carbons), 109.4 (2 carbons), 95.7 (2 carbons), 95.0 (2 carbons),
84.7 (2 carbons), 80.5 (2 carbons), 77.3 (2 carbons), 75.2 (2
carbons), 73.4 (2 carbons), 72.4 (2 carbons), 70.6 (2 carbons), 70.4
(2 carbons), 69.9 (2 carbons), 68.3 (2 carbons), 67.8 (2 carbons),
67.5 (2 carbons), 53.5 (2 carbons), 51.0 (2 carbons), 49.9 (2
carbons), 48.8 (2 carbons), 41.8 (2 carbons), 40.8 (2 carbons), 40.3
(2 carbons), 31.1 (2 carbons), 28.1 (2 carbons). ESI/APCI calcd
for C50H97N14O26 ([M + H]+) m/e 1309.6693; found m/e 1309.6684.
11403.
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1
Compound 5q. H NMR (D2O, 400 MHz) δ 5.95 (d, J ) 3.9
Hz, 1H), 5.33 (d, J ) 3.2 Hz, 1H), 5.20 (s, 1H), 4.42 (t, J ) 5.2
Hz, 1H), 4.28 (m, 2H), 4.14 (m, 3H), 3.8-4.0 (m, 7H), 3.7 (m,
2H), 3.3-3.5 (m, 11H), 2.42 (dt, J ) 12.4 Hz, J ) 4.0 Hz, 1H),
1.91 (dd, J ) 12.2 Hz, J ) 3.6 Hz, 1H). 13C NMR (D2O, 100
MHz) δ 172.0, 168.2, 109.7, 95.6, 94.8, 84.8, 80.2, 77.5, 75.1, 73.4,
72.4, 70.6, 70.4, 69.8, 68.3, 67.8, 67.4, 53.5, 51.0, 49.9, 48.8, 42.9,
41.9, 40.8, 40.7, 40.2, 28.2. ESI/APCI calcd for C27H54N9O14 ([M
+ H]+) m/e 728.3785; found m/e 728.3780.
Procedure for MIC Determination. A solution of selected
bacteria was inoculated in the Trypticase Soy broth at 35 °C for
1-2 h. After which, the bacteria concentration was found and
diluted with broth, if necessary, to an absorption value of 0.08 to
0.1 at 625 nm. The adjusted inoculated medium (100 µL) was
diluted with 10 mL of broth and then applied to a 96-well microtiter
plate (50 µL). A series of solutions (50 µL each in 2-fold dilution)
of the tested compounds was added to the testing wells. The 96-
well plate was incubated at 35 °C for 12-18 h. The minimum
inhibitory concentration (MIC) is defined as the minimum concen-
tration of compound needed to inhibit the growth of bacteria. The
MIC results are repeated at least three times.
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450.
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resistance gene cluster aadE-sat4-aphA-3 disseminated among mul-
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Chemother. 2001, 45, 3267–3269.
Acknowledgment. We acknowledge the National Institutes
of Health (Grant AI053138) for financial support. We thank
Prof. Mobashery of Notre Dame University for providing the
pTZ19U-3 and pSF815 plasmids.
Supporting Information Available: Table listing the appropriate
analytical data including spectra of 1H and 13C NMR for the
synthesized compounds and 1H and 13C NMR, HPLC, and HRMS
of the assayed compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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aminoglycoside phosphotransferase Type III from Enterococcus:
overexpression, purification, and substrate specificity. Biochemistry
1994, 33, 6936–6944.
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