Rational design and synthesis of potent aminoglycoside antibiotics against resistant bacterial strains

Article abstract of DOI:10.1016/j.bmc.2010.11.065

Based on the structural information of biomacromolecule-aminoglycoside complexes, a series of kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construction of kanamycin derivatives, in which the C4′-position on

Full text of DOI:10.1016/j.bmc.2010.11.065

Bioorganic & Medicinal Chemistry 19 (2011) 30–40
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rational design and synthesis of potent aminoglycoside antibiotics
against resistant bacterial strains
Ri-Bai Yan a, Min Yuan b, Yanfen Wu a, Xuefu You b, Xin-Shan Ye a,
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, Peking University, School of Pharmaceutical Sciences, Peking University, Xue Yuan Road No. 38, Beijing 100191, China
b Institute of Medicinal Biotechnology, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the structural information of biomacromolecule–aminoglycoside complexes, a series of
kanamycin B analogues were rationally designed and synthesized. A convenient approach to the construc-
tion of kanamycin derivatives, in which the C40-position on ring I of neamine moiety was modified, was
developed. Most synthetic analogues exhibited good to excellent antibiotic activity against some typical
drug-resistant bacteria. The disclosed results suggested that the C40-position of aminoglycosides such as
kanamycin may be an ideal site for modification to gain new modifying enzyme-resistant aminoglycoside
antibiotics.
Received 16 October 2010
Revised 27 November 2010
Accepted 30 November 2010
Available online 4 December 2010
Keywords:
Antibiotics
Aminoglycoside
Drug design
Ó 2010 Elsevier Ltd. All rights reserved.
Chemical modification
Drug resistance
1. Introduction
or drug-modifying enzymes in complex with aminoglycosides have
beendisclosed, theknowledgeabout theinteractiondetails between
Antibiotic resistance has become a severe problem in clinic
throughout the world nowadays. Since the first antituberculosis
agent, streptomycin, was discovered in 1940s, aminoglycoside anti-
biotics have been used in therapy over sixty years. Like other classes
of antibiotics, however, aminoglycosides also face the challenges of
resistance because of extensive use. Among resistance mechanisms,
the most common mode is overexpression of aminoglycoside-mod-
ifying enzymes.1 These enzymes include three groups: aminoglyco-
side phosphotransferases (APHs), aminoglycoside acetyltrans
ferases (AACs), and aminoglycoside nucleotidyltransferases (ANTs).
In order to surmount resistance from enzymatic modification,
researchers have developed two strategies so far. One way is to de-
velop enzyme inhibitors,2 and the other is to develop enzyme-resis-
tant analogues of natural aminoglycosides.3 Over the past decade
many structural analogues of natural aminoglycosides were synthe-
sized, and some of the designed structures showed considerable
antibacterial activities.4 But in some cases, the modified drugs lost
their activity.5 One possible reason resulting in antibacterial activity
failuremaybe thataminoglycosidesbindtodrugtarget (16SrRNAA-
site) in a highly sequence-selective and specified style, and inappro-
priate modification would bring adverse influence on the natural
binding mode, thus leading to the decrease or even complete loss
of activity. In recent years, since some 3D structures of drug target
aminoglycosides and biomacromolecules affords medicinal chem-
ists brand-new insights for rational design of new aminoglycoside
analogues, andsomeadvanceshavebeenachieved.4c,6 Herein, were-
port a new approach to aminoglycoside modifications based on the
structural information.
2. Results and discussion
2.1. Design
Neamine (1) is a conserved pseudodisaccharide which consti-
tutes the core structure of many aminoglycosides such as paromo-
mycin (2), neomycin B (3), tobramycin, and kanamycin A (4) and B
(5) (Fig. 1). Previousinvestigations proved that it is the optimalphar-
macophore to manifest the antibiotic activity.7 The studies on 3D
structures of RNA–aminoglycoside complexes revealed that these
aminoglycosides bind to RNA in a similar way: the sugar rings have
equivalent orientations and the relatively same binding site and
most of the conserved contacts involve the neamine moiety, even
in the case of apramycin (6), a larruping aminoglycoside.8 In contrast
to the neamine unit, the binding of the other segments of aminogly-
cosides looks disorderly (Fig. 2). All these results suggest that nea-
mine moiety might be an ideal target for modifications to gain new
antibacterial characteristics. Among the natural aminoglycosides
containing neamine core, the common substituent fashions are
4,5- and 4,6-disubstitution on 2-deoxystreptamine (2-DOS, ring II),
⇑
Corresponding author. Tel.: +86 10 62014949; fax: +86 10 82802724.
E-mail address: xinshan@bjmu.edu.cn (X.-S. Ye).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.065

R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
31
R
6'
NH2
O
4'
O
5'
HO
HO
HO
HO
I
I
3'
1'
H2N
H N
4 2
2'
2
NH2
4
NH2
3
O
O
HO
NH2
II
II
5
O
NH2
NH2
OH
HO
O
6
1
5
OH
OH
O
OH
Neamine, 1
O
OH
H2N
R=OH, Paromomycin, 2
R=NH2, Neomycin B, 3
OH
NH2
O
O
H2N
HO
HO
HO
I
CH3
6'
HN
H N
4 2
OH
OH
R
O
O
II
O
NH2
HO
O
I
4'
6
O
H2N
II
NH2
OH
OH
O
HO
O
NH2
HO
III
NH2
OH
Apramycin, 6
R=OH, Kanamycin A, 4
R=NH2, Kanamycin B, 5
Figure 1. Structures of several representative aminoglycosides. The neamine core is composed of ring I and ring II.
Despite the lack of resemblance among various enzymes, the bind-
ing specificity for neamine unit still could be observed, for instance
in the cases of AAC(20)-Ic or APH(30)-IIIa.10 Because of the strict spec-
ificityexisting in the recognition between substrates and enzymes, if
some modifications are made on the neamine segment, the intro-
duced functional groups would probably hinder the recognition be-
tween the resulting analogues and some resistant enzymes, thus
they could resist the attack from these enzymes and keep antibiotic
activity against those bacteria expressing aminoglycoside-modify-
ing enzymes. To verify these hypotheses, after considering the syn-
thetic accessibility, we designed and synthesized a series of
kanamycin B derivatives, in which the original 40-hydroxyl group
on neamine segment was replaced by some other functional groups.
In the crystal structure of kanamycin A–RNA complex, a H-bond can
be observed between the 40-hydroxyl group of kanamycin A and the
phosphate oxygen atom of A1493 (Fig. 3). In addition, the cavity
formed by A site can accommodate a bulky substituent on neamine
ring I of apramycin. In order to retain these specialties, a series of
amide functionalities including small and bulky groups were served
as substituents which were arbitrarily designated as acetamido,
4-amino-butanoylamino, cyclohexylcarbonylamino, 2-(2-methyl-
benzamido)-acetamido, and piperidinyl-4-carbonylamino groups.
Figure 2. Crystal structures of three neamine-containing aminoglycosides bound to
A site. The complexes of apramycin (magenta, 1YRJ), kanamycin A (gray, 2ESI) and
neomycin B (cyanic, 2ET4) with the decoding site are superimposed (based on
nucleic acid chains). Only the RNA (yellow) of the kanamycin A complex is shown.
The neamine moiety is marked with Roman numerals I and II. Molecular graphics
images were produced using the UCSF Chimera package from the Resource for
Biocomputing, Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR-01081).9
one component of neamine. An exceptional case is apramycin, in
which the substituents are attached to the C40- and C60-positions
of ring I, another component of neamine. Though the chemical struc-
ture of apramycin is so dissimilar to that of the other aminoglycoside
members, the 3D structure shows that the neamine moiety still
binds to A site with a very similar mode, whether the binding posi-
tion or the binding conformation. That is to say, substituents
attached to C40- or C60-position of ring I in neamine segment have lit-
tle effect on the binding between drugs (aminoglycosides) and the
drug target (RNA); so it is likely that modification at the correspond-
ing positions in other aminoglycosides would not affect the original
binding modes, thus the resulting analogues would keep antibacte-
rial activity. On the other hand, for those structures of inhomoge-
neous resistant enzymes in complex with similar aminoglycosides,
the binding modes are different. An obvious fact is that the neamine
moiety, in binding sites, binding groups, binding force, binding con-
formations, etc., makes different contacts with these enzymes.8a
Figure 3. The crystal structure of kanamycin A–A site complex. Hydrogen bonds
between aminoglycoside and RNA are shown with cyanic lines. Water molecules
are indicated as red balls.

32
R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
2.2. Chemistry
R =
Me2N
AcNH
7
8
After undergoing many frustrations in direct modifications on
kanamycin B, an alternative approach to modify the ring I of kana-
mycin was developed. Our modification strategy included the fol-
lowing steps: (1) disconnecting the ring I from kanamycin B to
achieve a glycosyl acceptor containing ring II and ring III, (2) pre-
paring 4-amino-protected thioglycoside donor as the building
block of ring I, (3) reconstructing kanamycin B skeleton by the cou-
pling of glycosyl donor and acceptor, and (4) further modifications
to yield the target compounds.
For this purpose, as shown in Scheme 1, the commercially avail-
able kanamycin B (5) was firstly converted into perazido species
13 by the modified diazotransfer procedure.11 The primary hydroxyl
group at the C600-position of 13 was selectively protected by
treatment with tert-butyldiphenylchlorosilane in pyridine to afford
compound 14. The protection of C600-OH was proved to be necessary
for decreasing byproducts and convenient purification in the next
step. The selective protection of 30,40-diol in 14 with isopropylidene
provided compound 15. The removal of silyl protection at C600-OH of
NH2
O
O
R
HO
H2N
9
N
H
H2N
NH2
HN
O
NH2
10
HO
O
O
OH
OH
O
O
H
N
HO
11
12
NH2
N
H
O
NH
HN
O
Figure 4. Designed kanamycin B analogues.
Besides, dimethylamino group was also used as a substituent to
investigate the influence of the positively charged replacement
(Fig. 4).
N3
O
NH2
O
N3
O
a
c
b
HO
HO
HO
HO
HO
HO
N3
H2N
N3
NH2
N3
N3
O
O
O
N3
NH2
N3
HO
HO
HO
O
O
O
OH
OH
OH
OH
OTBDPS
OH
O
O
O
HO
HO
HO
13
5
14
N3
NH2
N3
N3
O
N3
O
N3
O
f
e
d
O
O
HO
HO
O
O
N3
N3
N3
N3
N3
N3
O
O
O
N3
N3
N3
HO
BnO
BnO
O
O
O
OTBDPS
OH
OBn
OBn
OBn
OBn
O
O
O
HO
BnO
BnO
17
15
16
N3
N3
N3
N3
HO
BnO
N3
O
BnO
OBn
OBn
O
N3
18
Scheme 1. Synthesis of glycosyl acceptor 18. Reagents and conditions: (a) (i) TfN3, NEt3, Cu2+, CH3CN, H2O; (ii) acetic anhydride, pyridine; (iii) NaOMe, MeOH; (b) TBDPSCl,
pyridine, DMAP, 59% over four steps; (c) 2,2-dimethoxypropane, CH2Cl2, CSA, 52%; (d) (i) TBAF, THF; (ii) NaH, BnBr, DMF, 82% over two steps; (e) AcOH, MeOH, 100%; (f) (i)
NaIO4, MeOH; (ii) n-BuNH2, MeOH, 88% over two steps.
Ph
Ph
c
HO
HO
BnO
b
O
O
a
O
N
O
O
O
O
HO
BnO
N3
N3
3 STol
STol
21
24
20
23
19
22
STol
OH
N3
N3
N3
HO
BnO
e
d
O
O
O
TrocHN
BnO
BnO
N3
N3
N3
STol
STol
STol
Scheme 2. Synthesis of glycosyl donor 24. Reagents and conditions: (a) NaH, BnBr, DMF, 92%; (b) CSA, MeOH; (c) (i) TsCl, pyridine; (ii) NaN3, DMF, 81% over three steps; (d) (i)
Dess–Martin’s periodinane, CH2Cl2; (ii) NaBH4, MeOH, 95% based on the recovery of starting material; (e) (i) Tf2O, pyridine, CH2Cl2; (ii) NH3, MeOH; (iii) TrocCl, NaHCO3, THF,
55% over three steps.

R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
33
15 and the subsequentbenzylation of the free hydroxyl groups affor-
ded compound 16, which was treated with acetic acid to give the diol
17. Finally, the desired glycosyl acceptor 18 was prepared in 88% iso-
lated yield from 17 by two steps according to the reported
procedure.12
whereas compounds 8, 10, 11, 12 had MIC values close to that of
controls. The results showed that the antibacterial activities of
these compounds were retained or even enhanced in some cases.
The most significant fact was that these compounds can inhibit
the growth of drug-resistant bacteria, just as expected.
The synthesis of glycosyl donor was started from thioglycoside
19.13 As displayed in Scheme 2, benzylation of 19 gave compound
20 smoothly. Removal of the benzylidene group from 20 led to diol
21. Tosylation of the C6-hydroxyl group of 21 and the subsequent
nucleophilic displacement with sodium azide produced compound
22. Oxidation of 22 with Dess–Martin’s periodinane and subsequent
reduction of the resulting 4-ketone with sodium borohydride gave
the C4-configuration-inversed compound 23. Compound 23 reacted
with triflyl anhydride to afford a triflate intermediate, which was
successively treated with ammonia and 2,2,2-trichloroethoxycar-
bonyl chloride (TrocCl), providing the desired thioglycoside donor
24. This glycosyl donor possesses an azido group at the C-2 position,
which would facilitate forming a-glycosidic bond in the following
glycosylation under the influence of anomeric effect.
With the glycosyl donor and acceptor in hand, coupling was
conducted by N-iodosuccinimide (NIS)/TfOH-promoted glycosyla-
tion (Scheme 3). As expected, the a-anomeric product 25 was the
Klebsiella pneumoniae is a common nosocomial pathogen caus-
ing severe morbidity and mortality in paediatric patients. K. pneu-
moniae K6 (ATCC 700603) is a clinical isolate that is resistant to
some aminoglycosides (caused by ANT(200)).14 Our antimicrobial
susceptibility test showed that it was also resistant to kanamycin
A (MIC = 64
search, compounds 8 and 9 showed fairly good execution, with the
MIC values of 2 and 1 g/mL, respectively; while compound 12 had
lg/mL) and kanamycin B (MIC = 16 lg/mL). In our re-
l
somewhat better activity than kanamycin.
P. aeruginosa is an opportunistic human pathogen and the lead-
ing cause of nosocomial infection, which is frequently isolated
from patients with complicated urinary tract infections. The ubiq-
uity of genes encoding aminoglycoside-modifying enzymes includ-
ing aph(30)-IIb in P. aeruginosa leads to aminoglycoside
resistance.15 Using P. aeruginosa ATCC 27853 as evaluation object,
all newly synthesized compounds except 7 displayed better bacte-
ricidal effect than controls. Again, compound 9 showed preeminent
major product. Treatment of 25 with zinc powder in the presence
of acetic acid to remove Troc group unfortunately resulted in the
formation of a mess of complicated products. Therefore, compound
25 was subjected to hydrolysis under basic conditions to furnish
the key pseudotrisaccharide 26.
antibiotic efficacy (MIC = 0.5 lg/mL).
Methicillin-resistant S. aureus (MRSA) is the leading cause of
bacterial infections. Many MRSA strains contain genes encoded
for APH(30), ANT(40), and AAC(60)/APH(200), which render the bacte-
ria resistant to many aminoglycosides.16 Once again, the antimi-
crobial susceptibility test gave satisfactory results. With the
exception of 7, all new derivatives displayed better bactericidal ef-
fect against ATCC 33591 than controls. Compounds 9, 10, 12 were
able to inhibit the growth of the tested strains in low concentra-
Compound 26 was treated with iodomethane in the presence of
sodium hydride to obtain the dimethylated product 27, while acet-
ylation of 26 with acetic anhydride gave compound 28. Reaction of
26 with 4-chlorobutanoyl chloride using sodium bicarbonate as
acid scavenger followed by treatment with sodium azide provided
29 in 80% isolated yield. On the other hand, compound 26 was cou-
pled with different carboxylic acids using 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as the
condensation reagent in the presence of Hünig’s base (N,N-diiso-
propylethylamine, DIPEA), affording compounds 30, 31, and 32,
respectively (Scheme 3). Thus, all precursors for final deprotection
were achieved. After reduction of azido groups with H2S followed
by debenzylation via catalytic hydrogenolysis, all these precursors
were smoothly transformed into final products 7–12.
tions (2, 4 and 2 lg/mL, respectively).
Staphylococcus epidermidis 08–18 is a methicillin-resistant clin-
ical isolate obtained from a patient at the Beijing Friendship Hospi-
tal in 2008. The susceptibility test results showed that it was also
resistant to kanamycin B. Except 7, all newly synthesized deriva-
tives of kanamycin B showed good antibiotic effect, especially,
compounds 9 and 12 inhibited the growth of this strain at a very
low concentration (MIC = 0.25 lg/mL in both cases).
Staphylococcus epidermidis 07–9 is also a clinical isolate that was
obtained from a patient at the Beijing Friendship Hospital in 2007.
PCR assay indicated it contains aph(30)-IIIa gene. The susceptibility
test results showed that it was resistant to both new compounds
and controls. However, compared with controls, compound 9 still
showed moderate antibiotic activity.
In the case of E. coli BL21 (pET29a), a laboratory strain express-
ing APH(30)-Ia, the susceptibility test results clearly indicated that
two derivatives of kanamycin B, compounds 9 and 12, can tolerate
the attack from APH(30)-Ia.
2.3. Biological assay
All compounds were tested against American Type Culture Col-
lection (ATCC) reference strains as well as clinical isolates of both
Gram-positive and Gram-negative bacteria, including pathogenic
and resistant strains, and the minimum inhibitory concentrations
(MIC) in lg/mL were determined by the microdilution assay. Kana-
mycin A and B served as controls (Table 1). Resistant strains cov-
ered low to high level resistant microbe, including Klebsiella
pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC
27853), methicillin-resistant Staphylococcus aureus (ATCC 33591),
Enterococcus faecalis (ATCC 29212), Mortierella alpine (ATCC32221),
methicillin-resistant Staphylococcus epidermidis 08–18, Staphylo-
coccus epidermidis 07–9, and Escherichia coli BL21 (pET29a). Staph-
ylococcus epidermidis 08–18 and Staphylococcus epidermidis 07–9
are clinical isolated drug-resistant strains. E. coli BL21 (pET29a) is
a recombinant strain harboring pET29a expressing vector which
contains the gene encoding APH(30)-Ia as a marker.
As seen in Table 1, with the exception of 7, all new compounds
exhibited good to excellent antibacterial activity against nonresis-
tant bacteria. A remarkable 2- to 16-folds enhancement against the
selected four reference strains (E. coli ATCC 25922, E. coli ATCC
35218, S. aureus ATCC 29213 and S. epidermidis ATCC 12228) was
observed for compound 9 when compared with the controls,
Enterococcus faecalis ATCC 29212 is a microbe expressing efflux
pump responsible for resistance to many structurally unrelated
antimicrobial agents.17 Unfortunately, except 9, which showed
somewhat better activity, all new compounds showed similar or
higher MIC values when compared with kanamycin. It seemed that
the efflux pump could extrude all these compounds. The antifungal
activity against Mortierella alpine with ATCC 32221 as the reference
strain was also checked, however, none of the compounds showed
obvious inhibitory efficacy at the concentration of 128 lg/mL.
Generally speaking, all 40-amide derivatives displayed consid-
erable antibiotic activity. It indicated that the proper modification
at C40-position of neamine segment would not impart negative
effect on the bioactivity. Among these analogues, compounds 9
and 12 displayed the most potent activity. Interestingly, in spite
of the difference in structures of 40-substituents, these two com-
pounds have some commonness. That is to say, there is a nitrogen
atom at the end of substituent, and three carbon–carbon bonds

34
R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
N3 N3
TrocHN
BnO
O
H2N
BnO
O
a
b
18
24
N3
N3
N3
N3
O
BnO
O
BnO
N3
N3
O
BnO
O
OBn
OBn
OBn
OBn
O
26
O
25
BnO
N3
N3
N3
NH2
O
R
O
R
HO
BnO
c , d, e, or f
g
N3
H2N
NH2
N3
O
BnO
O
HO
N3
NH2
O
O
OBn
OBn
OH
OH
O
O
BnO
R =
HO
N3
NH2
R =
Me2N
AcNH
Me2N
27
7
8
AcNH
28
29
30
O
O
N3
H2N
9
N
N
H
H
HN
HN
10
O
O
O
O
H
N
H
N
31
32
11
12
N
H
N
H
O
O
NH
NH
CbzN
HN
O
O
Scheme 3. Reconstruction of kanamycin B skeleton and further modifications. Reagents and conditions: (a) NIS, TfOH, 4 Å molecular sieves, CH2Cl2, ꢀ70 °C to rt, 57%,
a/
b = 1.74/1; (b) NaOH, H2O, 1,4-dioxane, reflux, 64%; (c) NaH, MeI, DMF, 50% for 27; (d) Ac2O, pyridine, 80% for 28; (e) (i) 4-chloro-butanoyl chloride, NaHCO3, THF; (ii) NaN3,
DMF, 80% for 29; (f) TBTU, carboxylic acids, DIPEA, DMF, 78% for 30, 75% for 31, 81% for 32; (g) (i) H2S, pyridine, triethylamine, H2O; (ii) H2, Pd/C, MeOH, H2O, HCl, 95% for 7,
87% for 8, 66% for 9, 61% for 10, 64% for 11, 60% for 12.
Table 1
The results of antibacterial activities of target compoundsa
Compound
MIC (lg/mL)
E. coli (Gꢀ)
S. aureus (G+)
S. epidermidis (G+)
K. pneumoniae (Gꢀ)
P. aeruginosa (Gꢀ)
E. faecalis (G+)
Fungus
k
E. coli BL21
a
b
c
d
e
f
g
h
i
j
l
Kan A
Kan B
7
8
9
10
11
12
2
4
128
2
1
2
4
4
64
8
1
2
2
1
32
2
128
>128
>128
16
2
4
1
0.25
16
8
>128
>128
>128
>128
32
>128
>128
128
64
16
64
2
>128
64
>128
4
0.5
8
64
64
>128
>128
32
64
64
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
128
8
64
>128
4
32
32
2
0.25
1
0.5
0.25
<0.06
0.25
0.5
1
1
2
1
32
64
8
4
2
4
2
64
2
8
0.25
32
2
0.25
a
a: ATCC 25922; b: ATCC 35218; c: ATCC 29213; d: ATCC 33591; e: ATCC 12228; f: 08–18, methicillin-resistant clinical isolate; g: 07–9, clinical isolate expressing APH(30)-
IIIa; h: ATCC 700603; i: ATCC 27853; j: ATCC 29212; k: Mortierella alpine, ATCC 32221; l: pET29a, engineering strain expressing APH(30)-Ia. G+: Gram-positive; Gꢀ: Gram-
negative.
lie between the end nitrogen atom and carbonyl group in each
case. In contrast, compound 7, the sole 40-amine derivative of
kanamycin B, only showed poor activity occasionally. The possible
reason for this abnormality might be that the additional dimeth-
ylamino group can be protonated under test conditions, which
would result in a different binding mode to drug target (A site).
In the cases of 40-amide derivatives, however, the ability of 40-
N–H group forming hydrogen bond is still retained; as a result
these compounds would be able to bind to the A site in the sim-
ilar way to that of kanamycin, thus the antibiotic activity re-
mained. More importantly, several compounds exhibited
remarkable activity against some typical drug-resistant bacteria
that can express aminoglycoside-modifying enzymes. Obviously,
the introduced functional groups at C40 exerted some impact on
the interactions between these analogues and some resistant en-
zymes, probably by interference with the course of recognition.
Therefore, it would make the analogues no longer appropriate
substrates for the enzymes; as a result they could resist the at-
tack from these enzymes and keep antibiotic activity. The struc-
tural diversification of 40-amide manifested excellent latitude for
modification at this position, holding the potential to discover
more analogues with better activity.

R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
35
product was obtained as
a
colorless semisolid. 1H NMR
3. Conclusion
(500 MHz, CDCl3) d 7.70–7.65 (m, 4H), 7.47–7.39 (m, 6H), 5.54
(d, J = 3.5 Hz, 1H), 5.09 (d, J = 3.5 Hz, 1H), 4.13–4.09 (m, 1H),
4.01–3.90 (m, 4H), 3.84 (dd, J1 = 4.0 Hz, J2 = 11.0 Hz, 1H), 3.68–
3.45 (m, 9H), 3.42–3.27 (m, 4H), 2.94 (br s, 3H), 2.42 (ddd,
J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 1.77 (br s, 2H), 1.62 (ddd,
J1 = J2 = J3 = 12.5 Hz, 1H), 1.08 (s, 9H). 13C NMR (125 MHz, CDCl3)
d 135.60, 135.55, 132.74, 132.64, 130.00, 129.95, 127.88, 127.83,
99.40, 98.02, 84.06, 80.12, 75.29, 72.10, 72.01, 71.37, 71.22,
71.18, 70.03, 66.30, 63.80, 63.12, 58.90, 58.78, 51.17, 31.54,
26.82, 19.21. HRMS (ESI) calcd for C34H49N16O10Si (M+NH4+):
869.3581; found: 869.3584.
In conclusion, we have developed an accessible approach for the
construction of kanamycin derivatives based on the modifications
of ring I in neamine moiety. By using this protocol, a series of kana-
mycin analogues were rationally designed and synthesized. Among
these synthetic analogues, some compounds such as 9 and 12
exhibited remarkable antibiotic activity against drug-resistant bac-
teria. These results also designated that the C40-position on nea-
mine is
a noticeable site for modifications to gain new
antibacterial agents with resistance to aminoglycoside-modifying
enzymes.
4.1.2. 30,40-O-Isopropylidene-600-O-tert-butyldiphenylsilyl-
pentaazidokanamycin B (15)
4. Experimental
4.1. Chemistry
To a solution of 14 (0.71 g, 0.83 mmol) in dichloromethane
(20 mL) and 2,2-dimethoxypropane (20 mL) was added camphor-
sulfonic acid (10 mg, 0.04 mmol) and the mixture was stirred for
24 h at room temperature, then it was quenched with triethyl-
amine (0.5 mL). After removal of the solvent, the residue was puri-
fied by column chromatography on silica gel (petroleum ether/
ethyl acetate, 8:1 to 3:1) to afford three fractions: 0.24 g of 15,
the byproduct with a higher Rf value than 15 (which should be
the over-protected product based on the 1H NMR spectrum analy-
sis) and byproduct with a lower Rf value than 15. The byproducts
with higher Rf value was dissolved in the mixed solvent of dichlo-
romethane/methanol (15 mL/5 mL) and 20 mg of cation-exchange
resin was added into the mixture. The reaction mixture was stirred
at room temperature. After 3 h, TLC showed the reaction was com-
pleted. The mixture was filtered, and the filtrate was added trieth-
ylamine (0.5 mL). After removal of solvent, the crude product was
purified by column chromatography on silica gel using petroleum
ether/ethyl acetate (6:1 to 3:1) as eluent to give 0.14 g of 15. Thus,
0.38 g (0.43 mmol, 52% yield) of desired product was obtained in
all. 1H NMR (500 MHz, CDCl3) d 7.68–7.63 (m, 4H), 7.47–7.38 (m,
6H), 5.71 (d, J = 4.0 Hz, 1H), 5.02 (d, J = 3.0 Hz, 1H), 4.31–4.28 (m,
1H), 3.96–3.81 (m, 5H), 3.65–3.52 (m, 7H), 3.47–3.40 (m, 3H),
3.31–3.27 (m, 2H), 2.83 (d, J = 9.5 Hz, 1H), 2.60 (d, J = 3.0 Hz, 1H),
2.43 (ddd, J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 1.62 (ddd,
J1 = J2 = J3 = 13.0 Hz, 1H), 1.47 (s, 3H), 1.46 (s, 3H), 1.06 (s, 9H).
13C NMR (125 MHz, CDCl3) d 135.60, 135.55, 132.67, 132.60,
130.00, 129.94, 127.88, 127.83, 111.76, 100.06, 98.06, 85.37,
79.15, 75.68, 75.20, 74.98, 72.37, 72.07, 71.43, 69.92, 66.34,
63.81, 61.56, 59.05, 58.60, 51.68, 31.48, 26.81, 26.74, 26.43,
19.19. ESI-MS: 909 M+NH4+. Anal. Calcd for C37H49N15O10Si: C,
49.82; H, 5.54; N, 23.55. Found: C, 50.11; H, 5.46; N, 23.81.
General. Non-aqueous reactions were performed under nitrogen
atmosphere at room temperature, unless otherwise noted. All com-
mercial reagents were used without further purification unless
otherwise stated. Anhydrous pyridine and dichloromethane were
obtained by distilling commercial ones over CaH2 and anhydrous
DMF was obtained by distilling over P2O5 under reduced pressure.
Specific rotations were determined on RUDOLPH RESEARCH ANA-
LYTICAL AUTOPOL III automatic polarimeter at 20 °C. Routine 1H
and 13C nuclear magnetic resonance spectra were recorded on
the Varian INOVA-500. Samples were dissolved in deuterated chlo-
roform (CDCl3) or deuterium oxide (D2O) and tetramethylsilane
(TMS) was used as reference. Elemental analyses were performed
in PE-2400C analyzer. Mass spectra were recorded on PE SCIEX
QSTAR mass spectrometer (for low resolution ESI-MS) or Bruker
APEX IV mass spectrometer (for high resolution ESI-MS). Analytical
thin-layer chromatography (TLC) was performed on Merck Silica
Gel 60 F254. Compounds were visualized by UV light (254 nm)
and/or by staining with
a yellow solution containing Ce(N-
H4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24ꢁ4H2O (24.0 g) in 6% H2SO4
(500 mL) or ninhydrin solution in ethyl acetate (5%) followed by
heating.
4.1.1. 600-O-tert-Butyldiphenylsilyl-pentaazidokanamycin B (14)
To a solution of kanamycin B (2.00 g, 4.14 mmol) in water
(6 mL) was added CuSO4 (33 mg, 0.21 mmol) and the resulting
mixture was stirred for 15 min, then triethylamine (4.18 g,
41.4 mmol) was added in one portion. After the mixture was stir-
red for 20 min at ice-bath temperature, triflyl azide solution in ace-
tonitrile (21 mL, 1.5 N, 31.5 mmol) was added dropwise in a period
of 15 min. After 2 h, the ice-bath was removed and the mixture
was stirred for another 10 h at room temperature. Then the solvent
was removed and the crude product was treated with the mixture
of acetic anhydride (6 mL) and pyridine (9 mL). After stirring over-
night, the reaction mixture was poured into 150 mL of brine, and
the resulting precipitates were collected by filtration. The crude
product was purified by column chromatography on silica gel,
using petroleum ether/ethyl acetate (6:1 to 3:1) as eluent. The
resulting product was dissolved in 60 mL of methanol, and cata-
lytic amount of sodium methoxide was added. When TLC assay
showed that the reaction was completed, the reaction mixture
was neutralized with cation-exchange resin and the solvent was
removed. The yielded product (1.66 g, 2.71 mmol) was dissolved
in 30 mL of pyridine, tert-butyldiphenylchlorosilane (1.50 g,
5.5 mmol) and catalytic amount of DMAP were added to the solu-
tion. After stirring for 48 h at room temperature, the solvent was
removed and the crude product was purified by column chroma-
tography on silica gel using petroleum ether/ethyl acetate (3:1)
as eluent. Finally, 2.08 g (2.44 mmol, 59% overall yield) of title
4.1.3. 30,40-O-Isopropylidene-5,200,400,600-tetra-O-benzyl-pentaaz-
idokanamycin B (16)
To a solution of 15 (0.63 g, 0.71 mmol) in THF (15 mL) was
added TBAF (1.0 M in THF, 0.85 mL, 0.85 mmol). The resulting yel-
low solution was stirred for 30 min and then the solvent was re-
moved. The residue was purified by column chromatography on
silica gel using petroleum ether/ethyl acetate (3:1) as eluent to af-
ford a colorless semisolid, which was treated with sodium hydride
(0.16 g, 4.2 mmol, 60% dispersed in mineral oil) and benzyl
bromide (0.70 g, 4.1 mmol) in DMF (10 mL) for 4 h. The reaction
mixture was poured into 80 mL of water, and the aqueous phase
was extracted with ethyl acetate (3 ꢂ 10 mL). The combined
organic phase was washed with brine, dried over NaSO4 and
concentrated under vacuum. The crude product was purified by
column chromatography on silica gel (petroleum ether/ethyl
acetate 10:1) to give compound 16 (0.59 g, 0.58 mmol, 82%) as a
yellow semisolid. [a]
+133.3 (c 0.45, CHCl3). 1H NMR (500 MHz,
D
CDCl3) d 7.43–7.41 (m, 2H), 7.39–7.36 (m, 2H), 7.33–7.20 (m,
11H), 7.14–7.06 (m, 3H), 6.98–6.96 (m, 2H), 5.63 (d, J = 4.0 Hz,

36
R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
1H), 5.61 (d, J = 4.0 Hz, 1H), 4.95 (s, 2H), 4.81 (d, J = 12.0 Hz, 1H),
4.74 (d, J = 12.5 Hz, 1H), 4.62 (d, J = 11.0 Hz, 1H), 4.47 (d,
J = 12.5 Hz, 1H), 4.41–4.38 (m, 1H), 4.27–4.23 (m, 2H), 3.98 (dd,
J1 = 9.0 Hz, J2 = 11.0 Hz, 1H), 3.82–3.78 (m, 2H), 3.72 (t, J = 9.5 Hz,
1H), 3.65 (t, J = 9.5 Hz, 1H), 3.58–3.52 (m, 3H), 3.47–3.23 (m, 7H),
3.16 (dd, J1 = 3.0 Hz, J2 = 11.0 Hz, 1H), 2.40 (ddd, J1 = J2 = 4.5 Hz,
J3 = 13.0 Hz, 1H), 1.62 (ddd, J1 = J2 = J3 = 12.5 Hz, 1H), 1.45 (s, 3H),
1.43 (s, 3H). 13C NMR (125 MHz, CDCl3) d 137.85, 137.59, 137.33,
137.20, 128.49, 128.30, 128.23, 128.15, 128.10, 128.06, 127.75,
127.72, 127.47, 127.21, 126.16, 111.82, 97.74, 95.83, 83.23, 77.81,
77.48, 77.19, 75.89, 75.06, 74.99, 74.77, 74.46, 73.48, 73.08,
72.21, 70.10, 67.74, 65.31, 61.22, 60.18, 59.37, 51.70, 31.97,
4.1.6. p-Tolyl 2-deoxy-2-azido-3-O-benzyl-4,6-O-benzylidene-1-
thio- -glucopyranoside (20)
a-D
To a stirred solution of 19 (1.98 g, 4.95 mmol) in DMF (25 mL)
was added sodium hydride (0.40 g, 60% in mineral oil, 9.9 mmol)
and the resulting mixture was stirred for 20 min at room temper-
ature, then benzyl bromide (1.69 g, 10 mmol) was added dropwise.
After stirring for 3 h, the reaction mixture was poured into water
(150 mL) and the aqueous layer was extracted with ethyl acetate
(3 ꢂ 20 mL). The combined organic phase was washed with brine,
dried over Na2SO4 and concentrated under vacuum. The residue
was purified by column chromatography on silica gel using the
mixed solvent (petroleum ether/ethyl acetate/dichloromethane
8:1:1 to 5:1:1) as eluent to give product 20 (2.23 g, 4.55 mmol,
26.70, 26.38. ESI-MS: 1052 M+K+. Anal. Calcd for C49H55N15O10
C, 58.04; H, 5.47; N, 20.72. Found: C, 57.83; H, 5.55; N, 20.46.
:
92%) as a colorless oil. [
a]
+130.6 (c 0.75, CHCl3) 1H NMR
D
(500 MHz, CDCl3) d 7.52–7.49 (m, 2H), 7.42–7.28 (m, 10H), 7.13
(d, J = 8.5 Hz, 2H), 5.59 (s, 1H), 5.49 (d, J = 5.0 Hz, 1H), 4.97 (d,
J = 10.5 Hz, 1H), 4.83 (d, J = 11.0 Hz, 1H), 4.44 (dt, J1 = J2 = 5.0 Hz,
J3 = 10.0 Hz, 1H), 4.23 (dd, J1 = 5.0 Hz, J2 = 10.5 Hz, 1H), 3.99–3.93
(m, 2H), 3.78–3.73 (m, 2H), 2.34 (s, 3H). 13C NMR (125 MHz, CDCl3)
d 138.34, 137.65, 137.11, 133.10, 129.97, 129.08, 128.42, 128.30,
128.23, 127.94, 125.99, 101.47, 88.14, 82.74, 77.81, 75.17, 68.59,
63.69, 63.57, 21.14. ESI-MS: 528 M+K+. Anal. Calcd for
C27H27N3O4S: C, 66.24; H, 5.56; N, 8.58. Found: C, 66.08; H, 5.55;
N, 8.50.
4.1.4. 5,200,400,600-Tetra-O-benzyl-pentaazidokanamycin B (17)
A mixture of 16 (2.92 g, 2.88 mmol), acetic acid (1.5 mL) and
methanol (60 mL) was heated under reflux for 4 h. The solvent
was removed and the residue was purified by column chromatog-
raphy on silica gel (petroleum ether/ethyl acetate 6:1 to 3:1). Final-
ly, 2.80 g (2.87 mmol, 100%) of title compound was obtained as a
yellow semisolid. 1H NMR (500 MHz, CDCl3) d 7.42–7.36 (m, 4H),
7.34–7.18 (m, 11H), 7.12–7.07 (m, 3H), 6.96–6.94 (m, 2H), 5.59
(d, J = 4.0 Hz, 1H), 5.47 (d, J = 4.0 Hz, 1H), 4.98 (d, J = 12.0 Hz, 1H),
4.92 (d, J = 11.5 Hz, 1H), 4.80 (d, J = 11.5 Hz, 1H), 4.74 (d,
J = 12.0 Hz, 1H), 4.60 (d, J = 11.0 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H),
4.26 (d, J = 12.0 Hz, 1H), 4.22 (d, J = 11.0 Hz, 1H), 4.19–4.15 (m,
1H), 3.91 (dd, J1 = 9.0 Hz, J2 = 10.0 Hz, 1H), 3.81–3.77 (m. 2H),
3.67–3.63 (m, 2H), 3.58–3.33 (m, 8H), 3.29 (dd, J1 = 1.5 Hz,
J2 = 11.0 Hz, 1H), 3.13 (dd, J1 = 2.5 Hz, J2 = 11.0 Hz, 1H), 3.07 (dd,
J1 = 4.0 Hz, J2 = 10.5 Hz, 1H), 2.82 (br s, 1H), 2.66 (br s, 1H), 2.37
(ddd, J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 1.63 (ddd, J1 = J2 =
J3 = 12.5 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 137.79, 137.56,
137.30, 128.53, 128.33, 128.26, 128.20, 128.13, 128.10, 127.77,
127.52, 127.17, 125.92, 97.48, 95.90, 82.81, 77.78, 77.49, 75.92,
74.86, 74.47, 73.51, 73.18, 71.97, 71.20, 71.07, 70.09, 67.74,
65.28, 62.86, 60.07, 59.18, 51.21, 31.75. HRMS (ESI) calcd for
4.1.7. p-Tolyl 2,6-dideoxy-2,6-diazido-3-O-benzyl-1-thio-a-D-
glucopyranoside (22)
A mixture of 20 (4.25 g, 8.68 mmol) and camphorsulfonic acid
(100 mg) in dichloromethane (20 mL) and methanol (80 mL) was
heated under reflux for 6 h. The solvent was removed, and the res-
idue was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate (4:1 to 2:1) as eluent to give the cor-
responding diol intermediate, which was subsequently treated
with p-toluene sulfonyl chloride (2.37 g, 12.4 mmol) in the pres-
ence of pyridine (30 mL) overnight. Then the solvent was removed,
and the residue was purified by column chromatography on silica
gel using petroleum ether/ethyl acetate (6:1 to 4:1) as eluent to af-
ford the tosylate intermediate, which was mixed with sodium
azide (2.37 g, 12.4 mmol) and DMF (40 mL). After stirring at 60 °C
for 8 h, the reaction mixture was poured into 150 mL of water
and the aqueous layer was extracted with ethyl acetate
(3 ꢂ 40 mL). The combined organic phase was dried over Na2SO4
and concentrated under vacuum. The crude was purified by col-
umn chromatography on silica gel using petroleum ether/ethyl
acetate (8:1 to 4:1) as eluent to afford 22 (3.00 g, 7.03 mmol,
81%). 1H NMR (500 MHz, CDCl3) d 7.42–7.38 (m, 6H), 7.37–7.33
(m, 1H), 7.14 (d, J = 8.0 Hz, 2H), 5.52 (d, J = 5.5 Hz, 1H), 5.01 (d,
J = 11.0 Hz, 1H), 4.71 (d, J = 11.5 Hz, 1H), 4.33–4.29 (m, 1H), 3.89
(dd, J1 = 5.5 Hz, J2 = 10.5 Hz, 1H), 3.60 (t, J = 8.5 Hz, 1H), 3.54 (dt,
J1 = J2 = 3.5 Hz, J3 = 9.5 Hz, 1H), 3.49 (dd, J1 = 3.0 Hz, J2 = 13.0 Hz,
1H), 3.49 (dd, J1 = 5.5 Hz, J2 = 13.5 Hz, 1H), 2.34 (s, 3H), 2.15 (d,
J = 3.0 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 138.26, 137.69,
132.73, 129.98, 129.05, 128.85, 128.38, 128.15, 87.52, 81.33,
75.42, 71.55, 71.17, 63.78, 51.30, 21.11. ESI-MS: 444 M+NH4+. Anal.
Calcd for C20H22N6O3S: C, 56.32; H, 5.20; N, 19.70. Found: C, 56.30;
H, 4.98; N, 19.90.
C
46H52N15O10 (M+H+): 974.4016; found: 974.3997.
4.1.5. 1,3-Diazido-5-O-benzyl-6-O-(3-deoxy-3-azido-2,4,6-tri-O-
benzyl- -glucopyranosyl)-2-deoxystreptamine (18)
a-D
To a solution of 17 (4.60 g, 4.72 mmol) in methanol (80 mL)
was added NaIO4 (2.02 g, 9.44 mmol) in one portion at room tem-
perature. After stirring for 8 h, TLC showed that the starting mate-
rial was consumed. Then the reaction mixture was poured into
water (200 mL) and the aqueous layer was extracted with ethyl
acetate (3 ꢂ 40 mL). The combined organic phase was washed
with aqueous NaHCO3 and brine, dried over Na2SO4 and concen-
trated under vacuum. The crude product was dissolved in
methanol (200 mL), and n-butylamine (300 mg) was added. The
reaction was stirred overnight, and the solvent was removed.
The residue was purified by column chromatography on silica
gel using petroleum ether/ethyl acetate (6:1) as eluent to give
the desired product (3.18 g, 4.17 mmol, 88%) as
a colorless
semisolid. [
a]
+56.8 (c 0.95, CHCl3). 1H NMR (500 MHz, CDCl3)
D
d 7.43–7.09 (m, 20H), 5.62 (d, J = 3.5 Hz, 1H), 4.84–4.71 (m, 5H),
4.51 (d, J = 12.0 Hz, 1H), 4.35 (d, J = 11.0 Hz, 1H), 4.28 (d,
J = 12.5 Hz, 1H), 4.07–4.05 (m, 1H), 3.90 (t, J = 10.0 Hz, 1H),
3.60–3.53 (m, 2H), 3.47–3.30 (m, 7H), 2.48 (br s, 1H), 2.30–2.25
4.1.8. p-Tolyl 2,6-dideoxy-2,6-diazido-3-O-benzyl-1-thio-a-D-
galactopyranoside (23)
To a stirred solution of 22 (1.20 g, 2.81 mmol) in anhydrous
dichloromethane (30 mL) was added Dess–Martin’s periodinane
(1.44 g, 3.39 mmol) and the resulting mixture was stirred for 3 h
at room temperature. Subsequently, a solution of sodium dithio-
nite (1.50 g) and sodium bicarbonate (3.0 g) in water (30 mL)
was added. After stirring for 30 min, the organic layer was sepa-
rated out and the aqueous layer was extracted with dichlorometh-
(m, 1H), 1.53–1.45 (m, 1H). 13C NMR (125 MHz, CDCl3)
d
137.87, 137.66, 137.39, 137.28, 128.49, 128.32, 128.24, 128.18,
128.09, 127.97, 127.75, 127.70, 95.61, 82.45, 77.34, 76.91, 76.86,
76.32, 76.14, 74.80, 73.41, 72.93, 69.88, 67.74, 65.48, 60.72,
59.84, 31.76. ESI-MS: 784 M+Na+. Anal. Calcd for C40H43N9O7: C,
63.06; H, 5.69; N, 16.55. Found: C, 62.86; H, 5.69; N, 16.22.

R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
37
ane (2 ꢂ 10 mL).The combined organic phase was washed with
brine, dried over Na2SO4 and concentrated under vacuum. The
crude product was suspended in methanol (20 mL) and the result-
ing mixture was cooled by ice-bath, then sodium borohydride
(0.12 g, 3.16 mmol) was added in several portions. With the addi-
tion of sodium borohydride, the reaction mixture turned clear
soon. When TLC showed the reaction was completed, the solvent
was removed, and the residue was purified by column chromatog-
raphy on silica gel using petroleum ether/ethyl acetate (8:1 to 4:1)
as eluent to give product 23 (0.99 g, 2.32 mmol, 95% based on the
recovery of the starting material) as a colorless oil, and 0.16 g of 22
0.39 mmol), and 4Å molecular sieves (0.40 g) was added anhydrous
CH2Cl2 (5 mL) by a syringe. After the mixture was stirred for 20 min,
TfOH (10
l
L) was added. The mixture was stirred at ꢀ70 °C for
10 min and then gradually raised to room temperature over a period
of 1.5 h. Then triethylamine (0.2 mL) was added into the reaction
mixture, and the mixture was diluted with CH2Cl2 (10 mL), filtered,
and concentrated. Theresidue was purified by column chromatogra-
phy on silica gel (petroleum ether/ethyl acetate 10:1 to 5:1) to give
a-anomeric product (134 mg, 0.11 mmol, 36%) as well as b-anomer-
ic product (77 mg, 0.06 mmol, 21%) as white semisolids. For -ano-
a
mer 25: 1H NMR (500 MHz, CDCl3) d 7.43–7.19 (m, 20H), 7.13–7.09
(m, 3H), 6.95–6.93 (m, 2H), 5.60 (d, J = 3.5 Hz, 1H), 5.56 (d,
J = 3.5 Hz, 1H), 5.00 (d, J = 11.5 Hz, 1H), 4.94 (d, J = 12.0 Hz, 1H),
4.83–4.71 (m, 5H), 4.66 (d, J = 12.0 Hz, 1H), 4.61 (d, J = 11.0 Hz,
1H), 4.47 (d, J = 12.0 Hz, 1H), 4.35–4.32 (m, 1H), 4.26 (d,
J = 12.0 Hz, 1H), 4.23 (d, J = 11.0 Hz, 1H), 3.89 (t, J = 10.0 Hz, 1H),
3.81–3.77 (m, 2H), 3.70–3.61 (m, 3H), 3.58–3.27 (m, 10H), 3.14
(dd, J1 = 3.0 Hz, J2 = 11.0 Hz, 1H), 2.38 (ddd, J1 = J2 = 4.5 Hz,
J3 = 13.0 Hz, 1H), 1.63 (ddd, J1 = J2 = J3 = 12.0 Hz, 1H); 13C NMR
(125 MHz, CDCl3) d 154.02, 137.85, 137.59, 137.34, 137.26, 137.02,
128.55, 128.52, 128.32, 128.18, 128.15, 128.11, 127.72, 127.48,
127.21, 125.81, 97.14, 95.93, 82.93, 77.83, 77.77, 77.48, 76.15,
75.88, 75.04, 74.62, 74.51, 74.45, 73.51, 73.13, 70.79, 70.12, 67.75,
65.32, 63.42, 60.18, 59.31, 54.09, 51.58, 31.92. ESI-MS: 1259
M+Na+. Anal. Calcd for C56H59Cl3N16O11: C, 54.31; H, 4.80; N, 18.09.
Found: C, 54.59; H, 4.91; N, 17.88. For b-anomer: 1H NMR
(500 MHz, CDCl3) d 7.41–7.06 (m, 20H), 5.53 (d, J = 4.0 Hz, 1H),
4.90–4.85 (m, 3H), 4.81 (d, J = 11.5 Hz, 1H), 4.72–4.62 (m, 7H), 4.46
(d, J = 12.0 Hz, 1H), 4.27 (d, J = 11.0 Hz, 1H), 4.20 (d, J = 12.0 Hz,
1H), 3.93 (d, J = 10.0 Hz, 1H), 3.83 (t, J = 10.0 Hz, 1H), 3.73 (t,
J = 9.5 Hz, 1H), 3.61–3.51 (m, 4H), 3.44–3.33 (m, 5H), 3.29–3.23 (m,
1H), 3.19–3.12 (m, 2H), 3.06–3.03 (m, 1H), 2.37 (ddd,
J1 = J2 = 4.0 Hz, J3 = 13.5 Hz, 1H), 1.61 (ddd, J1 = J2 = J3 = 12.5 Hz,
1H); 13C NMR (125 MHz, CDCl3) d 154.06, 138.01, 137.88, 137.71,
137.42, 137.14, 128.68, 128.46, 128.26, 128.19, 128.10, 128.00,
127.89, 127.68, 127.59, 127.43, 101.03, 96.01, 80.47, 80.01, 77.71,
77.39, 76.04, 75.70, 74.69, 74.60, 74.13, 73.44, 72.86, 69.94, 67.48,
66.86, 65.47, 60.91, 60.44, 54.55, 51.22, 32.00. HRMS (ESI) calcd for
was also recovered. [a]
+87.6 (c 1.05, CHCl3). 1H NMR (500 MHz,
D
CDCl3) d 7.43–7.38 (m, 6H), 7.37–7.35 (m, 1H), 7.14 (d, J = 8.0 Hz,
2H), 5.54 (d, J = 5.5 Hz, 1H), 4.79 (d, J = 11.5 Hz, 1H), 4.69 (d,
J = 11.5 Hz, 1H), 4.44–4.42 (m, 1H), 4.22 (dd, J1 = 5.0 Hz,
J2 = 10.0 Hz, 1H), 3.99 (t, J = 1.5 Hz, 1H), 3.76 (dd, J1 = 3.5 Hz,
J2 = 10.5 Hz, 1H), 3.59 (dd, J1 = 7.5 Hz, J2 = 12.5 Hz, 1H), 3.36 (dd,
J1 = 5.0 Hz, J2 = 13.0 Hz, 1H), 2.41 (t, J = 1.5 Hz, 1H), 2.33 (s, 3H).
13C NMR (125 MHz, CDCl3) d 138.20, 136.75, 132.77, 129.97,
128.94, 128.78, 128.49, 128.10, 87.65, 77.54, 72.48, 69.95, 66.57,
+
59.52, 51.06, 21.12. ESI-MS: 444 M+NH4
. Anal. Calcd for
C20H22N6O3S: C, 56.32; H, 5.20; N, 19.70. Found: C, 56.39; H,
5.18; N, 19.85.
4.1.9. p-Tolyl 2,6-dideoxy-2,6-diazido-3-O-benzyl-4-deoxy-4-
(2,2,2-trichloroethoxycarbonylamino)-1-thio-
oside (24)
a-D-glucopyran-
To a solution of 23 (2.96 g, 6.94 mmol) in anhydrous dichloro-
methane (60 mL) was added pyridine (2.4 mL), then the solution
wascooledinaice-bathwhilestirring. After20 min, triflylanhydride
(3.92 g, 2.4 mL, 13.9 mmol) was added to the mixture dropwise over
2 h by a syringe. After completion of addition, the mixture was stir-
red for another 2 h. The reaction mixture was poured into the
ice–water mixture (200 mL), the pH was adjusted to ꢃ3 by the addi-
tion of solid KHSO4. The organic layer was separated out and the
aqueous layer was extracted with dichloromethane (2 ꢂ 10 mL).
The combined organic phase was washed with brine, dried over
Na2SO4 and concentrated under vacuum. The crude product was dis-
solved in THF (30 mL), then the resulting solution was transferred
into a sealed tube, and to the tube was added ammonia solution in
methanol (7 N, 10 mL). The sealed tube was kept at 50 °C for 48 h.
The solvent was removed, and the residue was purified by column
chromatography on silica gel using petroleum ether/ethyl acetate
(6:1 to 3:1) as eluent. The resulting product (1.55 g, 3.64 mmol)
was mixed with THF (30 mL), sodium bicarbonate (1.22 g,
14.52 mmol), and 2,2,2-trichloroethoxycarbonyl chloride (1.54 g,
7.28 mmol). The mixture was stirred for 4 h. After removal of the sol-
vent, the residue was purified by column chromatography on silica
gel using petroleum ether/ethyl acetate (10:1 to 8:1) as eluent to af-
ford the title compound 24 (2.07 g, 3.44 mmol, 55% based on the
recovery of the starting material) as a colorless syrup, and the start-
ing alcohol 23 (0.28 g) was also recovered. For compound 24: 1H
NMR (500 MHz, CDCl3) d 7.42–7.31 (m, 7H), 7.15 (d, J = 8.0 Hz, 2H),
5.55 (d, J = 5.0 Hz, 1H), 4.90 (d, J = 11.0 Hz, 2H), 4.77–4.66 (m, 3H),
4.49–4.46 (m, 1H), 3.96 (dd, J1 = 5.5 Hz, J2 = 10.0 Hz, 1H), 3.76 (t,
J = 10.0 Hz, 1H), 3.65 (t, J = 10.0 Hz, 1H), 3.44–3.36 (m, 2H), 2.34 (s,
3H); 13C NMR (125 MHz, CDCl3) d 154.10, 138.39, 137.01, 132.66,
130.04, 128.76, 128.65, 128.40, 128.29, 95.19, 87.30, 77.13, 74.96,
74.62, 71.13, 64.47, 54.55, 51.56, 21.12. Anal. Calcd for
C
56H59Cl3N16O11Na (M+Na+): 1259.3507, found: 1259.3517.
4.1.11. 40-Deoxy-40-amino-5,30,200,400,600-penta-O-benzyl-penta-
azido-kanamycin B (26)
To a solution of 25 (0.48 g, 0.39 mmol) in 1,4-dioxane (10 mL)
were added water (1.5 mL) and sodium hydroxide (0.10 g,
2.5 mmol), then the reaction mixture was heated under reflux for
3 h. After cooling to room temperature, the reaction mixture was
poured into brine (150 mL), and the aqueous layer was extracted
with dichloromethane (3 ꢂ 30 mL). The combined organic phase
was dried over Na2SO4, and concentrated in vacuo. The resulting
residue was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate as eluent (5:1 to 1:1) to afford the
title compound (0.26 g, 0.24 mmol, 64%) as a colorless semisolid.
1H NMR (500 MHz, CDCl3) d 7.43–7.23 (m, 16H), 7.22–7.19 (m,
4H), 7.14–7.08 (m, 3H), 6.96–6.93 (m, 2H), 5.61 (d, J = 3.5 Hz,
1H), 5.56 (d, J = 4.0 Hz, 1H), 5.03 (d, J = 12.0 Hz, 1H), 4.94 (d,
J = 12.0 Hz, 1H), 4.91 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 11.5 Hz, 1H),
4.74 (d, J = 11.5 Hz, 1H), 4.62 (d, J = 11.0 Hz, 1H), 4.60 (d,
J = 10.5 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.25 (d, J = 12.5 Hz, 1H),
4.22 (d, J = 11.5 Hz, 1H), 3.99–3.96 (m, 1H), 3.81–3.77 (m, 2H),
3.71–3.53 (m, 6H), 3.49–3.44 (m, 2H), 3.39 (dd, J1 = 3.5 Hz,
J2 = 10.5 Hz, 1H), 3.35 (t, J = 10.0 Hz, 1H), 3.29 (dd, J1 = 2.0 Hz,
J2 = 11.0 Hz, 1H), 3.21 (dd, J1 = 4.0 Hz, J2 = 10.0 Hz, 1H), 3.13 (dd,
J1 = 2.5 Hz, J2 = 11.0 Hz, 1H), 2.70 (t, J = 10.0 Hz, 1H), 2.37 (ddd,
J1 = J2 = 4.5 Hz, J3 = 13.5 Hz, 1H), 1.61 (ddd, J1 = J2 = J3 = 12.5 Hz,
1H). 13C NMR (125 MHz, CDCl3) d 137.87, 137.58, 137.35, 137.32,
128.64, 128.52, 128.31, 128.28, 128.18, 128.10, 127.74, 127.46,
C
23H24Cl3N7O4S: C, 45.97; H, 4.03; N, 16.32. ESI-MS: 622 M+Na+.
Found: C, 46.12; H, 4.16; N, 16.04.
4.1.10. 40-Deoxy-40-(2,2,2-trichloroethoxycarbonylamino)-
5,30,200,400,600-penta-O-benzyl-pentaazido-kanamycin B (25)
To a cold (ꢀ70 °C) mixture of glycosyl donor 24 (0.18 g,
0.30 mmol), acceptor 18 (0.30 g, 0.39 mmol), NIS (88 mg,

38
R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
127.15, 125.84, 97.54, 95.95, 83.04, 80.92, 77.87, 77.48, 75.89,
75.20, 75.00, 74.44, 73.51, 73.11, 73.03, 70.09, 67.74, 65.32,
63.69, 60.24, 59.49, 54.11, 51.83, 32.05. ESI-MS: 1063 M+H+. Anal.
Calcd for C53H58N16O9: C, 59.88; H, 5.50; N, 21.08. Found: C, 59.71;
H, 5.36; N, 20.99.
4.1.14. 40-Deoxy-40-(4-azidobutoylamino)-5,30,200,400,600-penta-O-
benzyl-pentaazido-kanamycin B (29)
To a stirred solution of 26 (85 mg, 0.08 mmol) was added so-
dium bicarbonate (27 mg, 0.32 mmol), followed by the addition
of 4-chlorobutanoyl chloride (45 mg, 0.32 mmol). After stirring
for 4 h, the reaction mixture was poured into saturated aqueous
NaHCO3 and stirred for 1 h. The aqueous layer was extracted with
ethyl acetate (3 ꢂ 15 mL). The combined organic layer was dried
over Na2SO4, filtered and concentrated in vacuo to afford crude
product, which was mixed with DMF (4 mL) and sodium azide
(20 mg, 0.31 mmol). After stirring at 80 °C overnight, the reaction
mixture was poured into brine (50 mL) while stirring and the
aqueous layer was extracted with ethyl acetate (3 ꢂ 15 mL). The
combined organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel using petroleum ether/ethyl acetate
(5:1 to 2:1) to give 29 (75 mg, 0.06 mmol, 80%) as a white semi-
solid. 1H NMR (500 MHz, CDCl3) d 7.42–7.11 (m, 23H), 7.12–7.00
(m, 4H), 6.95–6.94 (m, 2H), 5.60 (d, J = 3.5 Hz, 1H), 5.58 (d,
J = 3.5 Hz, 1H), 5.13 (d, J = 8.5 Hz, 1H), 5.00–4.93 (m, 2H),
4.82–4.69 (m, 3H), 4.61 (d, J = 11.0 Hz, 1H), 4.51 (d, J = 11.5 Hz,
1H), 4.47 (d, J = 12.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 1H), 4.23 (d,
J = 11.0 Hz, 1H), 4.18–4.14 (m, 1H), 3.93–3.77 (m, 4H), 3.71 (t,
J = 9.5 Hz, 1H), 3.65 (t, J = 9.5 Hz, 1H), 3.59–3.48 (m, 3H),
3.42–3.34 (m, 3H), 3.32–3.25 (m, 4H), 3.16–3.13 (m, 2H), 2.37
(ddd, J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 2.06–1.96 (m, 2H),
1.82–1.76 (m, 2H), 1.61 (ddd, J1 = J2 = J3 = 12.5 Hz, 1H). 13C NMR
4.1.12. 40-Deoxy-40-dimethylamino-5,30,200,400,600-penta-O-
benzyl-pentaazido-kanamycin B (27)
To a solution of 26 (64 mg, 0.06 mmol) in DMF (3 mL) was
added sodium hydride (60% in mineral oil, 20 mg, 0.5 mmol), fol-
lowed by the addition of iodomethane (60 mg, 0.42 mmol). After
stirring for 4 h, more sodium hydride (60% in mineral oil, 20 mg,
0.5 mmol) and iodomethane (60 mg, 0.42 mmol)) were added
and the mixture was stirred overnight. The reaction mixture was
poured into brine (50 mL), extracted with ethyl acetate
(3 ꢂ 15 mL). The combined organic phase was dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel using petroleum ether/ethyl
acetate (8:1 to 5:1) as eluent to give the title compound (33 mg,
0.03 mmol, 50%) as a white semisolid. 1H NMR (500 MHz, CDCl3)
d 7.43–7.18 (m, 15H), 7.11–7.07 (m, 3H), 6.95–6.93 (m, 2H), 5.60
(d, J = 3.5 Hz, 1H), 5.50 (d, J = 4.0 Hz, 1H), 5.02 (d, J = 11.5 Hz, 1H),
4.93 (d, J = 11.5 Hz, 1H), 4.83–4.72 (m, 4H), 4.59 (d, J = 11.0 Hz,
1H), 4.46 (d, J = 12.0 Hz, 1H), 4.37–4.33 (m, 1H), 4.24 (d,
J = 12.0 Hz, 1H), 4.21 (d, J = 11.0 Hz, 1H), 4.03 (t, J = 10.0 Hz, 1H),
3.80–3.76 (m, 2H), 3.68–3.63 (m, 2H), 3.59–3.45 (m, 5H), 3.39
(dd, J1 = 3.5 Hz, J2 = 10.5 Hz, 1H), 3.35 (t, J = 10.0 Hz, 1H), 3.28 (dd,
J1 = 2.0 Hz, J2 = 11.0 Hz, 1H), 3.24 (dd, J1 = 4.0 Hz, J2 = 10.0 Hz, 1H),
3.10 (dd, J1 = 11.0 Hz, J2 = 2.5 Hz, 1H), 2.64 (t, J = 10.0 Hz, 1H),
2.47 (s, 6H), 2.40 (ddd, J1 = J2 = 4.5 Hz, J3 = 13.5 Hz, 1H), 1.66 (ddd,
J1 = J2 = J3 = 12.5 Hz, 1H). 13C NMR (125 MHz, CDCl3) d 137.88,
137.71, 137.59, 137.34, 137.30, 128.52, 128.37, 128.31, 128.26,
128.19, 128.10, 127.97, 127.73, 127.45, 127.11, 125.78, 97.35,
95.97, 83.00, 77.85, 77.47, 75.88, 75.49, 75.02, 74.43, 73.51,
73.10, 70.11, 70.07, 67.72, 65.34, 65.13, 65.04, 60.24, 59.44,
52.67, 41.95, 32.10, 29.68. HRMS (ESI) calcd for C55H63N16O9
(M+H+): 1091.4958; found: 1091.4960.
(125 MHz, CDCl3)
d 171.87, 137.83, 137.57, 137.37, 137.32,
137.19, 128.64, 128.51, 128.44, 128.31, 128.17, 128.14, 128.09,
127.90, 127.73, 127.46, 127.23, 125.84, 96.96, 95.91, 82.97, 77.73,
77.47, 76.25, 75.86, 75.01, 74.44, 73.64, 73.50, 73.11, 71.60,
70.10, 67.72, 65.31, 63.12, 60.21, 59.49, 51.95, 51.52, 50.66,
32.91, 31.98, 24.33, 13.80. HRMS (ESI) calcd for C57H63N19O10Na
(M+Na+): 1196.4898; found: 1196.4894.
4.1.15. 40-Deoxy-40-cyclohexylcarbonylamino-5,30,200,400,600-
penta-O-benzyl-pentaazido-kanamycin B (30)
Compound 26 (65 mg, 0.06 mmol), cyclohexylcarboxylic acid
(16 mg, 0.12 mmol), DMF (4 mL), DIPEA (22 mg, 0.17 mmol), and
TBTU (47 mg, 0.17 mmol) were added to a flask in sequence,
and the mixture was stirred for 12 h. Then the reaction mixture
was poured into brine (30 mL) while stirring, and the aqueous layer
was extracted with ethyl acetate (3 ꢂ 6 mL). The combined organic
phase was dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel
using petroleum ether/ethyl acetate as eluent (4:1 to 1:1) to afford
the title compound (56 mg, 78%) as a white semisolid. 1H NMR
(500 MHz, CDCl3) d 7.43–7.19 (m, 20H), 7.14–7.08 (m, 3H), 6.95–
6.94 (m, 2H), 5.60 (d, J = 3.5 Hz, 1H), 5.59 (d, J = 4.0 Hz, 1H), 5.20 (d,
J = 8.0 Hz, 1H), 4.99 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 12.0 Hz, 1H),
4.81 (d, J = 12.0 Hz, 1H), 4.74 (d, J = 11.5 Hz, 1H), 4.71 (d,
J = 11.5 Hz, 1H), 4.61 (d, J = 11.0 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H),
4.47 (d, J = 12.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 1H), 4.22 (d,
J = 11.0 Hz, 1H), 4.18–4.15 (m, 1H), 3.95–3.86 (m, 2H), 3.81–3.77
(m, 2H), 3.72 (t, J = 9.5 Hz, 1H), 3.65 (d, J = 9.5 Hz, 1H), 3.58–3.48
(m, 3H), 3.40–3.34 (m, 3H), 3.31–3.24 (m, 3H), 3.14 (dd, J1 = 2.5 Hz,
J2 = 11.0 Hz, 1H), 2.36 (ddd, J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 1.86–
1.80 (m, 1H), 1.74–1.57 (m, 6H), 1.37–1.14 (m, 5H); 13C NMR
(125 MHz, CDCl3) d 176.14, 137.84, 137.57, 137.33, 137.28, 137.20,
128.51, 128.52, 128.31, 128.17, 128.14, 128.10, 127.73, 127.46,
127.22, 125.86, 97.01, 95.91, 83.00, 77.76, 77.47, 76.25, 75.86,
75.01, 74.44, 73.58, 73.50, 73.10, 71.76, 70.09, 67.71, 65.31, 62.97,
60.23, 59.53, 51.98, 51.25, 45.31, 32.01, 29.68, 29.18, 25.58, 25.53,
25.48. HRMS (ESI) calcd for C60H68N16O10Na (M+Na+): 1195.5197;
found: 1195.5177.
4.1.13. 40-Deoxy-40-acetamido-5,30,200,400,600-penta-O-benzyl-
pentaazido-kanamycin B (28)
Compound 26 (54 mg, 0.05 mmol) was dissolved in pyridine
(1.5 mL) and acetic anhydride (1 mL), and the mixture was stirred
for 6 h. The reaction mixture was poured into brine (30 mL) while
stirring, and the aqueous layer was extracted with ethyl acetate
(3 ꢂ 5 mL). The combined organic phase was dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel using petroleum ether/ethyl
acetate as eluent (4:1 to 1:1) to afford the title compound (45 mg,
0.04 mmol, 80%) as a white semisolid. 1H NMR (500 MHz, CDCl3) d
7.43–7.19 (m, 15H), 7.15–7.09 (m, 3H), 6.96–6.94 (m, 2H), 5.60 (d,
J = 3.5 Hz, 1H), 5.58 (d, J = 3.5 Hz, 1H), 5.06 (d, J = 8.5 Hz, 1H), 4.99
(d, J = 11.5 Hz, 1H), 4.94 (d, J = 11.5 Hz, 1H), 4.81 (d, J = 11.5 Hz,
1H), 4.77 (d, J = 11.5 Hz, 1H), 4.74 (d, J = 11.5 Hz, 1H), 4.61 (d,
J = 11.0 Hz, 1H), 4.51 (d, J = 11.5 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H),
4.25 (d, J = 12.5 Hz, 1H), 4.23 (d, J = 11.0 Hz, 1H), 4.17–4.13 (m,
1H), 3.89–3.77 (m, 4H), 3.71 (t, J = 9.5 Hz, 1H), 3.65 (t, J = 9.5 Hz,
1H), 3.58–3.47 (m, 3H), 3.40–3.34 (m, 3H), 3.31–3.27 (m, 3H), 3.14
(dd, J1 = 2.5 Hz, J2 = 11.0 Hz, 1H), 2.36 (ddd, J1 = J2 = 4.5 Hz,
J3 = 13.0 Hz, 1H), 1.76 (s, 3H), 1.61 (ddd, J1 = J2 = J3 = 12.5 Hz, 1H);
13C NMR (125 MHz, CDCl3) d 170.16, 137.83, 137.57, 137.38,
137.32, 137.19, 128.66, 128.50, 128.31, 128.17, 128.09, 127.72,
127.46, 127.22, 125.85, 96.93, 95.92, 82.99, 77.76, 77.65, 77.47,
76.22, 75.86, 75.02, 74.44, 73.75, 73.50, 73.10, 71.66, 70.10, 67.71,
65.30, 63.21, 60.23, 59.51, 51.94, 51.65, 31.99, 23.29. HRMS (ESI)
calcd for C55H61N16O10 (M+H+): 1105.4751, found: 1105.4746.

R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
39
4.1.16. 40-Deoxy-40-(2-(2-methylbenzamido)-acetamido)-
4.1.18. 40-Deoxy-40-dimethylamino-kanamycin B (7)
Yield 95%, white amorphous powder. [ +71.3 (c 0.75, H2O).
5,30,200,400,600-penta-O-benzyl-pentaazido-kanamycin B (31)
This compound was synthesized by the same procedure as de-
scribed in the preparation of 30, providing 31 as a crystalline pow-
der in 75% yield. 1H NMR (500 MHz, CDCl3) d 7.43–7.18 (m, 24H),
7.13–7.08 (m, 3H), 6.95–6.93 (m, 2H), 6.60 (d, J = 8.5 Hz, 1H),
6.48 (t, J = 4.5 Hz, 1H), 5.61 (d, J = 3.5 Hz, 1H), 5.51 (d, J = 3.5 Hz,
1H), 5.02 (d, J = 12.0 Hz, 1H), 4.92 (d, J = 12.0 Hz, 1H), 4.82–4.73
(m, 3H), 4.60 (d, J = 11.0 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H), 4.46 (d,
J = 12.0 Hz, 1H), 4.30–4.21 (m, 3H), 4.00–3.85 (m, 3H), 3.81–3.73
(m, 3H), 3.65–3.61 (m, 2H), 3.52–3.46 (m, 2H), 3.40–3.22 (m,
7H), 3.12 (dd, J1 = 3.0 Hz, J2 = 11.0 Hz, 1H), 2.45 (s, 3H), 2.27 (ddd,
J1 = J2 = 4.5 Hz, J3 = 13.0 Hz, 1H), 1.58 (ddd, J1 = J2 = J3 = 12.5 Hz,
1H); 13C NMR (125 MHz, CDCl3) d 170.36, 168.80, 137.84, 137.71,
137.56, 137.32, 136.47, 134.75, 131.23, 130.52, 128.51, 128.46,
128.30, 128.26, 128.17, 128.09, 127.91, 127.73, 127.70, 127.44,
127.20, 127.13, 125.88, 125.72, 97.28, 95.90, 82.87, 78.06, 77.71,
77.44, 77.09, 75.86, 75.02, 74.42, 74.24, 73.49, 73.10, 70.99,
70.06, 67.71, 65.32, 63.35, 60.17, 59.04, 51.88, 51.65, 43.84,
31.79, 20.05. HRMS (ESI) calcd for C63H67N17O11Na (M+Na+):
1260.5098; found: 1260.5088.
a]
D
1H NMR (500 MHz, D2O) d 6.09 (d, J = 4.0 Hz, 1H), 5.15 (d,
J = 4.0 Hz, 1H), 4.66 (dt, J1 = 3.0 Hz, J2 = J3 = 10.0 Hz, 1H), 4.58 (t,
J = 10.5 Hz, 1H), 4.16 (t, J = 10.0 Hz, 1H), 3.98–3.92 (m, 3H),
3.90–3.86 (m, 2H), 3.82–3.61 (m, 7H), 3.54–3.46 (m, 2H), 3.12 (s,
6H), 2.56 (ddd, J1 = 4.0 Hz, J2 = J3 = 12.5 Hz, 1H), 2.02 (ddd,
J1 = J2 = J3 = 12.5 Hz, 1H); 13C NMR (125 MHz, D2O) d 101.37,
96.34, 84.37, 79.03, 74.83, 73.74, 68.74, 67.13, 66.14, 65.48,
63.22, 60.66, 55.56, 55.02, 50.25, 48.68, 43.14, 41.06, 28.56. HRMS
(ESI) calcd for C20H43N6O9 (M+H+): 511.3086; found: 511.3067.
4.1.19. 40-Deoxy-40-acetamido-kanamycin B (8)
Yield 87%, white amorphous powder. [a] +80.6 (c 0.80, H2O).
D
1H NMR (500 MHz, D2O) d 6.06 (d, J = 3.5 Hz, 1H), 5.14 (d, 3.5 Hz,
1H), 4.25 (t, J = 10.0 Hz, 1H), 4.20–4.16 (m, 1H), 4.09 (t,
J = 10.0 Hz, 1H), 3.98–3.93 (m, 3H), 3.89–3.77 (m, 4H), 3.72 (t,
J = 10.0 Hz, 1H), 3.68–3.59 (m, 2H), 3.55 (dd, J1 = 4.0 Hz,
J2 = 10.5 Hz, 1H), 3.52 (t, J = 10.5 Hz, 1H), 3.36 (dd, J1 = 2.5 Hz,
J2 = 14.0 Hz, 1H), 3.16 (dd, J1 = 6.5 Hz, J2 = 14.0 Hz, 1H), 2.58–2.55
(m, 1H), 2.08 (s, 3H), 2.02 (ddd, J1 = J2 = J3 = 12.5 Hz, 1H); 13C
NMR (125 MHz, D2O) d 176.11, 101.39, 96.48, 84.44, 77.72, 75.04,
73.66, 68.87, 68.80, 66.13, 60.63, 55.60, 54.84, 53.27, 50.33,
49.09, 40.96, 28.53, 22.78. HRMS (ESI) calcd for C20H41N6O10
(M+H+): 525.2879; found: 525.2865.
4.1.17. 40-Deoxy-40-(N-benzoxycarbonyl-piperidinyl)-4-
carbonylamino-5,30,200,400,600-penta-O-benzyl-pentaazido-
kanamycin B (32)
This compound was synthesized by the same procedure as de-
scribed in the preparation of 30, providing 32 as a white semisolid
in 81% yield. 1H NMR (500 MHz, CDCl3) d 7.43–7.09 (m, 28H),
6.96–6.93 (m, 2H), 5.60 (d, J = 3.5 Hz, 1H), 5.59 (d, J = 3.5 Hz, 1H),
5.27 (d, J = 8.0 Hz, 1H), 5.11 (s, 2H), 4.98 (d, J = 12.0 Hz, 1H), 4.94
(d, J = 12.0 Hz, 1H), 4.81 (d, J = 12.0 Hz, 1H), 4.74 (d, J = 12.0 Hz,
1H), 4.73 (d, J = 11.5 Hz, 1H), 4.61 (d, J = 11.0 Hz, 1H), 4.49
(d, J = 12.0 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.25 (d, J = 12.0 Hz,
1H), 4.23 (d, J = 11.0 Hz, 1H), 4.17–4.14 (m, 3H), 3.94–3.84 (m, 2H),
3.81–3.77 (m, 2H), 3.72 (t, J = 9.5 Hz, 1H), 3.65 (t, J = 9.5 Hz, 1H),
3.58–3.48 (m, 3H), 3.40–3.28 (m, 5H), 3.24 (dd, J1 = 2.5 Hz,
J2 = 13.0 Hz, 1H), 3.14 (dd, J1 = 3.0 Hz, J2 = 11.0 Hz, 1H), 2.73 (br,
2H), 2.38–2.32 (m, 1H), 1.96–1.90 (m, 1H), 1.70–1.57 (m, 3H),
1.56–1.46 (m, 2H); 13C NMR (125 MHz, CDCl3) d 174.28, 155.04,
137.82, 137.55, 137.30, 137.16, 136.62, 128.58, 128.49, 128.30,
128.15, 128.07, 127.86, 127.70, 127.45, 127.22, 125.83, 96.95,
95.88, 82.95, 77.76, 77.70, 77.46, 76.30, 75.84, 74.99, 74.42, 73.48,
73.09, 71.65, 70.09, 67.71, 67.14, 65.29, 62.95, 60.19, 59.50, 51.93,
51.25, 43.21, 43.16, 42.85, 31.97, 28.38, 27.99. HRMS (ESI) calcd
for C67H73N17O12Na (M+Na+): 1330.5517; found: 1330.5516.
General procedure for deprotection of compounds 27–32. The
starting compound (0.03–0.08 mmol) was dissolved in the
mixture of pyridine (3 mL), triethylamine (2 mL), and water
(0.5 mL). Then, H2S gas was introduced into the mixture until it
turned deep-green. The mixture was stirred for 8–12 h at room
temperature. After TLC showed the reaction was completed, the
mixture was concentrated and the residue was passed through
a short column (silica gel) with eluents as the following: petro-
leum ether/ethyl acetate (25 mL/25 mL), ethyl acetate (50 mL),
ethyl acetate/methanol (25 mL/25 mL), methanol (50 mL), and
methanol/concentrated aqueous ammonia (100 mL/10 mL). The
crude product was then dissolved in 5 mL of methanol, and pH
value of the resulting solution was adjusted to 3–4 with hydro-
chloric acid (1 M). Then Pd/C (10%, 100 mg) was added. The mix-
ture was subjected to hydrogenolysis for 3–7 days. After TLC
showed that only one spot appeared, the mixture was filtered
through a pad of Celite. The filtrate was evaporated, and the
crude product was dissolved in deionized water (3 mL) and the
resulting solution was passed through a short column (packed
with C18 silica gel), the fraction was collected to yield the prod-
uct after lyophilization.
4.1.20. 40-Deoxy-40-(4-amino-butanoylamino)-kanamycin B (9)
Yield 66%, white amorphous powder. [a] +73.4 (c 0.35, H2O).
D
1H NMR (500 MHz, D2O) d 6.04 (d, J = 3.5 Hz, 1H), 5.14 (d, 3.5 Hz,
1H), 4.23 (t, J = 10.5 Hz, 1H), 4.21–4.18 (m, 1H), 4.08 (t, J = 9.0 Hz,
1H), 3.98–3.50 (m, 12H), 3.36 (dd, J1 = 3.0 Hz, J2 = 14.0 Hz, 1H),
3.16 (dd, J1 = 5.5 Hz, J2 = 13.5 Hz, 1H), 3.04 (t, J = 7.5 Hz, 2H),
2.58–2.55 (m, 1H), 2.49 (t, J = 7.5 Hz, 2H), 2.03–1.94 (m, 3H); 13C
NMR (125 MHz, D2O) d 176.80, 101.42, 96.75, 84.46, 78.08, 74.98,
73.70, 68.92, 68.76, 66.13, 60.61, 55.58, 54.91, 53.06, 50.28,
49.05, 40.89, 39.59, 33.10, 28.54, 23.40. HRMS (ESI) calcd for
C
22H46N7O10 (M+H+): 568.3301; found: 568.3300.
4.1.21. 40-Deoxy-40-cyclohexylcarbonylamino-kanamycin B (10)
Yield 61%, white amorphous powder. [ +67.2 (c 0.95, H2O).
a
]
D
1H NMR (500 MHz, D2O) d 6.04 (d, J = 4.0 Hz, 1H), 5.14 (d,
J = 3.5 Hz, 1H), 4.22 (t, J = 10.0 Hz, 1H), 4.20–4.17 (m, 1H), 4.07 (t,
J = 10.0 Hz, 1H), 3.99–3.78 (m, 7H), 3.73 (t, J = 10.0 Hz, 1H),
3.69–3.60 (m, 3H), 3.57–3.50 (m, 2H), 3.32 (dd, J1 = 2.5 Hz,
J2 = 13.5 Hz, 1H), 3.11 (dd, J1 = 5.5 Hz, J2 = 14.0 Hz, 1H), 2.59–2.55
(m, 1H), 2.37–2.31 (m, 1H), 2.02–1.95 (m, 1H), 1.84–1.75 (m,
4H), 1.67 (d, J = 12.0 Hz, 1H), 1.43–1.14 (m, 5H); 13C NMR
(125 MHz, D2O) d 182.22, 101.43, 96.85, 84.48, 78.12, 74.99,
73.72, 68.99, 68.75, 66.08, 63.27, 60.60, 55.59, 54.98, 52.69,
50.27, 49.05, 45.68, 40.79, 29.91, 28.56, 25.93, 25.80. HRMS (ESI)
calcd for C25H49N6O10 (M+H+): 593.3505; found: 593.3504.
4.1.22. 40-Deoxy-40-(2-(2-methylbenzamido)-acetamido)-
kanamycin B (11)
Yield 64%, yellow amorphous powder. [a
]D +64.9 (c 1.30, H2O). 1H
NMR (500 MHz, D2O) d7.47–7.42 (m, 2H), 7.35–7.29 (m, 2H), 6.07 (d,
J = 3.5 Hz, 1H), 5.14 (d, J = 3.0 Hz, 1H), 4.31(t, J = 10.0 Hz, 1H),
4.29–4.25 (m, 1H), 4.18–4.08 (m, 3H), 3.97–3.83 (m, 6H), 3.79 (dd,
J1 = 5.0 Hz, J2 = 12.5 Hz, 1H), 3.72 (t, J = 10.0 Hz, 1H), 3.70–3.61 (m,
2H), 3.59–3.50 (m, 2H), 3.40 (d, J = 12.0 Hz, 1H), 3.20 (dd,
J1 = 6.0 Hz, J2 = 13.5 Hz, 1H), 2.58–2.56 (m, 1H), 2.37 (s, 3H), 2.01
(ddd, J1 = J2 = J3 = 12.5 Hz, 1H); 13C NMR (125 MHz, D2O) d 174.76,
173.29, 136.59, 135.15, 131.66, 131.49, 127.83, 126.65, 101.42,
96.65, 84.47, 77.91, 75.03, 73.71, 68.79, 66.12, 65.98, 60.63, 55.60,
54.88, 53.46, 50.31, 49.09, 43.77, 40.91, 28.55, 19.44. HRMS (ESI)
calcd for C28H48N7O11 (M+H+): 658.3406; found: 658.3413.

40
R.-B. Yan et al. / Bioorg. Med. Chem. 19 (2011) 30–40
4.1.23. 40-Deoxy-40-(piperidinyl-4-carbonyl)amino-kanamycin B
(12)
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Yield 60%, white amorphous powder. [a]
D +65.6 (c 0.85, H2O). 1H
NMR (500 MHz, D2O) d 6.08 (d, J = 4.0 Hz, 1H), 5.14 (d, J = 3.5 Hz, 1H),
4.28 (t, J = 10.5 Hz, 1H), 4.24–4.17 (m, 1H), 4.09 (t, J = 10.0 Hz, 1H),
3.97–3.82 (m, 5H), 3.78 (dd, J1 = 5.0 Hz, J2 = 12.5 Hz, 1H), 3.71 (t,
J = 10.0 Hz, 1H), 3.67–3.59 (m, 3H), 3.55–3.44 (m, 4H), 3.33–3.26
(m, 1H), 3.18 (dd, J1 = 7.0 Hz, J2 = 14.5 Hz, 1H), 3.13–3.05 (m, 1H),
2.77–2.72 (m, 1H), 2.56–2.54 (m, 1 H), 2.10–1.98 (m, 2H), 1.91–
1.83 (m, 2H), 1.58–1.56 (m, 1H); 13C NMR (125 MHz, D2O) d
178.18, 101.42, 96.61, 84.47, 77.84, 75.03, 73.70, 68.89, 68.79,
66.12, 66.08, 60.63, 55.61, 54.92, 53.02, 50.31, 49.10, 43.81, 43.76,
40.97, 40.67, 28.55, 25.91, 25.60. HRMS (ESI) calcd for C24H48N7O10
(M+H+): 594.3457; found: 594.3453.
4.2. Antibacterial assay
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All newly synthesized compounds were tested in the form of
hydrochloride. The purity of all tested compounds was over 98%
according to the HPLC analysis results. Minimum inhibitory concen-
trations (MIC) in lg/mL were determined by the microdilution broth
method according to Clinical and Laboratory Standards Institute
(CLSI) guidelines. Inoculum was prepared by diluting actively grow-
ing broth cultures to a McFarland value of 0.5 (1–108 cfu/mL). Anti-
biotic dilutions were made by dispensing 0.1 mL into each of the 96
wells of a standard microtitre tray in 2-fold dilutions. The inoculum
was added to give a final concentration of 5–105 cfu/mL. The trays
were covered and incubated at 37 °C for 16–20 h. The MIC was de-
fined as the lowest concentration of drug inhibiting visible growth.
The microdilution broth susceptibility testing was performed in
triplicate.
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Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China and the grant (2009ZX09501-011)
from the Ministry of Science and Technology of China.
Supplementary data
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Supplementary data associated with this article can be found, in
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