Regioselective modification of amino groups in aminoglycosides based on cyclic carbamate formation

Article abstract of DOI:10.1016/j.tet.2008.07.022

Conditions for regioselective introduction of cyclic carbamate into per-N-Cbz neamine and per-N-Cbz kanamycin A have been found. The position and number of cyclic carbamate formed in these two aminoglycosides was controllable. On the base of selective cyclic carbamate formation, regioselective modification on N-1, N-6′ or both amino groups in neamine, and on N-6′, N-3″ or both amino groups in kanamycin A was achieved by ring-opening reaction with amines. A new neamine dimer linked at the N-1 was also synthesized with this method.

Full text of DOI:10.1016/j.tet.2008.07.022

Tetrahedron 64 (2008) 9078–9087
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Regioselective modification of amino groups in aminoglycosides based on
cyclic carbamate formation
*
Guihui Chen, Pan Pan, Yun Yao, Ying Chen, Xiangbao Meng, Zhongjun Li
Department of Chemical Biology, School of Pharmaceutical Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Conditions for regioselective introduction of cyclic carbamate into per-N-Cbz neamine and per-N-Cbz
kanamycin A have been found. The position and number of cyclic carbamate formed in these two
aminoglycosides was controllable. On the base of selective cyclic carbamate formation, regioselective
modification on N-1, N-60 or both amino groups in neamine, and on N-60, N-300 or both amino groups in
kanamycin A was achieved by ring-opening reaction with amines. A new neamine dimer linked at the
N-1 was also synthesized with this method.
Received 12 March 2008
Received in revised form 4 July 2008
Accepted 8 July 2008
Available online 12 July 2008
Keywords:
Cyclic carbamate
Regioselectivity
Ó 2008 Elsevier Ltd. All rights reserved.
Amino group modification
1. Introduction
1970s. They introduced several cyclic carbamates into different
aminoglycosides for the purpose of modifying specific hydroxyl
Aminoglycosides are potent antibacterial agents against serious
Gram-negative infections. This kind of antibiotics bind to the ri-
bosomal decoding site and reduce the fidelity of protein synthesis,
and eventually kill the bacteria.1–4 However, relative toxicity and
resistance development against aminoglycosides has significantly
limited their clinical applications.5–7 Many aminoglycosides that
are effective towards the resistant strain are semi-synthetic prod-
ucts. Many of them were developed by selective modification on
specific amino groups in naturally existing aminoglycosides, such
as neomycin B 1, neamine 2 or kanamycin A 3. The most successful
example is amikacin 4 that has an (S)-4-amino-2-hydroxybutyryl
(AHB) group at the N-1 position. This compound holds a wider
spectrum than its precursor kanamycin A8 (Fig. 1). Another nea-
mine derivative Gb-R-neamine 5 bearing a nucleobase with an
arginine as a linker at N-60 showed some inhibitory activity for HIV
TAR–Tat9 (Fig. 1).
groups.23,24 But they did not find the regioselectivity of cyclic
carbamate formation and made no attempt to modify the amino
groups in these aminoglycosides.
In order to extend the existing amino group modification
methods, we focused on the regioselective cyclic carbamate for-
mation in certain aminoglycosides. It is known that cyclic carba-
mate could form in N-Cbz protected aminoglycosides with the aid
of NaH.23,24 By careful adjustment of ring forming conditions, we
succeed in controlling the number and position of cyclic carbamate
formed in per-N-Cbz neamine and per-N-Cbz kanamycin A. The
mono and bicyclic carbamates formed this way could easily un-
dergo ring-opening reaction under the attack of amines to form
urea derivatives. It is a new method to achieve regioselective
modification of certain amino groups in aminoglycosides.
2. Results and discussion
In the search for more molecules that possess antibacterial and
antiresistance activities, regioselective modification on the amino
groups in aminoglycosides is an important strategy. Methods
for protecting unhindered amino groups have been extensively
studied, including metal-mediated chelation,10–14 regioselective
Staudinger reduction of azides,15–17 enzymatic approaches18 and
cyclic carbamate formation.19–24 The cyclic carbamate method in
aminoglycosides was first used by Kumar and Umezawa in late
2.1. Selective introduction of cyclic carbamate in
aminoglycosides
To achieve the regioselective modification of aminoglycosides,
we started to investigate the regioselectivity of cyclic carbamate
formation in per-N-Cbz neamine and per-N-Cbz kanamycin A. In
neamine, there are three amino groups, N-1, N-20 and N-60, with
adjacent hydroxyl group. Kumar and his co-workers had treated
compound 6 with NaH to give a bicyclic carbamate neamine
derivative 9, and under their reaction condition, no monocyclic
carbamate product had been found.23,24 By carefully adjusting the
* Corresponding author. Tel.: þ86 10 82801714; fax: þ86 10 62367134.
E-mail address: zjli@bjmu.edu.cn (Z. Li).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.07.022

G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
9079
NH2
O
NH2
O
HO
HO
HO
HO
H2N
O
N
HO
H2N
O
H2N
O
HO
NH2
N
N
O
HO
NH
NH2
NH2
NH
NH2
O
O
O
OH
OH
OH
NH2
O
HN
HO
OH
H
N
H
N
H2N
O
OH
O
OH
H2N
1
3
NH2
O
HO
HO
O
O
HO
HO
H2N
H2N
O
H2N
NH2
O
HO
OH
HO
NH2
O
H
N
HO
HO
OH
HO
NH2
5
H2N
H2N
O
O
O
OH
HO
NH2
O
HO
OH
OH
H2N
2
4
1. neomycin 2. neamine 3. kanamycin 4. amikacin 5. Gb-R-neamine
Figure 1. Structures of typical aminoglycosides.
Table 1
combination of NaH amount, reaction temperature and reaction
time, monocyclic carbamates 7, 8 and bicyclic carbamate 9 were
obtained, respectively, in considerable yields (Scheme 1). Due to
the poor solubility of the resulting cyclic carbamate derivatives in
organic solvents and subsequent difficulty of separation, the crude
products were acetylated, subjected to column chromatography
and then deacetylated to give the pure product. Detailed reaction
condition and yields were listed in Table 1.
As indicated by the results, cyclic carbamate formation was not
observed for N-20, O-3 in neamine, which was consistent with the
existing reports.22–24 This could be the result of ring strain and
steric hindrance.
In a similar way, cyclic carbamate could also form in per-N-Cbz
kanamycin A 10. Monocyclic carbamates 11 and 12 were obtained at
low temperature in 32% and 38%, respectively. At room tempera-
ture, bicyclic carbamate 14 was obtained in 63% yield.25 The
monocyclic six-membered-ring carbamate 15 couldn’t be synthe-
sized in one step. It was acquired by opening the carbamate on ring
I of compound 13 or 14 at a yield of 90% (Scheme 2).
Compounds 7, 8, 9, 11, 12, 13 and 14 are the key intermediates
for the further structure modification. Their structures were de-
termined by the NMR analysis of corresponding acetyl derivatives.
The positions of different protons and carbons were assigned by 1H
NMR, 13C NMR and 2D spectrum, and the chemical shift of sugar CH
or CH2 proton adjacent to the acetyl group had a significant
downfield shift. By comparing these shifts with their precursors,
the position of the acetyl groups could be determined, and sub-
sequently the position of cyclic carbamate could be confirmed.
We found that hydrogenolysis of the Cbz groups in compounds
11, 12, 13 and 14 could not be achieved. At the same time,
The yieldsa of cyclic carbamate derivatives of neamine
Entry
Condition
Compound (%)
7
8
9
1b
2
DMF, 6 equiv NaH, ꢀ40 ꢁC to ꢀ20 ꢁC, 72 h
DMF, 2 equiv NaH, 25 ꢁC, 6 h
DMF, 7 equiv NaH, 25 ꢁC, 2 h
53
10
4
4
51
7
3
8
62
3
a
The yields were calculated based on 6 after acetyl derivatizing purification.
Material 6 (32%) was recovered after purification.
b
hydrogenolysis of the Cbz groups in compound 15 was completed
smoothly under the same condition to give the desired product 16
in 98% yield (Scheme 2). We assumed that the existence of the five-
membered-ring carbamate brought about considerable ring strain
in ring III of kanamycin A, and it made the glycosidic bond of ring III
easy to cleave under the hydrogenolysis condition.
2.2. Regioselective modification of aminoglycosides by
ring-opening reaction
Opening the cyclic carbamate with amine could introduce
functional groups into aminoglycosides to produce urea de-
rivatives.26–28 On the base of regioselective cyclic carbamate
formation, we set out to synthesize derivatives with specific
structures. Neamine derivative 7 was deprotected by catalytic
hydrogenolysis in 96% yield. Derivative 17 underwent smooth ring-
opening reaction with different amines in water. The reaction was
completed within 2 h to furnish the urea derivatives 18, 19 and 20
(Scheme 3). This approach provided an effective alternative to
Cbz
HN
O
HO
HCl
CbzCl
Cbz
Ac2O
Pyridine
NaH
DMF
NaOH
dioxane
HO
HN
1
Cbz NH
MeOH
Na2CO3
H
N
O
HO
Cbz
HO
6(72%, two steps)
Cbz
H
N
H
N
O
O
+
HN
HO
HO
Cbz NH
O
O
O
O
O
Cbz
O
Cbz
Cbz
+
HN
HO
Cbz NH
HO
Cbz NH
HN
HN
H
N
O
O
O
NH
NH
O
HO
HO
HO
Cbz
HO
O
O
7
8
9
Scheme 1. Regioselective formation of cyclic carbamate in neamine.

9080
G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
Cbz
Cbz
HN
HN
O
O
HO
HO
HO
Cbz
Cbz
HO
HN
HN
HO
HO
H
N
H
N
O
O
HO
HO
NaH/DMF
-30 °C
Cbz
OH
Cbz
OH
O
O
Cbz
HN
+
O
O
O
HO
O
O
O
OH
HO
Cbz
HO
HN
NH
HN
HO
H
N
O
O
12(38%)
11 (32%)
HO
Cbz
OH
CbzCl
O
3
Na2CO3
O
O
H
N
O
HO
OH
H
N
Cbz
NH
O
O
O
O
Cbz
Cbz
10(84%)
HO
HO
HN
HN
HO
HO
H
H
N
O
O
NaH/DMF
r.t.
N
HO
HO
Cbz
+
Cbz
OH
O
O
OH
O
O
O
OH
HO
O
O
NH
HN
O
13 (12%)
14 (63%)
O
O
H
N
H
N
O
O
O
O
Cbz
HO
H2N
HO
HN
HO
HO
O
H
O
NH2
Na2CO3
dioxane
H2
Pd/C
N
HO
HO
13 or 14
Cbz
OH
O
O
OH
OH
O
O
HO
HO
OH
H2N
H2N
16(98%)
15(90%)
Scheme 2. Regioselective formation of cyclic carbamate in kanamycin A.
H2N
H2N
,
,
H
92%
18
R=
O
O
HO
HO
HO
H2N
HO
NH2
H2N
H2
H2N
RNH2
H2O
89%
94%
H2N
H
N
O
19 R=
O
NH
O
HO
Pd/C
HO
O
O
OH
,
20
NH2
R=
HN
R
17(96%)
Cbz
HN
HO
HO
Cbz NH
7
H2N
Cbz
HN
HO
HO
Cbz NH
O
O
HO
Cbz
HO
O
H2N
HN
Cbz
HN
H2N
H
N
H
N
O
O
HO
H
N
HO
O
HO
O
O
H2
O
O
HO
7
HO
pyridine
HO
O
HN
HN
dioxane
Pd/C
H2NCH2CH2NH2
HN
Cbz
H2N
HO
HO
HN
O
O
HO
Cbz
HN
NH
HN
H2N
HO
HN
NH2
H2N
Cbz NH
O
21(93%)
O
NH
HO
HO
HO
HO
23(87%)
22(92%)
Scheme 3. N-1 modification and synthesis of the neamine dimer.
modify the N-1 position of aminoglycosides, and previous re-
ports15,16 showed this to be a formidable task.
When bicyclic carbamate 9 was used as the substrate, the five-
membered-ring carbamate was opened selectively under mild
condition to produce N-1 modified monocyclic carbamates 24 and
25. The six-membered-ring was kept intact until the reaction
temperature rose up to 50 ꢁC. Under this condition, N-1 and N-60
modified compounds 28 and 29 were produced. In a same way,
compound 8 could be turned into N-60 modified derivatives 32 and
33 (Scheme 4).
Derivative 7 was treated with ethylene diamine in pyridine to
produce urea derivative 21 in 93% yield, which was subsequently
used in the second ring-opening reaction with compound 7 in
dioxane to furnish compound 22 in 92% yield. Hydrogenolysis
of the Cbz group in compound 22 gave desired new neamine
dimer 23 in 87% yield. To our knowledge, this is the first
reported dimer linked at the nitrogen atom of an aminoglyco-
side (Scheme 3).
After the synthesis of neamine derivatives, we began to in-
troduce amino groups into kanamycin A via the ring-opening

G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
9081
O
H
N
O
H
N
O
O
Cbz
O
O
HO
HO
HN
H2N
RNH2/pyridine
r.t.
H2
Cbz NH
H2N
H
N
H
N
O
O
Pd/C
HO
HO
OH
O
O
OH
HN
R
HN
R
24 (88%)
25 (84%)
26(93%)
27(91%)
9
HN R
HN R
HN
O
O
HN
O
O
Cbz
HO
HO
H2
Pd/C
RNH2/pyridine
50 °C
HO
HO
HN
H2N
Cbz NH
H2N
H
H
O
O
N
N
HO
HO
O
O
OH
OH
HN
R
HN
R
28(83%)
29(87%)
30(94%)
31(92%)
HN R
HN R
HN
O
O
HN
HO
HO
Cbz NH
O
O
Cbz
HN
RNH2/pyridine
50 °C
HO
H2
Pd/C
HO
8
H2N
H2N
H
O
O
N
NH2
HO
Cbz
HO
OH
OH
34 (94%)
35 (93%)
32 (87%)
33 (89%)
H
R =
R =
24, 26, 28, 30, 32, 34
25, 27, 29, 31, 33, 35
NH2
Scheme 4. Regioselective modification of neamine by ring open reaction.
reaction. As a result, all the expected products were produced
under different conditions in good yields (Scheme 5).
4.2. General procedure for hydrogenolysis
To a solution or suspension of glycoside (0.1 mmol) in MeOH/
H2O (10 ml/2 ml), HCl (1 M, 1 ml) and 10% Pd/C (50 mg) were
added. Evacuation was then carried out with hydrogen atmosphere
replacements (3ꢂ). The mixture was stirred for 8 h at room tem-
perature under an atmosphere of hydrogen at 5 atm. The catalyst
was removed by filtration and the filtrate was concentrated in
vacuo to furnish the product.
3. Conclusion
A versatile methodology to achieve the regioselective modifi-
cation of amino groups in per-N-Cbz neamine and per-N-Cbz
kanamycin A has been developed. The aminoglycoside derivatives
with desired cyclic carbamate structure were synthesized. The
regioselective modification at specific amino group of these two
aminoglycosides by ring-opening reaction was also achieved. These
results provided a valuable alternative to the structure modification
of certain aminoglycosides.
4.3. 1,3,20,60-Tetra-N-benzyloxycarbonyl-neamine (6)
To
a boiling solution of neomycin B trisulfate (20.00 g,
22.02 mmol) in methanol (500 ml), concentrated HCl (18.00 g,
12.1 N) was added dropwise in 30 min. The solution was then
refluxed until reactants were consumed. The light yellow reaction
solution was cooled to room temperature. This solvent was then
evaporated approximately half of its volume and the mixture was
cooled to 0 ꢁC. The solid precipitate formed was filtered to give
crude neamine hydrochloride (8.01 g).
4. Experimental
4.1. General
Proton magnetic resonance spectra were recorded in CDCl3 or
DMSO-d6 at 300 MHz with Jeol 300 spectrometer. 13C NMR spectra
were recorded at 75 Hz. Chemical shifts were reported in parts per
million downfield from tetramethylsilane. All NMR spectra were
recorded at ambient temperature. High-resolution electronspray
spectra were obtained from Bruker APEX IV-FTMS 7.0T Mass
spectrometer.
Reactions were monitored by thin layer chromatography on
silica gel 60 F254. Column chromatography was performed on silica
gel 60. Reagents and starting materials were purchased from
Aldrich or Acros and were used without further purification unless
otherwise noted. Chloroform was distilled from CaH2. Pyridine was
dried over CaH2.
A solution of CbzCl (16.00 g, 93.84 mmol) in acetone (30 ml) was
added dropwise to an aqueous saturated Na2CO3 solution (80 ml) of
acquired neamine hydrochloride at 0 ꢁC over 30 min. The reaction
mixture was vigorously stirred for 2 h in ice bath and 8 h at room
temperature to give a white precipitate. The precipitate was then
filtered and pulverized in 1 M HCl solution until the Na2CO3 was
fully neutralized. The white solid was then filtered again and dried
in vacuo (13.61 g, 72% for two steps).
1H NMR (300 MHz, DMSO-d6)
d 7.38–7.33 (m, 20H, Ph), 7.30–
7.14 (m, 2H, NHCO2), 6.96 (d, J¼9.9 Hz, 1H, NHCO2), 6.83 (s, 1H,
NHCO2), 5.13–4.86 (m, 11H), 3.63 (s, 1H), 3.44–3.10 (m, 8H), 1.75 (br
s, 1H, H-2eq), 1.31 (br s, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d
156.5, 156.1, 155.7, 137.2, 136.9, 128.3, 127.7, 127.3, 98.6, 81.5, 76.5,

9082
G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
Cbz
HN
HO
HO
H2N
HO
HO
O
O
Cbz
HN
H2N
HO
HO
H
N
O
O
NH2
OH
OH
H2
HO
RNH2/pyridine
r.t.
HO
Cbz
OH
O
11or12
O
Pd/C
O
O
HO
OH
HO
HN
HN
HN
HN
36 (91%)
37 (87%)
O
38 (93%)
39 (92%)
O
R
R
O
H
N
O
H
N
O
O
O
O
Cbz
HO
HN
HO
H2N
HO
H
HO
O
O
RNH2/pyridine
r.t.
N
H2
NH2
HO
Cbz
OH
HO
O
O
Pd/C
OH
O
HO
OH
O
HO
OH
HN
HN
HN
HN
O
O
40(84%)
41(93%)
R
R
13or 14
HN
R
HN R
O
O
HN
HO
HO
HN
HO
HO
O
O
Cbz
HN
H2N
HO
HO
H
RNH2/pyridine
O
O
H2
N
NH2
HO
HO
Cbz
OH
O
O
50 °C
Pd/C
OH
O
O
HO
OH
HO
OH
HN
HN
HN
HN
O
O
42 (92%)
43 (83%)
44(97%)
45(98%)
R
R
H
36, 38, 40, 41, 42, 44 R =
R =
37, 39, 43, 45
NH2
Scheme 5. Regioselective modification of kanamycin A by ring open reaction.
74.0, 71.4, 71.0, 70.6, 65.2, 65.1, 56.0, 51.1, 50.3, 41.9, 34.8; ESI-HRMS
solvent, ice water was added. The resulting white solid (0.23 g,
92.6%) was filtered and dried without further purification.
calcd for C44H51N4O14 ([MþH]þ) m/z 859.3396, found 859.3400.
1H NMR (300 MHz, DMSO-d6)
d 7.65 (s, 1H, NHCO2), 7.38–7.31
(m,15H, Ph), 6.98 (s, 1H, NHCO2), 6.73 (d, J¼7.5 Hz, 1H, NHCO2), 5.86
(d, J¼5.4 Hz, 1H, NHCO2), 5.17–4.88 (m, 8H), 3.91–3.83 (m, 1H),
3.62–3.10 (m, 9H), 1.98–1.94 (m, 1H, H-2eq), 1.47–1.35 (m, 1H, H-
4.4. 3,20,60-Tri-N-benzyloxycarbonyl-1,6-N,O-carbonyl-
neamine (7)
2ax); 13C NMR (75 MHz, DMSO-d6)
d 159.9, 156.5, 156.1, 155.7, 137.2,
To a solution of compound 6 (0.50 g, 0.58 mmol) in dry DMF
(20 ml), NaH (0.14 g, 3.50 mmol, 60% in mineral oil) was added
slowly at ꢀ40 ꢁC under vigorous stirring. After 12 h, the reaction
temperature was raised to ꢀ20 ꢁC, and the mixture was stirred for
60 h. HCl (1 M) was added to neutralize the solution. The solvent
was removed under reduced pressure, and the residue was pul-
verized in water to give a white solid (0.48 g). Due to the solubility
of the crude product, the purification of the product was achieved
through its acetyl derivative as described below.
To the solution of obtained solid in dry pyridine (15 ml), Ac2O
(5 ml) was added. The mixture was stirred for 24 h at room tem-
perature and MeOH (5 ml) was added to quench the reaction. The
solvent was removed and the residue was pulverized in water to
give a white solid. The solid was purified by chromatography to give
triacetyl derivative of compound 7.
136.8, 128.3, 127.7, 127.6, 127.5, 98.5, 83.3, 82.7, 72.4, 71.3, 71.1, 70.4,
65.4, 65.2, 55.9, 52.8, 51.6, 41.9, 32.5; ESI-HRMS calcd for
C
37H43N4O13 ([MþH]þ) m/z 751.2832, found 751.2804.
4.5. 1,3,20-Tri-N-benzyloxycarbonyl-60,40-N,O-carbonyl-
neamine (8)
To a solution of compound 6 (0.50 g, 0.58 mmol) in dry DMF
(20 ml), NaH (46 mg, 1.16 mmol, 60% in mineral oil) was added. The
mixture was stirred for 6 h at room temperature. HCl (1 M) was
added to neutralize the solution. The solvent was removed under
reduced pressure, and the residue was pulverized in water to give
a white solid (0.46 g). The product was purified through the same
process as compound 7 to give a white solid (0.22 g, 50.6%, yield
after three steps).
To a solution of compound 7 (0.29 g) in dioxane/H2O (30 ml/
5 ml), NaOH solution (1 M, 5 ml) was added at 0 ꢁC and the
resulting mixture was stirred for 45 min in ice bath. After com-
pletion of the reaction (monitored by TLC, CHCl3/MeOH 8:1), the
reaction was quenched with HCl (1 M, 4.8 ml). After removal of the
1H NMR (300 MHz, DMSO-d6)
d 7.35–7.31 (m, 17H, Ph and
NHCO2), 7.15 (br s, 1H, NHCO2), 6.99 (d, J¼8.7 Hz, 1H, NHCO2), 5.39
(d, J¼5.4 Hz, 1H), 5.13–4.73 (m, 9H), 3.88–3.01 (m, 8H), 1.75 (br s,
1H, H-2eq), 1.34–1.22 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)

G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
9083
d
156.2, 155.7, 155.5, 152.3, 137.2, 137.0, 129.0, 128.3, 127.7, 127.4,
d6) d 159.6, 156.6, 155.8, 155.6, 137.1, 137.0, 128.3, 127.8, 127.3, 101.1,
99.0, 82.3, 78.6, 76.5, 73.9, 67.8, 65.3, 65.1, 61.4, 55.9, 51.2, 50.0, 42.5,
34.8; ESI-HRMS calcd for C37H42N4O13Na ([MþNa]þ) m/z 773.2640,
found 773.2635.
97.6, 84.3, 79.0, 74.8, 74.3, 72.7, 72.3, 71.3, 70.7, 70.5, 65.3, 59.9, 58.5,
50.4, 49.6, 41.6, 34.7; ESI-HRMS calcd for C43H53N4O18 ([MþH]þ)
m/z 913.3360, found 913.3353.
Compound 12, white solid (135 mg, 38%). 1H NMR (300 MHz,
4.6. 3,20-Di-N-benzyloxycarbonyl-1,6:60,40-di-N,O-carbonyl-
DMSO-d6) d 7.89 (s, 1H, NHCO2), 7.33–7.30 (m, 17H, Ph and NHCO2),
neamine (9)
6.85 (br s, 1H, NHCO2), 5.56 (br s, 1H), 5.41 (d, J¼6.0 Hz, 1H), 5.37 (s,
1H), 5.30 (s, 1H), 5.09–4.85 (m, 9H), 4.35 (s, 1H), 3.81 (s, 2H), 3.56–
3.15 (m, 6H), 3.07 (br s, 1H), 1.70 (br s, 1H, H-2eq), 1.49–1.37 (m, 1H,
To a solution of compound 7 (0.50 g, 0.58 mmol) in dry DMF
(20 ml), NaH (0.16 g, 4.00 mmol, 60% in mineral oil) was added. The
mixture was stirred for 2 h at room temperature. HCl (1 M) was
added. The solvent was removed under reduced pressure, and the
residue was pulverized in water to give a white solid (0.48 g). The
product was purified through the same process as compound 7 to
give a white solid (0.23 g, 62.0%, yield after three steps).
H-2ax); 13C NMR (75 MHz, DMSO-d6)
d 159.4, 156.6, 155.7, 155.6,
137.2, 137.0, 128.4, 127.9, 127.8, 127.3, 101.3, 94.3, 84.7, 80.1, 76.8,
74.9, 74.8, 72.7, 72.3, 70.7, 70.5, 68.9, 65.4, 59.4, 56.8, 50.7, 49.8, 41.7,
34.8; ESI-HRMS calcd for C43H53N4O18 ([MþH]þ) m/z 913.3360,
found 913.3366.
1H NMR (300 MHz, DMSO-d6)
d
7.70 (s, 1H, NHCO2), 7.49 (d,
4.9. 1,3-Di-N-benzyloxycarbonyl-60,40:300,200-di-N,O-carbonyl-
kanamycin A (13) and 1,3-di-N-benzyloxycarbonyl-60,40:300,400-
di-N,O-di-carbonyl-kanamycin A (14)
J¼8.7 Hz, 1H, NHCO2), 7.36–7.31 (m, 11H, Ph and NHCO2), 6.95 (d,
J¼8.1 Hz, 1H, NHCO2), 5.75 (d, J¼5.1 Hz, 1H), 5.45 (s, 1H), 5.24 (s, 1H,
H-10), 5.13–4.96 (m, 4H), 4.76 (d, J¼12 Hz, 1H), 3.88–3.80 (m, 3H),
3.58–3.54 (m, 3H), 3.29 (br s, 1H), 3.03(t, J¼9.6 Hz, 1H), 1.93 (br s,
1H, H-2eq), 1.50–1.39 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
To a solution of per-N-Cbz kanamycin A (600 mg, 0.59 mmol) in
dry DMF (20 ml), NaH (235 mg, 5.88 mmol, 60% in mineral oil) was
added. The mixture was stirred for 2 h at room temperature. HCl
(1 M) was added to neutralize the solution. The solvent was re-
moved under reduced pressure, and the residue was pulverized in
water to give a white solid, most of which was the mixture of
compound 13 and 14. These two compounds could be separated by
chromatography on silica gel(CHCl3/MeOH/H2O 8:2:0.2).
d
160.0, 156.3, 155.6, 152.4, 137.1, 136.9, 128.4, 128.3, 127.8, 127.7,
127.6, 98.9, 83.4, 83.2, 78.5, 72.6, 67.7, 65.4, 65.3, 61.5, 55.8, 53.0,
51.4, 42.6, 32.5.
4.7. 1,3,60,200-Tetra-N-benzyloxycarbonyl-kanamycin A (10)
Solution of CbzCl (4.25 g, 24.92 mmol) in acetone (15 ml) was
added dropwise to an aqueous saturated Na2CO3 solution (40 ml) of
kanamycin A monosulfate (3.00 g, 5.15 mmol) at 0 ꢁC over 30 min.
This reaction mixture was vigorously stirred for 2 h in ice bath and
8 h at room temperature to give a white precipitate. The precipitate
was then filtered and pulverized in 1 M HCl until the Na2CO3 was
fully neutralized. The white solid was then filtered again and dried
in vacuo (4.14 g, 84%).
Compound 13, white solid (57 mg, 12%). 1H NMR (300 MHz,
DMSO-d6) d 7.86 (s, 1H, NHCO2), 7.34–7.29 (m, 13H, Ph and NHCO2),
5.68 (d, J¼6.0 Hz, 1H), 5.51 (d, J¼5.1 Hz, 1H), 5.39 (s, 1H), 5.09–4.87
(m, 5H), 4.76–4.68 (m, 2H), 4.58 (d, J¼8.4 Hz, 1H), 4.18 (br s, 1H),
3.86–3.19 (m, 10H), 2.96 (t, J¼9.6 Hz, 1H), 1.74–1.70 (m, 1H, H-2eq),
1.49–1.36 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d 159.6,
155.8, 155.5, 152.4, 137.1, 136.9, 128.4, 128.3, 127.8, 127.7, 101.3, 97.6,
84.8, 79.0, 78.0, 74.8, 74.2, 71.9, 71.3, 70.7, 69.9, 65.3, 62.8, 61.5, 59.9,
1H NMR (300 MHz, DMSO-d6)
d
7.37–7.31 (m, 21H, Ph and
58.5, 49.4, 42.6, 34.5; ESI-HRMS calcd for C36H44N4O17Na
NHCO2), 7.09 (d, J¼8.4 Hz, 1H, NHCO2), 7.01 (d, J¼9 Hz, 1H, NHCO2),
6.84 (br s, 1H, NHCO2), 5.47 (br s, 2H, H-10 and H-100), 5.00–4.80 (m,
11H), 4.22 (dd, J1¼4.8 Hz, J2¼5.4 Hz, 1H), 3.86–3.83 (m, 2H), 3.57–
3.25 (m, 10H), 3.08–3.06 (m, 1H), 1.83 (br s, 1H, H-2eq), 1.51–1.39 (m,
([MþNa]þ) m/z 827.2593, found 827.2618.
Compound 14, white solid (135 mg, 63%). 1H NMR (300 MHz,
DMSO-d6) d 7.92 (s, 1H, NHCO2), 7.40–7.29 (m, 13H, Ph and NHCO2),
5.79 (d, J¼6.0 Hz, 1H), 5.56 (d, J¼5.4 Hz, 1H), 5.44 (d, J¼6.3 Hz, 1H),
5.30 (s, 1H), 5.24 (s, 1H), 5.14–4.97 (m, 4H), 4.80 (d, J¼12.3 Hz, 1H),
4.38 (t, J¼5.4 Hz, 1H), 3.89–3.36 (m, 9H), 3.24 (br s, 1H), 3.01 (t,
1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d 156.6,156.5,155.8,155.6,
137.3, 137.1, 137.0, 128.3,127.7,127.5, 101.1, 98.5, 84.3, 80.2, 74.1, 72.8,
72.6, 72.3, 70.6, 70.4, 70.0, 67.0, 65.3, 65.0, 60.1, 56.5, 50.0, 49.6, 41.6,
34.4; ESI-HRMS calcd for C50H64N5O19 ([MþNH4]þ) m/z 1038.4190,
found 1038.4224.
J¼9.6 Hz, 1H), 1.72–1.68 (m, 1H, H-2eq), 1.56–1.42 (m, 1H, H-2ax); 13
C
NMR (75 MHz, DMSO-d6)
d 159.4, 155.7, 155.6, 152.4, 137.2, 136.9,
128.5, 128.4, 127.8, 127.7, 101.4, 94.3, 85.3, 80.0, 78.0, 76.7, 74.9, 74.6,
71.9, 70.0, 68.8, 65.4, 65.3, 61.5, 59.4, 56.7, 50.6, 49.5, 42.6, 34.6; ESI-
HRMS calcd for C36H45N4O17 ([MþH]þ) m/z 805.2774, found
805.2782.
4.8. 1,3,60-Tri-N-benzyloxycarbonyl-300,200-N,O-carbonyl-
kanamycin A (11) and 1,3,60-tri-N-benzyloxycarbonyl-
300,200-N,O-carbonyl-kanamycin A (12)
4.10. 1,3-Di-N-benzyloxycarbonyl-60,40-N,O-carbonyl-
To a solution of per-N-Cbz kanamycin A (400 mg, 0.39 mmol) in
dry DMF (20 ml), NaH (94 mg, 2.35 mmol, 60% in mineral oil) was
added slowly at ꢀ30 ꢁC under vigorous stirring and the mixture
was stirred for 72 h. HCl (1 M) was added to neutralize the solution.
The solvent was removed under reduced pressure, and the residue
was pulverized in water to give a white solid, most of which was the
mixture of compound 11 and 12. These two compounds could be
separated by chromatography on silica gel (CHCl3/MeOH/H2O
12:2:0.2).
kanamycin A (15)
To a solution of compound 14 (200 mg, 0.25 mmol) in dioxane/
H2O (20 ml/4 ml), saturated Na2CO3 solution (8 ml) was added. The
mixture was stirred for 12 h at 40 ꢁC. After the completion of the
reaction (monitored by TLC, CHCl3/MeOH/NH3$H2O 5:2:0.2), 0.2 M
HCl was added to adjust the pH to 8. After removal of the solvent,
ice water was added. The resulting white solid, compound 15
(175 mg, 90.3%), was filtered and dried without further purification.
Compound 15 could be prepared from compound 13 in the same
way.
Compound 11, white solid (113 mg, 32%). 1H NMR (300 MHz,
DMSO-d6) d 7.91 (s, 1H, NHCO2), 7.34–7.31 (m, 16H, Ph and NHCO2),
7.21 (d, J¼8.7 Hz, 1H, NHCO2), 6.87 (br s, 1H, NHCO2), 5.58 (br s, 1H),
5.53 (d, J¼5.7 Hz, 1H), 5.10–4.86 (m, 10H), 4.73 (dd, J1¼5.7 Hz,
J2¼5.1 Hz, 1H), 4.58 (d, J¼8.7 Hz, 1H), 4.48 (s, 2H), 4.24 (br s, 1H),
3.84–3.72 (m, 2H), 3.69–3.29 (m, 6H), 3.08–3.06 (m, 1H), 1.81–1.77
(m, 1H, H-2eq), 1.50–1.37 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-
1H NMR (300 MHz, DMSO-d6)
d 7.34 (s, 12H, Ph and NHCO2),
5.57–5.51 (m, 1H), 5.14–4.80 (m, 6H), 4.23 (br s, 1H), 3.86 (br s, 1H),
3.70 (br s, 2H), 3.49–3.04 (m, 6H), 2.79–2.72 (m, 1H), 1.88–1.83 (m,
1H, H-2eq), 1.23–1.17 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d
155.9, 155.5, 152.4, 137.1, 136.9, 128.3, 127.8, 101.2, 97.5, 84.9, 80.1,

9084
G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
77.9, 74.6, 72.7, 72.0, 69.9, 66.3, 65.2, 61.4, 60.3, 55.0, 50.0, 49.2, 42.6,
34.4; ESI-HRMS calcd for C35H47N4O16 ([MþH]þ) m/z 779.2992,
found 779.2990.
4.16. 3,20,60-Tri-N-benzyloxycarbonyl-1-N-{N-[2-amino-
ethyl]-amino-formyl}-neamine (21)
To a stirred solution of compound 7 (128 mg, 0.17 mmol) in
pyridine/MeOH (5 ml/1 ml), ethylene diamine (1 ml) was added at
room temperature. After 3 h, the reaction was complete according
to TLC (CHCl3/MeOH/NH3$H2O 4:3:1). The solvent was removed in
vacuo followed by addition of ice water (20 ml), and the resulting
white solid (129 mg, 94%) was filtered and dried without further
purification.
4.11. 60,40-N,O-Carbonyl-kanamycin A-trihydrochloride (16)
The title compound was prepared from compound 15 according
to Section 4.2 as a white solid (98%).
1H NMR (300 MHz, D2O)
d 5.34 (s, 1H), 4.92 (s, 1H), 4.10–4.05 (m,
1H), 3.94–3.76 (m, 3H), 3.64–3.37 (m, 10H), 3.12 (q, J¼10.2 Hz, 1H),
1H NMR (300 MHz, DMSO-d6)
NHCO), 5.08–4.85 (m, 7H), 3.49–2.97 (m, 15H), 1.88–1.84 (m, 1H, H-
eq), 1.24–1.12 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
158.8,
d 7.35–7.29 (m, 16H, Ph and
2.99–2.91 (m, 1H), 2.06–2.02 (m, 1H, H-2eq), 1.41–1.28 (m, 1H, H-
2
ax); 13C NMR (75 MHz, D2O)
d 155.2, 99.6, 98.8, 83.4, 81.1, 77.3,
2
d
73.3, 71.8, 70.6, 69.2, 68.9, 66.2, 60.7, 59.3, 54.2, 49.8, 48.1, 41.7, 31.9;
ESI-HRMS calcd for C19H35N4O12 ([MþH]þ) m/z 511.2246, found
511.2239.
156.9, 156.5, 156.1, 137.6, 137.5, 137.2, 128.7, 128.1, 127.9, 127.7, 98.9,
81.8, 76.9, 75.3, 71.6, 71.2, 70.8, 65.7, 65.6, 65.5, 56.3, 50.5, 50.4, 42.2,
41.8, 35.7; ESI-HRMS calcd for C39H51N6O13 ([MþH]þ) m/z 811.3519,
found 811.3509.
4.12. 1,6-N,O-Carbonyl-neamine-trihydrochloride (17)
4.17. The protected dimer of neamine (22)
The title compound was prepared from compound 7 according
to Section 4.2 (44 mg, 96%).
1H NMR (300 MHz, D2O)
d
5.86 (d, J¼3.3 Hz, 1H, H-10), 4.13 (t,
To a solution of compound 7 (75 mg, 0.1 mmol) in dioxane/H2O
(10 ml/3 ml), compound 21 (81 mg, 0.1 mmol) was added. The
resulting mixture was stirred for 6 h at 65 ꢁC. After removal of the
solvent, ice water was added. The resulting white solid (143 mg,
92%) was filtered and dried without further purification.
J¼9.0 Hz, 1H), 3.96–3.84 (m, 3H), 3.60–3.32 (m, 5H), 3.18–3.11 (m,
2H), 2.43–2.39 (m, 1H, H-2eq), 1.83–1.70 (m, 1H, H-2ax); 13C NMR
(75 MHz, D2O)
d 162.8, 96.4, 83.4, 80.0, 72.6, 71.1, 69.8, 68.7, 54.0,
53.6, 50.8, 40.6, 29.4; ESI-HRMS calcd for C13H25N4O7 ([MþH]þ) m/z
1H NMR (300 MHz, pyridine-d5)
d 8.03 (br s,1H, NHCO2), 7.68 (br
349.1717, found 349.1705.
s, 1H, NHCO2), 7.52 (br s, 1H, NHCO2), 7.21–6.91 (m, 16H, Ph and
NHCONH), 6.68 (br s, 1H, NHCONH), 5.75 (br s, 2H), 5.65 (br s, 1H),
5.13–4.80 (m, 6H), 4.26–4.18 (m, 3H), 3.75–3.67 (m, 7H), 3.39 (br s,
1H), 3.15 (br s, 2H), 2.16 (br s, 1H, H-2eq), 1.45–1.41 (m, 1H, H-2ax);
4.13. 1-N-[Amino-formyl]-neamine-trihydrochloride (18)
To a stirred solution of compound 17 (60 mg, 0.13 mmol) in H2O
(3 ml), ammonia (0.5 ml) was added at room temperature. After
2 h, the reaction was complete according to TLC (MeOH/NH3$ H2O
3:1). The mixture was concentrated in vacuo and dissolved in 1 M
HCl (0.5 ml). The resulting solution was put on a reversed-phase
column (H2O followed by 10:1 H2O/MeOH as eluent) to give the
urea derivative 18 as a viscous gum (57 mg, 92%).
13C NMR (75 MHz, pyridine-d5)
d 160.0, 157.5, 156.6, 137.6, 137.5,
128.4, 128.3, 128.0, 127.8, 127.7, 127.5, 100.4, 82.7, 77.7, 77.2, 72.8,
72.4, 72.2, 66.2, 65.9, 65.8, 57.5, 51.3, 51.2, 42.3, 41.0, 35.8; ESI-HRMS
calcd for C76H93N10O26 ([MþH]þ) m/z 1561.6268, found 1561.6210.
4.18. The deprotected dimer of neamine (23)
1H NMR (300 MHz, D2O)
3.52–3.45 (m, 2H), 3.31–3.10 (m, 6H), 2.11 (br s, 1H, H-2eq), 1.47 (br
s, 1H, H-2ax); 13C NMR (75 MHz, D2O)
161.5, 96.7, 79.5, 76.3, 75.0,
d
5.69 (s, 1H, H-10), 3.86–3.66 (m, 3H),
Compound 23 was obtained from compound 22 according to
d
Section 4.2 as solid trihydrochloride (87%).
1H NMR (300 MHz, D2O)
d
5.17 (d, J¼3.6 Hz, 2H, H-10), 3.85–3.72
71.3, 69.6, 69.3, 54.2, 50.3, 49.3, 40.7, 32.0; ESI-HRMS calcd for
C
13H28N5O7 ([MþH]þ) m/z 366.1983, found 366.1973.
(m, 6H), 3.51–3.46 (m, 4H), 3.30–3.27 (m, 8H), 3.12–3.05 (m, 4H),
3.09 (s, 4H), 2.12–2.08 (m, 1H, H-2eq), 1.57–1.44 (m, 1H, H-2ax); 13
NMR (75 MHz, D2O) 160.5, 96.5, 78.8, 76.3, 74.9, 71.2, 69.6, 68.8,
C
d
4.14. 1-N-{N-[2-Amino-ethyl]-amino-formyl}-neamine-
54.0, 50.2, 49.4, 40.7, 40.3, 31.8; ESI-HRMS calcd for C28H57N10O14
tetrahydrochloride (19)
([MþH]þ) m/z 757.4050, found 757.4064.
Compound 19 was synthesized through the similar process as
compound 18 with compound 17 and ethylene diamine as a viscous
gum (64 mg, 89%).
4.19. 3,20-Di-N-benzyloxycarbonyl-1-N-[amino-formyl]-60,40-
N,O-carbonyl-neamine (24)
1H NMR (300 MHz, D2O)
d
5.72 (d, J¼3.6 Hz, 1H, H-10), 3.84–3.71
(m, 2H), 3.51–3.45 (m, 2H), 3.40–2.91 (m, 10H), 2.13–2.09 (m, 1H, H-
To a solution of compound 9 (109 mg, 0.17 mmol) in pyridine/
MeOH (5 ml/1 ml), ammonia (2 ml) was added. The mixture was
stirred for 6 h at 25 ꢁC, and the reaction was complete according to
TLC (CHCl3/MeOH/NH3$H2O 4:3:1). The solvent was removed in
vacuo at low temperature (room temperature to 40 ꢁC) followed by
addition of ice water (20 ml), and the resulting white solid, com-
pound 24 (98 mg, 88%), was filtered and dried without further
purification.
2
eq), 1.57–1.44 (br s, 1H, H-2ax); 13C NMR (75 MHz, D2O)
d 160.5,
96.5, 78.9, 76.3, 74.9, 71.2, 69.6, 68.8, 54.0, 50.1, 49.3, 40.6, 40.4, 37.8,
31.7; ESI-HRMS calcd for C15H33N6O7 ([MþH]þ) m/z 409.2405,
found 409.2396.
4.15. 1-N-[Hydrazino-formyl]-neamine-trihydrochloride (20)
Compound 20 was synthesized through the similar process as
compound 18 with compound 17 and hydrazine hydrate as a vis-
cous gum (61 mg, 94%).
1H NMR (300 MHz, DMSO-d6)
d 7.37–7.26 (m, 11H, Ph and
NHCO2), 7.01 (d, J¼8.4 Hz, 1H, NHCO2), 5.89 (d, J¼6.3 Hz, 1H,
NHCONH2), 5.47 (s, 2H, NHCONH2), 5.40 (d, J¼8.7 Hz, 1H), 5.14–4.97
(m, 6H), 4.75 (d, J¼12.6 Hz, 1H), 3.91–3.86 (m, 1H), 3.78 (dd,
J1¼8.7 Hz, J2¼9.3 Hz, 1H), 3.62–3.30 (m, 5H), 3.05–2.98 (m, 2H),
1.89–1.85 (m, 1H, H-2eq), 1.25–1.12 (m, 1H, H-2ax); 13C NMR
1H NMR (300 MHz, D2O)
d
5.36 (s, 1H, H-10), 3.82 (s, 1H), 3.52–
3.17 (m, 7H), 2.93–2.81 (m, 3H), 1.86 (br s, 1H, H-2eq), 1.33–1.20 (m,
1H, H-2ax); 13C NMR (75 MHz, D2O)
d
162.5, 99.0, 83.6, 76.4, 75.4,
72.6, 71.8, 69.7, 55.1, 50.5, 41.3, 41.1, 34.6; ESI-HRMS calcd for
C
(75 MHz, DMSO-d6)
128.3, 127.7, 127.6, 127.5, 99.0, 82.4, 78.6, 76.8, 75.2, 67.8, 65.3, 65.1,
d 158.9, 156.3, 155.5, 152.4, 137.2, 137.0, 128.4,
13H29N6O7 ([MþH]þ) m/z 381.2092, found 381.2084.

G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
9085
61.4, 55.9, 50.1, 50.0, 42.6, 35.3; ESI-HRMS calcd for C30H38N5O12
ESI-HRMS calcd for C30H41N6O12 ([MþH]þ) m/z 677.2777, found
([MþH]þ) m/z 660.2511, found 660.2524.
677.2781.
4.24. 3,20-Di-N-benzyloxycarbonyl-1,60-di-N-{N-[2-amino-
ethyl]-amino-formyl}-neamine (29)
4.20. 3,20-Di-N-Benzyloxycarbonyl-1-N-{N-[2-amino-ethyl]-
amino-formyl}-60,40-N,O-carbonyl-neamine (25)
Compound 29 was synthesized through the similar process as
compound 28 with compound 9 and ethylene diamine as white
solid (93 mg, 87%).
Compound 25 was synthesized through the similar process as
compound 24 with compound 9 and ethylene diamine as white
solid (100 mg, 84%).
1H NMR (300 MHz, DMSO-d6)
d 7.33–7.31 (m, 10H, Ph), 7.12 (br s,
1H NMR (300 MHz, DMSO-d6)
d
7.29 (br s, 12H, Ph and NHCO2),
5.97 (br s, 2H,), 5.04 (br s, 8H), 3.75–2.49 (m, 13H), 1.83 (br s, 1H, H-
eq), 1.18 (br s, 1H, H-2ax); 1H NMR (300 MHz, pyridine-d5)
8.47 (d,
1H, NHCO2), 6.85 (br s,1H, NHCO2), 6.15 (br s,1H, NHCONH), 6.06 (br
s, 1H, NHCONH), 5.95 (br s, 2H, NHCONH), 5.06–4.83 (m, 4H), 3.59–
3.07 (m, 11H), 2.97 (s, 7H), 2.52 (br s, 2H), 1.86 (br s, 1H, H-2eq), 1.19–
2
d
J¼9.0 Hz,1H, NHCO2), 8.33 (s,1H, NHCO2), 7.42–7.19 (m,11H, Ph and
NHCO2), 6.92 (s, 1H, NHCONH2), 6.73 (s, 1H, NHCONH2), 6.18 (s, 1H),
5.56 (d, J¼12.3 Hz, 1H), 5.33 (d, J¼12.6 Hz, 1H), 5.15–4.61 (m, 9H),
4.37–4.34 (m, 1H), 4.18 (br s, 1H), 4.04 (s, 2H), 3.88 (s, 1H), 3.76–3.74
(m, 1H), 3.53 (s, 1H), 3.42 (s, 2H), 2.83 (t, J¼5.4 Hz, 2H), 2.57–2.54
(m, 1H, H-2eq), 1.89–1.77 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-
1.15 (m,1H, H-2ax);13C NMR(75 MHz, DMSO-d6)
d 159.2,158.5,156.3,
155.7,137.3,137.0,128.3,127.8,127.6,127.5, 98.8, 82.0, 76.8, 75.1, 71.4,
70.9, 70.4, 65.4, 65.2, 56.4, 50.2, 42.4, 42.3, 41.8, 41.5, 35.6; ESI-HRMS
calcd for C34H51N8O12 ([MþH]þ) m/z 763.3621, found 763.3626.
4.25. 1,60-Di-N-[amino-formyl]-neamine-dihydrochloride
(30)
d6) d 158.3, 156.2, 155.4, 152.3, 137.2, 137.0, 128.8, 128.7, 128.6, 128.2,
127.5, 127.4, 127.0, 126.9, 98.7, 82.1, 78.5, 76.6, 74.8, 67.6, 65.0, 61.2,
55.8, 50.0, 49.9, 42.5, 41.7, 41.5, 35.2; ESI-HRMS calcd for
C
32H43N6O12 ([MþH]þ) m/z 703.2933, found 703.2933.
Compound 30 was obtained from compound 28 according to
Section 4.2 as solid dihydrochloride (94%).
1H NMR (300 MHz, D2O) 5.37 (s, 1H, H-10), 3.67–3.11 (m, 10H),
d
4.21. 1-N-[Amino-formyl]-60,40-N,O-carbonyl-neamine-
dihydrochloride (26)
2.07–2.03 (m, 1H, H-2eq), 1.43–1.31 (m, 1H, H-2ax); 13C NMR
(75 MHz, D2O)
d 162.1, 161.6, 98.3, 82.9, 76.1, 75.0, 72.6, 71.2, 54.8,
50.4, 49.5, 40.8, 32.8; ESI-HRMS calcd for C14H29N6O8 ([MþH]þ) m/z
Compound 26 was obtained from compound 24 according to
409.2041, found 409.2030.
Section 4.2 as solid dihydrochloride (93%).
1H NMR (300 MHz, D2O)
d
5.34 (d, J¼3.0 Hz, 1H, H-10), 4.08–4.03
4.26. 1,60-Di-N-{N-[2-amino-ethyl]-amino-formyl}-neamine-
tetrahydrochloride (31)
(m, 1H), 3.90 (dd, J1¼9.0 Hz, J2¼9.9 Hz, 1H), 3.77 (t, J¼9.3 Hz, 1H),
3.47–3.33 (m, 4H), 3.21–2.92 (m, 4H), 2.00–1.95 (m, 1H, H-2eq),
1.35–1.23 (br s, 1H, H-2ax); 13C NMR (75 MHz, D2O)
d 161.6, 156.1,
Compound 31 was obtained from compound 29 according to
99.9, 83.3, 78.7, 76.1, 75.1, 69.7, 62.0, 54.8, 50.6, 49.2, 42.7, 33.4; ESI-
Section 4.2 as solid tetrahydrochloride (92%).
HRMS calcd for
392.1775.
C
14H26N5O8 ([MþH]þ) m/z 392.1775, found
1H NMR (300 MHz, D2O)
d
5.12 (s, 1H, H-10), 3.67–3.61 (m, 1H),
3.53–3.10 (m, 12H), 2.90–2.79 (m, 6H), 1.90–1.85 (m, 1H, H-2eq),
1.24–1.11 (m, 1H, H-2ax); 13C NMR (75 MHz, D2O)
d
161.0, 160.6,
4.22. 1-N-{N-[2-Amino-ethyl]-amino-formyl}-60,40-N,O-
carbonyl-neamine-trihydrochloride (27)
100.1, 86.5, 76.2, 75.3, 72.4, 71.6, 55.3, 50.7, 49.6, 41.0, 40.5, 37.9,
37.9, 34.6; ESI-HRMS calcd for C18H39N8O8 ([MþH]þ) m/z 495.2885,
found 495.2876.
Compound 27 was obtained from compound 25 according to
4.27. 1,3,20-Tri-N-benzyloxycarbonyl-60-N-[amino-formyl]-
neamine (32)
Section 4.2 as solid trihydrochloride (91%).
1H NMR (300 MHz, D2O)
d
5.40 (s, 1H, H-10), 4.10–4.04 (m, 1H),
3.96–3.80 (m, 2H), 3.47–2.92 (m, 12H), 2.03–1.99 (m, 1H, H-2eq),
1.41–1.29 (m, 1H, H-2ax); 13C NMR (75 MHz, D2O)
d
160.5, 156.1,
Compound 32 was synthesized through the similar process as
compound 28 with compound 8 and ammonia in 87% yield.
99.4, 82.4, 78.6, 76.1, 75.1, 69.2, 62.0, 54.6, 50.4, 49.2, 42.7, 40.5, 37.8,
33.0; ESI-HRMS calcd for C16H31N6O8 ([MþH]þ) m/z 435.2197,
found 435.2197.
1H NMR (300 MHz, DMSO-d6)
d 7.34 (s, 15H, Ph), 7.17 (br s, 2H,
NHCO2), 6.86 (br s, 1H, NHCO2), 6.00 (br s, 1H, NHCONH2), 5.63 (br s,
2H, NHCONH2), 5.04–4.89 (m, 8H), 3.76–3.09 (m, 8H), 1.78 (br s, 1H,
H-2eq), 1.39–1.26 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
4.23. 3,20-Di-N-benzyloxycarbonyl-1,60-di-N-[amino-formyl]-
neamine (28)
d 159.8, 156.1, 155.7, 155.5, 137.2, 137.1, 136.8, 128.2, 127.7, 127.5,
127.3, 98.9, 81.9, 76.5, 74.0, 71.3, 70.7, 70.2, 65.3, 65.0, 56.3, 51.1,
50.2, 34.9; ESI-HRMS calcd for C74H91N10O26 ([2MþH]þ) m/z
1535.6100, found 1535.6163.
To a solution of compound 9 (90 mg, 0.14 mmol) in pyridine/
MeOH (5 ml/1 ml), ammonia (2 ml) was added at room tempera-
ture. The mixture was stirred for 6 h at 50 ꢁC, and the reaction was
complete according to TLC (CHCl3/MeOH/NH3$H2O 4:3:1). The
solvent was removed in vacuo followed by addition of ice water
(20 ml), and the resulting white solid, compound 28 (77 mg, 83%),
was filtered and dried without further purification.
4.28. 1,3,20-Tri-N-benzyloxycarbonyl-60-N-{N-[2-amino-
ethyl]-amino-formyl}-neamine (33)
Compound 33 was synthesized through the similar process as
compound 28 with compound 8 and ethylene diamine in 89% yield.
1H NMR (300 MHz, DMSO-d6)
d 7.34 (s, 10H, Ph), 7.12 (br s, 1H,
NHCO2), 6.85 (br s, 1H, NHCO2), 5.97 (br s, 1H, NHCONH2), 5.91 (br s,
1H, NHCONH2), 5.62 (s, 2H, NHCONH2), 5.48 (s, 2H, NHCONH2),
5.03–4.84 (m, 7H), 3.55–3.06 (m, 8H), 1.86 (br s, 1H, H-2eq), 1.20–
1H NMR (300 MHz, DMSO-d6)
d 7.35–7.34 (m, 15H, Ph), 7.17 (d,
J¼6.0 Hz, 1H, NHCO), 6.85 (s, 1H, NHCO), 6.13 (s, 1H, NHCO), 5.95 (br
s, 1H, NHCO), 5.09–4.86 (m, 7H), 3.57 (br s, 1H), 3.43–3.00 (m, 14H),
2.56 (m, 2H), 1.79 (br s, 1H, H-2eq), 1.39–1.26 (m, 1H, H-2ax); 13C
1.16 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d 159.8, 159.0,
156.2, 155.6, 137.3, 137.0, 128.7, 128.3, 127.8, 127.6, 127.4, 99.0, 82.1,
76.8, 75.1, 71.4, 70.8, 70.3, 65.3, 65.1, 56.4, 50.2, 50.1, 43.8, 35.4;
NMR (75 MHz, DMSO-d6)
136.9, 128.3, 127.8, 127.6, 127.4, 98.9, 81.8, 76.6, 74.0, 71.4, 70.8, 70.3,
d 159.2, 156.2, 155.7, 155.6, 137.3, 137.2,

9086
G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
65.3, 65.1, 56.3, 51.2, 50.2, 42.3, 41.8, 34.9; ESI-HRMS calcd for
C
4.34. 300-N-{N-[2-Amino-ethyl]-amino-formyl}-kanamycin
A-tetrahydrochloride (39)
39H51N6O13 ([MþH]þ) m/z 811.3519, found 811.3519.
4.29. 60-N-[Amino-formyl]-neamine-trihydrochloride (34)
Compound 39 was obtained from compound 37 according to
Section 4.2 as solid tetrahydrochloride (92%).
Compound 34 was obtained from compound 32 according to
1H NMR (300 MHz, D2O)
d
5.38 (s,1H), 4.92 (s,1H), 3.60–3.01 (m,
21H), 2.09 (br s, 1H, H-2eq), 1.47–1.43 (m, 1H, H-2ax); 13C NMR
(75 MHz, D2O) 161.3, 101.2, 97.3, 85.6, 81.9, 73.9, 73.5, 72.7, 71.5,
Section 4.2 as solid tetrahydrochloride (94%).
1H NMR (300 MHz, D2O)
d
5.38 (s, 1H, H-10), 3.69 (m, 1H), 3.43–
d
3.08 (m, 9H), 2.19–2.15 (m, 1H, H-2eq), 1.53–1.43 (m, 1H, H-2ax); 13
NMR (75 MHz, D2O) 162.0, 97.9, 83.5, 75.6, 73.5, 72.7, 71.2, 70.0,
C
71.0, 69.0, 68.4, 60.9, 55.1, 50.8, 48.7, 41.0, 40.6, 38.5, 31.4; ESI-HRMS
d
calcd for C21H43N6O12 ([MþH]þ) m/z 570.2933, found 570.2938.
54.8, 50.7, 49.3, 40.8, 31.0; ESI-HRMS calcd for
C13H28N5O7
([MþH]þ) m/z 366.1983, found 366.1973.
4.35. 1,3-Di-N-benzyloxycarbonyl-300-N-[amino-formyl]-60,40-
N,O-carbonyl-kanamycin A (40)
4.30. 60-N-{N-[2-Amino-ethyl]-amino-formyl}-neamine-
tetrahydrochloride (35)
According to the procedure for compound 24, compound 40 was
obtained from compound 13 or compound 14 in 84% yield.
Compound 35 was obtained from compound 33 according to
1H NMR (300 MHz, DMSO-d6)
d 7.33 (s,13H, Ph and NHCO2), 6.06
Section 4.2 as solid tetrahydrochloride (93%).
(br s, 1H, NHCONH2), 5.80 (s, 2H, NHCONH2), 5.61 (d, J¼5.1 Hz, 1H),
5.52 (d, J¼3.9 Hz, 1H), 5.45 (br s, 1H), 5.13–4.76 (m, 5H), 4.55 (br s,
1H), 4.25 (br s, 1H), 3.83 (br s, 2H), 3.72–3.23 (m, 9H), 3.03 (t,
J¼9.6 Hz,1H),1.83 (brs,1H, H-2eq),1.54–1.42 (m,1H, H-2ax); 13C NMR
1H NMR (300 MHz, D2O)
d
5.49 (s, 1H, H-10), 3.73–3.66 (m, 3H),
3.48–3.17 (m, 8H), 2.90 (s, 3H), 2.54 (s,1H), 2.32–2.28 (m,1H, H-2eq),
1.74–1.62 (m, 1H, H-2ax); 13C NMR (75 MHz, D2O)
d
161.1, 97.3, 80.2,
75.4, 73.0, 72.8, 70.8, 69.3, 54.5, 50.3, 40.5, 37.9, 37.1, 29.1; ESI-HRMS
(75 MHz, DMSO-d6) d 161.4, 155.8, 155.5, 152.4, 137.1, 136.9, 128.4,
calcd for C15H33N6O7 ([MþH]þ) m/z 409.2405, found 409.2394.
128.3, 127.8, 127.6, 101.2, 97.2, 84.8, 80.1, 78.0, 74.3, 72.8, 72.0, 70.8,
70.0, 68.9, 65.3, 61.5, 60.1, 55.5, 50.2, 49.3, 42.6, 34.4; ESI-HRMS calcd
for C36H48N5O17([MþH]þ) m/z 822.3039, found 822.3029.
4.31. 1,3,60-Tri-N-benzyloxycarbonyl-300-N-[amino-formyl]-
kanamycin A (36)
4.36. 300-N-[Amino-formyl]-60,40-N,O-carbonyl-
According to the procedure for compound 24, compound 36 was
obtained from compound 11 or compound 12 in 91% yield.
kanamycin A (41)
1H NMR (300 MHz, DMSO-d6)
d
7.34–7.31 (m, 16H, Ph and
Compound 39 was obtained from compound 37 according to
NHCO2), 7.19 (d, J¼8.7 Hz, 1H, NHCO2), 6.86 (br s, 1H, NHCO2), 6.07
(br s, 1H, NHCONH2), 5.82 (s, 2H, NHCONH2), 5.49–5.44 (m, 2H),
5.10–4.86 (m, 8H), 4.52 (br s, 1H), 4.25 (br s, 1H), 3.84 (d, J¼9.3 Hz,
1H), 3.51–3.25 (m, 10H), 3.08 (br s, 1H), 1.84 (br s, 1H, H-2eq), 1.46–
Section 4.2 as solid tetrahydrochloride (93%).
1H NMR (300 MHz, D2O)
d
5.46 (s, 1H), 4.89 (s, 1H), 4.03 (br s,
1H), 3.93–3.24 (m, 14H), 3.09 (t, J¼9.3 Hz, 1H), 2.37–2.34 (m, 1H, H-
eq), 1.79–1.67 (m, 1H, H-2ax); 13C NMR (75 MHz, D2O)
162.5,
2
d
1.42 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
d
161.4, 156.5,
156.2, 101.7, 98.3, 84.4, 79.2, 78.3, 73.8, 73.5, 71.2, 71.1, 69.9, 68.1,
62.0, 60.8, 55.2, 50.2, 48.5, 42.8, 28.4; ESI-HRMS calcd for
155.8, 155.6, 137.1, 137.0, 128.3, 127.7, 127.6, 101.1, 97.1, 84.3, 80.2,
74.4, 72.7, 72.3, 70.8, 70.7, 70.5, 68.9, 65.3, 60.1, 55.5, 50.2, 49.6, 41.6,
34.9; ESI-HRMS calcd for C43H55N5O18 ([MþH]þ) m/z 930.3614,
found 930.3627.
C
20H36N5O13 ([MþH]þ) m/z 554.2304, found 554.2319.
4.37. 1,3-Di-N-benzyloxycarbonyl-300,60-N-[amino-formyl]-
kanamycin A (42)
4.32. 1,3,60-Tri-N-benzyloxycarbonyl-300-N-{N-[2-amino-
ethyl]-amino-formyl}-kanamycin A (37)
According to the procedure for compound 28, compound 42 was
obtained from compound 13 or compound 14 in 92% yield.
According to the procedure for compound 25, compound 37 was
obtained from compound 11 or compound 12 in 87% yield.
1H NMR (300 MHz, DMSO-d6)
d 7.34 (s, 11H, Ph and NHCO2), 7.20
(d, J¼8.4 Hz, 1H, NHCO2), 6.10 (br s, 1H, NHCONH2), 6.01 (br s, 1H,
NHCONH2), 5.82 (s, 2H, NHCONH2), 5.65 (s, 2H, NHCONH2), 5.49–
5.44 (m, 2H), 5.07–4.87 (m, 7H), 4.54 (br s, 1H), 4.25 (br s, 1H), 3.83
(d, J¼9.3 Hz, 1H), 3.51–3.25 (m, 10H), 3.01 (br s, 1H), 1.84 (br s, 1H,
H-2eq), 1.52–1.40 (m, 1H, H-2ax); 13C NMR (75 MHz, DMSO-d6)
1H NMR (300 MHz, DMSO-d6)
d 7.33–7.31 (m, 16H, Ph and
NHCO2), 7.20 (d, J¼7.8 Hz, 1H, NHCO2), 6.48 (br s, 1H, NHCONH),
6.22 (br s, 1H, NHCONH), 5.10–4.86 (m, 8H), 4.16 (br s, 2H, NH2),
3.85 (d, J¼9.0 Hz, 1H), 3.51–3.26 (m, 15H), 3.02 (br s, 3H), 2.60 (br s,
1H), 1.84 (br s, 1H, H-2eq), 1.50–1.38 (m, 1H, H-2ax); 13C NMR
d
161.4, 160.0, 155.8, 155.5, 137.2, 136.9, 128.3, 128.1, 127.8, 127.7,
(75 MHz, DMSO-d6)
d
160.7, 156.6, 155.8, 155.6, 137.2, 137.0, 128.7,
127.6, 101.5, 97.1, 84.9, 80.0, 74.4, 72.7, 72.4, 71.4, 70.8, 69.8, 68.9,
65.4, 65.3, 60.1, 55.5, 50.2, 49.5, 43.4, 34.5; ESI-HRMS calcd for
C
128.3, 127.7, 127.6, 101.1, 97.1, 84.2, 80.2, 74.3, 72.7, 72.3, 70.7, 70.5,
68.8, 65.3, 60.1, 55.6, 50.2, 49.6, 41.9, 41.7, 41.1, 34.6; ESI-HRMS calcd
for C45H60N6O18 ([MþH]þ) m/z 973.4036, found 973.4036.
36H50N6O17 ([MþH]þ) m/z 839.3305, found 839.3284.
4.38. 1,3-Di-N-benzyloxycarbonyl-300,60-N-{N-[2-amino-
ethyl]-amino-formyl}-kanamycin A (43)
4.33. 300-N-[Amino-formyl]-kanamycin A-trihydro-
chloride (38)
According to the procedure for compound 29, compound 43 was
obtained from compound 13 or compound 14 in 83% yield.
Compound 38 was obtained from compound 36 according to
Section 4.2 as solid tetrahydrochloride (93%).
1H NMR (300 MHz, DMSO-d6)
d 7.25 (s, 10H, Ph), 7.13 (br s, 1H,
1H NMR (300 MHz, D2O)
d
5.38 (s,1H), 4.91 (s,1H), 3.84–3.48 (m,
12H), 3.29–3.16 (m, 3H), 3.02–2.97 (m, 2H), 2.39–2.34 (m, 1H, H-
eq),1.82–1.70 (m,1H, H-2ax); 13C NMR (75 MHz, D2O)
162.5,101.6,
NHCO2), 6.48 (br s, 1H, NHCO2), 6.26 (br s, 1H, NHCONH2), 6.15 (br s,
1H, NHCONH2), 5.91 (br s, 1H, NHCONH2), 5.77 (br s, 1H, NHCONH2),
4.97–4.84 (m, 6H), 4.09 (br s, 7H), 3.73 (br s, 1H), 3.42–2.93 (m, 17H),
2.49 (br s, 2H), 1.76 (br s, 1H, H-2eq), 1.33 (br s, 1H, H-2ax); 13C NMR
2
d
95.8, 84.4, 78.5, 73.8, 72.7, 72.5, 71.3, 71.2, 71.0, 69.2, 68.2, 61.0, 55.2,
50.3, 48.0, 40.8, 28.0; ESI-HRMS calcd for C19H38N5O12 ([MþH]þ)
m/z 528.2511, found 528.2506.
(75 MHz, DMSO-d6)
d 160.7, 159.3, 155.7, 155.5, 137.1, 136.9, 128.3,
127.8,127.5,101.4, 97.0, 84.6, 79.9, 74.3, 72.7, 72.6, 72.4, 71.3, 70.6, 69.9,

G. Chen et al. / Tetrahedron 64 (2008) 9078–9087
9087
68.9, 65.3, 65.2, 60.1, 55.7, 50.1, 49.5, 42.0, 41.7, 41.5, 34.6; ESI-HRMS
References and notes
calcd for C40H61N8O17 ([MþH]þ) m/z 925.4149, found 925.4165.
1. Hermann, T.; Westhof, E. Curr. Opin. Biotechnol. 1998, 9, 66–73.
2. Walter, F.; Vicens, Q.; Westhof, E. Curr. Opin. Chem. Biol. 1999, 3, 694–704.
3. Hermann, T. Angew. Chem., Int. Ed. 2000, 39, 1891–1905.
4. Schroeder, R.; Waldsich, C.; Wank, H. EMBO J. 2000, 19, 1–9.
5. Neu, H. C. Science 1992, 257, 1064–1072.
4.39. 300,60-N-[Amino-formyl]-kanamycin A-dihydrochloride
(44)
6. Wright, G. D. Curr. Opin. Microbiol. 1999, 2, 499–503.
7. Wright, G. D.; Berghuis, A. M.; Mobashery, S. Adv. Exp. Med. Biol. 1998, 456,
27–69.
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2809.
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11. Nagabhushan, T. L.; Cooper, A. B.; Turner, W. N.; Tsai, H.; McCombie, S.;
Mallams, A. K.; Rane, D.; Wright, J. J.; Reichert, P.; Boxler, D. L.; Weinstein, J.
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Compound 44 was obtained from compound 42 according to
Section 4.2 as solid tetrahydrochloride (97%).
1H NMR (300 MHz, D2O)
d
5.26 (s, 1H), 4.89 (s, 1H), 3.73–3.16
(m, 17H), 2.34–2.30 (m, 1H, H-2eq), 1.75–1.63 (m, 1H, H-2ax); 13C
NMR (75 MHz, D2O) 162.5, 162.1, 101.6, 98.9, 84.1, 80.8, 73.7, 72.7,
d
72.3, 71.7, 71.1, 70.9, 68.1, 60.8, 55.2, 50.4, 49.0, 40.8, 28.7; ESI-
HRMS calcd for
571.2547.
C
20H39N6O13 ([MþH]þ) m/z 571.2569, found
12. Roestamadjli, J.; Grapsas, I.; Mobashery, S. J. Am. Chem. Soc. 1995, 117, 11060–
11069.
13. Umezawa, H.; Miyasaka, T.; Iwasawa, H.; Ikeda, D.; Kondo, S. J. Antibiot. 1981, 34,
1635–1640.
14. Shitara, T.; Umemura, E.; Tsuchiya, T.; Matsuno, T. Carbohydr. Res. 1995, 276,
75–89.
4.40. 300,60-N-{N-[2-Amino-ethyl]-amino-formyl}-kanamycin
A-tetrahydrochloride (45)
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3064.
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124, 10773–10778.
Compound 39 was obtained from compound 37 according to
Section 4.2 as solid tetrahydrochloride (98%).
1H NMR (300 MHz, D2O)
d
5.05 (s,1H), 4.88 (s,1H), 3.79–2.78 (m,
23H), 2.69 (t, J¼9.0 Hz, 1H), 1.87–1.84 (m, 1H, H-2eq), 1.19–1.07 (m,
1H, H-2ax); 13C NMR (75 MHz, D2O)
161.4, 160.9, 100.8, 100.7, 88.1,
18. Cappelletti, L. M.; Spagnoli, R.; Spa, P. J. Antibiot. 1983, 36, 328–330.
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d
86.7, 74.6, 73.2, 72.3, 72.2, 71.4, 71.0, 68.5, 60.7, 55.3, 50.9, 49.6, 41.1,
40.6, 40.4, 38.1, 34.3; ESI-HRMS calcd for C24H49N8O13 ([MþH]þ)
m/z 657.3413, found 657.3415.
Acknowledgements
We acknowledge the financial support from the National Basic
Research Program (973 Program, No. 2004CB518904).