Synthesis and antibacterial activity of pyranmycin derivatives with N-1 and O-6 modifications

Article abstract of DOI:10.1016/j.bmc.2007.08.059

Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1 positi

Full text of DOI:10.1016/j.bmc.2007.08.059

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7711–7719
Synthesis and antibacterial activity of pyranmycin derivatives
with N-1 and O-6 modifications
Jie Li, Fang-I Chiang, Hsiao-Nung Chen and Cheng-Wei Tom Chang*
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322-0300, USA
Received 2 July 2007; revised 24 August 2007; accepted 28 August 2007
Available online 1 September 2007
Abstract—Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant
bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1
positions. The revealed SAR results demonstrated that the antibacterial activity of pyranmycin can be modulated by different acylic
substituents at O-6. Among these results, the 6-O-aminoethyl derivative, JT050, showed effective activity against resistant strain
Escherichia coli (pTZ19U-3) and E. coli (pSF815), which provides insight into further structural modifications.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ture–activity relationship, we have noted that the attach-
ment of AHB group at N-1 can also revive the
antibacterial activity of pyranmycin against resistant
bacteria. Therefore, we began to explore other possible
site(s) for introducing further structural modifications
with the aim to enhance the activity.
Aminoglycoside antibiotics have been
a valuable
resource against infectious diseases due to their broad
spectrum activity against both Gram positive and nega-
tive bacteria.1–3 Nevertheless, the prevalence of amino-
glycoside resistant bacteria has significantly reduced
their effectiveness.4–6 Advancing discoveries from the
studies of resistant mechanism and the structural infor-
mation from the binding of aminoglycoside toward the
target, the A-site decoding region of 16S rRNA,7–9 have
inspired new strategies for structural modifications of
aminoglycosides aiming to revive the antibacterial activ-
ity against aminoglycoside resistant bacteria.10 For
example, attaching functionalities at the N-1 position
of the 2-deoxystreptamine of kanamycin A has led to
the development of semi-synthetic amikacin that has
an (S)-4-amino-2-hydroxybutanoyl (AHB) group at
N-1 position (Fig. 1). The added AHB group has been
suggested to cause steric interactions toward the amino-
glycoside-modifying enzymes leading to the regain of
antibacterial activity of aminoglycosides.13,19
Recently, there are two reports focusing on the struc-
tural modifications at O-5 and/or O-6 of 1-N-(S)-4-ami-
no-2-hydroxybutyryl neamine derivatives.13,14 The SAR
studies showed that both O-5 and O-6 positions can be
the effective sites for structural modifications, if appro-
priate functional groups were incorporated. Inspired
by the results, we began to investigate whether the addi-
tion of functional groups at O-6 can also be beneficial
for the antibacterial activity of pyranmycin containing
AHB group at N-1. Since trisaccharide-based aminogly-
coside, such as ribostamycin, is much more active than
disaccharide-based aminoglycoside like neamine, it is
expected that pyranmycin bearing both N-1 AHB group
and O-6 functionalities may yield improved antibacterial
activity as shown in previous reports. In this paper, we
describe the synthesis of the pyranmycin derivatives with
both O-6 and N-1 modifications and the antibacterial
activity study of these derivatives (Fig. 3).
Our laboratory has been working on the structural opti-
mization of a novel class of aminoglycoside antibiotic,
pyranmycin (Fig. 2).11,12 From the studies of struc-
2. Results
Keywords: Aminoglycoside; Neomycin; Pyranmycin; Aminoglycoside
resistant bacteria.
We have recently developed a protocol for regioselective
reduction of the N-1 azido group from polyazidoamino-
glycoside derivatives.15 The synthesis of pyranmycin
*
Corresponding author. Tel.: +1 435 797 3545; fax: +1 435 797
3390; e-mail: chang@cc.usu.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.059

7712
J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
NH2
O
NH2
O
HO
HO
HO
HO
HO
HO
HO
NH2
NH2
NH2
O
O
HO
OH
H
N
O
O
1
1
NH2
O
O
O
HO
OH
HO
OH
OH
OH
NH2
amikacin
NH2
kanamycin A
Figure 1. Structures of amikacin and kanamycin A.
NH2
NH2
I
O
I
O
HO
HO
HO
1'
1'
NH2
HO
NH2
IIH
OH
4
H2N
O
II
4
H2N
O
O
1
N
O
1
NH2
H3C
O
H3C
O
6
NH2
5
6
5
OH
H2N
HO
OH
H2N
HO
O
OH
OH
TC005
lead of
pyranmycin
III JT005
III
pyranmycin
with
N
-1 AHB
Figure 2. Design of pyranmycin with N-1 AHB.
NH2
NH2
I
O
I
HO
HO
O
HO
1'
1'
NH2
HO
NH2
OH
IIH
4
H2N
IIH
OH
4
H2N
O
O
1
N
O
1
N
H3C
O
O
H
3C
O
6
NH2
5
6
NH2
5
O
OH
H2N
HO
H2N
HO
O
OH
R
O
OH
III
III
O-6 Modified pyranmycin
with N-1 AHB
JT005
Figure 3. Design of pyranmycin with O-6 modification and N-1 AHB.
with both O-6 and N-1 AHB began with the synthesis of
neamine derivative bearing O-6 allyl group and N-1
AHB (Scheme 1). The N-1 AHB group can be installed
from deprotection of the Boc group of 1,15 followed by
coupling with the protected AHB derivative, 2, which
can be prepared using the reported procedure.16 Inter-
estingly, using the bulky base under the specific ratio
of mixture solvents for the allylation of 3, the O-6
mono-allylated adduct, 4, was obtained as the major
product.17 The regioselectivity was confirmed by the
acetylation of O-5 hydroxyl group of 4. The characteris-
tic down-field shift of H-5 of 5 can be discerned by
1H–1H COSY.
B, ribostamycin, and butirosin A as the controls
(Fig. 4).18 Butirosin A is the only control bearing N-1
AHB group. Aminoglycoside susceptible Escherichia
coli (ATCC 25922) was used as a standard reference
strain. E. coli (pTZ19U-3) and E. coli (pSF815) are lab-
oratory resistant strains using E. coli (TG1) as the host.
The first one is equipped with the pTZ19U-3 plasmid en-
coded for APH(30)-I, which catalyzes a phosphorylation
at the 30-OH of both neomycin and kanamycin classes of
aminoglycosides. The second one is equipped with the
pSF815 plasmid encoded for AAC(60) and APH(200),
which produces a bifunctional enzyme that catalyzes
acetylation of amino group at C-60 and phosphorylation
of hydroxyl group at C-200 position. These enzymes are
among the most prevalent modes of resistance found
in aminoglycoside resistant strains.
After verifying the regioselectivity of allylation, a glyco-
sylation of 4 with 612 followed by hydrolysis of acetyl
groups furnished 7, which led to the synthesis of JT052
after the global deprotection. In an alternative route,
compound 7 was subjected to ozonolysis and NaBH4
reduction (Scheme 2). The resulting compound 8 was con-
verted to 9 via substitution of hydroxyl group with azido
group. Both 8 and 9 were transformed to the correspond-
ing JT051 and JT050, respectively, in similar fashion.
3. Discussion
From the minimum inhibitory concentrations (MIC),
the synthesized aminoglycosides with the attachment
of AHB group at N-1 regain the activity against both
resistant strains of bacteria with the exception of
JT052, which has an n-propyl group attached at O-6
of 2-deoxystreptamine ring (Table 1). By comparing
The above synthesized aminoglycosides were assayed
against susceptible and resistant strains using neomycin

J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
7713
N3
N3
1) TFA, CH2Cl2
O
2) EDC, HOBt,
BnO
O
BnO
BnO
Et3N, DMF
N3
BnO
N3
NH
N
HO
3O
OBn
N
HO
3O
NH
Boc
OBn
HO
Z
HO
N
Z
HO
3
N
H
1
O
H
2
O
71%
N3
Allyl bromide
O
Ac2O, Et3N
BnO
BnO
TBAI, LiHMDS
THF/DMF=2/1, 0oC
DMAP, CH2Cl2
N3
NH
OBn
N
HO
3O
64%
Z
69%
N
O
H
O
4
N3
O
BnO
BnO
N3
NH
OBn
N
3O
AcO
Z
N
O
H
O
5
Scheme 1.
H3C
O
1)
N3
AcO
AcO
N3
6
OC(NH)CCl3
BF3-OEt2, CH2Cl2, 4A MS
N3
O
O
BnO
BnO
BnO
BnO
N3
NH
2) LiOH-H2O, THF:H2O=3:1
OBn
N3
NH
N
HO
3O
OBn
N
O
3O
Z
H3C
Z
65%
O
N
N3
O
N
H
H
O
HO
O
O
OH
4
7
1) PMe3, THF
0.1 M NaOH
2) Pd(OH)2/C
1) O3, CH2Cl2
2) NaBH4
MeOH
47%
HOAc/H2O (1/1)
70%
NH2
O
N3
O
HO
HO
NH2
NH
BnO
BnO
OH
H2N
O
O
H3C
N3
NH
O
OBn
H2N
N
O
3O
NH2
H3C
O
HO
Z
O
OH
N3
O
OH
N
O
HO
H
1) PMe3, THF
0.1 M NaOH
2) Pd(OH)2/C
HOAc/H2O (1/1)
O
JT052
OH
8
NH2
O
HO
HO
40%
NH2
NH
OH
H2N
O
H3C
O
O
H2N
NH2
O
1) TIBSCl, pyridine
2) NaN3, DMF, 80oC
HO
53%
O
OH
OH
JT051
NH2
O
N3
1) PMe3, THF
0.1 M NaOH
2) Pd(OH)2/C
HOAc/H2O (1/1)
O
HO
HO
BnO
BnO
N
NH2
NH
N3
NH
OH
H2N
O
OBn
3O
H3C
H3C
O
Z
O
O
O
H2N
N3
NH2
N
49%
O
HO
O
HO
H
O
OH
JT050
O
OH
NH2
N3
9
Scheme 2.

7714
J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
NH2
O
NH2
O
HO
HO
HO
HO
NH2
NH2
NH2
NH
H2N
O
OH
H2N
O
O
O
HO
HO
O
O
HO
NH2
HO
OH
OH
O
HO OH
ribostamycin
butirosin A
NH2
O
HO
HO
NH2
NH2
H2N
O
HO
O
O
HO
NH2
OH
O OH
neomycin B
O
H2N
OH
Figure 4. Structures of ribostamycin, butirosin A, and neomycin B.
Table 1. Minimum inhibitory concentrations (MIC)a
clear that N-1 AHB and O-6 modifications can function
cooperatively to achieve better antibacterial activity.
JT050 is slightly more active than JT005 suggesting a
possible beneficial role of 2-aminoethyl group at O-6,
which prompts the further molecular modeling investi-
gation that will be discussed later.
Compound
Strains of bacteria
E. coli E. coli
(TG1)c (pSF815)d (pTZ19U-3)e
E. colib
E. coli
Neomycin B
Ribostamycin
Butirosin A
TC005
4–8
4–8
4
4
Inactivef
Inactive
0.5–1
Inactive
4
8
4–8
4–8
0.5–1
8–16
2–4
2
1–2
8
Finally, even without the attachment of AHB group at
N-1, the neomycin class antibiotic, ribostamycin, seems
to be modestly active against the E. coli (pSF815). The
results imply that neomycin class antibiotics with the
AHB group at N-1 position represent a better template
for further modification. All the synthetic aminoglyco-
sides are less active than butirosin A, which may suggest
a furanose for ring III is superior to a pyranose. None-
theless, a pyranose scaffold as in pyranmycin is more
stable in acidic condition which could be advantageous
in generating orally active antibiotics.11
8–16
8
JT005
JT050
4
8
16
8
1
16
JT051
JT052
2
8
Inactive
Inactive
8
Inactive
N.D., not determined.
a Unit: lg/mL.
b Escherichia coli (ATCC 25922).
c E. coli (TG1) (aminoglycoside susceptible strain).
d E. coli (TG1) (pSF815 plasmid encoded for (AAC(60)/APH(200))).
e E. coli (TG1) (pTZ19U-3 plasmid encoded for APH(30)-I).
f Inactive is defined as MIC P 32 lg/mL.
JT050 and related pyranmycin adducts (TC005 and
JT005) were further assayed against other clinically sig-
nificant strains of bacteria (Table 2). None of the amino-
glycosides including butirosin A and clinically used
neomycin was active against methicillin-resistant Staph-
ylococcus aureus (ATCC 33591) (MRSA) although we
did notice that JT050 showed the lowest MIC of
32 lg/mL. Interestingly, the added O-6 functional group
the functionalities at O-6, 2-aminoethyl group as in the
case of JT050 appears to be the optimal structural scaf-
fold. In contrast, 2-hydroxyethyl and n-propyl groups as
in the case of JT051 and JT052, respectively, appear to
have negative effect leading to lower antibacterial activ-
ity. However, from the results of JT005 and JT050, it is
Table 2. MIC of JT050 against other strains of bacteriaa
Compound
Strains of bacteria
S. aureusb
K. pneumoniaec
K. pneumoniaed
P. aeruginosae
Neomycin B
Butirosin A
TC005
Inactivef
Inactive
Inactive
Inactive
Inactive
16–32
1–2
16–32
1
8
0.5
1
1–2
4
4
JT005
JT050
1–2
4
2
0.5–1
2
a Unit: lg/mL.
b Staphylococcus aureus (ATCC 33591) (MRSA).
c Klebsiella pneumoniae (ATCC 700603).
d K. pneumoniae (ATCC 13883).
e Pseudomonas aeruginosa (ATCC 27853).
f Inactive is defined as MIC P 32 lg/mL.

J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
7715
NH3
O
3'
5'
HO
G
G
C
C
G
C
HO
H3N
1403
OH
H
N
H3N
1404 C
O
H3C
G
U
O
NH3
O
U1495
H3N
O
O
G
A
HO
C
1408
OH
A
A
1492
1490
H
N
O
C
G
U
G
G
H
A
C
C
1410
3.25 A
2.65 A
H
3.81 A
2.72 A
U
G
U C
Model RNA construct
O
P
C1404
O
G1403
O
Figure 6. Interactions of the O-6 aminoethyl group of JT050 with
rRNA.
different acylic substituents at O-6 position. Based on the
revealed SAR and molecular modeling results, 2-amino-
ethyl group at O-6 is the optimal functionality. Further
modifications based on the lead, 2-aminoethyl group at
O-6, are currently being carried out.
Figure 5. Molecular modeling for the binding of JT050 toward model
rRNA. The hydrogen atoms on JT050 except for the ammonium
group at the O-6 aminoethyl group are omitted for clarity. The
phosphodiester backbone, C1404 and G1403 on the RNA backbones
are highlighted in wide stick.
5. Experimental
seems to slightly reduce the activity of JT050 against
Klebsiella pneumoniae albeit the difference was within
the margin of error. The addition of AHB is clearly
essential for reviving the activity against resistant
K. pneumoniae (ATCC 700603). Finally, JT050 mani-
fested the best activity against Pseudomonas aeruginosa
which suggest a collaboration effect of having N-1
AHB and O-6 2-aminoethyl group.
Proton nuclear magnetic resonance spectra were re-
corded using a Bruker ARX 400 spectrometer. Chemical
shifts were reported as parts per million (ppm) down-
field from tetramethylsilane in d unit, and coupling con-
stants were given in cycles per second (Hz). Splitting
patterns were designed as s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. 13C spectra were obtained using
a Bruker ARX 400 spectrometer at 100 MHz. Routine
13C NMR spectra were fully decoupled by broad-band
WALTZ decoupling. All NMR spectra were recorded
at ambient temperature unless otherwise noted. High-
resolution electrospray ionization (ESI) or atmospheric
pressure chemical ionization (APCI) was provided by
the Mass Spectrometry Facilities, University of Califor-
nia, Riverside.
Examination of the binding of JT050 toward rRNA
using molecular modeling based on the published
X-ray structural study reveals an interesting finding
(Fig. 5).19 The trisaccharide skeleton adopts similar con-
formation as what we have found previously. The N-1
AHB resides at the same location as reported by
Mobashery and co-workers.20 The terminal ammonium
group of O-6 aminoethyl group appears to interact clo-
sely with the phosphodiester backbone between C1404
and G1403 forming a hydrogen-bonding network which
may explain the improved activity of JT050 as com-
pared to its parent analog, JT005. In addition, the
hydroxyethyl group at O-6 of JT051 is expected to pro-
vide weaker interaction with the same phosphodiester
backbone leading to slightly lower antibacterial activity.
Finally, it is conceivable that a hydrophobic n-propyl
group at O-6 of JT052 will yield disfavored interaction
causing drastic decrease in activity (Fig. 6).
Chemical reagents and starting materials were pur-
chased from Aldrich Chemical Co. or Acros Chemical
Co. and were used without purification unless other-
wise noted. Dichloromethane was distilled over
CaH2. Other solvents were used without purification.
Column chromatography was carried out by using sil-
˚
ica gel (60 A, 230 · 450 mesh, Sorbent Tech.) unless
otherwise noted.
5.1. 30,40-Di-O-benzyl-1-N-[(S)-2-benzyloxy-4-(benzyl-
oxycarbonylamino)butanoyl]-3,20,60-triazidoneamine (3)
4. Conclusion
Compound 1 (670 mg, 0.98 mmol) was treated with a
mixture solution of 99% trifluoroacetic acid (5 mL) and
dichloromethane (5 mL) and the reaction mixture was
stirred for 1 h at room temperature. The reaction mixture
was quenched with triethylamine and concentrated to
dryness as crude product which was directly used for
peptide coupling. A mixture of (S)-2-benzyloxy-4-N-
In conclusion, we have completed the synthesis of several
pyranmycin adducts with N-1 AHB and various O-6
functional groups. The antibacterial assay reveals that
the antibacterial activity of 1-N-(S)-4-amino-2-hydroxy-
butyryl pyranmycin can be modulated and improved by

7716
J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
(benzyloxycarbonylamino)butyric
acid
(370 mg,
128.7, 128.4, 128.2, 127.9, 118.3, 99.6, 84.6, 80.9, 80.2,
78.9, 78.5, 77.1, 75.9, 75.4, 73.4, 73.3, 71.4, 66.8, 64.5,
59.0, 51.1, 48.3, 37.4, 32.9, 32.6. ESI/APCI Calcd for
C48H56N11O10 [M+H]+ m/z 946.4211; measure m/z
946.4219.
1.08 mmol) and EDC (280 mg, 1.48 mmol), HOBt
(200 mg, 1.48 mmol), Et3N (1 mL), and the above crude
product in anhydrous DMF (5 mL) was stirred under
nitrogen at room temperature for overnight. After com-
pletion of the reaction mixture (Rf = 0.42, monitored by
TLC, hexane/ethyl acetate = 35:65), the reaction mixture
was concentrated and extracted with EtOAc. The organ-
ic layer was washed by H2O, brine, and then dried with
Na2SO4(s). Removal of the solvent followed by purifica-
tion with gradient column chromatography (hexane/
ethyl acetate = 60:40 to 0:100) afforded the product as
white crystals after vacuum pump (640 mg, 0.71 mmol,
5.3. 5-O-Acetyl-6-O-allyl-30,40-di-O-benzyl-1-N-[(S)-2-
benzyloxy-4-(benzyloxy-carbonylamino)butanoyl]-3,20,60-
triazidoneamine (5)
To the solution of 4 (30 mg, 0.03 mmol) in anhydrous
CH2Cl2 (5 mL), Et3N (0.01 mL, 0.1 mmol) and
DMAP (50 mg, catalyst) were added slowly, followed
by Ac2O (6 lL, 0.06 mmol) at room temperature.
After completion of the reaction (Rf = 0.2, monitored
by TLC, EtOAc/hexane = 35:65), the reaction mixture
was quenched with saturated NaHCO3 and extracted
with EtOAc. The organic solution was washed with
water, brine and dried over Na2SO4(s). After removal
of the solvent, the crude product was subjected to a
1
71%). H NMR (400 MHz, CDCl3) d 7.3–7.4 (m, 20H),
6.74 (d, J = 7.4 Hz, 1H, NH), 5.34 (m, 1H, H-10), 5.2
(m, 1H), 5.05 (s, 2H, PhCH2O), 4.91 (s, 2H, PhCH2O),
4.90 (d, J = 10.1 Hz, 1H, PhCH2O), 4.65 (d,
J = 11.1 Hz, 1H, PhCH2O), 4.59 (d, J = 11.6 Hz, 1H,
PhCH2O), 4.49 (d, J = 11.6 Hz, 1H, PhCH2O), 4.19 (m,
1H, H-50), 3.9–4.0 (m, 2H), 3.8–3.9 (m, 2H), 3.6 (m,
1H), 3.60 (dd, J = 9.4, 9.4 Hz, 1H), 3.4–3.6 (m, 3H),
3.42 (dd, J = 13.3, 4.3 Hz, 1H), 3.2–3.3 (m, 3H), 2.2 (m,
1H, H-2eq), 2.0–2.1 (m, 2H), 1.3 (m, 1H, H-2ax); 13C
NMR (100 MHz, CDCl3) d 172.9, 156.9, 137.9, 137.8,
137.1, 136.9, 129.0, 128.8, 128.7, 128.4, 128.3, 128.2,
128.1, 128.0, 98.9, 82.1, 80.4, 78.9, 78.7, 77.0, 75.7,
75.3, 74.8, 73.2, 71.3, 66.8, 64.0, 59.4, 51.2, 48.8, 37.2,
32.9, 32.4; ESI/APCI Calcd for C45H52N11O10 [M+H]+
m/z 906.3898; measure m/z 906.3914.
gradient
column
chromatography
(hexane/
EtOAc = 100:0 to 50:50), afforded product (20 mg,
0.02 mmol, 64%). 1H NMR (400 MHz, CDCl3) d
7.2–7.3 (m, 20H), 6.74 (d, J = 7.4 Hz, 1H, NH), 5.8
(m,1H), 5.0–5.2 (m, 6H), 4.89 (d, J = 10.5 Hz, 1H,
PhCH2O), 4.88 (s, 2H, PhCH2O), 4.63 (d,
J = 11.1 Hz, 1H, PhCH2O), 4.62 (d, J = 11.2 Hz, 1H,
PhCH2O), 4.48 (d, J = 11.2 Hz, 1H, PhCH2O), 4.26
(ddd, J = 10.0, 3.4, 2.6 Hz, 1H, H-50), 3.9–4.0 (m,
4H), 3.8 (m, 1H, H-1), 3.5–3.6 (m, 4H), 3.3–3.4 (m,
4H), 3.2 (m, 1H), 2.39 (ddd, J = 12.7, 4.3, 3.9 Hz,
1H, H-2eq), 2.14 (s, 3H, CH3), 2.0 (m, 2H), 1.45
(ddd, J = 12.6, 12.4, 12.2 Hz, 1H, H-2ax). 13C NMR
(100 MHz, CDCl3) d172.4, 169.4, 156.5, 137.8 (s,
2C), 136.9, 136.8,133.9, 129.0, 128.7 (s, 2C), 128.5,
128.3, 128.2, 128.1, 128.0, 118.4, 99.1, 79.8, 79.2,
79.1, 78.9, 78.4, 77.4, 75.7, 75.4, 74.6, 73.4, 72.1,
71.5, 66.8, 63.5, 59.3, 51.1, 47.9, 37.5, 32.5, 30.0,
21.2. ESI/APCI Calcd for C50H58N11O11 [M+H]+ m/z
988.4317; measure m/z 988.4333.
5.2. 6-O-Allyl-30,40-di-O-benzyl-1-N-[(S)-2-benzyloxy-4-
benzyloxycarbonylamino-butanoyl]-3,20,60-triazidone-
amine (4)
To a solution of 3 (670 mg, 0.73 mmol) and tetrabutyl-
ammonium iodide (590 mg, 1.60 mmol) in a mixed
anhydrous solution of THF/DMF (2/1) was added ally-
lbromide (0.15 mL, 1.74 mmol). The reaction mixture
was stirred at room temperature for 5 min; while the
reaction mixture was vigorously stirred, lithium bis(tri-
methyl silyl) amide (1.9 mL, 1.89 mmol) was added at
once under ice-water bath. The reaction was monitored
by TLC until the complete consumption of the starting
material (ca. overnight, Rf = 0.20, EtOAc/hex-
ane = 35:65). The reaction was quenched with 1 N
AcOH and concentrated by compressed air. The crude
product was extracted with EtOAc, washed with H2O,
brine and dried over anhydrous Na2SO4. Removal of
the solvent followed by purification with gradient col-
umn chromatography (hexane/ethyl acetate = 85:15 to
45:55) afforded the product (400 mg, 0.42 mmol, 69%).
1H NMR (400 MHz, CDCl3) d 7.2–7.3 (m, 20H), 6.67
(d, J = 7.3 Hz, 1H, NH), 5.8 (m, 1H), 5.20 (d,
J = 3.6 Hz, 1H, H-10), 5.0–5.2 (m, 5H), 4.8–4.9 (m,
2H), 4.6 (m, 2H), 4.49 (d, J = 11.3 Hz, 1H, PhCH2O),
4.35 (dd, J = 10.5, 5.1 Hz, 1H), 4.15 (ddd, J = 9.8, 3.6,
2.7 Hz, 1H, H-50), 4.08 (dd, J = 12.6, 6.6 Hz, 1H), 4.00
(dd, J = 9.9, 9.1 Hz, 1H), 3.96 (dd, J = 5.5, 5.5 Hz,
1H), 3.7 (m, 1H), 3.5–3.6 (m, 4H), 3.4 (m, 2H), 3.2–3.3
(m, 3H), 3.15 (dd, J = 4.9, 4.7 Hz, 1H), 2.37 (ddd,
J = 13.2, 4.0, 4.0 Hz, 1H, H-2eq), 2.0 (m, 2H), 1.3 (m,
1H, H-2ax); 13C NMR (100 MHz, CDCl3) d 172.5,
156.6, 137.7, 137.6, 137.0, 136.8, 134.5, 129.0, 128.8,
5.4. 5-O-(4-Azido-4,6-dideoxy-b-D-glucopyranosyl)-6-O-
allyl-30,40-di-O-benzyl-1-N-[(S)-2-benzyloxy-4-(benzyl-
oxycarbonylamino)butanoyl]-3, 20, 60-triazidoneamine (7)
A solution of glycosyl trichloroacetimidate, 6 (250 mg,
0.58 mmol), neamine acceptor, 4 (460 mg, 0.48 mmol),
˚
and activated powder 4 A molecular sieve was stirred
in anhydrous CH2Cl2 (ca. 12 mL) at room temperature
for 1 h then cooled to ꢀ50 ꢁC. To this cloudy solution
was added BF3–OEt2 (0.07 mL, 0.58 mmol). The solu-
tion was stirred at low temperature until the complete
consumption of the glycosyl trichloroacetimidate (ca.
5 h). The reaction mixture was quenched by the addition
of NaHCO3 powder. After being stirred for 15 min, the
reaction mixture was filtered through Celite. The residue
was washed thoroughly with CH2Cl2 and ethyl acetate.
After removal of the solvents, the crude product was
directly used for the hydrolysis reaction.
A solution of above crude product and LiOH monohy-
drate (120 mg, 2.9 mmol) in THF/H2O (12 mL/4 mL)
was stirred at room temperature until the complete con-

J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
7717
sumption of starting material after overnight. (Rf = 0.15,
monitored by TLC, EtOAc/hexane = 35:65). The
reaction mixture was filtered through Celite and was
concentrated. After extracted with EtOAc, the organic
layer was washed by H2O, brine, and then dried with
Na2SO4(s). Removal of the solvent followed by purifica-
tion with gradient column chromatography (hexane/
ethyl acetate = 80:20 to 20:80) afforded the product
(380 mg, 0.03 mmol, 65%). 1H NMR (400 MHz, CDCl3)
d 7.2–7.4 (m, 20H), 6.91 (d, J = 8.8 Hz, 1H, NH), 5.8 (m,
1H), 5.67 (d, J = 3.4 Hz, 1H, H-10), 5.3 (m, 1H), 5.2 (m,
1H), 5.17 (s, 1H), 5.12 (d, J = 11.6 Hz, 1H, PhCH2O),
5.1 (s, 1H, H-100), 5.0 (m, 1H), 4.91 (d, J = 11.3 Hz,
1H, PhCH2O), 4.80 (m, 3H, PhCH2O), 4.5–4.6 (m,
3H), 4.49 (d, J = 11.2 Hz, 1H, PhCH2O), 4.2 (m, 2H),
3.9–4.0 (m, 5H), 3.8 (s, 1H), 3.70 (dd, J = 8.9, 7.9 Hz,
1H), 3.4–3.6 (m, 5H), 3.2–3.3 (m, 6H), 3.11 (dd,
J = 9.2, 7.9 Hz, 1H), 3.05 (dd, J = 9.6, 9.6 Hz, 1H),
2.24 (ddd, J = 13.2, 4.9, 4.4 Hz, 1H, H-2eq), 1.9–2.0
(m, 2H), 1.4 (m, 1H, H-2ax), 1.39 (d, J = 6.1 Hz, 3H,
CH3). 13C NMR (100 MHz, CDCl3) d 172.4, 156.8,
137.9 (s, 2C), 136.8 (s, 2C), 134.0, 129.1, 128.8, 128.7
(s, 2C), 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.7,
127.2, 118.5, 102.1, 96.9, 81.2, 79.7, 79.6, 78.9, 78.3,
76.6, 75.6, 75.2 (s, 2C), 73.5, 73.4, 71.4, 71.2, 69.9,
67.5, 66.9, 63.3, 59.6, 55.1, 51.4, 47.6, 37.6, 32.7, 32.4,
18.2. ESI/APCI Calcd for C54H65N14O13 [M+H]+ m/z
1117.4855; measure m/z 1117.4879.
128.3, 128.2, 128.1, 127.9, 102.3, 96.6, 83.1, 79.9, 79.7,
78.8, 78.2, 75.8, 75.6 (s, 2C), 75.3, 75.1, 73.9, 73.5, 71.3,
71.0, 67.6, 66.9, 63.3, 62.2, 59.9, 51.4, 48.1, 37.6, 32.8,
32.6, 18.1. ESI/APCI Calcd for C53H65N14O14 [M+H]+
m/z 1121.4804; measure m/z 1121.4816.
5.6. 5-O-(4-Azido-4,6-dideoxy-b-D-glucopyranosyl)-6-O-
(2-azidoethyl)-30,40-di-O-benzyl-1-N-[(S)-2-benzyloxy-4-
(benzyloxycarbonylamino)butanoyl]-3,20,60-triazidone-
amine (9)
To a solution of 8 (110 mg, 0.09 mmol) in anhydrous pyr-
idine (5 mL) was added into triisopropylbenzenesulfonyl
chloride (TIBSCl) (900 mg, 3.14 mmol) at room tempera-
ture. After the completion of the reaction (ca. 3 days,
Rf = 0.5, EtOAc/hexanes = 50:50), the reaction mixture
was extracted with EtOAc and was washed by 1 N HCl
(three times), water and brine, then dried over Na2SO4(s)
.
After removal of solvent, the crude product was dissolved
in DMF (5 mL), and added with NaN3 (60 mg,
0.98 mmol). The reaction was refluxed under 80 ꢁC oil
bath for overnight and TLC of the reaction showed the
complete consumption of the starting material (Rf = 0.3,
EtOAc/hexanes = 50:50). After removal of DMF, the
crude product was extracted with EtOAc and washed with
water, brine, then dried over anhydrous Na2SO4(s). Re-
moval of solvent followed by purification with gradient
column chromatography (hexane/ethyl acetate = 60:40
to 0:100) afforded the product (60 mg, 0.05 mmol,
53.4%). 1H NMR (400 MHz, CDCl3) d 7.3–7.4 (m,
20H), 7.14 (d, J = 8.8 Hz, 1H, NH), 5.68 (d, J = 3.1 Hz,
1H, H-10), 5.22 (dd, J = 5.5, 5.2 Hz, 1H), 5.1 (s, 2H),
4.90 (d, J = 13.1 Hz, 1H, PhCH2O), 4.8–4.9 (m, 3H),
4.5–4.7 (m, 2H), 4.51 (d, J = 11.0 Hz, 1H, PhCH2O), 4.2
(m, 1H), 3.9–4.1 (m, 5H), 3.8 (m, 1H), 3.74 (dd, J = 8.5,
7.4 Hz, 1H), 3.6 (m, 2H), 3.4–3.5 (m, 3H), 3.43 (dd,
J = 7.6, 7.4 Hz, 1H), 3.2–3.4 (m, 6H), 3.15 (dd, J = 8.6,
8.1 Hz, 1H), 3.06 (dd, J = 9.7, 9.6 Hz, 1H), 2.21 (ddd,
J = 13.4, 4.9, 4.7 Hz, 1H, H-2eq), 1.9–2.1 (m, 2H), 1.47
(ddd, J = 12.9, 10.9, 10.8 Hz, 1H, H-2ax),1.39 (d,
J = 6.0 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) d
172.2, 156.8, 137.9 (2C), 136.7 (2C), 129.2, 128.9, 128.8,
128.7, 128.5, 128.4, 128.3, 128.1, 128.0, 127.7, 101.9,
96.8, 81.9, 79.7, 78.9, 78.8, 78.4, 77.5, 76.3, 75.8, 75.6,
75.2, 74.8, 73.7 (2C), 67.5, 67.0, 63.3, 59.3, 51.4 (3C),
47.6, 37.6, 32.9, 29.9, 18.2. ESI/APCI Calcd for
C53H64N17O13 [M+H]+ m/z 1146.4869; measure m/z
1146.4895.
5.5. 5-O-(4-Azido-4,6-dideoxy-b-D-glucopyranosyl)-6-O-
(2- hydroxyethyl)-30,40-di-O-benzyl-1-N-[(S)-2-benzyl-
oxy-4- (benzyloxycarbonylamino)butanoyl]-3,20,60-triazi-
doneamine (8)
To a solution of 7 (210 mg, 0.19 mmol) in CH2Cl2 (6 mL),
under ꢀ35 ꢁC, O3 was bubbling through till the complete
consumption of starting material (1 min). After removal
of excess O3 by bubbling of N2, the reaction mixture
was cooled to 0 ꢁC, diluted with MeOH, and slowly added
with NaBH4 (excess). After being stirred for 3 h, TLC of
the reaction showed the complete consumption of the
starting material. (Rf = 0.2, monitored by TLC, EtOAc/
hexane = 65:35). The reaction mixture was concentrated,
diluted with EtOAc and was washed with 1 N HCl(aq)
,
H2O, saturated NaHCO3(aq) Brine, then dried with
Na2SO4(s). Removal of the solvent followed by purifica-
tion with gradient column chromatography (hexane/ethyl
acetate = 65:35 to 0:100) afforded the product (150 mg,
1
0.13 mmol, 71%). H NMR (400 MHz, CDCl3) d 7.2–
7.4 (m, 20H), 7.15 (d, J = 8.5 Hz, 1H, NH), 5.81 (d,
J = 3.6 Hz, 1H, H-10), 5.0–5.1 (m, 3H), 4.90 (d,
J = 11.9 Hz, 1H, PhCH2O), 4.89 (s, 2H, PhCH2O), 4.86
(d, J = 10.7 Hz, 1H, PhCH2O), 4.78 (d, J = 7.7 Hz, 1H,
H-100), 4.62 (d, J = 11.4 Hz, 1H, PhCH2O), 4.58 (d,
J = 15.0 Hz, 1H, PhCH2O), 4.47 (d, J = 11.1 Hz, 1H,
PhCH2O), 4.2 (m, 1H), 4.08 (dd, J = 10.1, 8.9 Hz, 1H),
4.00 (dd, J = 10.1, 8.2 Hz, 1H), 3.96 (dd, J = 6.5, 5.1 Hz,
1H), 3.7 (m, 1H), 3.6–3.7 (m, 3H), 3.4–3.5 (m, 4H), 3.2–
3.3 (m, 8H), 3.09 (dd, J = 9.7, 9.6 Hz, 1H), 2.14 (ddd,
J = 13.2, 4.8, 4.5 Hz, 1H, H-2eq), 1.9–2.0 (m, 2H), 1.39
(d, J = 6.1 Hz, 3H, CH3), 1.3 (m, 1H, H-2ax). 13C NMR
(100 MHz, CDCl3) d 172.5, 156.8, 137.9, 137.8, 136.7,
136.6, 129.1, 128.9, 128.7 (s, 2C), 128.6, 128.5, 128.4,
5.7. General procedure for the synthesis of pyranmycin
derivatives
To a starting material/THF solution in a reaction vial
equipped with a reflux condenser, 0.1 M NaOH(aq)
(0.5 mL ) and PMe3 (1 M in THF, 5–7 equiv) were added.
The reaction mixture was stirred at 50 ꢁC for 2 h. The
product has a Rf of 0 when eluted with EtOAc/MeOH
(9/1) solution and a Rf of 0.6 when eluted with i-PrOH/
1 M NH4OAc (2/1) solution. After completion of the
reaction, the solvents were removed, and the crude benzy-
lated aminoglycoside was added with catalytic amount of
Pd(OH)2/C (20% Degussa type) and 5 mL of degassed
HOAc/H2O (1/3). After being further degassed, the reac-

7718
J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
tion mixture was stirred at room temperature under atmo-
spheric H2pressure. After being stirred for 1 day, the reac-
tion mixture was filtered through Celite. The residue was
washed with water, and the combined solutions were con-
centrated. The crude product was purified with Amberlite
J = 13.7, 6.4 Hz, 1H), 3.06 (t, J = 7.3 Hz, 2H), 3.00 (dd,
J = 10.0, 10.0 Hz, 1H), 2.0–2.1 (m, 1H), 2.01 (ddd,
J = 12.9, 4.4, 3.8 Hz, 1H, H-2eq), 1.7–1.8 (m, 1H), 1.67
(ddd, J = 12.8, 12.7, 12.7 Hz, 1H, H-2ax), 1.3–1.5 (m,
2H), 1.31 (d, J = 6.2 Hz, 3H, CH3), 0.75 (t, J = 7.4 Hz,
3H); 13C NMR (100 MHz, D2O) d 175.4, 102.3, 96.3,
82.8, 79.4, 77.4, 75.1, 73.4, 72.3, 70.8, 70.2, 69.7, 69.5,
68.7, 57.2, 53.7, 49.3, 48.6, 40.2, 37.2, 31.2 (2C), 23.1,
16.9, 10.3. ESI/APCI Calcd for C25H51N6O11 [M+H]+
m/z 611.3615; measure m/z 611.3596.
þ
CG50 NH4 eluted with a gradient of NH4OH solution
(0–20%). The final product with Clꢀ salt can be prepared
with an ion-exchange column packed with Dowex 1X8-
200 (Clꢀ form) and eluting with water. After collection
of the desired fractions and removal of solvent, the final
products are subjected to bioassay directly. The reported
final products are characterized by 1H and 13C NMR at
this stage.
5.11. Procedure for MIC determination
A solution of selected bacteria was inoculated in the Try-
pticase Soy broth at 35 ꢁC for 1–2 h. After which, the bac-
teria concentration was measured, and diluted with broth,
if necessary, to an absorption value of 0.08–0.1 at 625 nm.
The adjusted inoculated medium (100 lL) was diluted
with 10 mL broth and then applied to a 96-well microtiter
plate (50 lL). A series of solutions (50 lL, each in 2-fold
dilution) of the tested compounds was added to the testing
wells. The 96-well plate was incubated at 35 ꢁC for 12–
18 h. The minimum inhibitory concentration (MIC) is de-
fined as the minimum concentration of compound needed
to inhibit the growth of bacteria. The MIC results are re-
peated at least twice.
5.8. 5-O-(4-Amino-4,6-dideoxy-b-D-glucopyranosyl)-6-O-
(2-aminoethyl)-1-N-[(S)-4-amino-2-hydroxybutanoyl]-
neamine (JT050)
Please refer to the general procedure for the preparation
1
of pyranmycin derivatives. H NMR (400 MHz, D2O)
(chloride salt) d 5.82 (d, J = 4.0 Hz, 1H, H-10), 5.00 (d,
J = 8.2 Hz,, 1H, H-100), 4.28 (dd, J = 9.7, 3.5 Hz, 1H),
4.17 (dd, J = 9.2, 9.2 Hz, 1H), 3.8–4.1 (m, 7H), 3.74 (dd,
J = 13.7, 5.8 Hz, 1H), 3.67 (dd, J = 10.1, 9.3 Hz, 1H),
3.5–3.6 (m, 4H), 3.39 (dd, J = 9.2, 9.2 Hz, 1H), 3.35 (dd,
J = 9.2, 9.2 Hz, 1H), 3.0–3.2 (m, 5H), 2.1–2.3 (m, 2H),
1.8 1.9 (m, 2H), 1.42 (d, J = 6.3 Hz, 3H, H-600). 13C
NMR (100 MHz, D2O) (chloride salt) d 175.7, 103.2,
96.4, 82.8, 80.1, 76.5, 73.5, 72.0, 70.4, 70.0, 69.80, 69.75,
68.9, 68.4, 57.0, 53.6, 49.4, 48.9, 40.0, 39.95, 37.2, 31.2,
30.3, 16.9. ESI/APCI Calcd for C24H50N7O11 [M+H]+
m/z 612.3568; measure m/z 612.3551.
Acknowledgments
We acknowledge National Institutes of Health
(AI053138) for financial support. We thank Prof.
Mobashery of Notre Dame University for providing
the pTZ19U-3 and pSF815 plasmids.
5.9. 5-O-(4-Amino-4,6-dideoxy-b-D-glucopyranosyl)-6-O-
(2-hydroxyethyl)-1-N-[(S)-4-amino-2-hydroxybutanoyl]-
neamine (JT051)
Supplementary data
Please refer to the general procedure for the preparation
of pyranmycin derivatives. 1H NMR (400 MHz, D2O) d
5.87 (d, J = 4.1 Hz, 1H), 5.17 (d, J = 8.2 Hz, 1H), 4.27
(dd, J = 9.4, 3.6 Hz, 1H), 4.17 (dd, J = 9.1, 9.0 Hz,
1H), 4.08 (dd, J = 9.8, 9.3 Hz, 1H), 4.0 (m, 1H), 4.01
(dd, J = 10.7, 9.0 Hz, 1H), 3.8–3.9 (m, 3H), 3.4–3.7 (m,
10H), 3.33 (dd, J = 13.8, 5.9 Hz, 1H), 3.1–3.2 (m, 3H),
2.20 (ddd, J = 12.8, 4.6, 4.3 Hz, 1H, H-2eq), 2.1 (m,
1H), 1.8–1.9 (m, 2H), 1.42 (d, J = 6.3 Hz, 3H, CH3).
13C NMR (100 MHz, D2O) d 175.7, 102.3, 96.1, 82.6,
79.5, 76.1, 73.9, 73.6, 72.0, 70.6, 69.9, 69.8 (2C), 68.3,
61.2, 57.1, 53.6, 49.3, 48.6, 40.1, 37.1, 31.1, 30.2, 16.9.
ESI/APCI Calcd for C24H49N6O12 [M+H]+ m/z
613.3408; measure m/z 613.3401.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.08.059.
References and notes
1. Haddad, J.; Kotra, L. P.; Mobashery, S. In Glycochem-
istry Principles, Synthesis, and Applications; Wang, P. G.,
Bertozzi, C. R., Eds.; Marcel Dekker: New York, 2001; pp
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4. Mingeot-Leclercq, M.-P.; Glupczynski, Y.; Tulkens, P. M.
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Agents Chemother. 2000, 44, 3249–3256.
6. Wright, G. D. Curr. Opin. Microbiol. 1999, 2, 499–503.
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5.10. 5-O-(4-Amino-4,6-dideoxy-b-D-glucopyranosyl)-6-
O-n-propyl-1-N-[(S)-4-amino-2-hydroxybutanoyl]-
neamine (JT052)
Please refer to the general procedure for the preparation
of pyranmycin derivatives. 1H NMR (400 MHz, D2O) d
5.72 (d, J = 3.9 Hz, 1H, H-10), 4.86 (d, J = 8.2 Hz, 1H,
H-100), 4.17 (dd, J = 9.6, 3.3 Hz, 1H), 4.01 (dd, J = 9.2,
9.1 Hz, 1H), 3.8–3.9 (m, 4H), 3.73 (dd, J = 6.9, 6.9 Hz,
1H), 3.70 (dd, J = 9.8, 3.2 Hz, 1H), 3.57 (dd, J = 9.9,
9.6 Hz, 1H), 3.3–3.5 (m, 6H), 3.2 (m, 1H), 3.19 (dd,
9. Fong, D. H.; Berghuis, A. M. EMBO J. 2002, 21, 2323–
2331.

J. Li et al. / Bioorg. Med. Chem. 15 (2007) 7711–7719
7719
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17. Various conditions were attempted. However, the poor
regioselectivity and yield were achieved: (1) Using
LiHMDS as the base and THF as the solvent, no reaction
occurred; (2) Using LiHMDS as the base and DMF as the
solvent, no mono-O-6 allylated product was obtained; (3)
Using NaH as the base, no mono-O-6 allylated product
was obtained.
13. Haddad, J.; Kotra, L. P.; Llano-Sotelo, B.; Kim, C.;
Azucena, E. F., Jr.; Liu, M.; Vakulenko, S. B.; Chow, C.
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18. The procedure was modified from Methods for Dilution
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Aerobically. Approved standard M7-A5, and Performance
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Approved standard M2-A7, National Committee for
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163–164.
19. Molecular modeling was conducted using HyperChem 7.0
based on the X-ray structure reported by Mobashery and
co-workers (Ref. 20).
20. Russell, R. J. M.; Murray, J. B.; Lentzen, G.; Haddad, J.;
Mobashery, S. J. Am. Chem. Soc. 2003, 125, 3410–3411.