Tobramycin analogues with C-5 aminoalkyl ether chains intended to mimic rings III and IV of paromomycin

Article abstract of DOI:10.1016/S0040-4020(02)01624-1

Based on available X-ray structural and modeling data, a series of tobramycin derivatives with C-5 ether chains bearing basic groups were synthesized. These were intended to be hybrid molecules that combine features of tobramycin and paromomycin. Their binding to ribosomes and their antibacterial activity were determined. The 5-O-(2-guanidylethyl) ether of tobramycin (9g) was the most active analogue in the series.

Full text of DOI:10.1016/S0040-4020(02)01624-1

Tetrahedron 59 (2003) 983–993
Tobramycin analogues with C-5 aminoalkyl ether chains intended
to mimic rings III and IV of paromomycin
a
b
Stephen Hanessian,a Martin Tremblay and Eric E. Swayze
,
*
a
´
´
´
Department of Chemistry, Universite de Montreal, C.P. 6128, Succ. Centre-Ville, Montreal, Que. P.Q., Canada H3C 3J7
bIbis Therapeutics, 2292 Faraday Av., Carlsbad, CA 92008, USA
Received 23 October 2002; revised 16 December 2002; accepted 16 December 2002
Abstract—Based on available X-ray structural and modeling data, a series of tobramycin derivatives with C-5 ether chains bearing basic
groups were synthesized. These were intended to be hybrid molecules that combine features of tobramycin and paromomycin. Their binding
to ribosomes and their antibacterial activity were determined. The 5-O-(2-guanidylethyl) ether of tobramycin (9g) was the most active
analogue in the series. q 2003 Elsevier Science Ltd. All rights reserved.
The binding of small organic molecules to RNAs of
different origins has been the subject of extensive research
on different fronts in recent years.1 The diversity of
structures, including biologically relevant compounds has
focused more attention to RNAs as targets for therapeutic
agents.2 The aminoglycoside family, long known for their
potent antibacterial action, are clinically used in a hospital
environment where they can be monitored.3 They are
particularly attractive ligands to ribosomal and other types
of RNAs, as exemplified by pioneering studies carried out in
several laboratories world-wide.4
Paromomycin 4, a member of the 4,5-linked aminoglyco-
sides has been a superb substrate for binding studies to
Figure 1. Structures of representative aminoglycosides. Roman numerals refer to individual rings.
Keywords: aminoglycoside; amidine; guanidine; ribosome; antibacterial activity.
*
Corresponding author. Tel.: þ1-514-3436738; fax: þ1-514-3435728; e-mail: stephen.hanessian@umontreal.ca
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01624-1

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S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
Figure. 2. Top panel. Superposition of paromomycin (pink) and tobramycin (yellow) in 16S RNA segment based on Puglisi6 and Westhof.8 Bottom panel.
Tobramycin analogue 9g with 5-O-(bis-guanidino)-alkyl chain (yellow), superposed on paromomycin (pink).

S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
985
ribosomal RNA5 (Fig. 1). Elegant NMR6 and X-ray7
crystallographic studies of paromomycin–RNA complexes
have delineated the origins of the interactions of each of the
four rings and their appended hydroxy and amino groups.
Puglisi’s NMR studies6 originally identified several impor-
tant contacts with specific nucleotide residues when bound
in the major groove of the A-site of the 16S subunit
ribosomal RNA. Rings I and II (paromamine subunit) make
specific contacts by H-bonded, charge, and stacking
interactions. Characteristically, ring I occupies the area
left by bulging out of the A1492 and A1493 base pair which
are critical for viable RNA function. Other contacts are
made by ring III inter- and intramolecularly, while ring IV
contributes in a less ordered manner by making H-bonds to
the phosphate backbone of U1406, C1407 and C1490. It is
reported that rings III and IV provide the requisite L-shape
of paromomycin within the RNA complex to properly
orient rings I and II for effective contacts.6b X-Ray
crystallographic studies of paromomycin complexed with
a sequence of oligonucleotides corresponding to the
paromomycin interact with the lower stem of the A-site
RNA,6 ring III of tobramycin and gentamicin C1a makes
contacts with the upper stem. Thus, we can qualitatively
assume that rings III in the 4,6-disubstituted, and rings III,
IV in the 4,5-disubstituted aminoglycosides, occupy dif-
ferent spaces while being anchored to their respective
pseudodisaccharides within the major groove. (Fig. 2, top
panel).
We reasoned that introduction of ether-linked basic groups
at C-5 in tobramycin, and of varying chain length could
produce hybrid molecules such that rings III and IV of
paromomycin could be mimicked. Molecular modelling
studies indicated that the amino or guanidino-containing
side-chains originating at C-5 of tobramycin might reach far
enough in the lower stem of the A-site of the 16S rRNA to
interact with backbone phosphate linkages at U1406, C1407
and C1490, or to alternative new contacts with C1498, C1490
,
,
and G1491 (Fig. 2, bottom panel). We report herein the
results of our studies toward this goal.13
˚
ribosomal A-site of E. coli at 2.5 A resolution by Vicens
and Westhof8 have corroborated the NMR results and
further characterized the nature of the contacts. For
example, direct contacts of N-2000 and O-4000 of ring IV
have been observed.8 The importance of water molecules in
H-bonding through hydration shells of paromomycin and
the RNA has been demonstrated. X-Ray crystal structures of
paromomycin complexed with mRNA and cognate tRNA in
Acetylation of penta N-Cbz tobramycin14 with excess acetic
anhydride in pyridine afforded a partially acetylated
derivative 5 in which the C-5 hydroxyl group of the
deoxystreptamine unit was free (Scheme 1). Allylation
under standard conditions afforded the C-5 allyl ether 6,
which was oxidatively cleaved to afford the aldehyde 7.
Detailed NMR studies on 6 indicated that acetyl migration
had not taken place during the O-allylation step of 5.
Scheme 2 shows the synthesis of the C-5 hydroxyethyl ether
10 as well as its utility in a Mitsunobu-type guanidylation15
reaction to afford the branched guanidino precursor 11.
Deprotection afforded the branched hybrid aminoglycoside
analogues 12 and 13. A series of reductive amination
reactions performed on 7 led to a set of acylic and cyclic
N-protected appendages in good to excellent yields
9
˚
the A-site at 3.1–3.3 A resolution have also been reported.
The interactions of 5,6-substituted aminoglycosides such as
tobramycin 110 and gentamicin C1a 211 with RNA sequences
have also been studied by NMR and X-ray crystal-
lography.8b,12 The constant ring I and ring II pseudodi-
saccharide subunits occupy the same space as for
paromomycin. However, whereas rings III and IV of
Scheme 1.

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S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
Scheme 2.
Table 1.
Amine useda
Product
R, (R0¼H)
Yield (%)
54
Amine useda
Product
R,R0
Yield (%)
46
21
8a
17
8g
23
8b
8c
61
92
–
14b
18
8h
8i
54
85
26
8d
90
27
20
8e
8f
26
81
19
8j
43
a
See Scheme 3.

S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
987
Table 2.
Starting material
Product
R, (R0¼H)
Yield
Starting material
Product
R,R0
Yield
8a
9a
Quant.
8g
9g
Quant.
8b
8c
9b
9c
Quant.
Quant.
Quant.
H
Quant.a
8h
8i
9h
9i
8d
9d
Quant.a
Quant.
8e
8f
9e
9f
Quant.a
Quant.
8j
9j
Quant.a
a
Deprotection conditions: (1) TFA/H2O (10:1) 1 h, (2) H2, Pd/C in H2O/dioxane/AcOH.
(Table 1). The low yield in the case of 8e was due to a
competing intramolecular cyclization to a cyclic guanidine.
Depending on the N-protecting group, the hybrid analogues
were first treated with NaOMe/MeOH to remove the
acetates, then subjected to hydrogenolysis to remove all
N-Cbz groups, or to TFA hydrolysis of N-Boc groups,
followed by hydrogenolysis as shown in Table 2. The
corresponding hybrid aminoglycosides were isolated as
amorphous white solids as acetate salts. The synthesis of
selected acyclic and cyclic amines used in the reductive
amination reactions is illustrated in Scheme 3.
constants (KD) for the binding of the analogues to a
prototypical segment of 16S rRNA.5,17
Some of the analogues were as active as tobramycin
exhibiting MIC values of 0.6–2.5 mM against E. coli.
(Table 3, compounds 9f–i). The guanidino analogues 9d
and 9g also showed potentially interesting activity against
P. aeruginosa (ATCC 27853) at MIC 12.5 mg/mL.18 In
general transcription/translation data and KD values com-
pared well with those obtained for tobramycin and
paromomycin. A clear SAR correlating the nature of the
appendage with MIC values could not be seen, although it
appeared that analogues with more basic groups such as 9g
favored ribosomal binding and antibacterial activity. Some
aminoglycosides such as paromomycin exhibit a good
relationship between their binding affinity toward the 16S
ribosome and transcription/translation activity.16 The
4,6-disubstituted series such as kanamycin and tobramycin
on the other hand show excellent MIC values, but do not
bind as tightly as paromomycin. Good IC50 values for
transcription/translation may reflect the efficiency of mis-
coding and the formation of non-functional proteins.
However low mM values for KD may not necessarily result
in potent compounds due to non-functional binding, efflux,
or mechanisms not related to A-site ribosomal binding. Low
1. Results
As mentioned above, the basic premise for synthesizing
hybrid aminoglycoside analogues using tobramycin as a
prototype was to incorporate an appendage at C-5 to
simulate the space and interactions made by rings III and IV
of paromomycin. The choice of basic appendages relied on
the diversity of pKa, chain-length, branching and flexible
topology. In Table 3 are listed the results of antibacterial
activities against strains of S. aureus, and E. coli, the IC50
values of transcription/ translation, and the dissociation

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S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
Scheme 3.
Table 3. Activities of aminoglycoside analogues
Entry
Compound
KD (mM)
Translation, T/T IC50 (mM)
E. colia MIC (mM)
S. aureusb MIC (mM)
1
2
3
4
5
6
7
8
Kanamycin A
Kanamycin B
Tobramycin
Amikacin
Paromomycin
Neamine
9a
9c
9d
9f
9g
9h
9i
9j
12
4.82
1.48
2.39
2.45
0.15
4.56
3.05
1.37
2.86
3.97
0.64
2.29
2.96
9.58
6.41
13.57
0.57
0.36
0.45
0.53
0.56
1.89
1.01
2.17
2.33
0.86
0.78
0.72
0.56
1.09
0.38
.10
3.62
.10
2.5–5
1.2–2.5
0.6–1.2
1.2–2.5
2.5–5
.10
2.5–5
5–10
2.5–5
1–2
0.6–1.2
1–2
1–2
2.5–5
1–2
.10
1.2–2.5
0.3–0.6
0.3–0.6
1.2–2.5
1.2–2.5
.10
.10
.10
.10
5–10
2.5–5
5–10
5–10
.10
2.5–5
.10
.10
9
10
11
12
13
14
15
16
17
18
28
29
30
1.15
c
.10
.10
.10
a
ATCC 25922.
ATCC 13709.
Not determined.
b
c

S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
989
Figure 3. Structure of paromamine analogues.
binding affinity for 300-OH substituted analogs of neomycin,
in spite of their good antibacterial activity has been reported
previously.19 The promising antimicrobial activity against
E. coli shown by some of the hybrid analogues, warrants
further exploitation of the ‘ring III/IV’ space by more
optimized appendages at C-5 of tobramycin.
Flash column chromatography was performed using (40–
60 mm) silica gel at increased pressure. Binding constant for
the 16S A-site model RNA, IC50s for inhibition of bacterial
transcription/translation, and MIC values were determined
as described previously.16,17
2.1.1. Compound 6
We have recently reported the synthesis of a series of
substituted analogues of paromamine 28–3120 (Fig. 3),
which were found to have no inhibitory activity against a
panel of sensitive strains (P. aeruginosa, S. aureus, E. coli,
and E. faecalis).17 These analogues resemble the 5-O-
substituted tobramycin analogues 9a and 9i, except for the
absence of the kanosamine ring, and the nature of ring I
which delineates the well known need for a pseudotri-
saccharide as a minimum structure if effective inhibition is
expected.13a Nevertheless, the branched paromamine ana-
logue 29 exhibited good ribosomal binding and/or tran-
scription/translation activity (Table 3). Clearly, there is a
need to better understand the structural and functional
requirements that correlate ribosomal binding and anti-
bacterial activity. Studies toward this goal are in progress in
our laboratories.
To a solution of 514 (1.0 g, 0.76 mmol) in DMF (7 mL) was
added allyl iodide (0.21 mL, 2.3 mmol). After stirring at 08C
for 15 min, a suspension of NaH (46 mg, 1.9 mmol) in DMF
(6 mL) was added dropwise and the resulting mixture was
stirred for a further 30 min, quenched with few drops of 1N
NH4Cl and the solvent was removed by evaporation. The
residue was dissolved in EtOAc and washed with H2O, brine
and dried over Na2SO4. The solvent was evaporated under
vacuum and the crude solid was purified by flash
chromatography (CH2Cl2/acetone, 85:15) to give 6
(0.51 g, 50%) as a white solid; 1H NMR (400 MHz,
CDCl3) d 7.45–7.20 (m, 25H), 5.82–5.60 (m, 1H), 5.50–
3.00 (m, 22H), 2.08 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H), 1.88
(s, 3H); 13C NMR (75 MHz, CD3OD) d 171.3, 171.1, 170.5,
170.4, 157.2, 156.5, 156.3, 155.8, 136.9, 136.7, 136.6,
133.7, 129.0, 128.9, 128.9, 128.7, 128.6, 128.5, 128.3,
128.2, 128.1, 117.8, 95.2, 83.6, 75.4, 72.0, 71.5, 70.9, 69.2,
68.6, 68.2, 67.5, 67.3, 67.2, 67.0, 66.9, 62.2, 52.2, 51.8,
50.4, 49.3, 32.1, 31.4, 31.2, 21.2, 21.1, 20.8, 19.7, 14.4;
[a]D¼þ37 (c 1.1, CHCl3); FAB m/z calcd (MþHþ) 1346.5;
found 1346.0.
2. General information
Solvents were distilled under positive pressure of dry
nitrogen before use and dried by standard methods; THF and
ether, from Na/benzophenone; and CH2Cl2, from CaCl2. All
commercially available reagents were used without further
purification. All reactions were performed under nitrogen
atmosphere. NMR (1H,13C) spectra were recorded on
AMX-300, ARX-400 and DMX-600 spectrometers. Low-
and high-resolution mass spectra were recorded on VG
Micromass, AEI-MS 902 or Kratos MS-50 spectrometers
using fast atom bombardement (FAB) or electrospray
techniques. Optical rotations were recorded on a Perkin–
Elmer 241 polarimeter in a 1 dm cell at ambient
temperature. Analytical thin-layer chromatography was
performed on Merck 60F254 pre-coated silica gel plates.
Visualization was performed by ultraviolet light and/or by
staining with ceric ammonium molybdate or ninhydrine.
2.1.2. Compound 7
A solution of 6 (345 mg, 0.260 mmol) in CH2Cl2 (14 mL)
was cooled at 2788C, ozone was bubbled until the solution
turned light blue, after which argon was bubbled through.
The solution was treated with PPh3 (134 mg, 0.510 mmol)
and warmed to room temperature. The solvent was removed
under vacuum and the crude solid purified by flash
chromatography (EtOAc/hexanes, 75:25) to give 7
(216 mg, 63%) as a white solid; 1H NMR (400 MHz,
CDCl3) d 8.90 (s, 1H), 7.40–7.10 (m, 25H), 5.60–3.00 (m,
22H), 2.09 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H);
13C NMR (75 MHz, CD3OD) d 197.1, 171.2, 170.3, 157.3,

990
S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
156.6, 155.9, 155.8, 136.9, 136.7, 132.6, 132.4, 128.9,
128.8, 128.6, 128.5, 128.3, 128.1, 128.0, 96.6, 94.8, 84.8,
79.2, 70.7, 68.6, 67.4, 67.0, 62.4, 51.9, 50.1, 49.3, 42.5,
35.6, 30.8, 21.1, 20.6, 19.5; [a]D¼þ65 (c 1.4, CHCl3); FAB
m/z calcd (MþHþ) 1348.5; found 1348.1.
15H), 2.70–2.12 (m, 6H), 2.05 (s, 3H), 1.97 (s, 3H), 1.86 (s,
6H); [a]D¼þ50 (c 1.2, CHCl3); FAB m/z calcd (MþHþ)
1703.7; found 1703.7.
2.2.9. Compound 8i. (85%); 1H NMR (400 MHz, CDCl3) d
7.40–7.10 (m, 30H), 5.60–4.55 (m, 22H), 4.40–3.20 (m,
18H), 3.02 (m, 2H), 2.50–2.10 (m, 4H), 2.04 (s, 3H), 1.98
(s, 3H), 1.88 (s, 3H), 1.80 (s, 3H); [a]D¼þ49 (c 1.4,
CHCl3); FAB m/z calcd (MþHþ) 1552.6; found 1553.0.
2.2.10. Compound 8j. (43%); 1H NMR (400 MHz, CDCl3)
d 7.40–7.20 (m, 25H), 5.70–4.50 (m, 20H), 4.40–3.20 (m,
16H), 3.10–2.85 (m, 2H), 2.60–2.30 (m, 4H), 2.05 (s, 3H),
1.98 (s, 3H), 1.89 (s, 3H), 1.80 (s, 3H), 1.49 (s, 18H);
[a]D¼þ46 (c 1.0, CHCl3); FAB m/z calcd (MþHþ) 1634.7;
found 1634.8.
2.2. General procedure for reductive amination (8a–j)
To a mixture of 7 (0.034 mmol) and the appropriate amine
(Scheme 3, 14b,21 18,22 20,23 21–23,24 2625 and 27,26)
(0.10 mmol) in MeOH (3 mL) was added AcOH (0.1 mL)
followed by NaBH3CN (1.0 M in THF, 0.1 mL). The
mixture was stirred at room temperature overnight until
disappearance of 7, diluted with EtOAc (15 mL), washed
with a solution of NaHCO3 (satd, 10 mL) and dried over
Na2SO4. After evaporation of the solvent, the residue was
purified by flash chromatography (CH2Cl2/MeOH, 95:5) to
give (8a–j) as white solids.
2.3. General procedure for deprotection (9a–c, 9f, 9h
and 9i)
2.2.1. Compound 8a. (54%); 1H NMR (400 MHz, CDCl3) d
7.40–7.10 (m, 30H), 5.50–4.50 (m, 17H), 4.40–2.80 (m,
12H), 2.05 (s, 3H), 2.00 (s, 3H), 1.84 (s, 6H); [a]D¼þ59 (c
1.7, CHCl3); FAB m/z calcd (MþHþ) 1526.6; found 1525.9.
2.2.2. Compound 8b. (61%); 1H NMR (400 MHz, CDCl3)
d 7.40–7.10 (m, 30H), 6.00–5.90 (large s, 1H), 5.50–4.50
(m, 17H), 4.40–3.00 (m, 12H), 2.70–2.45 (large, 2H), 2.07
(s, 3H), 2.03 (s, 3H), 1.84 (s, 6H), 1.70–1.40 (m, 4H), 1.35–
1.20 (m, 2H); [a]D¼þ56 (c 1.1, CHCl3); FAB m/z calcd
(MþHþ) 1568.7; found 1568.9.
2.2.3. Compound 8c. (92%); 1H NMR (400 MHz, CDCl3) d
7.42–7.00 (m, 29H), 5.85–5.70 (large s, 1H), 5.40–4.45
(m, 17H), 4.30–3.20 (m, 10H), 3.00 (m, 1H), 2.78 (m, 1H),
2.45 (m, 1H), 2.33 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.82 (s,
6H), 1.49 (s, 18H); [a]D¼þ49 (c 1.2, CHCl3); FAB m/z
calcd (MþHþ) 1634.7; found 1634.8.
2.2.4. Compound 8d. (90%); 1H NMR (400 MHz, CDCl3)
d 7.40–7.20 (m, 25H), 5.50–4.52 (m, 13H), 4.40–3.20 (m,
10H), 3.10–2.85 (m, 4H), 2.07 (s, 3H), 2.01 (s, 3H), 1.84 (s,
6H), 1.49 (s, 18H); [a]D¼þ46 (c 1.0, CHCl3); FAB m/z
calcd (MþHþ) 1634.7; found 1634.8.
2.2.5. Compound 8e. (26%); 1H NMR (400 MHz, CDCl3) d
7.44–7.21 (m, 25H), 5.80–4.60 (m, 13H), 4.30–2.60 (m,
16H), 2.04 (s, 6H), 1.80 (s, 6H), 1.53 (s, 18H); [a]D¼þ47 (c
1.0, CHCl3); FAB m/z calcd (MþHþ) 1648.7; found 1649.0.
2.2.6. Compound 8f. (81%); 1H NMR (400 MHz, CDCl3) d
7.45–7.15 (m, 35H), 5.70–4.42 (m, 20H), 4.38–2.80 (m,
16H), 2.70–2.60 (m, 2H), 2.05 (s, 3H), 1.99 (s, 3H), 1.84 (s,
3H), 1.82 (s, 3H); [a]D¼þ49 (c 1.2, CHCl3); FAB m/z calcd
(MþHþ) 1689.7; found 1689.7.
2.2.7. Compound 8g. (46%); 1H NMR (400 MHz, CDCl3) d
7.42–7.20 (m, 25H), 5.80–3.20 (m, 36H), 2.80–2.45 (m,
4H), 2.06 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H), 1.82 (s, 3H),
1.47 (s, 36H); [a]D¼þ48 (c 1.1, CHCl3); FAB m/z calcd
(MþHþ) 1919.9; found 1919.8.
The appropriate pseudo-disaccharide was treated with a
catalytic amount of NaOMe in MeOH (2 mL). The reaction
mixture was stirred at room temperature until completion
(approximately 2 h) then neutralized by addition of
Amberlite IR-120(Hþ), the resin was filtered off and the
filtrate was evaporated to dryness To the resulting residue
in AcOH (80% in H2O, 2 mL), was added 10% Pd/C
(approximately 10 mg) and the mixture was stirred under
1 atm of hydrogen overnight. The mixture was filtered
through a Celite pad, concentrated under vacuum, diluted
with water and lyophilized to afford (9a–c, 9f, 9h and 9i) as
fluffy white solids.
2.3.1. Compound 9a. (quant.); 1H NMR (400 MHz, D2O) d
5.25 (s, 1H), 5.07 (s, 1H), 4.20–3.00 (m, 22H), 2.30 (m,
1H), 2.10–1.90 (m, 3H), 1.78 (s, 21H); 13C NMR (75 MHz,
D2O) d 181.5, 101.2, 93.4, 82.8, 82.4, 77.7, 73.9, 73.6, 68.9,
68.3, 66.1, 64.3, 60.8, 54.9, 50.1, 48.7, 48.2, 47.8, 45.5,
39.4, 36.8, 29.4, 23.5; [a]D¼þ25 (c 0.9, H2O); MALDI-
FTMS calcd for C22H47N7O9Na (MþNaþ) 576.3327; found
576.3330.
2.3.2. Compound 9b. (quant.); 1H NMR (400 MHz, D2O) d
5.39 (s, 1H), 5.23 (s, 1H), 4.20–3.45 (m, 18H), 3.40–3.18
(m, 6H), 3.10–2.92 (m, 4H), 2.35 (m, 1H), 2.10–1.95 (m,
3H), 1.92 (s, 21H), 1.80–1.60 (m, 4H), 1.50–1.40 (m, 2H);
13C NMR (75 MHz, D2O) d 181.5, 101.2, 93.9, 83.0, 82.9,
78.1, 73.9, 73.2, 70.3, 68.9, 66.7, 64.5, 61.4, 54.7, 50.1,
48.9, 48.2, 47.9, 39.4, 30.6, 26.6, 25.5, 23.2,; [a]D¼þ16 (c
0.5, H2O); MALDI-FTMS calcd for C25H54N7O9 (MþHþ)
596.3977; found 596.3980.
2.3.3. Compound 9c. (quant.); 1H NMR (400 MHz, D2O) d
5.35 (s, 1H), 5.19 (s, 1H), 4.20–3.36 (m, 17H), 3.35–3.15
(m, 6H), 2.25 (m, 1H), 2.20 (m, 1H), 2.00 (m, 1H), 1.88 (s,
18H), 1.70 (m, 1H); 13C NMR (75 MHz, D2O) d 181.7,
100.9, 93.4, 82.9, 82.6, 77.6, 73.9, 73.3, 68.8, 67.0, 66.3,
64.3, 61.1, 54.7, 50.0, 48.8, 47.9, 39.8, 39.4, 30.3, 29.8,
23.5; [a]D¼þ26 (c 1.0, H2O); MALDI-FTMS calcd for
C20H42N6O9Na (MþNaþ) 533.2905; found 533.2909.
2.2.8. Compound 8h. (34%); 1H NMR (400 MHz, CDCl3)
d 7.42–7.10 (m, 35H), 5.50–4.50 (m, 17H), 4.40–2.80 (m,
2.3.4. Compound 9f. (quant.); 1H NMR (400 MHz, D2O) d
5.36 (s, 1H), 5.08 (s, 1H), 4.00–2.60 (m, 22H), 2.25 (m,

S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
991
1H), 2.27 (m, 1H), 1.92 (m, 1H), 1.92 (s, 1H), 1.78 (s, 24H)
1.62 (m, 1H); 13C NMR (100 MHz, D2O) d 182.1, 101.6,
93.5, 83.6, 83.1, 82.9, 82.7, 77.6, 74.1, 73.6, 71.8, 69.4,
66.1, 65.9, 64.8, 60.2, 55.5, 53.9, 50.5, 49.3, 48.4, 46.6,
45.7, 40.6, 40.5, 40.1, 30.2, 30.0, 29.9, 25.3, 24.0; [a]D¼
þ25 (c 1.0, H2O); MALDI-FTMS calcd for C23H50N8O9Na
(MþNaþ) 605.3593; found 605.3601.
2.3.5. Compound 9h. (quant.); 1H NMR (400 MHz, D2O) d
5.36 (s, 1H), 5.21 (s, 1H), 4.10–3.17 (m, 22H), 3.10–3.05
(m, 4H), 2.95–2.80 (m, 6H), 2.40–2.20 (m, 2H), 2.10–1.95
(m, 1H), 1.91 (s, 24H), 1.80–1.60 (m, 1H); 13C NMR
(75 MHz, D2O) d 182.1, 101.1, 94.3, 83.5, 82.3, 78.2, 74.0,
72.4, 69.5, 65.9, 65.4, 60.2, 55.7, 52.5, 51.3, 50.8, 49.4,
48.7, 40.5, 37.3, 31.5, 31.3, 31.0, 24.0; [a]D¼þ21 (c 0.90,
H2O); MALDI-FTMS calcd for C24H53N8O9 (MþHþ)
597.3930; found 597.3938.
1H), 2.10 (m, 2H), 1.90 (m, 1H); 13C NMR (75 MHz, D2O)
d 163.5, 163.0, 157.4, 118.6, 114.7, 101.1, 93.2, 83.6, 82.0,
78.4, 77.4, 74.3, 68.8, 65.8, 63.1, 54.9, 52.2, 49.6, 48.5,
47.4, 38.5, 36.9, 27.9; [a]D¼þ33 (c 1.1, H2O); MALDI-
FTMS calcd for C26H57N12O9 (MþHþ) 681.4366; found
681.4361.
2.4.4. Compound 9j. (quant.); 1H NMR (400 MHz, D2O) d
5.26 (s, 1H), 5.08 (s, 1H), 4.50–3.10 (m, 30H), 2.40 (m,
1H), 2.10 (m, 2H), 1.89 (m, 1H); 13C NMR (100 MHz, D2O)
d 163.9, 163.5, 163.1, 157.0, 118.2, 115.3, 101.4, 93.3, 83.1,
82.2, 78.5, 77.4, 74.4, 68.7, 68.5, 66.3, 63.1, 61.3, 57.5,
54.6, 52.6, 49.7, 48.5, 47.4, 43.1, 38.4, 27.9, 23.1; [a]D¼
þ33 (c 1.1, H2O); MALDI-FTMS calcd for C25H52N7O9
(MþHþ) 622.3882; found 622.3882.
2.4.5. Compound 10. A solution of 7 (200 mg, 0.148 mmol)
in MeOH (2 mL) was cooled at 08C and treated with NaBH4
(22 mg, 0.590 mmol). The mixture was stirred for 15–
20 min, few drops of AcOH were added to quench the
reaction and the mixture was warmed to rt. The solvents
were removed under vacuum, the residue was dissolved in
EtOAc, washed with water, brine and dried over Na2SO4.
The solvent was removed under vacuum and the crude solid
purified by flash chromatography (CH2Cl2/acetone, 85:15)
to give 10 (152 mg, 76%) as a white solid; 1H NMR
(400 MHz, CDCl3) d 7.40–7.20 (m, 25H), 5.60–5.40 (m,
4H), 5.30–4.70 (m, 16H), 4.60 (m, 1H), 4.40–4.20 (m, 2H),
4.00–3.25 (m, 13H), 3.05 (m, 1H), 2.85 (m, 1H), 2.08 (s,
3H), 2.00 (s, 3H), 1.84 (s, 6H), 1.70 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 171.2, 170.6, 157.3, 156.7, 156.4,
155.9, 136.9, 136.8, 136.7, 129.1, 129.0, 128.8, 128.6,
128.3, 128.2, 128.0, 95.8, 94.9, 84.6, 70.9, 68.9, 68.7, 67.5,
67.2, 67.0, 62.5, 61.7, 52.1, 51.7, 50.2, 49.3, 42.7, 35.6,
30.9, 21.2, 20.8; [a]D¼þ67 (c 1.4, CHCl3); FAB m/z calcd
(MþHþ) 1350.5; found 1350.4.
2.3.6. Compound 9i. (quant.); 1H NMR (400 MHz, D2O) d
5.30 (s, 1H), 5.05 (s, 1H), 3.92–3.00 (m, 24H), 2.75–2.52
(m, 8H), 2.17 (m, 2H), 1.88 (m, 1H), 1.76 (s, 21H) 1.55 (m,
1H); 13C NMR (100 MHz, D2O) d 181.7, 92.9, 82.7, 81.9,
77.0, 73.7, 71.9, 69.0, 66.5, 65.7, 64.8, 60.2, 57.0, 55.1,
50.4, 50.1, 48.7, 48.1, 43.1, 40.0, 30.7, 30.3, 23.5; [a]D¼
þ35 (c 1.0, H2O); MALDI-FTMS calcd for C24H49N7O9Na
(MþNaþ) 602.3484; found 602.3471.
2.4. General procedure for deprotection (9d,e, 9g and 9j)
The appropriate pseudo-disaccharide was treated with a
catalytic amount of NaOMe in MeOH (2 mL). The reaction
mixture was stirred at room temperature until completion
(approximately 2 h) then reaction mixture was neutralized
by addition of Amberlite IR-120(Hþ), the resin was filtered
off and the filtrate was evaporated to dryness The resulting
residue was dissolved in TFA (2 mL), stirred for 2 h and
concentrated under vacuum. To the resulting residue in
AcOH (80% in H2O, 2 mL), was added 10% Pd/C
(approximately 10 mg) and the mixture was stirred under
1 atm of hydrogen overnight. The mixture was filtered
through a Celite pad, concentrated under vacuum, diluted
with water and lyophilized to afford (9d,e, 9g and 9j) as
fluffy white solids.
2.4.6. Compound 11. To a suspension of 10 (50 mg,
0.037 mmol), tris(N,N0,N00-benzyloxycarbamate)guanidine15
(40 mg, 0.11 mmol) and PPh3 (15 mg, 0.056 mmol) in
toluene (1.5 mL) was added slowly diethylazodicarboxylate
(8.7 mL, 0.056 mmol). The mixture was heated at 708C
during 1 h, cooled down at room temperature and
concentrated under vacuum. The resulting residue was
purified by flash chromatography (EtOAc/hexanes, 45:55)
to give 11 (51 mg, 77%) as a white solid; 1H NMR
(400 MHz, CDCl3) d 7.35–7.20 (m, 40H), 5.80 (m, 1H),
5.50 (m, 2H), 5.35–5.76 (m, 20H), 4.45 (m, 2H), 4.25–3.30
(m, 12H), 3.00 (m, 2H), 2.20 (m, 2H), 2.05 (m, 3H), 1.99 (m,
3H), 1.89 (s, 3H), 1.74 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 170.4, 169.9, 169.7, 156.7, 155.7, 155.6, 155.5,
154.4, 136.6, 136.3, 134.4, 128.5, 128.4, 128.3, 128.0,
127.9, 127.8, 127.6, 95.7, 94.9, 70.1, 69.2, 68.5, 68.2, 67.8,
66.7, 66.5, 66.2, 61.8, 51.9, 49.3, 49.0, 41.6, 29.9, 29.5,
20.7, 20.5, 20.4, 20.1; [a]D¼þ62 (c 1.0, CHCl3); FAB m/z
calcd (MþHþ) 1793.7; found 1793.8.
2.4.1. Compound 9d. (quant.); 1H NMR (400 MHz, D2O) d
5.33 (s, 1H), 5.20 (s, 1H), 4.30 (m, 1H), 4.20–3.15 (m,
22H), 2.50 (m, 1H), 2.20 (m, 1H), 1.85 (s, 1H), 1.25 (m,
1H); 13C NMR (75 MHz, D2O) d 164.0, 163.5, 163.1, 162.6,
157.6, 122.5, 118.6, 114.7, 110.9, 101.5, 93.3, 82.6, 78.3,
77.3, 74.1, 68.7, 66.8, 63.1, 61.7, 54.5, 49.7, 48.5, 47.4,
46.9, 38.4, 38.1, 28.0, 27.8; [a]D¼þ27 (c 1.0, H2O);
MALDI-FTMS calcd for C23H49N9O9Na (MþNaþ)
618.3545; found 618.3548.
2.4.2. Compound 9e. (quant.); 1H NMR (400 MHz, D2O) d
5.35 (s, 1H), 5.15 (s, 1H), 4.15–2.85 (m, 30H), 2.42 (m,
1H), 1.90 (m, 3H); 13C NMR (75 MHz, D2O) d 165.8,
163.6, 115.3, 101.9, 94.1, 83.1, 82.3, 77.7, 74.4, 69.2, 67.1,
63.6, 55.1, 49.0, 47.9, 46.3, 38.9, 37.7, 32.2, 25.8; [a]D¼
þ29 (c 1.0, H2O).
2.4.7. Compound 12. A solution of 10 (50.0 mg,
0.037 mmol) in MeOH (2 mL) was treated with a catalytic
amount of NaOMe. The reaction mixture was stirred at
room temperature until completion (approximately 2 h),
neutralized by addition of Amberlite IR-120(Hþ), the resin
was filtered off and the filtrate was evaporated to dryness.
2.4.3. Compound 9g. (quant.); 1H NMR (400 MHz, D2O) d
5.27 (s, 1H), 5.05 (s, 1H), 4.50–3.05 (m, 30H), 2.40 (m,

992
S. Hanessian et al. / Tetrahedron 59 (2003) 983–993
To the resulting residue in AcOH (80% in H2O, 2 mL), was
added 10% Pd/C (approximately 10 mg) and the mixture
was stirred under 1 atm of hydrogen overnight. The mixture
was filtered through a Celite pad, concentrated under
vacuum, diluted with water and lyophilized to afford 12
(30 mg, quant.) as a fluffy white solid; 1H NMR (400 MHz,
D2O) d 5.45 (s, 1H), 5.06 (s, 1H), 4.03–3.53 (m, 16H),
3.50–3.30 (m, 4H), 3.20 (m, 2H), 2.37 (m, 1H), 2.10 (m,
1H), 2.01 (m, 1H), 1.84 (s, 15H), 1.78 (m, 1H); 13C NMR
(100 MHz, D2O) d 182.1, 101.8, 94.1, 82.8, 82.5, 78.7, 73.8,
72.4, 72.3, 69.3, 65.8, 65.1, 61.4, 60.1, 55.5, 50.9, 49.3,
48.5, 40.2, 31.1, 30.0, 23.9; [a]D¼þ42 (c 1.1, H2O);
MALDI-FTMS calcd for C20H41N5O10Na (MþNaþ)
534.2745; found 534.2745.
(400 MHz, CDCl3) d 8.50 (broad s, 2H), 7.40–7.23 (m, 5H),
5.16 (s, 2H), 3.63–3.42 (m, 8H), 1.49 (s, 36H); 13C NMR
(100 MHz, CDCl3) d 163.5, 156.8, 153.4, 136.6, 128.9,
128.5, 128.4, 83.8, 80.5, 80.0, 68.1, 47.1, 46.7, 28.4; FAB
m/z calcd (MþHþ) 722.4; found 722.4.
2.4.11. Compound 17. To a solution of 16 (383 mg,
0.530 mmol) in MeOH (8 mL) was added 10% Pd/C
(approximately 10 mg) and the mixture was stirred under
1 atm of hydrogen for 5 h, then few drops of AcOH were
added. The mixture was filtered through a Celite pad,
concentrated under vacuum, diluted with water and
lyophilized to afford 17 (340 mg, quant.) as a fluffy white
1
solid; H NMR (400 MHz, CD2OD) d 3.70 (m, 4H), 3.30
(m, 4H), 1.99 (s, 3H), 1.54 (s, 18H), 1.49 (s, 18H); 13C NMR
(100 MHz, CD2OD) d 162.5, 157.7, 152.6, 83.9, 80.1, 38.1,
27.6, 27.2, 20.0; HRMS calcd for C26H50N7O8 (MþHþ)
588.37208; found 588.37269.
2.4.8. Compound 13. A solution of 11 (50.0 mg,
0.028 mmol) in MeOH (2 mL) was treated with a catalytic
amount of NaOMe. The reaction mixture was stirred at
room temperature until completion (approximately 2 h),
neutralized by addition of Amberlite IR-120(Hþ), the resin
was filtered off and the filtrate was evaporated to dryness.
To the resulting residue in AcOH (80% in H2O, 2 mL), was
added 10% Pd/C (approximately 10 mg) and the mixture
was stirred under 1 atm of hydrogen overnight. The mixture
was filtered through a Celite pad, concentrated under
vacuum, diluted with water and lyophilized to afford 13
(25 mg, quant.) as a fluffy white solid; 1H NMR (400 MHz,
D2O) d 5.30 (s, 1H), 5.04 (s, 1H), 4.00–3.27 (m, 23H),
3.22–3.00 (m, 3H), 2.20 (m, 1H), 2.10 (m, 1H), 1.95 (m,
1H), 1.76 (s, 15H), 1.60 (m, 1H); 13C NMR (100 MHz,
D2O) d 181.6, 164.1, 156.5, 101.2, 92.8, 83.2, 82.5, 77.2,
73.6, 72.9, 69.1, 65.6, 64.8, 64.4, 61.6, 60.1, 55.2, 54.1,
53.3, 50.2, 48.9, 48.1, 41.3, 30.7, 29.8, 23.5; [a]D¼þ52 (c
1.0, H2O).
2.4.12. Compound 19. To a solution of 18 (203 mg,
0.921 mmol) in CHCl3 (10 mL) was added successively
Et3N (0.26 mL, 1.8 mmol) followed by N,N0-di-Boc-N00-
trifluoromethanesulfonylguanidine15 (360 mg, 1.01 mmol).
The reaction mixture was stirred at room temperature for
1.5 h, diluted with CH2Cl2, washed with HCl 1N, NaHCO3
(sat.), brine, and dried over Na2SO4. The solvent was
removed under vacuum and the crude oil was purified by
flash chromatography (EtOAc/hexanes, 20:80). To a
solution of the resulting oil in MeOH (6 mL) was added
10% Pd/C (approximately 10 mg), the mixture was stirred
under 1 atm of hydrogen for 24 h, filtered through a Celite
pad, concentrated under vacuum, diluted with water and
lyophilized to afford 19 (180 mg, 60%) as a fluffy white
1
solid; H NMR (400 MHz, CD2OD) d 3.77 (m, 4H), 3.31
(m, 4H), 1.49 (s, 18H); 13C NMR (100 MHz, CD2OD) d
152.8, 81.2, 43.4, 43.1, 40.2, 27.5; HRMS calcd for
C15H28N4O4 (MþHþ) 328.21105; found 328.21155.
2.4.9. Compound 15. To a solution of 14a21 (300 mg,
0.989 mmol) in CH2Cl2 (6 mL) was added successively
Et3N (0.15 mL, 1.1 mmol), CbzCl (0.16 mL, 1.1 mmol) at
08C and the solution was stirred overnight at room
temperature. The solvent was removed under vacuum, the
residue was dissolved in EtOAc/H2O, the aqueous layer was
extracted with EtOAc, the combined organic extracts were
washed with water, brine and dried over Na2SO4. The
solvent was removed under vacuum and the crude oil was
purified by flash chromatography (EtOAc/hexanes, 30:70)
to give 15 (307 mg, 71%) as a colorless oil; 1H NMR
(400 MHz, CDCl3) d 7.40–7.20 (m, 5H), 5.11 (s, 2H),
3.40–3.08 (m, 8H), 1.41 (s, 18H); 13C NMR (100 MHz,
CDCl3) d 157.2, 156.6, 156.4, 136.8, 129.0, 128.5, 128.3,
79.8, 67.9, 48.2, 39.8, 28.8; HRMS calcd for C22H36N3O6
(MþHþ) 438.26041; found 438.26025.
Acknowledgements
We thank NSERC for financial assistance through the
Medicinal Chemistry Chair Program. M. T. acknowledges
scholarships from NSERC and FCAR. We thank Dr
William Baker (Pathogenesis) for his support at the
inception of this project. We acknowledge the services of
Lynn Barker (Pathogenesis, Seattle, WA, now Chiron
Corporation, Emeryville, CA) for MIC determinations and
Dr Kristin A. Sannes-Lowery for KD determinations, and
Ms L. M. Risen for T/T and MIC evaluations.
2.4.10. Compound 16. To a solution of 15 (244 mg,
0.558 mmol) in CH2Cl2 (12 mL) was added TFA (0.86 mL,
11 mmol) and stirred at room temperature for 2 h. The
solvent was evaporated under vacuum, the residue was
dissolved in CH2Cl2 (7 mL), then00 Et3N (0.39 mL,
2.8 mmol) followed by N,N0-di-Boc-N -trifluoromethane-
sulfonylguanidine26 (424 mg, 1.12 mmol) were added. The
reaction mixture was stirred at room temperature for 1 h,
and concentrated under vacuum. The resulting residue was
purified by flash chromatography (EtOAc/hexanes, 25:75)
to give 16 (384 mg, 95%) as a colorless oil; 1H NMR
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