Bacterial transferase MraY inhibitors: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2010.04.023

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino,

Full text of DOI:10.1016/j.bmc.2010.04.023

Bioorganic & Medicinal Chemistry 18 (2010) 4560–4569
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bacterial transferase MraY inhibitors: Synthesis and biological evaluation
Delphine Lecerclé a, Anthony Clouet a, Bayan Al-Dabbagh b, , Muriel Crouvoisier b, Ahmed Bouhss b,
a,
Christine Gravier-Pelletier a, , Yves Le Merrer
*
*
a Université Paris Descartes, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Pères, 75006 Paris, France
b Université Paris-Sud, IBBMC, UMR 8619 CNRS, Enveloppes Bactériennes et Antibiotiques, Bât. 430, 91405 Orsay, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 December 2009
Revised 6 April 2010
Accepted 7 April 2010
Available online 13 April 2010
New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribo-
syl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with pro-
line or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a
triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interest-
ing inhibitory activity for both amino compounds.
Ó 2010 Published by Elsevier Ltd.
Keywords:
Bacterial transferase MraY
Inhibition
Bis-epoxide
Triazole
Glycosylation
Cycloaddition
1. Introduction
On the one hand, for this new pharmacophore, crucial residues
such as aminoribosyl and uracil moieties have been retained. On
In an ongoing programme, undertaken to develop new potential
antibiotics,1 we are targeting the bacterial transferase MraY which
is an essential enzyme2 in peptidoglycan biosynthesis.3 The latter
is a unique feature in bacteria cell wall, without equivalent in
eukaryotic cells. Enzymes involved in this biopolymer biosynthesis
have been demonstrated to be ubiquitous and essential to bacterial
growth3a and therefore represent challenging targets for the search
of new antibiotics. The transferase MraY, which catalyses the first
membrane step of peptidoglycan biosynthesis (Fig. 1), has been lit-
tle exploited due to its transmembrane localisation,4 so that it is
currently the target of no antibiotics in clinical use. It catalyses
the transfer of uridine mono-phosphate-N-acetyl-muramoyl-pen-
tapeptide from its precursor UDP-Mur-N-Ac-pentapeptide onto
undecaprenyl phosphate, resulting in the formation of lipid I.
We recently developed the synthesis of a new pharmacophore,5
whose structure is bioinspired from both the south part of liposid-
omycins6 (Fig. 2), which are naturally occurring inhibitors of MraY,
and of a pharmacophore developed by Aventis.7
the other hand, an inversion of the absolute configuration at one
chiral center (C2) and insertion of an extra methylene group be-
tween the sugar and the pyrimidic base, expected to enhance the
stability of the resulting inhibitors,8 have been introduced. Such
an inversion of the absolute configuration at C2 should not be prej-
udiciable to biological activity in reference to a structure–activity
relationships study developed by Aventis9 and related to their
apparented pharmacophore. Our strategy (Fig. 3) presents the
advantage that only two main key steps allow the obtention of
CYTOPLASM
UDP-Mur-N-Ac-pentapeptide
UMP
MraY
Undecaprenyl-P
Bac A
Lipid I
Pi
UDP-GlcNAc
UDP
MurG
Undecaprenyl-PP
Lipid II
MEMBRANE
PERIPLASM
* Corresponding authors. Tel.: +33 142862181; fax: +33 142868387 (C.G.-P.); tel.:
+33 142862176; fax: +33 142868387 (Y.L.M.).
E-mail addresses: christine.gravier-pelletier@parisdescartes.fr (C. Gravier-Pelle-
tier), yves.le-merrer@parisdescartes.fr (Y. Le Merrer).
Present address: Department of Internal Medicine, Faculty of Medicine and
Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, United Arab
Emirates.
PBPs
PBPs
Peptidoglycan
Polymer
Acceptor
Figure 1. Role of MraY in bacterial peptidoglycan biosynthesis.
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.04.023

D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4561
R2
The present study is directed towards the introduction of struc-
tural diversity on the amino end of the scaffold expected to im-
prove the resulting inhibitors’ biological activity. Accordingly,
compounds including other basic residues such as proline or gua-
nidine have been targeted. Furthermore, to increase the number
of compounds in this series, it was decided to take advantage of
the Huisgen 1,3-dipolar cycloaddition11,12 that involves the azido
group, a precursor of the scaffold amino group, and various al-
kynes. The chosen alkynes were substituted by the same amine,
proline and guanidine substituents, which resulted in the insertion
of a triazole linker in the previous compounds. Indeed, numerous
examples of interesting biological properties13 displayed by such
triazole-containing compounds have already been demonstrated.
Such a unit is not just a passive linker but a rather active pharma-
cophore that may significantly contribute to protein binding.
R3O2C
CO2H
N
O
O
O
O
O
NH
O
N
O
O
N
O
O
H2N
OH
HO
R1
HO
R1 = OH or OSO3H
R2 = lipid side chain
R3 = H (Liposidomycins)
R3 = methyl glucoside (Caprazamycins)
O
NH
N
O
O
3'
O
O
2
H2N
HO
OH
2. Results and discussion
HO
OH
O
N
Aventis pharmacophore
According to the proposed retrosynthetic plan, the azidophar-
NH
macophore
(Scheme 1) from the C2-symmetrical 1,2:5,6-dianhydro-3,4-di-O-
benzyl- -iditol readily available from
-mannitol.14
4 was efficiently obtained in only two steps
O
O
O
O
L
D
H2N
Tandem nucleophilic opening—O-cyclisation15,16 of this bis-
epoxide by bis-(trimethylsilyl) uracil, in situ prepared from uracil
and N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA), was per-
formed in the presence of magnesium(II) perchlorate in refluxing
acetonitrile. The resulting uridine-like derivative 2 was obtained
in 46% yield. The latter was then glycosylated with 5-azido-1,5-
HO
OH
HO
Targeted pharmacophore
OH
Figure 2. Natural inhibitors of MraY and pharmacophores.
di-deoxy-2,3-di-O-ethylpropylidene-1-fluoro-D 3
-ribofuranose17,18
in the presence of boron trifluoride etherate leading to the major
formation of the protected b-glycosylated azido scaffold 4. Indeed,
the presence of the hindered isopentylidene 1,2-diol protecting
O
O
N
NH
NH
group on the sugar
a face promotes major glycosylation on its b
N
O
O
O
O
face (b/a = 13/1). The configuration in anomeric position was
O
O
O
unambiguously assigned by 1H NMR analysis: singlet or doublet
O
A
N3
00
for H1 in the b- or
a
-anomer, respectively.18 The pure b-anomer
HO
OH
PO
OP
HO
OH
PO
OP
was easily separated from its a-anomer by chromatographic puri-
fication and was isolated in 87% yield.
O
N
We next turned to the preparation of the first series of inhibitors
(Scheme 2). Acidic hydrolysis of the di-O-ethylpropylidene protec-
tive group of 4 was carried out in the presence of aqueous trifluoro-
acetic acid giving the corresponding diol 5 in 80% yield. Finally,
hydrogenolysis of the benzyl ethers and concomitant azido reduc-
tion were achieved in the presence of palladium black in acetic acid.
It has to be pointed out that the partial reduction of the uracil
:
H2N
A
NH
O
O
N
O
O
X
H
N3
NH
HO
+
NH
PO
OP
O
PO
OP
H2N
N
H
H2N
O
O
O
O
N
N
N
O
NH
N
N
+
NH
NH
N
H
N
H
O
PO
OP
NH
O
O
N
O
N
O
N
O
H
NH
HO
a
BnO
OBn
H2N
N
H
BnO
OBn
2
1
N
N
N
O
N
O
NH
F
Figure 3. Retrosynthetic analysis.
N3
3
O
O
O
O
O
O
the azido-pharmacophore,5 namely a tandem N-alkylation—O-cyc-
N3
BnO
OBn
lisation of the C2-symmetrical
L
-ido-bis-epoxide 1 with uracil, and
b
O
O
O-glycosylation with a masked aminoribosyl derivative.5 Its depro-
tection results in the amino scaffold whose biological evaluation on
MraY from Bacillus subtilis10 causes an inhibition of the enzymatic
4
Scheme 1. Reagents and conditions: (a) BSTFA, CH3CN then Mg(ClO4)2,
(b) BF3ꢀOEt2, CH2Cl2, M.S., ꢁ78 °C, 87%.
D, 46%;
activity with an IC50 of 580 lM.

4562
D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
O
N
O
N
NH
NH
O
O
O
O
O
O
O
O
b
N3
H2N
a
BnO
OBn
HO
OH
HO
OH
HO
OH
O
N
O
N
O
N
O
N
5
6
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
e
O
a
O
O
O
O
O
d
A
N
H
N
H
N
H
NBoc
NBoc
NH
BnO
OBn
BnO
OBn
O
HO
OH
HO
OH
O
c
O
O
O
O
O
HO
OH
9
10
11
O
O
4 : A = N3
8 : A = NH2
NP
NBoc
NH
N
O
N
O
a
N
O
NBoc
NBoc
N
NH
O
O
O
O
h
O
O
O
O
O
f
BocNP
N
P
Boc2N
H2N
N
Boc
H
BnO
OBn
HO
OH
HO
OH
O
O
O
O
HO
OH
12 : P = H
13 : P = Boc
14
15
g
Scheme 2. Reagents and conditions: (a) TFA, H2O, 80%, 70% and 60%, respectively, for 5, 11 and 15; (b) H2, Pd, AcOH, 20%; (c) (Ph2PCH2)2, CH2Cl2, H2O, 90%; (d) N-Boc-
L-
proline, HATU, Et3N, DMF, 68%; (e) H2, Pd(OH)2, EtOAc, EtOH, 96%; (f) BocNHC(SMe)NBoc, HgCl2, CH2Cl2, Et3N, 90%; (g) Boc2O, DMAP, Et3N, THF, 92%; (h) H2, Pd/C, MeOH, 99%.
double bond was observed during this step affording 7 (6/7 = 85/
15). Nevertheless, the pure amino pharmacophore 6 could be iso-
lated after careful flash chromatographic purifications. In a comple-
mentary manner, azido group reduction under Staudinger
conditions, in the presence of bis-1,2-diphenyl phosphinoethane,19
led to the corresponding amine 8 and was followed by coupling
with N-Boc-proline in the presence of HATU as coupling agent20
giving the protected proline derivative 9 (61% overall yield). Then,
benzyl ethers hydrogenolysis in the presence of Pearlman’s catalyst
and acidic hydrolysis with aqueous trifluoroacetic acid of both pro-
line N-Boc and isopentylidene diol protecting groups gave the pro-
line targeted inhibitor 11 in good yield. Finally, guanylation of the
amino scaffold 8 was carried out with N,N0-di-Boc-S-methyl isot-
hiourea in the presence of mercuric(II) chloride giving 12 (90%
yield). However, as precedently observed for the preparation of 6,
subsequent O-benzyl ethers deprotection by hydrogenolysis in
the presence of Pearlman’s catalyst, led to a slight reduction of
the uracil double bond and total deprotection of benzyl ethers could
not be achieved, whatever the conditions. To avoid this side reac-
tion, the uracil and guanidine moieties were first protected as the
N-Boc derivative 13 prior to hydrogenolysis, which could then be
efficiently performed in the presence of 10% Pd/C in MeOH, without
any reduction of the uracil double bond, giving 14. Then, final acidic
hydrolysis of both isopentylidene and N-Boc protecting groups was
carried out to afford the expected guanidine inhibitor 15.
the corresponding alkynes (Scheme 3). They resulted from propar-
gylamine by its bis-N-Boc protection with tert-butyldicarbonate for
16, coupling with N-Boc-L-Pro-OH in the presence of HATU for 17
and reaction with N,N0-bis-(tert-butyloxycarbonyl)-1H-pyrazole-
1-carboxamidine for 18, respectively.
The 1,3-dipolar cycloaddition involving azido scaffold 4 and al-
kynes 16, 17 or 18 was then carried out in the presence of cop-
per(II) sulfate pentahydrate and sodium ascorbate in t-BuOH/H2O
to yield the expected 1,4-di-substituted triazoles 19, 20 or 21 in
moderate yield (Scheme 4). Then, two different pathways towards
the final compounds were designed depending on the eventual
uracil protection as its tert-butyl carbamate. On the one hand,
hydrogenolysis of benzyl ethers of 19 and 20 could be efficiently
carried out in the presence of 10% Pd on charcoal affording 22
and 23, respectively. Final acidic hydrolysis with aqueous trifluoro-
acetic acid of both N-Boc and the ketal protecting groups afforded
the targeted triazole substituted by an amine or a proline moiety,
24 or 25. On the other hand, direct hydrogenolysis of benzyl ethers
from 21 could not be achieved, since all the tested conditions re-
sulted in the recovery of starting material, probably due to catalyst
poisoning. Consequently, first N-Boc protection of both uracil and
guanidine was performed which gave 26, prior to benzyl ethers
hydrogenolysis which led to 27 and subsequent acidic hydrolysis
which afforded the targeted triazole substituted by a guanidine
moiety 28.
We next tackled the synthesis of the triazole-containing inhib-
itors from the azido scaffold 4. It first required the preparation of
The in vitro biological evaluation of the resulting compounds 6,
7, 11, 15, 24, 25, 28 on purified MraY was carried out as described
in the experimental section (Table 1). The residual activity of the
enzyme was measured in the presence of 2 mM concentration of
the tested compounds. For the more active compounds, the IC50
values were determined.
H2N
c
a
b
The results indicate that the amino scaffold shows an interest-
ing IC50 equal to 580 lM. The inhibition observed for compound
7 confirms that the uracil double bond is important for biological
activity as already observed by Aventis.21 However, the amine sub-
stitution with either a proline or a guanidine moiety led to the loss
of activity. Interestingly, the triazole insertion improves activity for
the inhibitor 25 or 28 as compared to the apparented compounds
11 or 15 that do not contain this linker. Nevertheless, the activity
of the triazole-containing inhibitors remains comparable to that
O
BocN
NH
NH
(Boc)2N
BocNH
NBoc
16
17
18
Scheme 3. Reagents and conditions: (a) Boc2O, DMAP, Et3N, THF; (b) N-Boc-L-
proline, HATU, Et3N, DMF, 99%; (c) N,N0-di-Boc-1H-pyrazole-1-carboxamidine,
(iPr)2NEt, DMF, 59%.

D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4563
O
Boc2N
19 : A =
20 : A =
NH
O
A
N
O
O
a
N
H
O
N
N
O
4
A
NBoc
N
BnO
OBn
NBoc
O
O
16, 17 or 18
21 : A =
BocNH
N
H
d
b
O
N
O
N
NBoc
NH
NBoc
A
Boc2N
N
O
O
Boc
Boc2N
22 : A =
23 : A =
O
O
O
O
N
N
O
N
N
O
O
N
N
HO
OH
BnO
OBn
N
H
O
O
O
O
NBoc
26
c
O
N
b
NBoc
NBoc
O
O
N
O
Boc2N
N
O
NH
NH
Boc
O
O
N
N
O
H2N
N
H
O
N
O
O
N
O
NH
O
O
N
N
O
N
N
O
HO
c
OH
N
N
O
O
HO
OH
HO
OH
27
O
N
HO
OH
HO
OH
25
NH
NH
24
H2N
N
O
H
O
O
N
N
O
N
HO
OH
28
HO
OH
Scheme 4. Reagents and conditions: (a) CuSO4ꢀ5H2O, sodium ascorbate, t-BuOH, H2O, 29%, 29% and 57%, respectively, for 19, 20 and 21; (b) H2, Pd/C, MeOH, 79%, 79% and 90%
for, respectively, 22, 23 and 26; (c) TFA, H2O, 47%, 64% and 50% for, respectively, 24, 25 and 28; (d) Boc2O, DMAP, Et3N, THF, 79%.
Table 1
prolino or guanidino derivatives showed no or weak inhibitory
Inhibitory activities of synthetized compounds on the MraY enzyme
activity. Nevertheless, the introduction of a triazole linker on the
Compound
IC50
M) or RAa (%)
6
7
11
15
24
25
28
previous compounds improved the inhibitory activity, but that
activity remained comparable to that of the amino scaffold, so that,
future work in this series will be based on that scaffold.
(l
580
946
—61%
1790
595
820
600
a
Residual activity (RA) of MraY in the presence of compound 11 at a final con-
centration of 2 mM.
4. Experimental
of the amino scaffold. Furthermore, it has to be pointed out that the
observed activity seems to be selective towards MraY, since testing
of these compounds at 1 mM on the MurG enzyme, catalyzing lipid
II formation (Fig. 1) revealed no inhibition.
4.1. Chemical synthesis
1H NMR (250 MHz) and 13C NMR (63 MHz) spectra were re-
corded on a Bruker AM250 in CDCl3 (unless indicated). 1H NMR
(500 MHz) and 13C NMR (125 MHz) spectra were recorded on a
Bruker Avance or Avance II. Chemical shifts (d) are reported in
ppm and coupling constants are given in Hz. Mass spectra, electro-
spray (ESI) and high resolution (HRMS) were recorded by the ser-
vice de Spectrométrie de Masse, ICSN Gif sur Yvette. Optical
rotations were measured with Perkin–Elmer 241C polarimeter
with a sodium (589 nm) or a mercury (365 nm) lamp at 20 °C. All
reactions were carried out in anhydrous solvents under a nitrogen
atmosphere (except for hydrogenolysis), and were monitored by
thin-layer chromatography with Merck 60F-254 precoated silica
(0.2 nm) on glass. Flash chromatography was performed with
3. Conclusions
In this study, we have developed a straightforward synthetic
route towards new inhibitors of the bacterial transferase MraY
based on an aminoribosyl-O-uridine like scaffold. The latter is read-
ily obtained through a tandem nucleophilic opening O-cyclisation of
L-ido-bis-epoxide by uracil and subsequent O-glycosylation of the
resulting primary alcohol with an azidoribosyl derivative. On the
one hand, azide reduction led to the amino scaffold, which can be
either coupled with proline or guanylated. On the other hand, the
azido scaffold can be submitted to cycloaddition with several amino,
prolinyl or guanidinyl substituted alkynes. Biological evaluation of
these compounds on MraY from B. subtilis revealed that the amino
scaffold presents an interesting inhibitory activity. However, the
Merck Kieselgel 60 (40–63 lm) or octadecyl-functionalized silica
gel (column C18). Spectroscopic 1H and 13C NMR, MS and/or ana-
lytical data were obtained using chromatographically homoge-
neous samples.

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D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4.1.1. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-
D
-
3.99–4.27 (m, 8H, H1, H2, H3, H4, H5, H2 , H3 , H4 ), 4.61–4.76 (m,
00
00
00
00
0
0
0
glucitol 2
4H, HCH2Ph), 5.01 (s, 1H, H1 ), 5.51 (d, JH5 -H6 = 7.9 Hz, 1H, H5 ),
7.38 (m, 11H, HAr, H6 ); 13C NMR ((CD3)2CO, 62.5 MHz): d 48.6
(C1), 54.5 (C5 ), 69.3 (C6), 72.1, 72.2 (CCH2Ph), 73.0 (C2 ), 75.6 (C3 ),
79.3 (C2), 82.7, 83.2 (C3, C4), 83.8 (C4 ), 84.0 (C5), 101.4 (C5 ),
108.8 (C1 ), 128.7, 128.9, 129.3, 129.4, 138.8, 139.1 (CAr), 147.0
(C6 ), 151.9 (C2 ), 164.5 (C4 ); HRMS calcd for [M+Na] 618.2176,
found 618.2170.
0
To a solution of uracil (560 mg, 5 mmol) in acetonitrile (5.0 mL),
was added bis-(trimethylsilyl)trifluoroacetamide (BSTFA) (6.6 mL,
25 mmol). The mixture was refluxed for 30 min. The silylated ura-
cil obtained after concentration in vacuo was dissolved in acetoni-
trile (5.0 mL), then a solution of bis-epoxide 1 (326 mg, 1 mmol) in
acetonitrile (5.0 mL) was added. The mixture was heated to 85 °C,
and magnesium(II) perchlorate (334 mg, 1.5 mmol) was added. The
reaction was stirred overnight, then concentrated in vacuo. The
residue was dissolved in EtOAc, washed with brine, dried (MgSO4),
and the solvent was evaporated in vacuo. Flash chromatography of
the crude (EtOAc/cyclohexane/MeOH 6:4:0.3) afforded 2 (202 mg,
46%) as a foamy solid. Rf 0.37 (EtOAc/cyclohexane/MeOH 5:4:1);
00
00
00
00
0
00
+
0
0
0
4.1.4. 2,5-Anhydro-1-deoxy-1-(uracil-10-yl)-6-(500-amino-100,500-
dideoxy-D D-glucitol 6
-ribos-100-yl)-
Palladium black (150 mg) was added to a stirred solution of 5
(105 mg, 0.18 mmol) in acetic acid (10 mL). The mixture was stir-
red under H2 for 6 h at rt. Acetic acid was removed in vacuo. The
crude was purified first with a column HyperSep C18 with H2O,
then H2O/MeOH (1:1). The cleanest fraction (first one) was purified
on silica gel chromatography, using different eluent system; start-
ing with CH2Cl2, then CH2Cl2/MeOH (8:2), then iPrOH, then iPrOH/
NH3 (100:25), and finally iPrOH/NH3:MeOH (90:25:10) to afford
½
a 2D0
ꢂ
ꢁ8 (c 1.0, MeOH); IR (neat):
m ;
2924, 1678, 1455, 1250 cmꢁ1
1H NMR ((CD3)2CO, 250 MHz): d 3.68–3.75 (m, 2H, H6), 3.90 (dd,
JH1-H1 = 14 Hz, JH1-H2 = 9 Hz, 1H, H1), 4.00–4.05 (m, 1H, H5), 4.22
(dd, JH1-H1 = 14 Hz, JH1-H2 = 4 Hz, 1H, H1), 4.25–4.29 (m, 2H, H3,
H4), 4.36 (dd, JH2-H1 = 9 Hz, JH2-H1 = 4 Hz, 1H, H2), 4.63 (d,
J = 12 Hz, 1H, HCH2Ph), 4.72 (s, 1H, HCH2Ph), 4.73 (s, 1H, HCH2Ph),
pure 6 (13.9 mg, 20%). Rf 0.27 (iPrOH/NH3 100:25); ½a D20
ꢁ33 (c
ꢂ
0
0
0
4.76 (d, J = 12 Hz, 1H, HCH2Ph), 5.54 (d, JH5 -H6 = 8 Hz, 1H, H5 ),
1.0, H2O); 1H NMR (D2O, 500 MHz): d 3.13 (dd, JH5 -H5 = 14 Hz, J
7.33–7.48 (m, 10H, HAr), 7.57 (d, JH6 -H5 = 8 Hz, 1H, H6 ); 13C NMR
((CD3)2CO, 62.5 MHz): d 48.8 (C1), 62.8 (C6), 71.7 (CCH2Ph), 78.8
00
00
0
0
0
00
00
00
00
00
H500-H400
= 6 Hz, 1H, H5 ), 3.43 (dd, JH5 -H5 = 14 Hz, JH5 -H4 = 3 Hz,
1H, H5 ), 3.73 (dd, JH6-H6 = 11 Hz, JH6-H5 = 8 Hz, 1H, H6), 3.92–3.98
(m, 2H, H5, H1), 3.99 (dd, JH6-H6 = 11 Hz, JH6-H5 = 3 Hz, 1H, H6),
4.06 (dd, JH3-H2 = 4 Hz, JH3-H4 = 3 Hz, 1H, H3), 4.16 (d, JH2 -H3 = 5 Hz,
00
0
(C2), 83.2 (C3, C4), 85.4 (C5), 101.1 (C5 ), 127.9, 128.0, 128.1,
128.4, 128.7, 128.8, 138.4, 138.8 (CAr), 146.5 (C6 ), 151.7 (C2 ),
0
0
+
00
00
0
163.9 (C4 ); HRMS calcd for [M+Na] 461.1689, found 461.1676.
00
1H, H 0 0 ), 4.17–4.22 (m, 2H, H1, H4 ), 4.26 (dd, JH4-H5 = 5 Hz, JH4-
H3 = 3 2Hz, 1H, H4), 4.28 (dd, J = 7 Hz, J = 5 Hz, 1H, H3 ), 4.35 (ddd,
4.1.2. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
00
(500-azido-100,500-dideoxy-200,300-O-isopentylidene-b- -ribos-100-
D
00
JH2-H1 = 9 Hz, JH2-H1 = JH2-H3 = 4 Hz, 1H, H2), 5.13 (s, 1H, H1 ), 5.86
(d, JH5 -H6 = 8 Hz, 1H, H5 ), 7.68 (d, JH6 -H5 = 8 Hz, 1H, H6 ); 13C
0
0
0
0
0
0
yl)-D-glucitol 4
00
NMR (CDCl3, 125 MHz): d 43.8 (C5 ), 48.9 (C1), 69.3 (C6), 73.1
(C3 ), 74.8 (C2 ), 77.2 (C4), 78.7, 78.8 (C2, C3), 79.1 (C4 ), 84.1 (C5),
To a solution of 2 (350 mg, 0.799 mmol) in CH2Cl2 (20 mL) were
added the fluorinated ribose 3 (295 mg, 1.2 mmol) and molecular
sieves 4 Å. The mixture was stirred for 1 h, then cooled to ꢁ78 °C
00
00
00
0
00
0
0
0
102.1 (C5 ), 108.7 (C1 ), 148.4 (C6 ), 153.0 (C2 ), 167.5 (C4 ); HRMS
calcd for [M+Na]+, 412.1332, found 412.1327.
and BF3ꢀOEt2 (152
lL, 1.2 mmol) was added. The mixture was stirred
at rt overnight. The mixture was diluted with CH2Cl2, washed with
saturated aqueous NaHCO3, and concentrated in vacuo. Flash chro-
matography of the crude (acetone/cyclohexane 4:6) afforded 4 as a
4.1.5. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
(500-amino-100,500-dideoxy-200,300-O-isopentylidene-b- -ribos-100-
D
white solid (462 mg, 87%). ½a D20
ꢂ
ꢁ4 (c 1.0, MeOH); 1H NMR (CDCl3,
yl)-D-glucitol 8
To a solution of 4 (148 mg, 0.22 mmol) in THF (10 mL) was added
1,2-bis-(diphenylphosphino)ethane (53 mg, 0.13 mmol) and water
(1 mL). The mixture was stirred overnight at rt, and then concen-
trated in vacuo. Flash chromatography of the crude (CH2Cl2/MeOH
500 MHz): d 0. 90, 0.93 (2t, J = 7 Hz, 6H, HCH3CH2), 1.57, 1.71 (2qd,
00
00
00
00
J = 7 Hz, 4 H, HCH2CH3), 3.13 (dd, JH5 -H5 = 13 Hz, JH5 -H4 = 7 Hz, 1H,
00
00
00
00
00
00
H5 ), 3.35 (dd, JH5 -H5 = 13 Hz, JH5 -H4 = 8 Hz, 1H, H5 ), 3.58 (dd,
JH6-H6 = 11 Hz, JH6-H5 = 7 Hz, 1H, H6), 3.72 (dd, JH1-H1 = 14 Hz,
JH1-H2 = 8 Hz, 1H, H1), 3.80 (dd, JH6-H6 = 11 Hz, JH6-H5 = 5 Hz, 1H,
H6), 3.89 (d, JH4-H3 = 3 Hz, 1H, H4), 4.03 (d, JH3-H4 = 3 Hz, 1H, H3),
95:5) afforded 8 as a white solid (128 mg, 90%). ½a D20
ꢁ29 (c 1.0,
ꢂ
MeOH); 1H NMR (CDCl3, 500 MHz):d 0.88, 0.92 (2t, J = 7 Hz, 6H,
H
CH3CH2), 1.56, 1.70 (2qd, J = 7 Hz, 4H, HCH2CH3), 2.68–2.71 (m, 2H,
00
4.05–4.07 (m, 1H, H4 ), 4.27–4.31 (m, 2H, H2, H5), 4.35 (dd,
JH1-H1 = 14 Hz, JH1-H2 = 3 Hz, 1H, H1), 4.41–4.48 (m, 2H, H2 , HCH2Ph),
00
H5 ), 3.57 (dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.75 (dd,
JH1-H1 = 14 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.78 (dd, JH6-H6 = 11 Hz,
JH6-H5 = 5 Hz, 1H, H6), 3.96 (d, JH4-H3 = 3 Hz, 1H, H4), 4.02 (d,
JH3-H4 = 3 Hz, 1H, H3), 4.17 (t, JH4 -H5 = 7 Hz, 1H, H4 ), 4.26–4.30
(m, 2H, H2, H5), 4.33 (dd, JH1-H1 = 14 Hz, JH1-H2 = 3 Hz, 1H, H1),
00
00
00
4.50–4.62 (m, 3H, H3 , HCH2Ph); 5.16 (s, 1H, H1 ), 5.63 (d, J = 8 Hz,
1H, H5 ), 7.27–7.41 (m, 11H, HAr, H6 ), 9.24 (s, 1H, H3 ); 13C NMR
0
0
0
00
00
00
(CDCl3, 125 MHz): d 7.6, 8.6 (CCH3CH2), 29.1, 29.6 (CCH2CH3), 48.5
(C1), 53.6 (C5 ), 68.5 (C6), 71.8, 71.9 (CCH2Ph), 79.0 (C2), 82.4 (C2 ),
82.8, 82.9 (C3, C4), 83.4 (C4 ), 85.6 (C3 ), 85.9 (C5); 101.8 (C5 ), 109.3
00
00
00
00
4.41–4.45 (m, 3H, H2 ,HCH2Ph), 4.49–4.62 (m, H3 , 3 H, HCH2Ph), 5.10
00
00
0
00
0
(s, 1H, H1 ), 5.61 (d, J = 8 Hz, 1H, H5 ), 7.25–7.38 (m, 10H, HAr), 7.42
00
(C1 ), 117.3 (CCEt2), 127.9, 128.3, 128.4, 128.7, 128.8, 137.3, 137.5
(d, J = 8 Hz, 1H, H6 ); 13C NMR (CDCl3, 125 MHz): d 7.5, 8.5 (CCH3CH2),
+
0
0
0
0
(CAr), 145.8 (C6 ), 151.2 (C2 ), 164.0 (C4 ); HRMS calcd for [M+Na]
686.2802, found 686.2814.
00
29.0, 29.5 (CCH2CH3), 45.3 (C5 ), 48.4 (C1), 68.0 (C6), 71.6, 71.9 (CCH2Ph),
79.0 (C2), 82.4, 82.7, 82.9, 83.3 (C3, C4, C5, C2 ), 86.0 (C3 ), 89.3 (C4 ),
00
00
00
4.1.3. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
0
00
101.7 (C5 ), 109.0 (C1 ), 116.7 (CCEt2), 127.8, 127.8, 128.1, 128.3,
(500-azido-100,500-dideoxy-
D
-ribos-100-yl)-
D
-glucitol 5
To a solution of 4b (20 mg, 0.03 mmol) in CH2Cl2 (1.0 mL) were
added TFA (30 L) and water (two drops). The mixture was stirred
0
0
0
128.7, 128.7, 137.4, 137.5 (CAr), 146.1 (C6 ), 151.3 (C2 ), 164.2 (C4 );
HRMS: Calcd for [M+Na]+ 660.2897, found 660.2914.
l
4.1.6. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
at rt for 48 h, then concentrated, diluted with EtOAc, washed with
brine, dried (MgSO4), and concentrated in vacuo. The crude was
purified by flash chromatography (CH2Cl2/MeOH 95:5), yielding 5
as a colourless oil (11 mg, 62%). Rf 0.27 (CH2Cl2/MeOH 95:5);
(500-(N-Boc-
lidene-b-
To a solution of Boc-
(4 mL) was added HATU (82 mg, 0.22 mmol) and TEA (40
L
-prolinyl-N0-amido)-100,500-dideoxy-200,300-O-isopenty
D
-ribos-100-yl)-
D
-glucitol 9
-Pro-OH (47 mg, 0.22 mmol) in DMF
L,
L
a 2D0
ꢂ
ꢁ6 (c 1.0, MeOH); IR (neat):
m 3381, 2924, 2102, 1676,
l
½
1455m, 1250 cmꢁ1
;
1H NMR ((CD3)2CO, 250 MHz): d 3.34 (m, 1H,
0.28 mmol). To the mixture was added a solution of 8 (92 mg,
0.14 mmol) in DMF (2 mL). The mixture was stirred overnight at
00
00
H5 ), 3.41 (m, 1H, H5 ), 3.58 (m, 1H, H6), 3.81 (m, 2H, H1, H6),

D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4565
rt, and then concentrated in vacuo. Flash chromatography (cyclo-
4.1.9. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
(500-(N,N0-bis-(tert-butyloxycarbonyl)guanidino)-100,500-dideoxy-
200,300-O-isopentylidene-b- -ribos-100-yl)-
-glucitol 12
hexane/acetone 65:35) afforded 9 as a white solid (81 mg, 68%).
½
a 2D0
ꢂ
ꢁ49 (c 1.0, MeOH); 1H NMR (CDCl3, 250 MHz): d 0.87, 0.91
D
D
(2t, J = 7.4 Hz, 6H, HCH3CH2), 1.46 (s, 9H, HBoc), 1.54, 1.69 (2qd,
To a solution of 8 (91 mg, 0.14 mmol) in CH2Cl2 (10 mL) were
J = 7.4 Hz, 4H, HCH2CH3), 1.76–1.88 (m, 2H, H ), 1.92–2.02 (m, 1H,
successively added N,N0-bis-(tert-butyloxycarbonyl)-S-methyl-
c
Hb), 2.06–2.24 (m, 1H, Hb), 3.38–3.52 (m, 5H, H5”, Hd, H1), 3.60
(dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.78–3.84 (m, 1H, H6),
3.96 (d, JH3-H4 = 3 Hz, 1H, H3), 4.03 (d, JH4-H3 = 3 Hz, 1H, H4), 4.24–
isothiourea (49 mg, 0.17 mmol), triethylamine (40 lL, 0.28 mmol)
and HgCl2 (58 mg, 0.21 mmol). The mixture was stirred for 36 h
at rt, then filtered through a Celite pad, washed with CH2Cl2 and
the filtrate was concentrated in vacuo. Flash column chromatogra-
phy (cyclohexane/acetone 6:4) afforded 12 as a white solid
00
00
00
00
4.33 (m, 4H, H1, H2, H5 ), 4.35–4.62 (m, 8H, H2 , H3 , H4 , H5,
00
0
H
CH2Ph), 5.21 (s, 1H, H1 ), 5.62 (d, J = 8 Hz, 1H, H5 ), 7.30–7.41 (m,
11H, HAr, H6 ), 9.4 (s, 1H, H3 ); 13C NMR (CDCl3, 125 MHz): d 7.8,
(113 mg, 90%). ½a D20
ꢂ
+5 (c 1.0, MeOH); 1H NMR (CDCl3, 500 MHz):
d 0.87, 0.91 (2t, J = 7 Hz, 6H, HCH3CH2), 1.46, 1.48 (2s, 18H, HBoc),
0
0
8.8 (CCH3CH2), 28.8 (CBoc), 28.8–29.7 (CCH2CH3, C , Cb), 42.5, 42.6
c
00
00
00
(Cd, C5 ), 47.5 (C1), 68.5 (C6), 72.0, 72.3 (CCH2Ph), 79.3 (C2), 82.7,
1.54, 1.69 (2qd, J = 7 Hz, 4H, HCH2CH3), 3.26 (ddd, JH5 -H5 = 14 Hz,
00
00
00
0
00
00
00
00
82.7, 83.3, 83.6 (C3, C4, C5, C2 , C3 , C4 ), 86.1 (CBoc), 102.0 (C5 ),
JH5 -H4 = 9 Hz, JH5 -NH = 4 Hz,1H, H5 ), 3.61 (dd, JH6-H6 = 11 Hz,
00
109.4 (C1 ), 117.0 (CCEt2), 128.1, 128.1, 128.4, 128.5, 128.9, 129.0,
JH6-H5 = 6 Hz, 1H, H6), 3.67–3.69 (m, 1H, H1), 3.76–3.84 (m, 2H,
0
0
0
00
137.7, 137.8 (CAr), 146.0 (C6 ), 151.5 (C2 ), 164.4 (C4 ); HRMS calcd
H6, H5 ), 3.93 (d, JH4-H3 = 4 Hz, 1H, H4), 4.01 (d, JH3-H4 = 4 Hz, 1H,
for [M+Na]+ 857.3949, found 857.3945.
H3), 4.05–4.07 (m, 1H, H5), 4.27–4.35 (m, H2, 2H, H1, H4 ), 4.41
00
00
00
(d, J = 12 Hz, 1H, HCH2Ph), 4.46–4.58 (m, 5H, HCH2Ph, H2 , H3 ), 5.14
00
0
0
0
0
(s, 1H, H1 ), 5.58 (dd, JH5 -H6 = 8 Hz, JH5 -NH = 2 Hz, 1H, H5 ), 7.27–
4.1.7. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-6-
0
7.36 (m, 11H, H6 , HAr), 8.57–8.59 (m, 1H, NH), 9.16–9.18 (m, 1H,
0
(500-(N-Boc-
L
-prolinyl-N0-amido)-100,500-dideoxy-200,300-O-isopenty
H3 ), 11.48 (s, 1H, NH); 13C NMR (CDCl3, 125 MHz): d 7.5, 8.5
(CCH3CH2), 28.2, 28.4 (CBoc), 29.0, 29.5 (CCH2CH3), 42.3 (C5 ), 48.6
lidene-b-
D
-ribos-100-yl)-
D-glucitol 10
00
A suspension of 20% Pd(OH)2 (6 mg) and 9 (33 mg, 0.04 mmol)
in a mixture of EtOAc (2 mL) and EtOH (0.75 mL) was saturated
with dihydrogen and stirred for 3 h. The reaction mixture was then
filtered through a Celite pad and the residue was washed with
MeOH. The filtrate was concentrated in vacuo to afford 10 as a
(C1), 68.1 (C6), 71.6, 72.0 (CCH2Ph), 78.9 (C2), 80.9, 81.7 (CBoc), 82.5
00
00
00
(C3 ), 82.9, 83.0 (C3, C4), 83.3 (C5), 85.3 (C4 ), 85.8 (C2 ), 101.7
0
00
(C5 ), 109.1 (C1 ), 116.9 (CCEt2), 127.8, 127.9, 128.0, 128.2, 128.7,
0
0
128.8, 137.4, 137.4 (CAr), 146.0 (C6 ), 151.2 (C2 ), 153.1 (Cgua),
+
0
156.3 (CBoc), 163.9 (C4 ); MS: m/z 880 (100%, [M+1] ).
white solid (25 mg, 96%). ½a D20
ꢂ
ꢁ77 (c 1.0, MeOH); 1H NMR (CDCl3,
4.1.10. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(30-(N-tert-
butyloxycarbonyl)-uracil-10-yl)-6-(500-(N,N0,N00-tetra(tert-butyl
oxycarbonyl)guanidino)-100,500-dideoxy-200,300-O-isopentylidene-
500 MHz): d 0.84, 0.88 (2t, J = 7.4 Hz, 6H, HCH3CH2), 1.44 (s, 9H,
HBoc), 1.54, 1.67 (2qd, J = 7.4 Hz, 4H, HCH2CH3), 1.84–1.88 (m, 2H,
H ), 1.96–2.08 (m, 1H, Hb), 2.12–2.21 (m, 1H, Hb), 3.36–3.46 (m,
c
b-
D
-ribos-100-yl)-
D
-glucitol 13
To a solution of 12 (105 mg, 0.12 mmol) in THF (10 mL) were
added, di-tert-butyldicarbonate (78 mg, 0.36 mmol), DMAP (5 mg,
0.024 mmol) and triethylamine (50 L, 0.36 mmol). The solution
4H, H5”, Hd), 3.55–3.60 (m, 1H,H6), 3.75 (dd, JH1-H1 = 13 Hz,
JH1-H2 = 6.5 Hz, 1H, H1), 3.90–3.94 (m, 2H, H5, H6), 4.04–4.06 (m,
000
00
,
2H, H4, H2), 4.20–4.25 (m, 2H, H1, H2 ), 4.28–4.30 (m, 1H, H4
)
00
00
00
00
00
4.65 (d, JH2 -H3 = 5.5 Hz, 1H, H2 ), 4.58 (d, JH3 -H2 = 5.5 Hz, 1H,
l
0
00
00
0
0
H3 ), 5.12 (s, 1H, H1 ), 5.66 (d, JH5 -H6 = 8.5 Hz, 1H, H5 ), 7.39 (d, J
was stirred overnight at rt, then concentrated in vacuo. Flash col-
umn chromatography (cyclohexane/acetone 6:4) afforded 13 as a
= 8.5 Hz, 1H, H6 ); 13C NMR (CDCl3, 125 MHz): d 7.3, 8.4
0
H60-H50
colourless oil (129 mg, 92%). ½a D20
ꢂ
ꢁ23 (c 1.0, CH2Cl2); 1H NMR
(CCH3CH2), 22.7 (C ), 28.4 (CBoc), 28.8 (CCH2CH3), 29.0 (Cb), 29.3
c
00
(CCH2CH3), 42.7 (Cd), 47.3 (C5 ), 47.9 (C1), 60.4 (C ), 68.4 (C6), 79.0
a
(CDCl3, 500 MHz): d 0.86, 0.91 (2t, J = 7 Hz, 6H, HCH3CH2), 1.47,
1.50, 1.52 (3s, 36H, HBoc), 1.53 (qd, J = 7 Hz, 2H, HCH2CH3), 1.62 (s,
9HBoc), 1.67 (qd, J = 7 Hz, 2H, HCH2CH3), 3.56 (dd, JH6-H6 = 11 Hz,
JH6-H5 = 7 Hz, 1H, H6), 3.68 (dd, JH1-H1 = 15 Hz, JH1-H2 = 9 Hz, 1H,
H1), 3.79 (dd, JH6-H6 = 11 Hz, JH6-H5 = 5 Hz, 1H, H6), 3.87 (dd,
00
00
(C3), 79.5 (C4), 80.9 (CBoc), 82.2 (C3 ), 84.6 (C5), 85.6 (C2 ), 85.8
00
0
00
0
0
(C4 ), 102.0 (C5 ), 109.1 (C1 ), 116.9 (CCEt2), 146.0 (C6 ), 151.5 (C2 ),
+
0
155.8 (CBoc), 164.0 (C4 ); HRMS calcd for [M+Na] 677.3010, found:
677.3000.
00
00
00
00
00
JH5 -H5 = 14 Hz, JH5 -H4 = 4 Hz, 1H, H5 ), 3.93 (d, JH3-H4 = 4 Hz, 1H,
H4), 4.00 (d, JH3-H4 = 4 Hz, 1H, H3), 4.05–4.08 (m, 1H, H5), 4.25
4.1.8. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-6-
00
00
00
00
00
(dd, JH5 -H5 = 14 Hz, JH5 -H4 = 10 Hz, 1H, H5 ), 4.32–4.35 (m, 2H,
H1, H2), 4.42 (d, J = 12 Hz, 1H, HCH2Ph), 4.49–4.55 (m, 3H, HCH2Ph
(500-(N-
L D-
-prolinyl-N0-amido)-100,500-dideoxy-200,300-dihydroxy-b-
,
ribos-100-yl)-
D
-glucitol 11
00
00
00
H4 ), 4.59 (d, J = 12 Hz, 1H, HCH2Ph), 4.65 (d, JH2 -H3 = 6 Hz, 1H,
H2 ), 4.93 (d, JH3 -H2 = 6 Hz, 1H, H3 ), 5.11 (s, 1H, H1 ), 5.67 (d,
JH5 -H6 = 8 Hz, 1H, H5 ), 7.28 (d, JH6 -H5 = 8 Hz, 1H, H6 ), 7.28–7.34
(m, 6H, HAr), 7.36–7.39 (m, 4H, HAr); 13C NMR (CDCl3, 125 MHz):
d 7.6, 8.6 (CCH3CH2), 27.6, 28.1, 28.2 (CBoc), 29.1, 29.4 (CCH2CH3),
At 0 °C, to a solution of 10 (25 mg, 0.04 mmol) in water
(0.55 mL) was added TFA (2.2 mL). The mixture was stirred for
1 h at rt, and then concentrated in vacuo. The residue was diluted
with water and aqueous NH4OH (28%, 6 mL) was added at 0 °C. The
mixture was then concentrated in vacuo. Chromatography on re-
verse phase (C18) with H2O, H2O/MeOH (1:1) and then MeOH
00
00
00
00
00
0
0
0
0
0
0
00
48.8 (C5 ), 49.3 (C1), 68.0 (C6), 71.7, 72.1 (CCH2Ph), 78.7 (C2), 82.3
00
00
00
(C3 ), 82.5 (C3), 83.4 (C4, C5), 84.2 (C4 ), 84.3 (C2 ), 84.6, 85.9, 86.8
(CBoc), 101.6 (C5 ), 109.3 (C1 ), 116.5 (CCEt2), 127.9, 128.0, 128.2,
128.3, 128.7, 128.8, 137.6, 137.3 (CAr), 144.2 (CBoc), 145.4 (C6 ),
147.6, 148.1 (CBoc), 149.4 (C2 ), 151.6 (Cgua), 157.4 (CBoc), 161.1
afforded 11 as a white solid (13 mg, 70%). ½a D20
ꢂ
ꢁ1 (c 1.0, MeOH);
0
00
1H NMR (D2O, 500 MHz): d 1.91–1.99 (m, 2H, H ), 2.07–2.16 (m,
c
0
1H, Hb), 2.21–2.29 (m, 1H, Hb), 3.37–3.49 (m, 4H, H5”, Hd), 3.54–
3.61 (m, 1H, H6), 3.77 (dd, JH1-H1 = 13 Hz, JH1-H2 = 6.5 Hz, 1H, H1),
3.90–3.95 (m, 2H, H5, H6), 4.06–4.13 (m, 2H, H4, H2), 4.21–4.28
0
+
0
(C4 ); HRMS calcd for [M+Na] 1202.5736, found 1202.5734.
000
00
00
00
(m, 2H, H1, H2 ), 4.30–4.34 (m, 1H, H4 ), 4.65 (d, JH2 -H3 = 5.5 Hz,
4.1.11. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(30-(N-tert-buty
loxycarbonyl)-uracil-10-yl)-6-(500-(N-N0-N00-tetra(tert-butyloxy
00
00
00
00
00
1H, H2 ), 4.61 (d, JH3 -H2 = 5.5 Hz, 1H, H3 ), 5.02 (s, 1H, H1 ), 5.68
(d, JH5 -H6 = 8.5 Hz, 1H, H5 ), 7.41 (d, JH6 -H5 = 8.5 Hz, 1H, H6 ); 13C
0
0
0
0
0
0
carbonyl)guanidino)-100,500-dideoxy-200,300-O-isopentylidene-b-
D-
00
NMR (D2O, 125 MHz): d 22.4 (C ), 29.1 (Cb), 42.7 (Cd), 47.6 (C5 ),
47.9 (C1), 59.5 (C ), 68.5 (C6), 79.0 (C3), 79.8 (C4), 82.3 (C3 ), 84.8
ribos-100-yl)-
D-glucitol 14
A suspension of 10% Pd/C (40 mg) and 13 (40 mg, 0.04 mmol) in
MeOH (2 mL) was saturated with dihydrogen and stirred for 3 h.
The reaction mixture was then filtered through a Celite pad, the
c
00
a
00
00
0
00
0
(C5), 85.6 (C2 ), 85.8 (C4 ), 101.9 (C5 ), 109.1 (C1 ), 146.6 (C6 ),
+
0
0
153.2 (C2 ), 164.8 (C4 ); MS: m/z 487 (100%, [M+1] ).

4566
D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
residue was washed with MeOH and the filtrate was concentrated
(CDCl3, 125 MHz): d 23.8ꢁ24.7 (Cd), 28.4 (CBoc), 29.1 (Cc), 30.9–
in vacuo affording 14 as a colourless oil (34 mg, 99%). ½a D20
ꢂ
ꢁ43 (c
31.1 (Cb), 47.2 (Cd), 59.9–61.1 (C ), 71.4–71.8 (Ca), 79.6 (CBoc),
a
1.0, MeOH); 1H NMR (CDCl3, 500 MHz): d 0.82, 0.87 (2t, J = 7 Hz,
6H, HCH3CH2), 1.45, 1.47, 1.50 (3s, 36H, HBoc), 1.50 (qd, J = 7 Hz,
2H, HCH2CH3), 1.58 (s, 9H, HBoc), 1.65 (qd, J = 7 Hz, 2H, HCH2CH3),
80.7 (Cb), 154.8–156.1 (CBoc), 172.0–172.5 (CCO-NH); MS: m/z 275
(100%, [M+Na]+).
3.49 (dd, JH6-H6 = 11 Hz, JH6-H5 = 3 Hz, 1H, H6), 3.73 (dd, JH1-H1
=
4.1.15. N,N0-di-(tert-Butyloxycarbonyl)guanidine 18
13 Hz, JH1-H2 = 6 Hz, 1H, H1), 3.85–3.88 (m, 2H, H5
,
H4),
To a solution of N,N0-bis-(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine (579 mg, 2.6 mmol) in DMF (2 mL) were added
00
00
3.94–3.99 (m, 2H, H6, H3), 4.19–4.25 (m, 4H, H5 , H1, H2, H5),
0
00
0
4.52 (dd, J = 11 Hz, J = 5 Hz, 1H, H4 ), 4.64 (d, JH200ꢁH3 = 6 Hz, 1H,
N,N-diisopropylethylamine (446
lL, 2.6 mmol) and propargyl-
00
00
00
00
0
H2 ), 4.93 (d, JH3 -H2 = 6 Hz, 1H, H3 ), 5.10 (s, 1H, H1 ), 5.68 (d,
amine (50 L, 2.4 mmol). The mixture was stirred overnight at rt,
l
JH5 -H6 = 8 Hz, 1H, H5 ), 7.32 (d, JH6 -H5 = 8 Hz, 1H, H6 ); 13C NMR
(CDCl3, 125 MHz): d 7.4, 8.5 (CCH3CH2), 27.6, 28.1 (CBoc), 28.8, 29.4
then extracted with EtOAc. Flash chromatography (cyclohexane/
acetone 7:3) afforded 17 as a white solid (418 mg, 59%). 1H NMR
(CDCl3, 500 MHz): d 1.44, 1.48 (2s, 18HBoc), 2.25 (t, JHa-Hc = 2 Hz,
1H, Ha), 4.22 (dd, JHc-Hc = 5 Hz, JHc-Ha = 2 Hz, 2H, Hc), 8.43, 11.42
(s, 1H, NH), 13C NMR (CDCl3, 125 MHz): d 28.2 (CBoc), 28.4 (CBoc),
30.9 (Cc), 72.4 (Ca), 79.7 (CBoc), 83.6 (Cb), 153.2 (Cgua), 155.8,
163.4 (CBoc).
0
0
0
0
0
0
00
(CCH2CH3), 48.3 (C5 ), 48.7 (C1), 67.4 (C6), 77.5 (C4), 79.1 (C2), 79.3
00
00
00
(C5), 82.6 (C3 ), 84.4 (C3), 84.7 (C4 ), 84.8 (C2 ), 85.0 (CBoc), 85.9,
0
00
86.8 (CBoc), 101.5 (C5 ), 109.3 (C1 ), 116.7 (CCEt2), 145.0 (CBoc),
0
0
145.9 (C6 ), 148.1 (CBoc), 149.5 (C2 ), 151.3 (Cgua), 157.4 (CBoc),
+
0
161.1 (C4 ); HRMS calcd for [M+Na] 1022.4797, found 1022.4802.
4.1.12. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-6-
4.1.16. 2,5-Anhydro-3,4-di-O -benzyl-1-deoxy-1-(uracil-10-yl)-
(500-guanidino)-100,500-dideoxy-200,300-dihydroxy-b-
D
-ribos-100-yl)-
6-[500-(4000-(N,N-di-tert -butyloxyaminomethyl)-1H,1000,2000,3000
-
D-glucitol 15
triazol-1000-yl)-100,500-dideoxy-200,300-O -isopentylidene-b-
100-yl]-
-glucitol 19
D-ribos-
At 0 °C, to a solution of 14 (30 mg, 0.03 mmol) in water
D
(0.3 mL) was added TFA (1.2 mL). Then, the mixture was stirred
for 1 h at rt and concentrated in vacuo. The residue was diluted
with water and aqueous NH4OH (28%, 6 mL) was added at 0 °C
prior to concentration in vacuo. Chromatography on reverse phase
(C18) using H2O, H2O/MeOH (1:1), and then MeOH afforded 15 as
A mixture of azide 4 (119 mg, 0.179 mmol), alkyne 16 (55 mg,
0.215 mmol), sodium ascorbate (11 mg, 0.054 mmol) and Cu-
SO4ꢀ5H2O (9 mg, 0.036 mmol) in t-BuOH/H2O (2:1, 4.5 mL) was
vigorously stirred at rt for 3 days. After disappearance of azide,
the mixture was diluted with H2O and extracted with CH2Cl2.
The combined organic layers were washed with H2O, dried
(MgSO4) and concentrated in vacuo. Flash column chromatography
a white solid (8 mg, 60%). ½a D20
ꢂ
ꢁ8 (c 1.0, MeOH); 1H NMR (D2O,
00
00
00
00
00
500 MHz): d 3.32 (dd, JH5 -H5 = 15 Hz, JH5 -H4 = 6 Hz, 1H, H5 ),
00
00
00
00
00
3.55 (dd, JH5 -H5 = 15 Hz, JH5 -H4 = 3 Hz, 1H, H5 ), 3.66 (dd, JH6-H6
=
(cyclohexane/acetone 1:1) afforded 19 as a white powder (48 mg,
20
12 Hz, JH6-H5 = 8 Hz, 1H, H6), 3.85–3.90 (m, 4H, H6, H5, H1),
3.96 (dd, JH3-H2 = 4 Hz, JH3-H4 = 3 Hz, 1H, H3), 4.03–4.06 (m, 2H,
H4 , H2 ), 4.13 (dd, JH1-H1 = 15 Hz, JH1-H2 = 4 Hz, 1H, H1), 4.16 (dd,
JH3 -H2 = 7 Hz, JH3 -H4 = 5 Hz, 1H, H3 ), 4.19 (dd, J H4-H5 = 4 Hz,
JH4-H3 = 3 Hz, 1H, H4), 4.29 (ddd, JH2-H1 = 9 Hz, JH2-H1 = JH2-H3
29%). ½
aꢂ
ꢁ12 (c 1.0, MeOH); 1H NMR (CDCl3, 500 MHz): d 0.83,
Hg
0.86 (2t, J = 7 Hz, 6H, HCH3CH2), 1.47 (s, 18H, HBoc), 1.50, 1.64 (2qd,
J = 7 Hz, 4H, HCH2CH3), 3.54 (dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H,
H6), 3.70 (dd, JH1-H1 = 14 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.82 (dd,
JH6-H6 = 11 Hz, JH6-H5 = 4 Hz, 1H, H6), 3.96 (d, JH4-H3 = 3 Hz, 1H, H4),
00
00
00
00
00
00
00
=
00
0
0
0
00
4 Hz, 1H, H2), 5.03 (s, 1H, H1 ), 5.79 (d, JH5 -H6 = 8 Hz, 1H, H5 ),
3.99 (d, JH3-H4 = 3 Hz, 1H, H3), 4.03–4.06 (m, 1H, H4 ), 4.24–4.32
7.62 (d, JH6 -H5 = 8 Hz, 1H, H6 ); 13C NMR (D2O, 125 MHz): d 45.2
(m, 3H, H2, H5 , H1), 4.38–4.45 (m, 3H, H5, H5 , HCH2Ph), 4.49–4.60
0
0
0
00
00
00
00
00
00
00
000
00
(C5 ), 49.9 (C1), 70.5 (C6), 73.0 (C3 ), 75.6 (C4 ), 78.0 (C4), 79.5,
(m, 5H, H2 , H3 , HCH2Ph), 4.88 (s, 2H, H6 ), 5.11 (s, 1H, H1 ), 5.55
00
0
00
0
0
79.5 (C2, C3), 82.0 (C2 ), 84.8 (C5), 102.9 (C5 ), 109.4 (C1 ), 149.1
(d, J = 8 Hz, 1H, H5 ), 7.2 (d, J = 8 Hz, 1H, H6 ), 7.24–7.37 (m, 10H,
+
HAr), 7.54 (s, 1H, H5 ), 8.96 (s, 1H, H3 ); 13C NMR (CDCl3,
125 MHz): d 7.4, 8.5 (CCH3CH2), 28.2 (CBoc), 28.9, 29.5 (CCH2CH3),
0
0
0
000
0
(C6 ), 154.1 (C2 ), 158.8 (Cgua), 168.7 (C4 ); HRMS calcd for [M+1]
432.1731, found 432.1737.
000
00
41.8 (C6 ), 48.4 (C1), 53.1 (C5 ), 68.2 (C6), 71.6, 72.1 (CCH2Ph), 79.1
00
4.1.13. N,N-bis-(tert-Butyloxycarbonyl)allylamine 16
(C2), 82.1 (CBoc), 82.7 (C2 ), 82.9 (CBoc), 83.0 (C3, C4), 83.5 (C2),
00
0
00
To a solution of propargylamine (250
l
L, 3.9 mmol) in THF
85.5 (C3 ), 85.8 (C5), 101.9 (C5 ), 109.2 (C1 ), 117.4 (CCEt2), 122.6
000
(30 mL) were successively added, di-tert-butyl dicarbonate (2 g,
9.4 mmol), DMAP (476 mg, 3.9 mmol) and triethylamine (1.3 mL,
9.4 mmol) and the solution was stirred overnight. After concentra-
tion in vacuo, flash column chromatography (cyclohexane/acetone
6:4) afforded 16 as a colourless oil (89 mg, 90%). 1H NMR (CDCl3,
250 MHz): d 1.49 (s, 18H, HBoc); 2.15 (t, JHa-Hc = 3 Hz, 1H, Ha),
4.31 (d, JHc-Ha = 3 Hz, 2H, Hc); 13C NMR (CDCl3, 125 MHz): d 28.2
(CBoc), 35.8 (Cc), 70.6 (Ca), 79.7 (CBoc), 83.0 (Cb), 151.6 (CBoc); MS:
m/z 278 (100%, [M+Na]+).
(C5 ), 127.8, 127.9, 128.3, 128.4, 128.8, 128.8, 137.4, 137.5 (CAr),
000
0
0
0
145.7, 145.8 (C4 , C6 ), 151.1 (C2 ), 152.5 (CBoc), 163.7 (C4 ); HRMS
calcd for [M+Na]+ 941.4273, found 941.4281.
4.1.17. 2,5-Anhydro-3,4-di-O -benzyl-1-deoxy-1-(uracil-10-yl)-
6-[500-(4000-(N-Boc- -prolinyl-N 0-amidomethyl)-1H ,1000,2000,3000
L -
triazol-1000-yl)-100,500-dideoxy-200,300-O -isopentylidene-b-
100-yl]-
-glucitol 20
D-ribos-
D
A mixture of 4 (120 mg, 0.18 mmol), 17 (55 mg, 0.216 mmol),
sodium ascorbate (11 mg, 0.054 mmol), CuSO4ꢀ5H2O (9 mg,
4.1.14. (S)-tert-Butyl 2(pro-2-ynylcarbamoyl)pyrrolidine-1-
0.036 mmol) and N,N-diisopropylethylamine (93 lL, 0.54 mmol)
carboxylate 17
in t-BuOH/H2O (2:1; 9 mL) was vigorously stirred at 50 °C for 2 h.
After disappearance of azide, the mixture was diluted with H2O
and extracted with CH2Cl2. The combined organic layers were
washed with H2O, dried (MgSO4) and concentrated in vacuo. Flash
To a solution of N-Boc-
(10 mL) were added HATU (290 mg, 0.76 mmol) and TEA (141
1 mmol). To this mixture was added a solution of propargylamine
(33 L, 0.5 mmol) in DMF (5 mL). The mixture was stirred over-
L
-proline (164 mg, 0.76 mmol) in DMF
lL,
l
column chromatography (cyclohexane/acetone 1:1) afforded 20 as
20
Hg
night at rt, and then concentrated in vacuo. Flash chromatography
(cyclohexane/acetone 65:35) afforded 17 as a colourless solid
(128 mg, 99%). 1H NMR (CDCl3, 500 MHz, rotamers mixture): d
1.1 (s, 9H, HBoc), 1.79–1.90 (m, 2H, Hd), 1.98–2.21 (m, 2H, Hb),
a white powder (44 mg, 29%). ½
aꢂ
ꢁ46 (c 1.0, MeOH); 1H NMR
(CDCl3, 500 MHz, rotamers mixture): d 0.85, 0.86 (2t, J = 7 Hz, 6H,
HCH3CH2), 1.34 (s, 9H, HBoc), 1.50, 1.65 (2qd, J = 7 Hz, 4H, HCH2CH3),
1.83–1.89 (m, 4H, Hb, H ), 3.32–3.42 (m, 1H, H ), 3.43–3.51 (m,
c
a
2.23–2.35 (m, 1H, Ha), 3.19–3.39 (m, 2H, H , 3.85–4.1 (m, 2H,
2H, Hd), 3.62 (dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.73 (dd,
JH1-H1 = 14 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.83 (dd, JH6-H6 = 11 Hz,
c
Hc), 4.10–4.30 (m, 1H, H ), 6.39, 7.20 (br s, 1H, NH); 13C NMR
a

D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4567
000
000
0
0
JH6-H5 = 4 Hz, 1H, H6), 3.89–3.96 (m, 1H, H4), 4.02 (d, JH3-H4 = 3 Hz,
122.9 (C5 ), 145.6, 146.2 (C4 , C6 ), 152.0 (C2 ), 152.6 (CBoc), 164.7
+
00
00
0
1H, H3), 4.08–4.12 (m, 1H, H4 ), 4.20–4.35 (m, 3H, H2, H5 , H1),
(C4 ); HRMS calcd for [M+Na] 761.3334, found 761.3348.
00
00
00
4.40–4.46 (m, 3H, H5, H5 ,HCH2Ph), 4.54–4.65 (m, 6H, H2 , H3
,
00
00
0
H
CH2Ph, H6 ), 5.14 (s, 1H, H1 ), 5.46–5.50 (m, 1H, H5 ), 7.16–7.2
4.1.20. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-
(m, 1H, H6 ), 7.26–7.37 (m, 10H, HAr), 7.62 (br s, 1H, H5 ), 8.52,
6-[500-(4000-(N -Boc-
L
-prolinyl-N0-amidomethyl)-1H ,1000,2000,3000
-
0
000
8.64 (br s, 1H, H3 ); 13C NMR (CDCl3, 125 MHz): d 8.7, 9.8 (CCH3CH2),
triazol-1000-yl)-100,500-dideoxy-200,300-O -isopentylidene-b-
D
-ribos-
0
100-yl]-
D
-glucitol 23
000
28.3 (Cb, C ), 29.8 (CBoc), 30.2, 30.8 (CCH2CH3), 44.9 (C6 ), 48.5, 48.7
c
00
(C , Cd), 49.6 (C1), 54.1 (C5 ), 70.0 (C6), 73.0, 73.4 (CCH2Ph), 78.7 (C2),
A suspension of 10% Pd/C (25 mg) and 20 (24 mg, 0.026 mmol)
in MeOH (2 mL) was saturated with dihydrogen and stirred for
12 h. The reaction mixture was then filtered through a Celite pad,
the residue was washed with MeOH and the filtrate was concen-
trated in vacuo affording 23 as a colourless oil (15 mg, 79%). 1H
NMR (MeOD, 500 MHz): d 0.88, 0.90 (2t, J = 7 Hz, 6H, HCH3CH2),
1.34 (s, 9H, HBoc), 1.61, 1.69 (2qd, J = 7 Hz, 4H, HCH2CH3), 1.83–
1.95 (m, 3H, Hb, H ), 2.17–2.24 (m, 1H, H , 3.40–3.45 (m, 1H, Hd),
a
00
00
00
81.9 (CBoc), 83.5, 84.1, 84.6 (C2 , C3, C4, C4 ), 86.8, 87.0 (C3 , C5),
103.0 (C5 ), 111.0 (C1 ), 118.6 (CCEt2), 129.1, 129.2, 129.6, 129.7,
130.0, 130.1 (CAr), 132.3 (C5 ), 138.6, 138.7 (CAr), 146.9 (C6 ),
152.3 (C2 ); HRMS calcd for [M+Na] 938.4276, found 938.4258.
0
00
000
0
+
0
4.1.18. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(uracil-10-yl)-6-
[500-(4000-(N-N0-bis-(tert-butyloxycarbonyl)guanidinomethylene)-
1H, 1000,2000,3000-triazol-1000-yl)-100,500-dideoxy-200,300-O-isopentylidene-
-glucitol 21
A mixture of 4 (108 mg, 0.163 mmol), 18 (58 mg, 0.196 mmol),
sodium ascorbate (10 mg, 0.054 mmol), CuSO4ꢀ5H2O (8 mg,
0.033 mmol) and diisopropylethylamine (84 L, 0.49 mmol) in t-
BuOH/H2O (2:1, 9 mL) was vigourously stirred at 50 °C for 3 h.
After disappearance of azide, the mixture was diluted with H2O
and extracted with CH2Cl2. The combined organic layers were
washed with H2O, dried (MgSO4) and concentrated in vacuo. Flash
c
c
3.51–3.55 (m, 1H, Hd), 3.65–3.69 (m, 1H, H6), 3.80 (dd, JH1-H1
=
-ribos-100-yl]-
D
15 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.89–3.93 (m, 2H, H4 , H6), 3.98
00
b-D
(br s, 1H, H4), 4.04 (br s, 1H, H3), 4.14–4.16 (m, 1H, H ), 4.19–
a
00
00
00
00
4.26 (m, 4H, H1, H2, H5), 4.39–4.67 (m, 6H, H2 , H3 , H5 , H6 ),
00
0
l
5.21 (s, 1H, H1 ), 5.56 (d, J = 8 Hz, 1H, H5 ), 7.54 (d, J = 8 Hz, 1H,
H6 ), 8.0 (s, 1H, H5 ); 13C NMR (MeOD, 125 MHz): d 7.8, 8.8
(CCH3CH2), 24.7 (Cb), 28.7 (CBoc), 29.9, 30.3 (CCH2CH3), 32.5 (C ),
0
000
c
000
00
35.5 (C6 ), 47.9 (Cd), 48.2 (C1), 54.8 (C5 ), 61.9 (C ), 70.0 (C6),
a
00
00
00
78.5 (C3), 80.2, 80.3 (C2, C4, C5), 83.5 (C4 ), 85.9, 85.9 (C2 , C3 ),
0
00
000
column chromatography (cyclohexane/acetone 1:1) afforded 21 as
86.8 (CBoc), 102.1 (C5 ), 111.0 (C1 ), 118.1 (CCEt2), 125.3 (C5 ),
20
a white powder (89 mg, 57%). ½ ꢂ
a
ꢁ8 (c 1.0, MeOH); 1H NMR
146.0, 147.7 (C4 , C6 ), 156.0 (C2 ), 169.1 (C4 ).
000
0
0
0
Hg
(CDCl3, 500 MHz): d 0.84, 0.85 (2t, J = 7 Hz, 6H, HCH3CH2), 1.45,
1.49 (2s, 18H, HBoc), 1.51, 1.64 (2qd, J = 7 Hz, 4H, HCH2CH3), 3.54
(dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.68 (dd, JH1-H1 = 14 Hz,
JH1-H2 = 8 Hz, 1H, H1), 3.82 (dd, JH6-H6 = 11 Hz, JH6-H5 = 4 Hz, 1H,
H6), 3.94 (d, JH3-H4 = 3 Hz, 1H, H3), 3.98 (d, JH4-H3 = 3 Hz, 1H, H4),
4.1.21. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-6-
[500-(4000-aminomethyl-1H,1000,2000,3000-triazol-1000-yl)-100,500-dideoxy-
200,300-dihydroxy-b- -ribos-100-yl]-
D D-glucitol 24
At 0 °C, to a solution of 22 (30 mg, 0.04 mmol) in water (0.3 mL)
was added TFA (1.2 mL). The mixture was stirred for 1 h at rt, and
then concentrated in vacuo. The residue was diluted with water
and aqueous NH4OH (28%, 6 mL) was added at 0 °C. The mixture
was concentrated in vacuo. Chromatography on reverse phase
(C18) using H2O, H2O/MeOH (1:1), and then MeOH afforded 24 as
00
00
4.03–4.07 (m, 1H, H4 ), 4.23–4.33 (m, 3H, H2, H5 , H1), 4.38–4.42
00
(m, 2H, H5 , HCH2Ph), 4.46 (dd, JH5-H6a = 7 Hz, JH5-H6b = 7 Hz, 1H,
00
00
000
H5), 4.49–4.59 (m, 5H, H2 , H3 , HCH2Ph), 4.88 (br s, 2H, H6 ), 5.12
00
0
0
(s, 1H, H1 ), 5.54 (d, J = 8 Hz, 1H, H5 ), 7.16 (d, J = 8 Hz, 1H, H6 ),
000
0
7.23–7.34 (m, 10H, HAr), 7.57 (s, 1H, H5 ), 8.82 (s, 1H, H3 ), 9.39,
11.43 (2s, 2H, NH); 13C NMR (CDCl3, 125 MHz): d 7.4, 8.5 (CCH3CH2),
a white solid (19 mg, 47%). ½a D20
ꢂ
ꢁ2 (c 1.0, MeOH); 1H NMR (D2O,
000
28.2, 28.4 (CBoc), 28.9, 29.5 (CCH2CH3), 36.6 (C6 ), 48.3 (C1), 53.1
500 MHz): d 3.65 (dd, JH6-H6 = 12 Hz, JH6-H5 = 9 Hz, 1H, H6), 3.83–
00
00
00
00
00
(C5 ), 68.4 (C6), 71.6, 72.0 (CCH2Ph), 79.0 (C2), 79.6 (CBoc), 82.1
3.90 (m, 3H, H1, H6, H5), 3.98 (dd, JH4 -H5 = 4 Hz, JH4 -H5 = 2 Hz,
00
00
00
00
(C2 ), 82.7, 82.8 (C3, C4), 83.4, 83.5 (C4 , CBoc), 85.5 (C5), 85.6 (C3 ),
1H, H4 ), 4.08 (d, JH3-H4 = 4 Hz, 1H, H3), 4.16 (dd, JH1-H1 = 14 Hz,
0
00
000
101.8 (C5 ), 109.3 (C1 ), 117.3 (CCEt2), 122.5 (C5 ), 127.7, 127.9,
JH1-H2 = 4 Hz, 1H, H1), 4.22 (dd, JH2-H1 = 4 Hz, JH2-H1 = 2 Hz, 1H, H2),
000
00
00
000
128.2, 128.3, 128.7, 128.8, 137.3, 137.5 (CAr), 144.3 (C4 ), 145.7
4.28–4.34 (m, 3H, H3 , H4, H2 ), 4.36 (s, 2H, H6 ), 4.61 (dd,
0
0
0
00
00
00
00
00
00
00
(C6 ), 151.2 (C2 ), 153.0 (Cgua), 156.0 (CBoc), 163.9 (C4 ); HRMS calcd
JH5 -H5 = 14 Hz, JH5 -H4 = 7 Hz, 1H, H5 ), 4.80 (dd, JH5 -H5 = 14 Hz,
for [M+Na]+ 983.4491, found 983.4464.
JH5 -H4 = 3 Hz, 1H, H5 ), 5.04 (s, 1H, H1 ), 5.77 (d, J = 8 Hz, 1H, H5 ),
00
00
00
00
0
7.63 (d, J = 8 Hz, 1H, H6 ), 8.14 (s, 1H, H5 ); 13C NMR (D2O,
0
000
4.1.19. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-
125 MHz): d 35.5 (C6 ), 49.9 (C1), 54.7 (C5 ), 70.5 (C6), 73.4 (C3 ),
75.5 (C3), 78.1 (C2), 79.5, 79.6 (C4 , C2 ), 84.8 (C4), 86.6 (C5), 102.7
000
00
00
6-[500-(4000-(N,N-di-ter t-butyloxyaminomethyl)-1H,1000,2000,3000
-
00
00
triazol-1000-yl)-100,500-dideoxy-200,300-O -isopentylidene-b-
D
-ribos-
(C5 ), 109.5 (C1 ), 127.2 (C5 ), 141.3 (C4 ), 149.2 (C6 ), 153.6 (C2 ),
0
00
000
000
0
0
+
100-yl]-
D
-glucitol 22
168.1 (C4 ); HRMS calcd for [M+Na] 493.1659, found 493.1664.
0
A suspension of 10% Pd/C (45 mg) and 19 (47 mg, 0.05 mmol) in
MeOH (2 mL) was saturated with dihydrogen and stirred for 3 h.
The reaction mixture was then filtered through a Celite pad, the
residue was washed with MeOH and the filtrate was concentrated
4.1.22. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-6-
[500-(4000-(N- -prolinyl-N0-amidomethyl)-1H,1000,2000,3000-triazol-1000-
L
yl)-100,500-dideoxy-200,300-dihydroxy-b- -ribos-100-yl]-
D D-glucitol 25
in vacuo affording 22 as a colourless oil (30 mg, 79%). ½a D20
ꢂ
ꢁ16 (c
At 0 °C, to a solution of 23 (15 mg, 0.002 mmol) in water
(0.1 mL) was added TFA (0.4 mL). The mixture was stirred at rt
for 1 h, and then concentrated in vacuo. The residue was diluted
with water and aqueous NH4OH (28%, 6 mL) was added at 0 °C.
The mixture was then concentrated in vacuo and chromatography
on reverse phase (C18) using H2O, H2O/MeOH (1:1), and then
1.0, CH2Cl2); 1H NMR (CDCl3, 500 MHz): d 0.83, 0.86 (2t, J = 7 Hz,
6H, HCH3CH2), 1.46 (s, 18H, HBoc), 1.53, 1.65 (2qd, J = 7 Hz, 4H,
HCH2CH3), 3.56 (dd, JH6-H6 = 10 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.71 (dd,
JH1-H1 = 15 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.84 (dd, JH6-H6 = 10 Hz,
00
JH6-H5 = 3 Hz, 1H, H6), 3.91–3.96 (m, 1H, H4 ), 4.06 (br s, 1H, H4),
4.09 (br s, 1H, H3), 4.16–4.24 (m, 2H, H2, H1), 4.43–4.60 (m, 3H,
MeOH afforded 25 as a white solid (7 mg, 64%). ½a D20
ꢁ6 (c 1.0,
ꢂ
H5, H5 ), 4.72–4.76 (m, 2H, H2 , H3 ), 4.86 (s, 2H, H6 ), 5.16 (s,
MeOH); 1H NMR (D2O, 500 MHz): d 1.99–2.04 (m, 3H, Hb, H ),
c
00
00
00
000
00
0
0
1H, H1 ), 5.60 (d, J = 8 Hz, 1H, H5 ), 7.34 (d, J = 8 Hz, 1H, H6 ), 7.66
2.38–2.40 (m, 1H, H , 3.36–3.40 (m, 2H, Hd), 3.62 (dd, JH6-H6 =
11 Hz, JH6-H5 = 8 Hz, 1H, H6), 3.78–3.86 (m, 3H, H1, H6, H5),
c
(s, 1H, H5 ); 13C NMR (CDCl3, 125 MHz): d 7.5, 8.6 (CCH3CH2), 28.2
000
000
00
00
(CBoc), 29.0, 29.6 (CCH2CH3), 41.8 (C6 ), 48.2 (C1), 53.4 (C5 ), 68.9
3.92–3.95 (m, 1H, H4 ), 4.01 (d, JH3-H4 = 4 Hz, 1H, H3), 4.12–4.31
00
00
00
(C6), 77.3 (C3), 79.1 (C2), 79.4 (C4), 82.2 (C2 ), 83.3 (CBoc), 84.6
(m, 5H, H4, H2, H1, H2 , H3 ), 4.34–4.37 (m, 1H, H ), 4.49 (s, 2H,
a
00
00
0
00
000
00
00
00
00
00
(C4 ), 85.5 (C3 ), 85.8 (C5), 102.2 (C5 ), 109.5 (C1 ), 117.5 (CCEt2),
H6 ), 4.53 (dd, JH5 -H5 = 14 Hz, JH5 -H4 = 7 Hz, 1H, H5 ), 4.71–4.73

4568
D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
00
00
0
0
0
(m, 1H, H5 ), 5.0 (s, 1H, H1 ), 5.72 (d, J = 8 Hz, 1H, H5 ), 7.58 (d,
148.0 (CBoc), 149.6 (C2 ), 151.0 (Cgua), 158.2 (CBoc), 161.0 (C4 );
J = 8 Hz, 1H, H6 ), 7.91 (s, 1H, H5 ); 13C NMR (D2O, 125 MHz): d
HRMS calcd for [M+1]+ 1081.5305, found 1083.5311.
0
000
000
00
25.2 (Cb), 31.0 (C ), 36.0 (C6 ), 47.9 (Cd), 49.9 (C1), 54.4 (C5 ), 61.3
c
00
00
(C ), 70.6 (C6), 73.3 (C2), 75.5 (C3), 78.1 (C4), 79.6, 79.6 (C2 , C4 ),
4.1.25.
6-[500-(4000-guanidinomethylene)-1H,1000,2000,3000-triazol-1000-yl)-100,
500-dideoxy-200,300-dihydroxy-b- -ribos-100-yl]-
-glucitol 28
2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(uracil-10-yl)-
a
00
0
00
000
81.8 (C3 ), 84.9 (C5), 102.7 (C5 ), 109.5 (C1 ), 125.9 (C5 ), 145.5
(C4 ), 149.2 (C6 ), 153.6 (C2 ), 168.1 (C4 ); HRMS calcd for [M+1]
568.2367, found 568.2378.
+
000
0
0
0
D
D
At 0 °C, to a solution of 27 (39 mg, 0.036 mmol) in water
(0.3 mL) was added TFA (1.2 mL). The mixture was stirred at rt
for 1 h, and then concentrated in vacuo. The residue was diluted
with water and aqueous NH4OH (28%, 6 mL) was added at 0 °C.
The mixture was then concentrated in vacuo and chromatography
on reverse phase (C18) using H2O, H2O/MeOH (1:1) and then
4.1.23. 2,5-Anhydro-3,4-di-O-benzyl-1-deoxy-1-(30-(N-tert-
butyloxycarbonyl)-uracil-10-yl)-6-[500-(4000-(N-N0-N00-tetra(tert-
butyloxycarbonyl)guanidinomethylene)-1H,1000,2000,3000-triazol-
1000-yl)-100,500-dideoxy-200,300-O-isopentylidene-b- -ribos-100-yl]-
D D-
glucitol 26
MeOH afforded 28 as a white solid (9 mg, 50%). ½a D20
ꢂ
+3 (c 1.0,
To a solution of 21 (41 mg, 0.042 mmol) in THF (5 mL) were
added, di-tert-butyl dicarbonate (47 mg, 0.21 mmol), DMAP (5 mg,
0.042 mmol), then triethylamine (30 lL, 0.21 mmol) and the solu-
MeOH); 1H NMR (D2O, 500 MHz): d 3.63 (dd, JH6-H6 = 11 Hz, JH6-
H5 = 8 Hz, 1H, H6), 3.80 (dd, JH6-H6 = 11 Hz, JH6-H5 = 3 Hz, 1H, H6),
3.81–3.83 (m, 1H, H5), 3.87 (dd, JH1-H1 = 11 Hz, JH1-H2 = 8 Hz, 1H,
00
00
00
00
00
tion was stirred overnight at rt. After concentration in vacuo, flash
H1), 3.95 (dd, JH4 -H5 = 4 Hz, JH4 -H5 = 2 Hz, 1H, H4 ), 4.02 (d,
column chromatography (cyclohexane/acetone 7:3) afforded 26 as
JH3-H4 = 4 Hz, 1H, H3), 4.11–4.23 (m, 3H, H1, H2, H4), 4.25–4.30
a colourless oil (51 mg, 79%). ½a D20
ꢂ
+2 (c 1.0, MeOH); 1H NMR (CDCl3,
(m, 2H, H3 , H2 ), 4.50 (s, 2H, H6 ), 4.56 (dd, JH5 -H5 = 14 Hz,
00
00
000
00
00
00
00
00
00
00
00
00
500 MHz): d 0.83, 0.84 (2t, J = 7 Hz, 6H, HCH3CH2), 1.46, 1.48, 1.49 (3s,
36H, HBoc), 1.51 (qd, J = 7 Hz, 2H, HCH2CH3), 1.59 (s, 9H, HBoc), 1.63 (qd,
J = 7 Hz, 2H, HCH2CH3), 3.54 (dd, JH6-H6 = 11 Hz, JH6-H5 = 6 Hz, 1H, H6),
3.65 (dd, JH1-H1 = 14 Hz, JH1-H2 = 8 Hz, 1H, H1), 3.87 (dd,
JH6-H6 = 11 Hz, JH6-H5 = 4 Hz, 1H, H6), 3.95 (d, JH3-H4 = 3 Hz, 1H, H3),
JH5 -H4 = 7 Hz, 1H, H5 ), 4.76 (dd, JH5 -H5 = 14 Hz, JH5 -H4 = 3 Hz,
00
00
0
1H, H5 ), 5.01 (s, 1H1 ), 5.73 (d, J = 8 Hz, 1H, H5 ), 7.59 (d, J =
8 Hz, 1H, H6 ), 7.98 (s, 1H, H5 ); 13C NMR (D2O, 125 MHz): d 37.7
0
000
000
00
(C6 ), 49.9 (C1), 54.4 (C5 ), 70.6 (C6), 73.3 (C2), 75.5 (C3), 78.1
00
00
00
0
00
(C4), 79.5 (C4 ), 81.8 (C2 , C3 ), 84.8 (C5), 102.7 (C5 ), 109.5 (C1 ),
00
000
000
0
0
3.97 (d, JH4-H3 = 3 Hz, 1H, H4), 4.04–4.07 (m, 1H, H4 ), 4.26–4.34
126.0 (C5 ), 144.6 (C4 ), 149.1 (C6 ), 153.6 (C2 ), 158.4 (Cgua),
+
00
00
0
(m, 3H, H2, H5 , H1), 4.37–4.42 (m, 3H, H5, H5 ,HCH2Ph), 4.51–4.59
168.1 (C4 ); HRMS calcd for [M+1] 513.2057, found 513.2045.
00
00
000
000-H6000
(m, 5H, H2 , H3 , 3HCH2Ph), 5.06 (d, JH6
= 15 Hz 1H, H6 ), 5.12
00
000
000-H6000
(s, 1H, H1 ), 5.13 (d, JH6
0
= 15 Hz 1H, H6 ), 5.57 (d, J = 8 Hz, 1H,
4.2. Enzymatic assays
0
H5 ), 7.17 (d, J = 8 Hz, 1H, H6 ), 7.16–7.38 (m, 10H, HAr), 7.80 (s, 1H,
H5 ); 13C NMR (CDCl3, 125 MHz): d 8.7, 9.8 (CCH3CH2), 28.3, 28.4,
000
The activities of the compounds against MraY from B. subtilis
were tested as previously described.10,22 The assay was performed
000
29.3, 29.4 (CBoc), 30.2, 30.8 (CCH2CH3), 43.9 (C6 ), 50.2 (C1), 54.3
(C5 ), 69.6 (C6), 72.9, 73.3 (CCH2Ph), 80.2 (C2), 83.4, 83.5 (C2 , CBoc),
84.0, 84.1 (C3, C4), 84.8, 85.1 (CBoc), 85.6 (C4 ), 86.8, 87.0 (C5, C3 ),
88.1 (CBoc), 102.8 (C5 ), 110.5 (C1 ), 118.5 (CCEt2), 125.1 (C5 ), 129.1,
129.2, 129.5, 129.7, 130.0, 130.1, 138.6, 138.8 (CAr), 145.4, 145.7
(C4 CBoc), 146.3 (C6 ), 148.9, 149.3 (CBoc), 150.6 (C2 ), 152.3 (Cgua),
158.8 (CBoc), 162.2 (C4 ); HRMS calcd for [M+Na] 1283.6063, found
1283.6051.
00
00
in a reaction mixture of 10 lL containing, in final concentrations,
00
00
100 mM Tris–HCl, pH 7.5, 40 mM MgCl2, 1.1 mM C55-P, 250 mM
NaCl, 0.25 mM UDP-MurNAc-[14C]pentapeptide (337 Bq), and
8.4 mM N-lauroyl sarcosine. The reaction was initiated by the
addition of MraY enzyme, and the mixture was incubated for
30 min at 37 °C under shaking with a thermomixer (Eppendorf).
The reaction was stopped by heating at 100 °C for 1 min. The
compounds were also tested against MurG from Escherichia coli
as previously described.23,24 Reaction mixtures contained, in a fi-
0
00
000
000
0
0
,
+
0
4.1.24. 2,5-Anhydro-3,4-dihydroxy-1-deoxy-1-(30-(N-tert-
butyloxycarbonyl)-uracil-10-yl)-6-[500-(4000-(N-N0-N00-tetra(tert-
butyloxycarbonyl)guanidinomethylene)-1H,1000,2000,3000-triazol-
nal volume of 12.5
l
L, 200 mM Tris–HCl, pH 7.5, 10 mM MgCl2,
M lipid I analogue, 35%
16
l
M UDP-[14C]GlcNAc (1.7 kBq), 16
l
1000-yl)-100,500-dideoxy-200,300-O-isopentylidene-b- -ribos-100-yl]-
D D-
(v/v) dimethyl sulfoxide and MurG. After 30 min at 37 °C, it was
stopped by boiling for 3 min. Then, the mixture was lyophilised
and taken up in 10 lL of 2-propanol/ammonium hydroxide/water
glucitol 27
A suspension of 10% Pd/C (50 mg) and 26 (51 mg, 0.04 mmol) in
MeOH (2 mL) was saturated with dihydrogen and stirred for 3 h.
The reaction mixture was then filtered through a Celite pad, the
residue was washed with MeOH and the filtrate was concentrated
(6:3:1; v/v/v). In both cases, the radiolabeled substrates (UDP-
MurNAc-pentapeptide in the case of MraY, UDP-GlcNAc in the
case of MurG) and reaction product (lipid I, product of MraY
and lipid II, product of MurG) were separated by TLC on silica
gel plates LK6D (Whatman) using 2-propanol/ammonium hydrox-
ide/water (6:3:1; v/v/v) as a mobile phase. The radioactive spots
were located and quantified with a radioactivity scanner (model
Multi-Tracemaster LB285; EG&G Wallac/Berthold). For MraY
activity, residual activities and IC50 values were calculated with
respect to a control assay without the inhibitors. Data represent
the mean of independent triplicate determinations, and the stan-
dard deviation was less than 10%.
in vacuo affording 27 as a colourless oil (39 mg, 90%). ½a D20
ꢂ
+6 (c 1.0,
MeOH); 1H NMR (CDCl3, 500 MHz): d 0.85, 0.86 (2t, J = 7 Hz, 6H,
H
H
CH3CH2), 1.43, 1.45, 1.49 (3s, 36H, HBoc), 1.56 (qd, J = 7 Hz, 2H,
CH2CH3), 1.58 (s, 9HBoc), 1.66 (qd, J = 7 Hz, 2H, HCH2CH3), 3.55 (dd,
JH6-H6 = 10 Hz, JH6-H5 = 6 Hz, 1H, H6), 3.72 (dd, JH1-H1 = 14 Hz,
JH1-H2 = 6 Hz, 1H, H1), 3.84 (dd, JH6-H6 = 10 Hz, JH6-H5 = 4 Hz, 1H,
H6), 3.93–3.97 (m, 1H, H5), 4.05 (d, JH4-H3 = 3 Hz, 1H, H4), 4.09 (d,
JH3-H4 = 3 Hz, 1H, H3), 4.21–4.24 (m, 1H, H2), 4.27 (dd, JH1-H1
14 Hz, JH1-H2 = 5 Hz, 1H, H1), 4.44 (dd, JH5 -H5 = 14 Hz, JH4 -H5
10 Hz, 1H, H4 ), 4.5 (dd, JH5 -H5 = 14 Hz, JH5 -H4 = 5 Hz, 1H, H5 ),
=
=
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
4.60 (dd, JH4 -H5 = 10 Hz, JH4 -H5 = 5 Hz, 1H, H4 ), 4.72–4.74 (m,
00
00
000
00
Acknowledgements
3H, H2 , H3 ), 5.08 (s, 2H, H6 ), 5.14 (s, 1H, H1 ), 5.66 (d, J = 8 Hz,
1H, H5 ), 7.31 (d, J = 8 Hz, 1H, H6 ), 7.87 (s, H5 ); 13C NMR (CDCl3,
125 MHz): d 7.5, 8.6 (CCH3CH2), 27.7, 28.0, 28.2 (CBoc), 29.0, 29.7
0
0
000
We gratefully acknowledge the European Community for the
financial support of the Eur-INTAFAR integrated project within
the 6th PCRDT framework (contract N° LSHM-CT-2004-512138)
and for post-doctoral Grants to D.L., A.C. and B.A. We thank L. Le
Corre for his generous gift of compound 17.
000
00
(CCH2CH3), 43.2 (C6 ), 48.5 (C1), 53.6 (C5 ), 68.9 (C6), 77.5 (C4),
79.1 (C2), 79.9 (C3), 82.2 (C2 ), 83.0, 84.3, 84.7 (CBoc), 85.1 (C5),
85.5 (C3 ), 85.9 (C3 ), 87.0 (CBoc), 101.9 (C5 ), 109.3 (C1 ), 117.6 (CC
Et2), 124.0 (C5 ), 144.1 (C4 ), 145.0 (CBoc), 145.1 (C6 ), 147.9,
00
00
00
0
00
000
000
0

D. Lecerclé et al. / Bioorg. Med. Chem. 18 (2010) 4560–4569
4569
13. For antibacterial activity of triazole compounds, see: Genin, M. J.; Allwine, D.
A.; Anderson, D. J.; Barbachyn, M. R.; Emmert, D. E.; Garmon, S. A.; Graber, D.
R.; Grega, K. C.; Hester, J. B.; Hutchinson, D. K.; Morris, J.; Reischer, R. J.; Ford, C.
W.; Zurenko, G. E.; Hamel, J. C.; Schaadt, R. D.; Stapert, D.; Yagi, B. H. J. Med.
Chem. 2000, 43, 953.
14. Poitout, L.; Le Merrer, Y.; Depezay, J.-C. Tetrahedron Lett. 1994, 35, 3293.
15. For analogous tandem reaction on bis-epoxides, see: (a) Poitout, L.; Le Merrer,
Y.; Depezay, J.-C. Tetrahedron Lett. 1995, 36, 6887; (b) Le Merrer, Y.; Sanière, M.;
McCort, I.; Dupuy, C.; Depezay, J.-C. Tetrahedron Lett. 2001, 42, 2661; (c)
Saladino, R.; Ciambecchini, U.; Hanessian, S. Eur. J. Org. Chem. 2003, 4401; (d)
Busca, P.; McCort, I.; Prange´, T.; Le Merrer, Y. Eur. J. Org. Chem. 2006, 2403.
16. There are some typographical errors in Ref. 15c. For example, in Scheme 1, bis-
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Int. Appl. 2001, pp 73, WO 2001012643.; (c) Igarashi, M.; Nakagawa, N.; Doi, N.;
Hattori, S.; Naganawa, H.; Hamada, M. J. Antibiot. 2003, 56, 580.
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Brosch, G. J. Med. Chem. 2003, 46, 512; (b) Narsinghani, T.; Chaturvedi, S. C.
Bioorg. Med. Chem. Lett. 2006, 16, 461.
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epoxide 3 and related compounds 4–9 are described with a
gulo configuration, respectively, instead of a -manno or a -gulo configuration
and in Scheme 2, compounds 13–18 should have an -gluco configuration.
L-manno and a D-
D
L
L
17. Hirano, S.; Ichikawa, S.; Matsuda, A. Angew. Chem., Int. Ed. 2005, 44, 1854.
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Le Merrer, Y. Tetrahedron: Asymmetry 2006, 17, 142.
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