Synthesis and biological evaluation of a diazepanone-based library of liposidomycins analogs as MraY inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.02.006

New inhibitors of the bacterial tranferase MraY are described. A scaffold strategy based on the diazepanone central core of liposidomycins, natural inhibitors of MraY has been developed. It involves the introduction of key structural fragments required fo

Full text of DOI:10.1016/j.ejmech.2011.02.006

European Journal of Medicinal Chemistry 46 (2011) 1582e1592
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a diazepanone-based library
of liposidomycins analogs as MraY inhibitors
,
,
Janez Mravljak a 1, Olivier Monasson a, Bayan Al-Dabbagh b 2, Muriel Crouvoisier b, Ahmed Bouhss b,
Christine Gravier-Pelletier a , Yves Le Merrer
,
a
*
a Université Paris Descartes, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Pères, 75006 Paris, France
b Univ Paris-Sud, UMR 8619, Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Orsay 91405, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New inhibitors of the bacterial tranferase MraY are described. A scaffold strategy based on the dia-
zepanone central core of liposidomycins, natural inhibitors of MraY has been developed. It involves the
introduction of key structural fragments required for biological activity on enantiopure diazepanones by
reductive amination, esterification and glycosylation. Biological evaluation of these compounds on MraY
enzyme revealed interesting inhibitory activity for compounds displaying three fragments on the scaf-
fold: a palmitoyl chain, an aminoribose part and an alkyluracil moiety. The inhibitors were also evaluated
Received 9 December 2010
Received in revised form
1 February 2011
Accepted 3 February 2011
Available online 5 March 2011
on MurG enzyme. The best compounds resulted in inhibition with IC50 values in the 100
one or the other enzyme.
mM range for
Keywords:
Membrane transferase MraY
Glycosyltransferase MurG
Diazepanone scaffolds
Reductive amination
Glycosylation
Ó 2011 Elsevier Masson SAS. All rights reserved.
Inhibition tests
1. Introduction
address the problem of bacterial resistance is to focus on targets
that have not been explored as much as before to delay the
occurrence of resistance. Owing to its trans-membrane localization
[4], the MraY transferase, which is an essential enzyme [5] that
catalyzes the first membrane-associated step of peptidoglycan
biosynthesis, has only recently been purified to homogeneity and
characterized [6], with tests allowing high throughput screening of
inhibitors being achieved [7]. MraY catalyzes the transfer of uridine
di-phosphate-N-acetyl-muramoyl-pentapeptide from the cyto-
plasm to the membrane (Fig. 1) through its fixation on the unde-
caprenyl phosphate lipid carrier, leading to the formation of lipid I
(undecaprenylpyrophosphate-N-acetyl-muramoyl-pentapeptide)
and to the release of uridine monophosphate (UMP).
At the moment, neither is a crystal structure of MraY available,
nor are there any MraY-directed antibiotics in clinical use as
a consequence of the trans-membrane localization of this enzyme.
Nevertheless, several families of natural antibiotics including lip-
osidomycins [8], caprazamycins [9] or tunicamycins [10] have been
identified (Fig. 2) which display high in vitro inhibition of the
enzymatic activity, but modest antibacterial activity probably due
to their high hydrophilicity limiting their passive diffusion through
membranes [11]. Based on the structure of these inhibitors and in
the context of a program directed to the synthesis and biological
Multidrug resistant pathogens such as methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus
(VRSA) set a severe public health problem [1]. Therefore, designing
and synthesizing new antibacterial agents is a priority and a chal-
lenge for the scientific community. Among the most validated
targets for developing new antibacterial compounds are enzymes
involved in the biosynthetic pathway of peptidoglycan. This poly-
mer constitutes an essential part of the bacterial cell wall and
protects the cell from osmotic pressure [2]. Indeed, enzymes
involved in its biosynthesis have been demonstrated to be ubiqui-
tous and essential to bacterial growth [3] and any anomaly in this
biosynthetic process leads to cell lysis. Furthermore, peptidoglycan
has no counterparts in eukaryotic cells. Of particular interest to
* Corresponding author. Tel.: þ33 142862181; fax: þ33 142868387.
E-mail address: christine.gravier-pelletier@parisdescartes.fr (C. Gravier-Pelletier).
Present address: Faculty of Pharmacy, University of Ljubljana, Askerceva, 1000
Ljubljana, Slovenia.
1
ꢀ
ꢀ
2
Present address: Department of Internal Medicine, Faculty of Medicine and
Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, United Arab
Emirates.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.006

J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
1583
UDP-Mur-N-Ac-pentapeptide
R
O
CYTOPLASM
HO
N
UMP
MraY
OBn
N
H
Undecaprenyl-P
Lipid I
TBDPSO
Pi
UDP-GlcNAc
UDP
Bac A
MurG
1a : R = Me
1b : R = H
MEMBRANE
Undecaprenyl-PP
Lipid II
Fig. 3. Exemples of synthesized diazepanones.
PBPs
PBPs
PERIPLASM
Peptidoglycan
Polymer
Acceptor
Taking these results into account, we hypothesized that only the
uracil template and not the uridine one, should be crucial to ensure
a possible recognition of the resulting inhibitors by the enzyme and
chose to replace the ribose part of uridine by a simple C5-acyclic
alkyl chain. Therefore, alkyluracil, aminoribose and fatty acid chain,
all three mimicking or being key fragments of the naturally
occurring inhibitors, were retained in the targeted compounds.
Either one, two, three or four of these fragments were introduced
on scaffold 1a or 1b (Fig. 5) in which the intracyclic nitrogen of the
amine function was substituted or not by a methyl group.
Sequential introduction of these fragments and biological evalua-
tion of the corresponding inhibitors on the MraY transferase
allowed the identification of their role in the biological activity.
Fig. 1. Role of MraY in bacterial peptidoglycan biosynthesis.
evaluation of new antibacterials [12], our goal is to develop efficient
access to libraries of compounds that inhibit the MraY enzymatic
activity. The finality is to contribute not only to the discovery of new
antibacterials, but also to the elucidation of the structure of the
enzyme’s active site through structureeactivity relationships study
of families of inhibitors [12,13]. A key point of this study is the
development of scaffolds allowing structural changes that could
permit further optimization to generate “lead” compounds [14].
Toward that goal, we recently described [15] straightforward
access to enantiopure 1,4-diazepanone scaffolds (Fig. 3) that
display differentiated functions such as amine, amide, primary and
secondary alcohols and for which different configurations at
asymmetric carbons are available.
2. Results and discussion
Such heterocycles are the central core of liposidomycins, MraY
natural inhibitors isolated from Streptomyces griseosporeus. With
caprazamycins, they display a complex nucleosidic structure based
on that heterocycle and also involve a uridine moiety, an amino-
ribosyl part, fatty acid chains and a methyl glucoside unit for cap-
razamycins. Knapp et al. [16] have determined the absolute
configuration of these natural compounds as being 2S,5S,6S. Our
goal is to develop a library of simplified analogs of liposidomycins
based on the diazepanone scaffold. The choice of key fragments to
be introduced on this polyfunctionalized platform took advantage
of the structureeactivity relationships study carried out by Aventis
and involving an aminoribosyl uridine pharmacophore [17] (Fig. 4).
Conclusions of this study were notably that the aminoribosyl
moiety and the uracil part are important for activity [18]. Indeed,
either uracil N-alkylation or double bond reduction led to loss of
activity. Furthermore, it was shown that removal of both hydroxyl
groups of uridine has no drastic impact on biological activity [19].
2.1. Chemistry
From the N-methyl diazepanone 1a (Scheme 1), easily obtained
from L-ascorbic acid and L-serine [15], it was possible to introduce
either one or two key fragments. First, O-acylation of the free
secondary alcohol function by palmitic acid in the presence of
dicyclohexylcarbodiimide afforded the ester
2 in 93% yield.
Successive deprotections of the primary alcohol functions involved
hydrogenolysis of benzyl ether in the presence of palladium black
in acetic acid giving 3 followed by silyl ether cleavage by ammo-
nium fluoride affording 4 in 76% overall yield from 2. It has to be
noted that carrying out this reaction in the presence of tetrabuty-
lammonium fluoride led to partial migration of the palmitoyl chain
to the primary alcohol function. Thanks to ammonium fluoride, the
intermediate alcoholate was directly protonated by the ammonium
counter ion avoiding the chain migration. We next turned to the
introduction of the aminoribosyl moiety through O-glycosylation
[20, 13b, 13e] of the primary alcohol function of 3 by the fluo-
roazidoribose derivative 5 which was carried out in the presence of
boron trifluoride etherate and molecular sieves in excess leading to
6 in 87% yield. The fluororibose 5 was readily obtained in four steps
HO3SO OH
NH2
O
O
R
O
O
O
Me
from
D
-ribose [13b, 13e]. The presence of the isopentylidene moiety
-face of this sugar derivative allows steric control of the
-selectivity during the ribosylation reaction. Indeed, only the
-anomer is formed during this reaction. Next, azide reduction by
O
N
on the
b
b
b
O
O
N
NH
R1O2C
6
5
2
O
N
Me
HO2C
O
HO OH
hydrogenation in the presence of palladium on charcoal (10%)
afforded the corresponding primary amine 7 in 83% yield. Synthesis
of the targeted inhibitor
Liposidomycins : R1 = H
Caprazamycins : R1 = methyl glucoside
O
9
first involved acidolysis of the
O
OH
OH
O
HO
O
R
N
H
N
NH
O
NH
N
O
O
O
O
O
AcHN
O
H2N
HO OH
Tunicamycins
HO
HO
OH
HO
O
HO
OH
HO
Fig. 4. Structure of Aventis pharmacophore.
Fig. 2. Natural inhibitors of MraY.

1584
J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
Then, the removal of benzyl ether protection on the primary alcohol
R1 = H, Me,
O
function was studied. Catalytic hydrogenation in various conditions
(palladium on charcoal, palladium black, palladium dihydroxide,
.) either failed or resulted in partial reduction of the uracil double
bond, the obtained aminoalcohol probably poisoning the catalyst
[22]. The best conditions for this reaction revealed to be boron
trichloride in dichloromethane at low temperature [23]. In these
conditions, the pure alcohol 15 could be isolated in 92% yield.
Introduction of the aminoribose was then carried out by glycosyl-
ation with the azidofluororibose derivative 5, as above, and affor-
ded the protected inhibitor 16 displaying three structural
fragments (97% yield). To overcome the already observed uracil
double bond reduction in hydrogenolysis conditions, the azido
group reduction was achieved in Staudinger conditions in the
presence of 1,2-bis(diphenylphosphino)ethane [24] yielding 17
(92%). Acidolysis of the isopentylidene ketal, followed by silyl
deprotection respectively gave 18 (57%) and 19 (87%).
R
2 = COC15H31
NH
N
O
R1
R2O
N
OR3
N
H
O
R4O
NH2
O
R4 = H,
R3
=
NH2
O
HO
OH
HO
OH
Alternatively, taking into account both the structure of the diol
20 and the presence of several sugars in some natural inhibitors of
MraY such as tunicamycins (Fig. 2), we have been aiming at intro-
ducing two ribose derivatives on the scaffold. Thus, we turned to
silyl ether deprotection of 15 that could be efficiently achieved in
the presence of ammonium fluoride, as above, yielding 20 in 96%
yield. The resulting diol 20 was then involved in a double glyco-
sylation reaction with the fluoroazidoribose 5 affording 21 in 62%
yield. Azide reduction in Staudinger conditions gave the bis-amino
derivative 22 (96%) and was followed by acidic hydrolysis of both
isopentylidene ketals giving the targeted inhibitor 23 in 57% yield.
Fig. 5. Proposed scaffold strategy.
isopentylidene protecting group of the diol leading to 8 in 65%
yield, followed by silyl ether deprotection as above giving 9 in 50%
yield. In a complementary manner, in order to evaluate the bio-
logical activity of the diazepanone scaffold itself, and to compare its
activity with that of the decorated scaffold with the different
fragments, 1a was submitted to sequential deprotections of its
primary alcohol functions. Thus, silyl deprotection by tetrabuty-
lammonium fluoride afforded 10 in quantitative yield and subse-
quent benzyl ether hydrogenolysis gave 11.
Alternatively, toward the possible introduction of three key
fragments, we embarked on chemical manipulation of the dia-
zepanone 1b (Scheme 2). Introduction of the alkyluracilyl moiety
involved reductive amination of the diazepanone 1b with uracil
pentanal 12 [12a] in the presence of sodium triacetoxyborohydride
[21] yielding 13 in 87% yield. Acylation of the secondary alcohol
function with palmitic acid, as above, gave the ester 14 in 87% yield.
2.2. Biological studies
The in vitro biological evaluation of the resulting compounds 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 1 mM of the tested compounds. For the most
active inhibitors, the IC50 values were determined. Furthermore,
Me
C15H31CO2
TBDPSO
HO
Me
N
N
O
C15H31CO2H
OBn
OBn
HN
DCC, DMAP
93%
N
H
TBDPSO
O
H2
Pd black
AcOH
Me
1a
2
nBu4NF
THF
C15H31CO2
TBDPSO
O
C15H31CO2
HO
Me
N
N
O
NH4F
OH
OH
HO
DMF
76% from 2
HN
HN
Me
N
O
OP
3
4
HN
HO
O
F
N3
BF3.OEt2
5
10 : P = Bn (100%)
11 : P = H
H2, Pd/C
MeOH
O
O
C15H31CO2
TBDPSO
C15H31CO2
Me
Me
N
N
O
O
O
O
A
NH2
HN
HN
TFA
H2O
PO
O
O
O
O
HO
OH
6 : A = N3 (87%)
8 : P = TBDPS (65%)
9 : P = H (50%)
H2, Pd/C
MeOH
NH4F
DMF
7 : A = NH2 (83%)
Scheme 1. Synthesis of inhibitors with two key fragments from diazepanone 1a.

J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
1585
O
NH
N
( )5
O
HO
C15H31CO2H
DCC, DMAP
HO
12
NH
O
N
O
NaBH(OAc)3
87%
OBn
OBn
HN
13
N
H
TBDPSO
TBDPSO
1b
O
N
O
NH
NH
O
N
O
C15H31CO2
HO
C15H31CO2
TBDPSO
O
N
( )5
( )5
nBu4NF
N
O
N
O
NH
THF
96%
12
OH
OP
O
HN
HN
(CH2)4
CHO
O
20
14 : P = Bn (87%)
BCl3
CH2Cl2
F
N3
5
15 : P = H (92%)
O
O
BF3.OEt2
5
BF3.OEt2
5
O
O
N
NH
O
NH
O
N
( )5
C15H31CO2
C15H31CO2
TBDPSO
( )5
N
O
N
O
O
O
O
O
A
HN
HN
A
O
O
A
O
O
O
O
O
O
16 : A = N3 (97%)
17 : A = NH2 (92%)
21 : A = N3 (62%)
22 : A = NH2 (96%)
(Ph2P-CH2)2
THF, H2O
H2O (57%)
TFA
H2O
TFA
O
N
O
N
NH
O
NH
O
C15H31CO2
C15H31CO2
( )5
( )5
N
O
N
O
O
O
O
O
HN
NH2
HN
NH2
O
O
PO
H2N
HO
OH
HO
OH
18 : P = TBDPS (57%)
19 : P = H (87%)
NH4F
DMF
HO
OH
23
Scheme 2. Synthesis of inhibitors with three or four key fragments from diazepanone 1b.
the inhibitors were also tested on the MurG enzyme which cata-
lyzes the second membrane step of peptidoglycan biosynthesis
(Fig. 1), leading to lipid II formation.
of a supplementary aminoribose on the scaffold seems to lead to
weakest inhibitors (compare the respective activities of 21 and 23
to that of 16 and 19). Concerning MurG inhibition, several
Concerning MraY inhibition, comparison of the results for both
N-Me and N-alkyluracil-inhibitors shows that the latter are better
inhibitors than the former as exemplified by the respective activi-
ties of 20 and 4, 17 and 7, 18 and 8, 19 and 9. In the N-alkyluracil
series, the introduction of an aminoribose moiety on the dia-
zepanone scaffold improves the inhibition. Indeed, compounds 16
and 18 exhibited better inhibitory activities than 15, and 19 was
a bit better than 20. Finally, the best MraY inhibitor 18 resulted in
compounds caused enzymatic activity inhibition in the 100 mM
range. It appears that reduction of the azido group into the corre-
sponding amine(s) is crucial for inhibition as demonstrated by the
respective activities of 17 and 16, 22 and 21.
3. Conclusion
The synthesis of a small library of MraY inhibitors has been
developed in high yields according to a scaffold strategy based on
diazepanone platforms. Indeed, such an heterocycle is the central
core of liposidomycins, known natural inhibitors of MraY. Key
structural fragments were introduced on these scaffolds to get
simplified analogs of the natural compounds. Well-differentiated
protected functions within these structures allowed sequential
introduction of either one, two, three or four key fragments
an IC50 value of 120 mM and presented three key fragments on the
scaffold: a palmitoyl chain, alkyluracil and aminoribose moieties,
the remaining primary alcohol function being protected as a bulky
hydrophobic silyl ether. Surprisingly, alcohol deprotection of
compound 18 slightly decreased the inhibitory activity as
compared to that of the parent compound, showing that a hydro-
phobic residue is better in this position. Furthermore, introduction

1586
J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
Table 1
including a fatty acid chain, an alkyluracil residue mimicking the
uridine present in liposidomycins and either one or two amino-
ribose moieties. Biological evaluation of the resulting inhibitors on
the ribose seems to be crucial to inhibit the MurG activity. There-
fore, this study led to reaching some insight into the requirements
for inhibition of the trans-membrane protein MraY.
both MraYand MurG activities resulted in IC50 values in the 100 mM
range for the best compounds and showed that among the intro-
duced fragments, three of them are important for activity since
their absence led to lower activities, while the presence of a second
aminoribose leads to weaker inhibitors. Interestingly the biological
results suggest that the presence of a hydrophobic residue on the
primary alcohol function of the scaffold is preferable to that of a free
alcohol for MraY inhibition. Furthermore, a free primary amine on
4. Experimental
4.1. Chemical synthesis
1H NMR (250 MHz) and 13C NMR (63 MHz) spectra were
recorded on a Bruker AM250 in CDCl3 (unless otherwise indicated).
1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded

J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
1587
(110 mg, 0.14 mmol) in acetic acid (1 mL) was then added and the
mixture was stirred under dihydrogen atmosphere for 20 h. The
catalyst was removed by filtration and the filtrate was concentrated
in vacuo. Flash chromatography of the residue (cyclohexane/EtOAc
6:4) gave 74 mg (76%) of the alcohol 3 as a colorless oil. Rf 0.5
O
4'
3' NH
5'
6'
2'
1'
N
O
5"
4"
(cyclohexane/EtOAc 1:1); 1H NMR
d 7.57e7.38 (m, 10H, Har), 6.03
(d,1H, JH1eH7 ¼ 5 Hz, H1), 5.04 (dl, 1H, H6), 4.01 (m, 1H, H7), 3.86 (d,
2H, JCH2OHeH3 ¼ 5 Hz, CH2OH), 3.71 (ABX, 2H, JAx ¼ 4 Hz,
JAB ¼ 10.6 Hz, CH2OSi), 3.31 (t, 1H, JH3eCH2OH ¼ 3.3 Hz, H3), 3.12 (dd,
1H, JH5aeH5b ¼ 15.7 Hz, JH5aeH6 ¼ 3.3 Hz, H5a), 2.85 (dd, 1H,
JH5aeH5b ¼ 15.7 Hz, JH5beH6 ¼ 1 Hz, H5b), 2.39 (s, 3H, NMe), 2.10 (m,
2H, Hb), 1.70e1.50 (m, 2H, Hc), 1.24 (sl, 24H, (CH2)12), 1.03 (s, 9H,
3"
2"
c
a
1"
O
b
12
5
4 N
6
O
3
7
1
O
O
2
5r
NH2
HN
4r
1r
2r
PO
O
3r
tBu), 0.86 (t, 3H, JCH3eCH2 ¼ 6.7 Hz, CH3); 13C NMR
d 173.5 (C2), 172.9
HO
OH
(Ca),135.7,132.5,130.2, 128.0 (Car), 73.3 (C3), 71.3 (C6), 62.5 (CH2OH ,
CH2OSi), 58.0 (C5), 53.7 (C7), 44.5 (NMe), 35.0 (Cb), 32.1, 29.7, 25.0,
22.8 (-(CH2)13), 27.2, 19.5 (tBu), 14.5 (CH3); MS (ESIþ) 681.4
(M þ H)þ, 703.3 (M þ Na)þ; HRMS (ESIþ) for C40H65N2O5Si
(M þ H)þ calcd 681.4663, found 681.4639.
Fig. 6. Numbering of the compound 18.
on a Bruker Avance or Avance II. Chemical shifts (
d) are reported in
ppm and coupling constants are given in Hz. To facilitate the
understanding of NMR spectroscopic data, the numbering of atoms
for the following representative compound 18 is as indicated (Fig. 6).
Optical rotations were measured on a Perkin-Elmer 341 polar-
imeter with sodium (589 nm) or mercury (365 nm) lamp at 20 ꢀC.
Mass spectra, electrospray, chemical ionization (CI) and high
resolution (HRMS) were recorded by the Service de Spectrométrie
de Masse, ICSN Gif sur Yvette or Ecole Normale Supérieure, Paris. All
reactions were carried out under a nitrogen atmosphere, and were
monitored by thin-layer chromatography with Merck 60F-254
precoated silica (0.2 mm) on glass. Flash chromatography was
4.1.3. (3S,6S,7R)-3,7-Dihydroxymethyl-4-N-methyl-6-
palmitoyloxy-1,4-diazepan-2-one (4)
To a solution of silyl ether 3 (50 mg, 72
mmol) in DMF (5 mL) was
added ammonium fluoride (9 mg, 210 mol) and the mixture was
m
stirred for 18 h at r.t. After concentration in vacuo, trituration of the
residue in diethyl ether led to 32 mg (100%) of the diol 4 as a hygro-
scopic solid. [
a]
D þ10 (c 1.0, CH2Cl2); 1H NMR (MeOD)
d 4.99 (dt, 1H,
JH6-H7 ¼ 8.3 Hz, JH6eH5 ¼ 2.7 Hz, H6), 4.17 (dt, 1H, JH7eH6 ¼ 8.3 Hz,
JH7eH9 ¼ 4.5 Hz, H7), 3.95 (dd,1H, JH3eH8a ¼ 4.5 Hz, JH8aeH8b ¼ 11.8 Hz,
H8a), 3.85 (dd,1H, JH3eH8b ¼ 4.5 Hz, JH8aeH8b ¼ 11.8 Hz, H8b), 3.75 (dd,
1H, JH7eH9a ¼ 4.3 Hz, JH9aeH9b ¼ 11.8 Hz, H9a), 3.67 (dd, 1H,
JH7eH9b ¼ 5.3 Hz, JH9aeH9b ¼ 11.8 Hz, H9b), 3.31 (t, 1H, JH3eH8 ¼ 4.4 Hz,
H3), 3.17 (dd, 1H, JH5aeH6 ¼ 2.7 Hz, JH5beH5a ¼ 15.5 Hz, H5a), 3.01 (dd,
1H, JH5beH6 ¼ 2.7 Hz,JH5beH5a ¼ 15.5 Hz, H5b), 2.47(s, 3H, NMe), 2.37(t,
2H, JCH2beCH2c ¼ 7.3 Hz, Hb), 1.70e1.50 (m, 2H, Hc), 1.32 (s, 24H,
performed with Merck Kieselgel 60 (200e500
mm); the solvent
systems were given v/v. Spectroscopic 1H and 13C NMR, MS and/or
analytical data were obtained using chromatographically homo-
geneous samples.
4.1.1. (3S,6S,7R)-3-Benzyloxymethyl-7-tert-
butyldiphenylsilyloxymethyl-4-N-methyl-6-palmitoyloxy-1,4-
diazepan-2-one (2)
To a solution of the alcohol 1a (670 mg, 1.26 mmol) in CH2Cl2
(15 mL) were successively added palmitic acid (645 mg, 2.51 mmol),
dicyclohexylcarbodiimide (579 mg, 2.51 mmol) and dimethylami-
nopyridine (37 mg, 0.25 mmol) and the mixture was stirred for 20 h
at r.t. After filtration through a celite pad and concentration in vacuo,
flash chromatography of the residue (cyclohexane/EtOAc 8:2)
afforded 902 mg (93%) of the palmitic ester 2 as a colorless oil. Rf 0.25
(CH2)12), 0.93 (t, 3H, JCH3eCH2 ¼ 6.6 Hz, CH3); 13C NMR (MeOD)
d 176.8
(C2), 174.5 (Ca), 74.8 (C3), 72.7 (C6), 62.0 (C8), 61.1 (C9), 58.6 (C5), 55.1
(C7), 42.7 (NMe), 35.3 (Cb), 33.1, 30.8, 26.0, 23.7 (e(CH2)13),14.4 (CH3);
MS(ESIþ) 465.3 (M þ Na)þ; HRMS(ESIþ) for C24H46N2O5Na (M þ Na)þ
calcd 465.3304, found 465.3297.
4.1.4. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-methyl-
6-palmitoyloxy-3-(50-azido-50-deoxy-20,30-O-isopentylidene-
b-D-
ribos-10-yl-methyl)-1,4-diazepan-2-one (6)
To a solution of alcohol 3 (377 mg, 554
mmol) in CH2Cl2 (16 mL)
(cyclohexane/EtOAc 8:2);
[
a
]
þ19 (c 1.0, CH2Cl2); 1H NMR
at r.t. was added the fluororibose derivative 5 (204 mg, 831 m
mol)
D
d
7.70e7.24 (m, 15H, Har), 6.07 (d,1H, JH1eH7 ¼ 6 Hz, H1), 5.02 (dl, 1H,
and molecular sieves 4 Å (1.5 g). After 1 h stirring, the mixture was
H6), 4.52 (AB, 2H, JAB ¼ 15 Hz, CH2Ph), 4.35 (m, 1H, H7), 3.82 (d, 2H,
JCH2OBneH3 ¼ 3.3 Hz, CH2OBn), 3.63 (dd, 1H, JCH2aOSieH7 ¼ 3.3 Hz,
JCH2OSi ¼ 11.4 Hz, CH2aOSi), 3.61 (dd, 1H, JCH2bOSieH7 ¼ 3.3 Hz,
JCH2OSi ¼ 11.4 Hz, CH2bOSi), 3.31 (t, 1H, JH3eCH2OBn ¼ 3.3 Hz, H3), 3.12
cooled to ꢁ78 ꢀC and boron trifluoride etherate (104
mL, 830 mmol)
was dropwise added. The mixture was slowly warmed to 20 ꢀC in
5 h and was hydrolyzed by the addition of a saturated NaHCO3
aqueous solution. After CH2Cl2 extractions, the combined organic
layers were washed (H2O), dried (MgSO4), filtered and concen-
trated in vacuo. Flash chromatography of the residue (cyclohexane/
EtOAc 7:3) afforded 435 mg (87%) of the ribosyl diazepanone 6 as an
(dd, 1H, JH5aeH5b ¼ 15.7 Hz ,3JH5aeH6 ¼ 3.3 Hz, H5a), 2.85 (dd, 1H,
2
JH5aeH5b ¼ 15.7 Hz, JH5beH6 ¼ 1 Hz, H5b), 2.39 (s, 3H, NMe), 2.10 (t, 2H,
JCH2beCH2c ¼ 7.7 Hz, Hb), 1.52 (m, 2H, Hc), 1.24 (sl, 24H, (CH2)12), 1.03
(s, 9H, tBu), 0.86 (t, 3H, JCH3eCH2 ¼ 6.7 Hz, CH3); 13C NMR
d
173.5 (C2),
oil. Rf 0.37 (cyclohexane/EtOAc 7:3); [
a
]
þ13 (c 1.0, CH2Cl2); 1H
D
173.1 (Ca), 138.2, 135.7, 130.2, 128.4, 128.1, 127.7 (Car), 74.6 (C3), 73.6
(CH2Ph), 71.3 (C6), 70.2 (CH2OBn ), 60.9 (CH2OSi), 58.0 (C5), 52.3 (C7),
44.6 (NMe), 32.1, 29.6, 29.9, 25.0, 22.9 (e(CH2)14), 27.0, 19.3 (tBu),
14.3 (CH3); MS (ESIþ) 771.5 (M þ H)þ, 793.5 (M þ Na)þ; HRMS (ESIþ)
for C47H71N2O5Si (M þ H)þ calcd 771.5132, found 771.5151.
NMR
d
7.62e7.36 (m, 10H, Har), 6.01 (d, 1H, JH1eH7 ¼ 6.2 Hz, H1), 5.14
0
(s, 1H, H1 ), 4.97 (dd, 1H, JH6eH7 ¼ 9.5 Hz, JH6eH5 ¼ 2 Hz, H6), 4.53 (d,
0
0
0
0
0
0
1H, JH3 eH2 ¼ 6 Hz, H3 ), 4.45 (d, 1H, JH2 eH3 ¼ 6 Hz, H2 ), 4.27 (t, 1H,
0
0
0
JH4 eH5 ¼ 7.7 Hz, H4 ), 4.19 (m,1H, H7), 4.01 (dd,1H, JCH2OR ¼ 10.6 Hz,
JCH2aOReH3 ¼ 3.6 Hz, CH2aOR), 3.74e3.66 (m, 3H, CH2OSi, CH2bOR),
0
0
0
0
0
3.41 (dd, 1H, JH5 aeH5 b ¼ 12.8 Hz, JH5 aeH4 ¼ 7.7 Hz, H5 a), 3.29 (t, 1H,
0
4.1.2. (3S,6S,7R)-3-Hydroxymethyl-7-tert-
butyldiphenylsilyloxymethyl-4-N-methyl-6-palmitoyloxy-1,4-
diazepan-2-one (3)
A suspension of palladium black (100 mg) in acetic acid (5 mL)
was saturated with dihydrogen. A solution of the benzyl ether 2
JH3eCH2aOR ¼ 3.6 Hz, H3), 3.17e3.06 (m, 2H, H5b, H5 b), 2.83 (dd, 1H,
JH5aeH5b ¼ 15.7 Hz, H5a), 2.38 (s, 3H, NMe), 2.15 (m, 2H, Hb); 1.67,
1.46 (2q, 4H, JCH2eCH3 ¼ 6.7 Hz, C(CH2CH3)2), 1.55e1.45 (m, 2H, Hc),
1.24 (sl, 24H, (CH2)12), 1.04 (s, 9H, tBu), 0.90 (2t, 6H,
JCH3eCH2 ¼ 6.7 Hz, C(CH2CH3)2), 0.86 (t, 3H, JCH3eCH2 ¼ 7.5 Hz, CH3);

1588
J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
13C NMR
d
173.3, 173.0 (C2, Ca), 136.0, 135.9, 132.9, 132.6, 130.5,
4.1.7. (3S,6S,7R)-3-(50-Amino-50-deoxy-
hydroxymethyl-4-N-methyl-6-palmitoyloxy-1,4-diazepan-2-one (9)
To a solution of the silyl ether 8 (46 mg, 56 mol) in DMF
(4 mL) was added NH4F (2.1 mg, 56 mol) and the reaction
b-D
-ribos-10-yl-methyl)-7-
0
0
128.4, 128.3 (Car), 117.5 (C(CH2CH3)2), 109.4 (C1 ), 86.2, 85.9 (C4 ,
0
0
C2 ), 82.8 (C3 ), 74.2 (C3), 71.7 (C6), 67.4 (CH2OR), 61.7 (CH2OSi ),
m
0
58.0 (C5), 53.6 (C7), 46.2 (C5 ), 44.8 (NMe), 32.3 (Cb), 30.5e23.1
m
(e(CH2)13, C(CH2CH3)2), 27.3, 19.6 (tBu), 14.3 (CH3), 8.8, 7.8 (C
(CH2CH3)2); MS (ESIþ) 687.4 (100%), 928.6 (M þ H)þ, 703.3
(M þ Na)þ; HRMS (ESIþ) for C50H79N5O8NaSi (M þ Na)þ calcd
928.5596, found 928.5523.
mixture was stirred at r.t. for 15 h NH4F was filtered off and the
reaction mixture was concentrated under reduced pressure. The
residue was than carefully rinsed with ether (3 ꢂ 2 mL) and dried
in vacuo to afford the alcohol 9 (16 mg, 50%) as an oil. [
a
]
þ32 (c
D
1.0, MeOH); 1H NMR (500 MHz, MeOD, 330 K)
d
4.94 (s, 1H, H1 ),
0
4.1.5. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-methyl-
4.87 (dt, 1H, JH6eH7 ¼ 9.2 Hz, JH6eH5 ¼ 2 Hz, H6), 4.18 (ddd, 1H,
6-palmitoyloxy-3-(50-amino-50-deoxy-20,30-O-isopentylidene-
b-D-
JH7eH6 ¼ 9.2 Hz, JH7eCHbOH ¼ 6.2 Hz, JH7eCHaOH ¼ 3.6 Hz, H7),
ribos-10-yl-methyl)-1,4-diazepan-2-one (7)
4.12e4.04 (m, 2H, H3 , H2 ), 4.01 (m, 2H, CH2aOr, H4 ), 3.88 (dd, 1H,
0
0
0
To a solution of the azido-ribosyl-diazepanone 6 (305 mg,
337 mol) in methanol (7 mL) was added palladium black
JCH2Or
¼
10.5 Hz, JCHbOreH3
¼
3.7 Hz, CH2bOR), 3.69 (dd,
m
1H, JCH2OH ¼ 11.5 Hz, JCHaOHeH7 ¼ 3.7 Hz, CHaOH), 3.62 (dd, 1H,
(200 mg) and the mixture was stirred under dihydrogen atmo-
sphere for 1 h. The mixture was then filtered through a celite pad
and concentrated in vacuo. Flash chromatography of the residue
(CH2Cl2/MeOH/Et3N 9:1:3&) gave 245 mg (83%) of the expected
amine 7 as a colorless oil. Rf 0.39 (CH2Cl2/MeOH/Et3N 9:1:3&);
JCH2OH
¼
11.5 Hz, JCHbOHeH7
¼
6.1 Hz, CHbOH), 3.35 (t,
0
1H, JH3eCH2OR ¼ 3.7 Hz, H3), 3.19 (sl, 1H, H5 a), 3.07 (dd, 1H,
JH5aeH5b ¼ 15.5 Hz, JH5aeH6 ¼ 3.7 Hz, H5a), 3.00 (m, 1H, H5b), 2.89
(dd, 1H, JH5aeH5b ¼ 15.5 Hz, JH5beH6 ¼ 2 Hz, H5b), 2.41 (s, 3H, NMe),
2.34 (t, 2H, JCH2beCH2c
¼
7.3 Hz, Hb), 1.63 (tt, 2H, Hc,
[
a
]
D þ14 (c 1.0, CH2Cl2); 1H NMR
d
7.60e7.34 (m, 10H, Har), 6.07 (d,
JHceHb ¼ JHceHd ¼ 7.5 Hz), 1.28 (s, 24H, (CH2)12), 0.88 (t, 3H,
1H, JH1eH7 ¼ 6.0 Hz, H1), 5.07 (s, 1H, H1 ), 4.97 (dd, 1H,
JCH3eCH2 ¼ 7.5 Hz, CH3); 13C NMR (125 MHz, MeOD)
d 176.2 (C2),
0
0
0
0
0
0
0
JH6eH7 ¼ 9.5 Hz, JH6eH5 ¼ 3.4 Hz, H6), 4.51 (d, 1H, JH3 eH2 ¼ 6 Hz,
174.6 (Ca), 109.3 (C1 ), 80.9 (C3 ), 76.4 (C4 ), 74.6 (C2 , C3), 73.6 (C6),
0
0
0
0
0
H3 ), 4.42 (d, 1H, JH2 eH3 ¼ 6 Hz, H2 ), 4.24 (m, 1H, H7), 4.17 (t, 1H,
68.2 (CH2OR), 61.4 (CH2OH), 59.4 (C5), 55.3 (C7), 44.9 (C5 ), 44.1
JH4 eH5
JCH2aOReH3 ¼ 3.6 Hz, CH2aOR), 3.75e3.62 (m, 3H, CH2OSi, CH2bOR),
3.26 (t, 1H, JH3eCH2OR 3.6 Hz, H3), 3.07 (dd, 1H,
JH5aeH5b 15.6 Hz, JH5aeH6 3.4 Hz, H5a), 2.83 (dl, 1H,
¼
7.1 Hz, H4 ), 4.00 (dd, 1H, JCH2OR
¼
10.5 Hz,
(NMe), 35.6 (Cb), 33.4e24.1 (-(CH2)13), 14.8 (CH3); MS (ESIþ) 574.2
(100%, M þ H)þ; HRMS (ESIþ) for C29H55N3O8Na (M þ Na)þ calcd
596.3887, found 596.3890.
0
0
0
¼
¼
¼
3
0
0
0
JH5aeH5b ¼ 15.6 Hz, H5b), 2.66 (dl, 2H, JH5 eH4 ¼ 7.1 Hz, H5 ), 2.37
(s, 3H, NMe), 2.13 (AB of ABX, 2H, JAB ¼ 15.8 Hz, JBx ¼ JAx ¼ 7.7 Hz,
Hb), 1.63, 1.47 (2q, 4H, JCH2eCH3 ¼ 7.4 Hz, C(CH2CH3)2), 1.58e1.38
(m, 2H, Hc), 1.22 (sl, 24H, (CH2)12), 1.03 (s, 9H, tBu), 0.87, 0.78 (2t,
6H, JCH3eCH2 ¼ 7.4 Hz, C(CH2CH3)2), 0.84 (t, 3H, JCH3eCH2 ¼ 7.5 Hz,
4.1.8. (3S,6S,7R)-3-(Benzyloxymethyl)-7-(hydroxymethyl)-6-
hydroxy-4-N-methyl-1,4-diazepan-2-one (10)
To a solution of the diazepanone 1a (147 mg, 276
CH2Cl2 (6.3 mL) at r.t. was added a solution of tetrabutylammonium
fluoride in THF (1 M, 328 L, 1.1 mmol). After 2 h stirring, the
mmol) in
m
CH3); 13C NMR
d
173.0 (Ca), 170.7 (C2), 135.7, 135.6, 132.6, 132.4,
mixture was concentrated in vacuo and flash chromatography of
the residue (CH2Cl2/MeOH/Et3N 9:1:3&) afforded 81 mg (100%) of
the diol 10 as a white foam. Rf 0.3 (CH2Cl2/MeOH/Et3N 9:1:3&);
0
0
130.2, 128.1 (Car), 116.7 (C(CH2CH3)2), 109.2 (C1 ), 88.7 (C4 ), 86.0
0
0
(C3 ), 82.7 (C2 ), 73.9 (C3), 71.4 (C6), 67.4 (CH2OR), 61.4 (CH2OSi ),
58.1 (C5), 52.5 (C7), 45.3 (C5 ), 44.2 (NMe), 34.4 (Cb), 32.03, 29.5,
[a
]
þ13 (c 1.0, CH2Cl2); 1H NMR
d 7.29 (m, 5H, Har), 6.75 (d, 1H,
0
D
25.0, 22.8 (e(CH2)13, C(CH2CH3)2), 27.0, 19.3 (tBu), 14.3 (CH3), 8.5,
7.5 (C(CH2CH3)2); MS (ESIþ) 681.4 (100%), 880.5 (M þ H)þ, 902.5
(M þ Na)þ; HRMS (ESIþ) for C50H81N3O8NaSi (M þ Na)þ calcd
902.5691, found 902.5569.
JH1eH7 ¼ 3.8 Hz, H1), 4.54 (s, 2H, CH2Ph); 3.92 (m,1H, H7), 3.84e3.75
(m, 4H, H6, CH2OBn, CH2aOH), 3.77 (dd, 1H, JCH2OH ¼ 11.3 Hz,
JCH2bOHeH7 ¼ 3.4 Hz, CH2bOH), 3.34 (t, 1H, JH3eCH2OBn ¼ 3.7 Hz, H3),
3.09 (dd, 1H, JH5aeH5b ¼ 14.8 Hz, JH5aeH6 ¼ 3.4 Hz, H5a), 2.79 (dd, 1H,
JH5aeH5b ¼ 14.8 Hz, JH5beH6 ¼ 2.0 Hz, H5b), 2.44 (s, 3H, NMe); 13C
4.1.6. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-methyl-
NMR d 174.4 (C2), 137.9, 128.5, 127.8, 127.7 (Car), 74.1 (C3), 73.5
6-palmitoyloxy-3-(50-amino-50-deoxy-
b
-D
-ribos-10-yl-methyl)-1,4-
(CH2Ph), 69.4 (CH2OBn), 68.2 (C6), 60.2 (C5, CH2OH), 56.4 (C7), 45.0
diazepan-2-one (8)
(NMe); MS (ESIþ) 295 (M þ H)þ, 317 (M þ Na)þ.
At 20 ꢀC, the aminoribosyl-diazepanone 7 was stirred for
1.5 h in a 4/1 mixture of trifluoroacetic acid (4 mL) and water
(1 mL). The mixture was then concentrated in vacuo and
purification on Waters SEP-PAKÒ cartridge (CH2Cl2/MeOH 8:2)
afforded 30 mg (65%) of 8 as a colorless oil. Rf 0.23 (CH2Cl2/
4.1.9. (3S,6S,7R)-3,7-Dihydroxymethyl-6-hydroxy-4-N-methyl-1,4-
diazepan-2-one (11)
To a solution of benzyl ether 10 (40 mg, 136
mmol) in THF
(5 mL) was added palladium black (80 mg) and the mixture was
stirred under dihydrogen atmosphere for 24 h. Monitoring of the
reaction by thin-layer chromatography (CH2Cl2/MeOH 9:2)
revealed that the conversion was not complete. The mixture was
filtered through a celite pad, concentrated in vacuo and the same
procedure was repeated. TLC control showed that the starting
material was less than 10%. The mixture was filtered through
a celite pad and concentrated in vacuo. Addition of cold CH2Cl2
allowed partial recovery of pure triol 11 as a solid. 1H NMR
MeOH 8:2); 1H NMR (250 MHz, MeOD)
d
7.67e7.37 (m, 10H,
0
Har), 5.06 (dl, 1H, JH6eH7 ¼ 8.6 Hz, H6), 4.92 (s, 1H, H1 ), 4.17 (m,
0
0
0
1H, H7), 4.04e3.86 (m, 4H, CH2aOR, H4 , H3 , H2 ), 3.83e3.74 (m,
3H, CH2OSi, CH2bOR), 3.38 (t, 1H, JH3eCH2OR ¼ 4.1 Hz, H3), 3.07
0
(m, 2H, H5a
,
H5 a), 2.92 (dd, 1H, JH5beH5a
¼
15.4 Hz,
0
0
JH5beH6 ¼ 2.0 Hz, H5b), 2.82 (dd, 1H, JH5 beH5 a ¼ 13.1 Hz,
0
0
0
JH5 beH4 ¼ 8.5 Hz, H5 b), 2.40 (s, 3H, NMe), 2.20 (AB of ABX2, 2H,
JCH2b ¼ 15.7 Hz, JCH2beCH2c ¼ 7.5 Hz, Hb), 1.52 (m, 2H, Hc), 1.28 (s,
24H, (CH2)12), 1.07 (s, 9H, tBu), 0.89 (t, 3H, JCH3eCH2 ¼ 7.5 Hz,
(500 MHz, D2O)
d
4.05 (dd, 1H, JH8aeH8b
¼
12.2 Hz,
CH3); 13C NMR (63 MHz, MeOD)
d
175.8 (C2), 174.2 (Ca), 136.8,
JH8aeH3 ¼ 5.6 Hz, H8a), 3.97e3.77 (m, 5H, H8b, H6, H7, H9), 3.42 (t,
1H, JH3eH8 ¼ 5.6 Hz, H3), 3.13 (dd, 1H, JH5aeH5b ¼ 15.4 Hz,
JH5aeH6 ¼ 3.0 Hz, H5a), 2.99 (dd, 1H, JH5aeH5b ¼ 15.4 Hz,
0
0
133.9, 133.7, 131.2, 129.1, 129.0 (Car), 109.2 (C1 ), 81.7 (C3 ), 76.2
0
0
(C4 ), 74.1 (C2 ), 73.6 (C6), 72.4 (C3), 67.7 (CH2OR), 63.1 (CH2OSi),
58.6 (C5), 54.9 (C7), 44.9 (C5 ), 42.8 (NMe), 35.2 (Cb), 33.2e23.8
JH5beH6 ¼ 3.9 Hz, H5b), 2.50 (s, 3H, NMe); 13C NMR (D2O)
d 177.4
0
(-(CH2)13), 27.5, 20.1 (tBu), 14.5 (CH3); MS (ESIþ) 812.4 (100%,
M þ H)þ; HRMS (ESIþ) for C45H74N3O8Si (M þ H)þ: calcd
812.5245, found 812.5209.
(C2), 73.0 (C3), 67.4 (C6), 60.7 (C9), 59.9 (C8), 58.9 (C5), 56.6 (C7),
42.5 (NMe); MS (ESIþ) 227.1 (M þ Na)þ; HRMS (ESIþ) for
C8H16N2O4Na (M þ Na)þ calcd 227.1008, found 227.1005.

J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
1589
þ
00
4.1.10. (3S,6S,7R)-3-(Benzyloxymethyl)-7-(tert-
butyldiphenylsilyloxymethyl)-4-N-(500-(uracil-10-yl)pentyl)-6-
hydroxy-1,4-diazepan-2-one (13)
24.8 (Cc), 23.7 (C3 ), 22.7 (CH3-CH2), 14.1 (CH3); MS (ESI ) 937.5
(M þ H)þ, 959.6 (M þ Na)þ; HRMS (ESIþ) calcd. for C55H80N4O7SiNa
(M þ Na)þ 959.5694, found 959.5690.
To a solution of the aldehyde 12 (353 mg, 1.02 eq., 1.80 mmol)
in 1,2-dichloroethane (18.6 mL) was added sodium sulfate
(5.11 g, 20 eq., 36.0 mmol). After stirring under argon atmo-
sphere at r.t. for 10 min a solution of the diazepanone 1b
(917 mg, 1 eq., 1.77 mmol) in 1,2-dichloroethane (36.7 mL) was
added and the reaction mixture was stirred for additional 19 h.
Sodium triacetoxyborohydride (1.12 g, 3 eq., 5.26 mmol) was
then added and the reaction mixture was stirred for additional
24 h. The resulting suspension was filtered through a celite pad
and the reaction was quenched by addition of saturated aqueous
solution of NaHCO3 (20 mL). Layers were separated and the
aqueous one was extracted with CH2Cl2 (3 ꢂ 20 mL). The
combined extracts were dried (MgSO4), filtered and concen-
trated in vacuo. Flash chromatography of the residue (AcOEt/
MeOH/Et3N 96:4:3&) afforded 13 (1.07 g, 87%) as a white foam.
4.1.12. (3S,6S,7R)-7-(tert-Butyldiphenylsilyloxymethyl)-3-
(hydroxymethyl)-4-N-(500-(uracil-10-yl)pentyl)-6-palmitoyloxy-1,4-
diazepan-2-one (15)
To a solution of 14 (800 mg, 854 mmol) in anhydrous CH2Cl2
(17 mL) was added dropwise BCl3 (1.0 M in CH2Cl2, 3.42 mL) at
ꢁ78 ꢀC under argon atmosphere. The resulting solution was
allowed to warm to ꢁ65 ꢀC in 3 h and was stirred for additional 2 h
at the same temperature. The reaction mixture was quenched by
the slow addition of a saturated aqueous NaHCO3 solution (20 mL)
at ꢁ78 ꢀC, diluted with CH2Cl2 (17 mL), and warmed to room
temperature. The layers were separated, and the aqueous layer was
further extracted with CH2Cl2 (3 ꢂ 20 mL). The combined organic
fractions were dried (MgSO4) and concentrated in vacuo. Flash
chromatography of the residue (CH2Cl2/MeOH 97:3 to 94:6) affor-
ded 15 (666 mg, 92%) as a white stable foam. Rf 0.18 (CH2Cl2/MeOH
Rf 0.26 (AcOEt/MeOH/Et3N, 96:4:3&); [
1H NMR
(500 MHz) 8.62 (bs, 1H, NHuracil), 7.67e7.24 (m, 15H,
Har), 7.06 (d, 1H, JH6 eH5
a
]
þ17 (c 1.0, CH2Cl2);
D
d
95:5); [
a
]
D þ39 (c 1.0, CH2Cl2); 1H NMR (500 MHz, CDCl3)
d
8.83 (s,
0
0
0
0
0
0
0
¼
8.0 Hz, H6 ), 6.08 (d, 1H,
1H, H3 ), 7.64e7.38 (m, 10H, Har), 7.13 (d, 1H, JH6 eH5 ¼ 7.9 Hz, H6 ),
0
0
0
0
0
JH1eH7 ¼ 5.4 Hz, H1), 5.65 (d, 1H, JH5 eH6 ¼ 7.9 Hz, H5 ), 4.56, 4.52
6.15 (d, 1H, JH1eH7 ¼ 5.4 Hz, H1), 5.70 (dd, 1H, JH5 eH6 ¼ 7.9 H,
0
0
0
(AB, 2H, JAB ¼ 12.1 Hz, CH2Ph), 3.96e3.92 (m, 1H, H7), 3.90e3.75
JH5 eH3 ¼ 1.7 Hz, H5 ), 5.03 (td, 1H, JH6eH7 ¼ 9.1 Hz, JH6eH5a ¼ JH6eH5b
z2.8 Hz, H6), 4.09e4.05 (m, 1H, H7), 3.92e3.90 (m, 2H, CH2OH),
3.79 (dd, 1H, JCH2OSi ¼ 10.9 Hz, JCHaOSieH7 ¼ 3.5 Hz, CHaOSi),
00
00
(m, 6H, CH2OSi, CH2OBn, H6, OH), 3.67 (t, 2H, J
¼ 7.3 Hz,
H5 eH4
00
H5 ), 3.60 (t, 1H, JH3eCH2OBn ¼ 3.8 Hz, H3), 3.06 (dd, 1H,
JH5aeH5b ¼ 15.0 Hz, JH5beH6 ¼ 3.2 Hz, H5b), 2.84 (dd, 1H,
JH5aeH5b ¼ 14.9 Hz, JH5aeH6 ¼ 2.2 Hz, H5a), 2.64e2.58 (m, 1H,
00
3.72e3.67 (m, 3H, CHbOSi, H5 ), 3.50 (t, 1H, JH3eCH2OH ¼ 4.9 Hz, H3),
3.09 (dd, 1H, JH5aeH5b ¼ 15.7 Hz, JH5aeH6 ¼ 3.2 Hz, H5a), 3.01 (dd, 1H,
00
00
00
00
H1 b), 2.51e2.45 (m, 1H, H1 a), 1.73e1.60 (m, 2H, H4 ), 1.57e1.42
JH5beH5a ¼ 15.7 Hz, JH5beH6 ¼ 2.6 Hz, H5b), 2.68e2.63 (m, 2H, H1b
,
(m, 2H, H2 ), 1.41e1.22 (m, 2H, H3 ), 1.09 (s, 9H, tBu); 13C NMR
-OH), 2.54e2.49 (m, 1H, H1a ), 2.19e2.09 (m, 2H, Hb), 1.74e1.64 (m,
00
00
00
0
0
0
00
00
00
d
173.8 (C2), 163.6 (C4 ), 150.8 (C2 ), 144.3 (C6 ), 138.0, 135.7, 132.6,
2H, Hc), 1.54e1.43 (4H, H2 , H4 ), 1.39e1.26 (m, 26H, (CH2)12, H3 ),
1.08 (s, 9H, tBu), 0.90 (t, 3H, JCH3eCH2 ¼ 6.9 Hz, CH3); 13C NMR
0
130.1, 128.4, 128.0, 127.9, 127.6 (Car), 102.2 (C5 ), 73.3 (CH2Ph),
72.2 (C3), 70.4 (CH2OBn), 69.4 (C6), 61.8 (CH2OSi), 57.0 (C5), 55.5
0
0
(125 MHz, CDCl3)
d
174.3(C2), 172.6(Ca), 163.7 (C4 ), 150.9 (C2 ), 144.2
00
00
00
00
0
0
(C7), 54.7 (C1 ), 48.5 (C5 ), 29.6 (C4 ),þ28.7, 19.3 (tBu), 2þ6.9 (C2 ),
(C6 ), 135.6, 135.5, 132.4, 132.2, 130.0, 127.9, 127.8 (Car), 102.2 (C5 ),
þ
00
23.8 (C3 ); MS (ESI ) 699.3 (M þ H) , 721.3 (M þ Na) ; HRMS
72.1 (C3), 70.8 (C6), 61.5 (CH2OH), 61.4 (CH2OSi), 53.4 (C5), 53.2 (C7),
00 00
52.6 (C1 ), 48.7 (C5 ), 34.2 (Cb), 31.9e28.6 ((CH2)12), 26.8, 19.1 (tBu),
26.7 (C2 ), 24.7 (C4 ), 23.6 (C3 ), 22.6 (CH3eCH2), 14.1 (CH3); MS
(ESIþ) 847.4 (M þ H)þ, 1694.1 (2M þ 2H)þ; HRMS (ESIþ) calcd. for
C48H74N4O7NaSi 869.5224 (M þ Na)þ, found 869.5212.
(ESIþ) calcd for C39H51N4O6Si (M
þ
H)þ 699.3578, found
00
00
00
699.3571.
4.1.11. (3S,6S,7R)-3-(Benzyloxymethyl)-7-(tert-
butyldiphenylsilyloxymethyl)-4-N-(500-(uracil-10-yl)pentyl)-6-
palmitoyloxy-1,4-diazepan-2-one (14)
4.1.13. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-(500-
(uracil-10-yl)pentyl)-6-palmitoyloxy-3-(5-azido-5-deoxy-2,3-O-
isopentylidene-
A mixture of the hydroxymethyl-diazepanone 15 (100 mg,
118 mol), the azidofluororibose derivative 5 (43.5 mg, 177 mol)
To a solution of the secondary alcohol 13 (1.06 g, 1.52 mmol) in
CH2Cl2 (18 mL) was added palmitic acid (778 mg, 3.04 mmol), N,N0-
dicyclohexylcarbodiimide (626 mg, 3.04 mmol) and 4-dimethyla-
minopyridine (37 mg, 0.30 mmol). After stirring at r.t. overnight
and under argon atmosphere, the reaction mixture was filtered
through a celite pad and concentrated under reduced pressure.
Flash chromatography of the residue (EtOAc/cyclohexane 8:2)
afforded 14 (1.24 g, 87%) as a white stable foam. Rf 0.18 (EtOAc/
b-D-ribos-1-yl-methyl)-1,4-diazepan-2-one (16)
m
m
and molecular sieves 4 Å (400 mg) in CH2Cl2 (3.7 mL) was stirred at
r.t. for 1 h under argon atmosphere. The suspension was than
cooled down to ꢁ78 ꢀC and boron trifluoride etherate (100
mL,
0.81 mmol) was added dropwise. The reaction mixture was allowed
to slowly warm up to r.t. and was stirred overnight. Saturated
aqueous NaHCO3 (20 mL) was added and the mixture was extracted
with CH2Cl2 (3 ꢂ 10 mL). The organic phase was dried (MgSO4),
filtered, and concentrated in vacuo. Flash chromatography of the
residue (CH2Cl2/MeOH 97:3 to 94:6) afforded 16 (123 mg, 97%) as
cyclohexane 7:3); [
a
]
D
þ47 (c 1.0, CH2Cl2); [
a
]
365
þ 161 (c 1.0,
CH2Cl2); 1H NMR (500 MHz, CDCl3)
d
8.58 (s, 1H, H3 ), 7.64e7.24 (m,
0
0
0
0
15H, Har), 7.02 (d, 1H, JH6 eH5 ¼ 7.9 Hz, H6 ), 6.12 (d, 1H,
0
0
0
JH1eH7 ¼ 4 Hz, H1), 5.63 (d, 1H, JH5 eH6 ¼ 7.9 Hz, H5 ), 5.02e4.99 (m,
1H, H6), 4.56, 4.54 (AB, 2H, JAB ¼ 12.0 Hz, CH2Ph), 4.33e4.28 (m, 1H,
H7), 3.85 (d, 2H, JCH2OBn ¼ 3.8 Hz, CH2OBn), 3.70e3.62 (m, 4H,
a colorless solid. Rf 0.40 (CH2Cl2/MeOH 95:5); [
a
]
D
þ28 (c 1.0,
CH2Cl2); 1H NMR (500 MHz, CDCl3)
d
8.62 (s, 1H, H3 ), 7.65e7.38 (m,
00
0
CH2OSi and H5 ), 3.60 (t, 1H, JH3eCH2OBn ¼ 3.6 Hz, H3), 3.07 (dd, 1H,
0
0
0
JH5b,H5a
¼
15.6 Hz, JH5b,H6
¼
3.4 Hz, H5b), 2.97 (dd, 1H,
10H, Har), 7.14 (d, 1H, JH6 eH5 ¼ 7.9 Hz, H6 ), 6.10 (d, 1H,
00
0
0
0
JH5a,H5b ¼ 15.7 Hz, JH5a,H6 ¼ 2.3 Hz, H5a), 2.63e2.58 (m, 1H, H1b ),
JH1eH7 ¼ 5.9 Hz, H1), 5.69 (d, 1H, JH5 eH6 ¼ 7.9 Hz, H5 ), 4.98 (td, 1H,
JH6eH7 ¼ 9.8 Hz, JH6eH5a ¼ JH6eH5b z2.7 Hz, H6), 5.18 (s, 1H, H1r),
4.59 (d, 1H, JH2reH3r ¼ 6.0 Hz, H2r), 4.53 (d, 1H, JH3reH2r ¼ 6.1 Hz,
H3r), 4.34 (t, 1H, JH4reH5r ¼ 7.6 Hz, H4r), 4.17e4.14 (m, 1H, H7), 4.02
(dd, 1H, JCH2OSi ¼ 10.7 Hz, JCHaOSieH7 ¼ 4.02 Hz, CHaOSi), 3.77e3.65
00
2.52e2.47 (m, 1H, H1a ), 2.16e2.06 (m, 2H, Hb), 1.66e1.61 (m, 2H,
00
00
00
Hc), 1.52e1.50 (m, 2H, H4 ), 1.45e1.26 (m, 28H, (CH2)12, H3 ,H2 ),
1.08 (s, 9H, tBu), 0.90 (t, 3H, JCH3eCH2 ¼ 6.9 Hz, CH3); 13C NMR
0 0
(125 MHz, CDCl3) d 173.7(C3), 172.7(Ca), 163.1 (C4 ), 150.4 (C2 ), 144.2
0
00
(C6 ), 138.0, 135.6, 135.5, 132.5, 132.4, 130.1, 130.0, 128.3, 127.9, 127.8,
127.6, 127.4, (Car), 102.0 (C5 ), 77.2 (C6), 73.3 (CH2Ph), 71.8 (C3), 70.5
(CH2OBn), 61.0 (CH2OSi), 53.8 (C5), 53.6 (C1 ), 52.6 (C7), 48.7 (C5 ),
34.3 (Cb), 31.9e29.2 (-(CH2)12), 28.8 (C2 ), 26.9, 19.2 (tBu), 26.7 (C4 ),
(m, 5H, CHbOSi, H5 , CH2OR), 3.54 (t, 1H, JH3eCH2OR ¼ 4.0 Hz, H3),
0
3.45 (dd, 1H, JH5areH5br ¼ 12.6 Hz, JH5areH4r ¼ 8.0 Hz, H5ar), 3.20 (dd,
1H, JH5breH5ar ¼ 12.7 Hz, JH5breH4r ¼ 6.9 Hz, H5br), 3.05 (dd, 1H,
00
00
00
00
JH5aeH5b
¼
15.7 Hz, JH5a,H6
¼
3.3 Hz, H5a), 2.94 (dd, 1H,

1590
J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
00
0
0
0
JH5beH5a ¼ 15.7 Hz, JH5beH6 ¼ 2.1 Hz, H5b), 2.59e2.54 (m, 1H, H1a ),
JH6 eH5 ¼ 7.8 Hz, H6 ), 7.49e7.41 (m, 6H, Har), 5.65 (d, 1H,
00
0
0
0
2.50e2.45 (m, 1H, H1b ), 2.17e2.08 (m, 2H, Hb), 1.73e1.65 (m, 4H,
JH5 eH6 ¼ 7.8 Hz, H5 ), 5.03 (td, 1H, JH6eH7 ¼ 9.3 Hz, JH6eH5a ¼ JH6eH5b
z2.6 Hz, H6), 4.95 (s, 1H, H1r), 4.24e4.23 (m, 1H, H7), 4.02e3.99 (m,
2H, CHaOR, H4r), 3.96e3.91 (m, 2H, H2r, H3r), 3.87e3.78 (m, 3H,
00
H4 , C(CH2CH3)2), 1.56e1.51 (m, 4H, Hc, C(CH2CH3)2), 1.46e1.36 (m,
00
00
2H, H2 ), 1.35e1.26 (m, 26H, (CH2)12, H3 ), 1.08 (s, 9H, tBu), 0.92 (t,
3H, JCH3eCH2 ¼ 7.4 Hz, C(CH2CH3)2), 0.90 (t, 3H, JCH3eCH2 ¼ 7.0 Hz,
CH3), 0.84 (t, 3H, JCH3eCH2 ¼ 7.5 Hz, C(CH2CH3)2); 13C NMR
00
00
00
CH2OSi, CHbOR), 3.75 (t, 2H, JH5 eH4 ¼ 7.2 Hz, H5 ), 3.50 (t, 1H,
JH3eCH2OR ¼ 3.9 Hz, H3), 3.04 (dd, 1H, JH5aeH5b ¼ 13.1 Hz,
JH5aeH6 ¼ 3.1 Hz, H5a), 2.97 (d, 2H, J ¼ 11.6 Hz, H5r), 2.81 (dd, 1H,
0 0
(125 MHz, CDCl3) d 172.9 (C2) 172.6 (Ca),163.2 (C4 ),150.5 (C2 ),144.1
0
00
(C6 ), 135.6, 135.5, 132.4, 132.2, 130.0, 127.9, 127.8 (Car), 117.2 (C
JH5beH5a ¼ 8.4 Hz, JH5beH6 ¼ 13.1 Hz, H5b), 2.68e2.62 (m, 1H, H1b ),
0
00
(CH2CH3)2), 109.0 (C1r), 102.1 (C5 ), 85.7 (C4r), 85.5 (C2r), 82.3 (C3r),
71.5 (C3), 71.1 (C6), 67.1 (CH2OSi), 61.2 (CH2OR), 53.7 (C5), 53.3 (C7,
2.45e2.40 (m, 1H, H1a ), 2.23e2.11 (m, 2H, Hb), 1.73e1.62 (m, 2H,
00
00
H4 ), 1.54e1.51 (m, 2H, Hc), 1.47e1.39 (m, 2H, H2 ), 1.35e1.27 (m,
00
00
00
C5r), 52.7 (C1 ), 48.7 (C5 ), 34.2 (Cb), 31.9e29.6 ((CH2)8), 29.5 (C
26H, (CH2)12, H3 ), 1.09 (s, 9H, tBu), 0.91 (t, 3H, JCH3eCH2 ¼ 6.9 Hz,
CH3); 13C NMR (125 MHz, CD3OD)
d
176.2 (C2), 174.3 (Ca), 166.7 (C4 ),
0
(CH2CH3)2), 29.4, 29.3, 29.2, 28.9 ((CH2)4), 28.8 (C(CH2CH3)2), 26.9,
00
00
00
0
0
19.2 (tBu), 26.8 (C2 ), 24.8 (C4 ), 23.8 (C3 ), 22.7 (CH3-CH2), 14.1
(CH3), 8.3, 7.3 (C(CH2CH3)2); MS (ESIþ) 1072.4 (M þ H)þ, 1094.6
(M þ Na)þ; HRMS (ESIþ) calcd. for C58H89N7O10Si 1072.6518
(M þ H)þ, found 1072.6514.
152.8 (C2 ), 147.3 (C6 ), 136.8, 136.7, 133.9, 133.7, 131.3, 129.2, 129.1
0
(Car),109.1 (C1r),102.3 (C5 ), 82.8 (C4r), 76.5 (C2r), 74.1 (C3r), 72.9 (C3),
72.8 (C6), 68.4 (CH2OR), 62.8 (CH2OSi), 54.7 (C5r), 54.6 (C7), 54.1
(C1 ), 49.6 (C5 ), 45.4 (C5), 35.2 (Cb), 33.1e30.3 ((CH2)11), 29.9 (C4 ),
28.3 (C2 ), 27.5, 20.1 (tBu), 25.9 (Cc), 24.8 (Cd), 23.8 (C3 ), 14.5 (CH3);
MS (ESIþ) 978.5 (M þ H)þ; HRMS (ESIþ) calcd. for C53H84N5O10Si
978.5987 (M þ H)þ, found 978.5986.
00
00
00
00
00
4.1.14. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-(500-
(uracil-10-yl)pentyl)-6-palmitoyloxy-3-(5-amino-5-deoxy-2,3-O-
isopentylidene-
A solution of 16 (100 mg, 93.2
phosphino)ethane (20 mg, 51.0 mol) in THF (330
(33 L) was stirred at r.t. overnight. Resulting suspension was
b
-D
-ribos-1-yl-methyl)-1,4-diazepan-2-one (17)
mol) and 1,2-bis(diphenyl-
L) and H2O
m
4.1.16. (3S,6S,7R)-3-(5-Amino-5-deoxy-b-D-ribos-1-yl-methyl)-7-
hydroxymethyl-4-N-(500-(uracil-10-yl)pentyl)-6-palmitoyloxy-1,4-
diazepan-2-one (19)
m
m
m
concentrated in vacuo and the residue was suspended in ether.
White precipitate was filtered off and the filtrate was concentrated
in vacuo. Flash chromatography of the residue (CH2Cl2/MeOH/Et3N
95:5:3&) afforded 17 (90 mg, 92%) as a colorless solid. Rf 0.30
To a solution of 18 (18 mg, 18.4
mmol) in DMF (1.3 mL) was added
NH4F (0.7 mg, 0.0184 mol) and the reaction mixture was stirred at
m
r.t. overnight. Reaction mixture was then concentrated in vacuo and
the residue was carefully rinsed with ether (3 mL). Product was
dissolved in methanol, filtered through the cotton and concen-
trated in vacuo to afford 19 (12 mg, 87%) as a colorless solid. Rf 0.15
(CH2Cl2/MeOH 92:8); [
CDCl3)
a
]
þ26 (c 1.0, CH2Cl2); 1H NMR (500 MHz,
D
0
0
0
d
7.64e7.38 (m, 10H, Har), 7.15 (d, 1H, JH6 eH5 ¼ 7.9 Hz, H6 ),
6.11 (d, 1H, JH1eH7 ¼ 5.7 Hz, H1), 5.67 (d, 1H, JH5 eH6 ¼ 7.9 Hz, H5 ),
(CH2Cl2/MeOH/NH4OH 7:3:0.3); [a]
þ25 (c 1.0, MeOH); 1H NMR
0
0
0
D
0
0
0
5.12 (s, 1H, H1r), 4.99 (td, 1H, JH6eH7 ¼ 9.8 Hz, J
¼ JH6eH5b
(CD3OD)
d
7.60 (d, 1H, JH6 eH5 ¼ 7.8 Hz, H6 ), 5.68 (d, 1H,
H6eH5a
0
0
0
z2.7 Hz, H6), 4.56 (d, 1H, JH2reH3r ¼ 6.0 Hz, H2r), 4.51 (d, 1H,
JH3reH2r ¼ 6.1 Hz, H3r), 4.24e4.21 (m, 2H, H7, H4r), 4.03 (dd, 1H,
JCH2OSi ¼ 10.7 Hz, JCHaOSieH7 ¼ 3.1 Hz, CHaOSi), 3.78e3.65 (m, 5H,
JH5 eH6 ¼ 7.8 Hz, H5 ), 4.97 (s, 1H, H1r), 4.83 (td, 1H, JH6eH7 ¼ 9.8 Hz,
JH6eH5a ¼ JH6eH5b z2.5 Hz, H6), 4.35e4.31 (m,1H, H7), 4.16e4.13 (m,
1H, H3r), 4.12e4.08 (m, 1H, H4r), 4.05e4.02 (m, 2H, CHaOR, H2r),
3.91 (dd, 1H, JCH2OR ¼ 10.6 Hz, JCHbOReH3 ¼ 3.2 Hz, CHbOr), 3.79 (t,
00
CHbOSi, H5 , CH2OR), 3.54 (t, 1H, JH3eCH2OR ¼ 3.3 Hz, H3), 3.04 (dd,
00
00
00
1H, JH5aeH5b ¼ 15.7 Hz, JH5a,H6 ¼ 3.3 Hz, H5a), 2.94 (dd, 1H,
JH5beH5a ¼ 15.7 Hz, JH5beH6 ¼ 1.5 Hz, H5b), 2.77e2.70 (m, 2H, H5br),
2H, JH5 eH4 ¼ 7.2 Hz, H5 ), 3.68 (dd, 1H, JCH2OH ¼ 11.5 Hz,
JCHaOHeH7 ¼ 2.8 Hz, CHaOH), 3.61 (dd, 1H, JCH2OH ¼ 11.5 Hz, J
00
00
2.60e2.54 (m, 1H, H1b ), 2.49e2.44 (m, 1H, H1a ), 2.19e2.08 (m, 2H,
¼ 6.2 Hz, CHbOH), 3.48 (t, 1H, JH3eCH2OR ¼ 3.1 Hz, H3), 3.23
CHbOHeH7
00
Hb), 1.72e1.64 (m, 4H, H4 , C(CH2CH3)2)), 1.56e1.52 (m, 4H, Hc, C
(dd, 1H, JH5aeH5b ¼ 11.4 Hz, JH5aeH6 ¼ 2.4 Hz, H5a), 3.00 (dd, 1H,
JH5beH5a ¼ 11.4 Hz, JH5beH6 ¼ 2.1 Hz, H5b), 2.96 (d, 2H, J ¼ 2.2 Hz,
00
00
(CH2CH3)2),1.46e1.36 (m, 2H, H2 ),1.33e1.27 (m, 26H, (CH2)12, H3 ),
1.08 (s, 9H, tBu), 0.92 (t, 3H, JCH3eCH2 ¼ 7.4 Hz, C(CH2CH3)2), 0.90 (t,
3H, JCH3eCH2 ¼ 6.0 Hz, CH3), 0.84 (t, 3H, JCH3eCH2 ¼ 7.5 Hz, C
00
00
H5r), 2.69e2.63 (m, 1H, H1b ), 2.43e2.31 (m, 3H, Hb, H1a ), 1.78e1.69
00
00
(m, 2H, H4 ), 1.67e1.61 (m, 2H, Hc), 1.56e1.45 (m, 2H, H2 ),
(CH2CH3)2); 13C NMR (63 MHz, CDCl3)
d
173.2 (C2), 172.7 (Ca), 163.4
1.42e1.31 (m, 26H, (CH2)12, H3 ), 0.92 (t, 3H, JCH3eCH2 ¼ 7.0 Hz, CH3);
00
(C4 ), 150.7 (C2 ), 144.1 (C6 ), 135.6, 135.5, 132.4, 132.2, 130.0, 127.9,
13C NMR (125 MHz, CD3OD)
d
176.4 (C2), 174.6 (Ca), 166.7 (C4 ), 152.8
0
0
0
0
0
0
0
0
127.8 (Car), 116.7 (C(CH2CH3)2), 109.0 (C1r), 102.1 (C5 ), 89.0 (C4r),
(C2 ), 147.3 (C6 ), 109.0 (C1r), 102.2 (C5 ), 80.3 (C4r), 76.3 (C2r), 74.3
(C3r), 73.6 (C3, C6), 68.4 (CH2OR), 61.0 (CH2OH), 55.3 (C5r), 54.6 (C7),
85.8 (C2r), 82.5 (C3r), 71.5 (C3), 71.1 (C6), 67.4 (CH2OSi), 61.2 (CH2OR),
00
00
00
00
53.7 (C5), 53.5 (C7), 52.5 (C1 ), 48.5 (C5 ), 45.1 (C5r), 34.2 (Cb), 31.9
54.1 (C1 ), 49.7 (C5 ), 44.5 (C5), 35.2 (Cb), 33.0e30.3 ((CH2)10), 29þ.9
00
00
00
((CH2)8), 29.6 (C(CH2CH3)2), 29.4, 29.3, 29.3, 29.1 ((CH2)4), 28.9 (C
(C4 ), 28.3 (C2 ), 26.0 (Cc), 24.8 (Cd), 23.6 (C3 ), 14.4 (CH3); MS (ESI )
740.5 (M þ H)þ; HRMS (ESIþ) calcd. for C37H66N5O10 740.4810
(M þ H)þ, found 740.4814.
00
00
00
(CH2CH3)2), 26.7, 19.2 (tBu), 26.6 (C2 ), 24.8 (C4 ), 23.6 (C3 ), 22.6
(CH3eCH2), 14.1 (CH3), 8.3, 7.4 (C(CH2CH3)2); MS (ESIþ) 1046.7
(M þ H)þ; HRMS (ESIþ) calcd. for C58H91N5O10Si 1046.6613
(M þ H)þ, found 1046.6658.
4.1.17. (3S,6S,7R)-3,7-Dihydroxymethyl-6-palmitoyloxy-4-N-(500-
(uracil-10-yl)pentyl)-1,4-diazepan-2-one (20)
To a solution of 15 (176 mg, 208
added NH4F (24 mg, 0.645 mol) and the reaction mixture was
4.1.15. (3S,6S,7R)-7-tert-Butyldiphenylsilyloxymethyl-4-N-(500-
(uracil-10-yl)pentyl)-6-palmitoyloxy-3-(5-amino-5-deoxy-
mmol) in DMF (15 mL) was
b
-D
-
m
ribos-1-yl-methyl)-1,4-diazepan-2-one (18)
stirred at r.t. overnight. NH4F was filtered off and the reaction
mixture was then concentrated in vacuo. The residue was carefully
rinsed with ether (3 ꢂ 2 mL) and dried in vacuo to afford 20
A solution of 17 (57 mg, 53.0 mmol) in TFA (4 mL) and H2O (1 mL)
was stirred at r.t. for 1.5 h. The reaction mixture was then
concentrated in vacuo. Saturated aqueous NaHCO3 (5 mL) was
added to the residue and the mixture was extracted with CH2Cl2
(3 ꢂ 10 mL). The organic phase was dried (MgSO4), filtered and
concentrated in vacuo. Purification on Waters SEP-PAKÒ cartridge
(CH2Cl2/MeOH 8:2) afforded 18 (30 mg, 57%) as a colorless solid. Rf
(122 mg, 96%) as a colorless solid. Rf 0.38 (CH2Cl2/MeOH 9:1); [a]
D
þ68 (c 1.0, CH2Cl2); 1H NMR (500 MHz)
d
10.05 (s, 1H, H3 ), 7.33 (d,
0
0
0
0
1H, JH1eH7 ¼ 4.5 Hz, H1), 7.22 (d, 1H, JH6 eH5 ¼ 7.9 Hz, H6 ), 5.73 (d,
0
0
0
1H, JH5 eH6 ¼ 7.8 Hz, H5 ), 4.87 (td, 1H, JH6eH7 ¼ 5.3 Hz,
JH6eH5a ¼ JH6eH5b z2.8 Hz, H6), 4.26e4.21 (m, 1H, H7), 4.01e3.70
þ35 (c 1.0, CH2Cl2); 1H NMR
(m, 6H, CH2OH, H5 ), 3.44 (t, 1H, JH3eCH2OH ¼ 4.0 Hz, H3), 3.05 (dd,
00
0.37 (CH2Cl2/MeOH 8:2); [
a
]
D
(500 MHz, CD3OD)
d
7.69e7.63 (m, 4H, Har), 7.58 (d, 1H,
1H, JH5aeH5b ¼ 15.4 Hz, JH5aeH6 ¼ 2.8 Hz, H5a), 2.93 (dd, 1H,

J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
1591
00
JH5beH5a ¼ 15.4 Hz, JH5beH6 ¼ 2.6 Hz, H5b), 2.67e2.62 (m, 2H, H1b
,
H
1r1), 5.02 (s, 1H, H1r2), 4.88 (td, 1H, JH6eH7
¼
9.9 Hz,
00
-OH), 2.47e2.43 (m, 1H, H1a ), 2.35e2.24 (m, 3H, Hb, -OH),
1.82e1.73 (m, 1H, H4 a), 1.71e1.651 (m, 1H, H4 b), 1.63e1.58 (m, 2H,
Hc), 1.47e1.42 (m, 4H, H2 , H3 ), 1.30e1.27 (m, 24H, (CH2)12,), 0.89 (t,
175.5(C2), 173.2 (Ca), 164.4
(C4 ), 151.3 (C2 ), 144.8 (C6 ), 102.1 (C5 ), 73.4 (C3), 71.5 (C6), 61.8
(CH2OH), 60.6 (CH2OH), 54.0 (C5), 53.7 (C7), 52.5 (C1 ), 48.8 (C5 ),
JH6eH5a ¼ JH6eH5b z2.6 Hz, H6), 4.64e4.57 (m, 4H, H3r1, H3r2, H2r1
,
00
00
H2r2), 4.31e4.29 (m, 1H, H7), 4.28e4.21 (m, 2H, H4r1, H4r2), 4.03 (dd,
1H, J ¼ 10.5 Hz, J ¼ 3.3 Hz, CHaOR1), 3.80 (dd, 1H, J ¼ 10.5 Hz,
00
00
3H, JCH3eCH2 ¼ 6.9 Hz, CH3); 13C NMR
d
0
J ¼ 2.7 Hz, CHaOR2), 3.77e3.74 (m, 3H, CHbOR1, H5 ), 3.52 (dd, 1H,
00
0
0
0
J ¼ 10.3 Hz, J ¼ 4.2 Hz, CHbOR2), 3.48 (t, 1H, JH3eCH2Or ¼ 3.2 Hz, H3),
3.23 (bs, 4H, NH2), 2.98 (dd, 1H, JH5aeH5b ¼ 15.6 Hz, JH5aeH6 ¼ 3.3 Hz,
H5a), 2.91 (d, 1H, JH5beH5a ¼ 15.6 Hz, JH5beH6 ¼ 1.9 Hz, H5b),
00
00
00
þ 00
34.3 (Cb), 31.9e29.2 ((CH2)11), 28.4 (Cc), þ26.6 (C2 ), 24.8 (C4 ), 23.2
00
00
(C3 ), 22.6 (CH3-CH2), 14.1 (CH3); MS (ESI ) 631.4 (M þ Na) ; HRMS
2.88e2.75 (m, 4H, H5r1, H5r2), 2.59e2.54 (m, 1H, H1b ), 2.47e2.42
(ESIþ) calcd. for C32H56N4O7Na 631.4047 (M
631.4065.
þ
Na)þ, found
(m, 1H, H1a ), 2.35e2.24 (m, 2H, Hb), 1.73e1.54 (m, 10H, Hc, C
00
00
00
(CH2CH3)2), 1.49e1.37 (m, 4H, H2 , H4 ), 1.30e1.26 (m, 26H, (CH2)12
,
H3 ), 0.93e0.86 (m, 15H, C(CH2CH3)2, CH3); 13C NMR (125 MHz,
00
0
0
0
4.1.18. (3S,6S,7R)-3,7-Di-(5-azido-5-deoxy-2,3-O-isopentylidene-
-ribos-1-yl-methyl)-4-N-(500-(uracil-10-yl)pentyl)-6-palmitoyloxy-
1,4-diazepan-2-one (21)
A mixture of the dihydroxymethyl-diazepanone 20 (102 mg,
167 mol), the fluororibose derivative 5 (123 mg, 0.502 mmol) and
b
-
CDCl3)
d
173.6 (C2), 172.8 (Ca), 163.7 (C4 ), 150.9 (C2 ), 144.1 (C6 ),
0
D
116.7, 116.6 (C(CH2CH3)2), 109.0 (C1r), 102.1 (C5 ), 89.0, 88.3 (C4r),
85.9, 85.5 (C2r), 82.6, 82.3 (C3r), 71.7 (C3), 71.3 (C6), 67.4, 65.7
(CH2OR), 53.8 (C5), 53.3 (C1 ), 51.2 (C7), 48.6 (C5 ), 45.1, 44.8 (C5r),
00
00
m
34.4 (Cb), 31.8e29.4 ((CH2)9), 29.3, 29.3 (C(CH2CH3)2), 29.2, 29.0
00
00
molecular sieves 4 Å (900 mg) in CH2Cl2 (8 mL) was stirred at r.t. for
30 min under argon atmosphere. The suspension was then cooled
((CH2)2), 28.9, 28.8 (C(CH2CH3)2), 26.7 (C2 ), 24.9 (Cc), 2þ3.7 (C4 ),
00
22.6 (C3 ), 14.0 (CH3), 8.3, 7.4, 7.28 (C(CH2CH3)2); MS (ESI ) 1007.7
down to ꢁ78 ꢀC and boron trifluoride etherate (80
mL, 0.65 mmol)
(M þ H)þ; HRMS (ESIþ) calcd. for C52H91N6O13 1007.6644 (M þ H)þ,
was added dropwise. The reaction mixture was allowed to slowly
warm up to r.t. and was stirred overnight. Saturated aqueous
NaHCO3 (20 mL) was added and the mixture was extracted with
CH2Cl2 (3 ꢂ 10 mL). The organic phase was dried (MgSO4), filtered,
and concentrated in vacuo. Flash chromatography of the residue
(CH2Cl2/MeOH 99:1 to 92:8) afforded 21 (110 mg, 62%) as a color-
found 1007.6629.
4.1.20. (3S,6S,7R)-3,7-Di-(5-amino-5-deoxy-b-D-ribos-1-yl-
methyl)-4-N-(500-(uracil-10-yl)pentyl)-6-palmitoyloxy-1,4-
diazepan-2-one (23)
A solution of 22 (46.3 mg, 46 mmol) in TFA (4 mL) and H2O (1 mL)
less solid. Rf 0.32 (CH2Cl2/MeOH 95:5); [
a
]
D þ14 (c 1.0, CH2Cl2); 1H
was stirred at r.t. for 2 h. The reaction mixture was then concen-
trated in vacuo. The crude product was purified by column chro-
matography on octadecyl-functionalized silica gel (Aldrich) by
using gradient elution (MeOH/H2O/AcOH 4:4:2 to 7:1:2) to afford
23 (30 mg, 57%) as a diacetate. Rf 0.20 (CH2Cl2/MeOH/NH4OH
0
NMR (500 MHz, CDCl3)
d
9.45 (s, 1H, H3 ), 7.14 (d, 1H,
0
0
0
JH6 eH5 ¼ 7.9 Hz, H6 ), 6.48 (d, 1H, JH1eH7 ¼ 5.1 Hz, H1), 5.68 (d, 1H,
0
0
0
JH5 eH6 ¼ 7.8 Hz, H5 ), 5.15 (s, 1H, H1r1), 5.08 (s, 1H, H1r2), 4.85 (td,
1H, JH6eH7 ¼ 10.0 Hz, JH6eH5a ¼ JH6eH5b z2.6 Hz, H6), 4.67 (d, 1H,
JH2eH3 ¼ 6.0 Hz, H2r1), 4.63e4.58 (m, 3H, H2r2, H3r1, H3r2), 4.38 (t,1H,
JH4eH5 ¼ 6.4 Hz, H4r1), 4.32 (t, 1H, JH4eH5 ¼ 7.5 Hz, H4r2), 4.28e4.25
(m, 1H, H7), 4.03 (dd, 1H, J ¼ 10.5 Hz, J ¼ 3.6 Hz, CHaOR1), 3.86 (dd,
6:4:0.5); [
a
]
D þ26 (c 1.0, MeOH); 1H NMR (500 MHz, MeOD)
d 7.61
0
0
0
0
0
0
(d, 1H, JH6 eH5 ¼ 7.8 Hz, H6 ), 5.69 (d, 1H, JH5 eH6 ¼ 7.8 Hz, H5 ), 4.99
(s, 1H, H1r1), 4.94 (s, 1H, H1r2), 4.83 (bs, 1H, H6), 4.43e4.40 (m, 1H,
H7), 4.13e4.07 (m, 4H, H4r1, H4r2, H3r1, H3r2), 4.05e4.00 (m, 3H,
CHaOR1, H2r1, H2r2), 3.93e3.86 (m, 2H, CHbOR1, CHaOR2), 3.82e3.75
00
1H, J ¼ 10.4 Hz, J ¼ 2.8 Hz, CHaOR2), 3.78e3.64 (m, 3H, CHbOR1, H5 ),
3.53 (t, 1H, JH3eCH2OR ¼ 3.5 Hz, H3), 3.48e3.33 (m, 4H, H5ar1, H5ar2
,
00
H5br1, CHbOR2), 3.18 (dd, 1H, JH5beH5a ¼ 12.6 Hz, JH5beH4 ¼ 7.8 Hz,
H5br2), 3.01 (dd, 1H, JH5aeH5b ¼ 15.5 Hz, JH5aeH6 ¼ 3.2 Hz, H5a), 2.91
(dd,1H, JH5beH5a ¼ 15.6 Hz, JH5beH6 ¼ 2.0 Hz, H5b), 2.60e2.54 (m,1H,
(m, 2H, H5 ), 3.55 (dd, 1H, J ¼ 10.6 Hz, J ¼ 6.7 Hz, CHbOR2), 3.48 (t,
1H, JH3eCH2OR ¼ 3.0 Hz, H3), 3.25 (dd, 1H, JH5aeH5b ¼ 12.9 Hz,
JH5aeH6 ¼ 4.3 Hz, H5a), 3.08e2.92 (m, 5H, H5r1, H5r2, H5b), 2.69e2.63
00
00
00
00
H1b ), 2.48e2.42 (m, 1H, H1a ), 2.36e2.25 (m, 2H, Hb), 1.72e1.55 (m,
(m, 1H, H1b ), 2.42e2.30 (m, 3H, Hb, H1a ), 1.96 (s, 6H,
AcOꢁ),1.77e1.69 (m, 2H, H4 ), 1.67e1.61 (m, 2H, Hc), 1.56e1.45 (m,
00
00
00
10H, Hc, C(CH2CH3)2), 1.44e1.38 (m, 4H, H2 , H4 ), 1.29e1.26 (m,
26H, (CH2)12, H3 ), 0.93e0.86 (m, 15H, C(CH2CH3)2, CH3); 13C NMR
2H, H2 ), 1.34e1.31 (m, 26H, (CH2)12
, H3 ), 0.92 (t, 3H,
00
00
00
0
0
(125 MHz, CDCl3)
d
173.4 (C2), 172.8 (Ca), 163.7 (C4 ), 150.7 (C2 ),
JCH3eCH2 ¼ 6.9 Hz, CH3); 13C NMR (125 MHz, MeOD)
d 178.8 (C3),
ꢁ
0
0
0
0
0
144.2 (C6 ), 117.3, 117.2 (C(CH2CH3)2), 109.7, 108.8 (C1r), 102.1 (C5 ),
85.9, 85.5 (C4r), 85.4 (C2r), 82.4, 82.0 (C3r), 71.2 (C6), 71.1 (C3), 67.0,
176.4 (CH3COO ),174.7 (Ca), 166.8 (C4 ), 153.0 (C2 ), 147.5 (C6 ), 110.2,
109.0 (C1r), 81.1, 80.8 (C4r), 76.4, 76.0 (C2r), 74.6, 74.4 (C3r), 73.9 (C3),
00
00
66.4 (CH2OR), 53.8 (C5), 53.7, 53.5 (C5r), 52.9 (C7), 51.5 (C1 ), 48.7
73.8 (C6), 68.6, 68.3 (CH2OR), 55.4 (C5), 54.2 (C1 ), 52.8 (C7), 49.7
00
00
00
(C5 ), 34.3 (Cb), 31.8e29.4 ((CH2)9), 29.4, 29.3 (C(CH2CH3)2), 29.3,
(C5 ), 44.9, 44.8 (C5r), 35.3 (Cb), 33.2e28.5 ((CH2)12), 26.1 (C2 ), 24þ.9
(Cc), 23.9 (AcOꢁ), 23.2 (C4 ), 23.2 (C3 ), 14.6 (CH3); HRMS (ESI )
00
00
00
29.2 28.9 ((CH2)3), 28.9, 28.8 (C(CH2CH3)2), 26.8 (C2 ), 24.9 (Cc), 23.7
(C4 ), 22.6 (C3 ), 14.0 (CH3), 8.3 (C(CH2CH3)2), 7.3 (C(CH2CH3)2); MS
(ESIþ) 1081.7 (M þ Na)þ; HRMS (ESIþ) calcd. for C52H86N10O13Na
1081.6274 (M þ Na)þ, found 1081.6267.
calcd. for C42H75N6O13 871.5392 (M þ H)þ, found 871.5406.
00
00
4.2. Enzymatic assays
4.1.19. (3S,6S,7R)-3,7-Di-(5-amino-5-deoxy-2,3-O-isopentylidene-
b-D
-ribos-1-yl-methyl)-4-N-(500-(uracil-10-yl)pentyl)-6-
The activities of the compounds against MraY transferase were
tested as previously described [6,25]. The assay was performed in
palmitoyloxy-1,4-diazepan-2-one (22)
A solution of 21 (62.0 mg, 58.5
a reaction mixture of 10 mL containing, in final concentrations,
m
mol) and 1,2-bis(diphenyl-
mol) in THF (0.7 mL) and H2O
L) was stirred at r.t. overnight. The resulting suspension was
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 as previously described
phosphino)ethane (25.6 mg, 64.4
(70
m
m
concentrated in vacuo and the residue was suspended in ether
(3 mL). White precipitate was filtered off and the filtrate was
concentrated in vacuo. Flash chromatography of the residue
(CH2Cl2/Et3N 1:3& to CH2Cl2/MeOH/Et3N 80:20:3&) afforded 22
(56.9 mg, 96%) as a colorless solid. Rf 0.21 (CH2Cl2/MeOH 9:1); [
a
]
[26,27]. Reaction mixtures contained, in a final volume of 12.5
200 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 16
M UDP-[14C]GlcNAc
(1.7 kBq), 16 M lipid I analog, 30% (v/v) dimethyl sulfoxide and
ml,
D
þ11 (c 1.0, CH2Cl2); 1H NMR (500 MHz, CDCl3)
d
7.15 (d, 1H,
m
0
0
0
0
0
0
JH6 eH5 ¼ 7.9 Hz, H6 ), 5.67 (d, 1H, JH5 eH6 ¼ 7.8 Hz, H5 ), 5.11 (s, 1H,
m

1592
J. Mravljak et al. / European Journal of Medicinal Chemistry 46 (2011) 1582e1592
MurG. After 30 min at 37 ꢀC, it was stopped by boiling for 3 min.
Then, the mixture was lyophilized and taken up in 10 l of
[11] For reviews on MraY inhibitors, see: (a) K.-I. Kimura, T.D.H. Bugg, Nat. Prod.
Rep. 20 (2003) 252e273;
m
(b) C. Dini, Curr. Top. Med. Chem. 5 (2005) 1221e1236;
(c) T.D.H. Bugg, A.J. Lloyd, D.I. Roper, Infect. Disord. Drug Targets 6 (2006)
85e106.
2-propanol/ammonium hydroxide/water (6:3:1; v/v/v). In both
cases, the radiolabeled substrate (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 hydroxide/water (6:3:1; v/v/v) as the
mobile phase. The radioactive spots were located and quantified
with a radioactivity scanner (model Multi-Tracemaster LB285;
EG&G Wallac/Berthold). Residual activities were calculated with
respect to a control assay without inhibitors. IC50 values were
determined with 7 inhibitor concentrations. Data represent the
mean of independent triplicate determinations, and the standard
deviations were less than 10%.
[12] (a) L. Le Corre, C. Gravier-Pelletier, Y. Le Merrer, Eur. J. Org. Chem. (2007)
5386e5394;
(b) O. Monasson, M. Ginisty, G. Bertho, C. Gravier-Pelletier, Y. Le Merrer,
Tetrahedron Lett. 48 (2007) 8149e8152;
(c) A. Clouet, C. Gravier-Pelletier, B. Al-Dabbagh, A. Bouhss, Y. Le Merrer,
Tetrahedron: Asymmetry 19 (2008) 397e400;
(d) N. Auberger, C. Gravier-Pelletier, Y. Le Merrer, Eur. J. Org. Chem. (2009)
3323e3326;
(e) D. Lecerclé, A. Clouet, B. Al-Dabbagh, A. Bouhss, C. Gravier-Pelletier, Y. Le
Merrer, Bioorg. Med. Chem. 18 (2010) 4560e4569.
[13] For recent and selected synthetic approaches to MraY inhibitors:
(a) S. Ichikawa, A. Matsuda, Nucleos Nucleot Nucleic Acids 24 (2005)
319e329;
(b) S. Hirano, S. Ichikawa, A. Matsuda, Angew. Chem. Int. Ed. Engl. 44 (2005)
1854e1856;
(c) F. Sarabia, L. Martin-Ortiz, Tetrahedron 61 (2005) 11850e11865;
(d) Y. Bourdreux, B. Drouillat, C. Greck, Lett. Org. Chem. 3 (2006) 368e370;
(e) S. Hirano, S. Ichikawa, A. Matsuda, J. Org. Chem. 72 (2007) 9936e9946;
(f) M. Kurosu, S. Mahapatra, P. Narayanasamy, D.C. Crick, Tetrahedron Lett. 48
(2007) 799e803;
(g) B. Drouillat, Y. Bourdreux, D. Perdon, C. Greck, Tetrahedron: Asymmetry 18
(2007) 1955e1963;
(h) S. Hirano, S. Ichikawa, A. Matsuda, Tetrahedron 63 (2007) 2798e2804;
(i) X.H. Xu, A.E. Trunkfield, T.D.H. Bugg, F.-L. Quing, Org. Biomol. Chem. 6
(2008) 157e161;
Acknowledgments
We gratefully acknowledge the European Community for the
financial support of the Eur-INTAFAR integrated project within the
6th PCRDT framework (contract No. LSHM-CT-2004-512138) for
a PhD studentship to O.M and for a post-doctoral fellowship
granted to B.A. We thank the “Ville de Paris” for a post-doctoral
grant to J.M.
(j) S. Hirano, S. Ichikawa, A. Matsuda, Bioorg. Med. Chem. 16 (2008) 428e436;
(k) S. Hirano, S. Ichikawa, A. Matsuda, Bioorg. Med. Chem. 16 (2008)
5123e5133;
(l) K. Li, M. Kurosu, Heterocycles 76 (2008) 455e469;
(m) M. Kurosu, K. Li, Heterocycles 77 (2009) 217e225;
(n) T. Tanino, S. Ichikawa, M. Shiro, A. Matsuda, J. Org. Chem. 75 (2010)
1366e1377;
(o) T. Tanino, S. Ichikawa, B. Al-Dabbagh, A. Bouhss, H. Oyama, A. Matsuda,
ACS Med. Chem. Lett. 1 (2010) 258e262.
Appendix. Supplementary material
Supplementary material related to this article can be found
online at doi:10.1016/j.ejmech.2011.02.006.
[14] D.J. Payne, M.N. Gwynn, D.J. Holms, D.L. Pompliano, Nat. Rev. Drug Discover 6
(2007) 29e40.
References
[15] O. Monasson, M. Ginisty, J. Mravljak, G. Bertho, C. Gravier-Pelletier, Y. Le
Merrer, Tetrahedron: Asymmetry 20 (2009) 2320e2330 (and references cited
therein).
[16] S. Knapp, G.J. Moriello, G.A. Doss, Org. Lett. 4 (2002) 603e606.
[17] C. Dini, P. Collette, N. Drochon, J.C. Guillot, G. Lemoine, P. Mauvais, J. Aszodi,
Biorg. Med. Chem. Lett. 10 (2000) 1839e1843.
[18] C. Dini, N. Drochon, S. Feteanu, J.C. Guillot, C. Peixoto, J. Aszodi, Biorg. Med.
Chem. Lett. 11 (2001) 529e531.
[19] C. Dini, N. Drochon, J.C. Guillot, P. Mauvais, P. Walter, J. Aszodi, Biorg. Med.
Chem. Lett. 11 (2001) 533e536.
[1] For example, see: (a) C. Walsh, G. Wright, Chem. Rev. 105 (2005) 391 and all
references of this volume dedicated to antibiotic resistance;
(b) K.M. Overbye, J.F. Barrett, Drug Discov. Today 10 (2005) 45e52;
(c) R.L. Monaghan, J.F. Barrett, Biochem. Pharmacol. 71 (2006) 901e909;
(d) G.H. Talbot, J. Bradley, J. Edwards Jr., D. Gilbert, M. Scheld, J.G. Bartlett, Clin.
Infect. Dis. 42 (2006) 657e668.
[2] (a) H. Barreteau, A. Kovac, A. Boniface, M. Sova, S. Gobec, D. Blanot, FEMS
Microbiol. Rev. 32 (2008) 168e207;
(b) R.S. Narayan, M.S. VanNieuwenhze, Eur. J. Org. Chem. (2007) 1399e1414;
(c) A. Bouhss, A.E. Trunkfield, T.D.H. Bugg, D. Mengin-Lecreulx, FEMS Micro-
biol. Rev. 32 (2008) 208e233.
[20] M. Ginisty, C. Gravier-Pelletier, Y. Le Merrer, Tetrahedron: Asymmetry 17
(2006) 142e150.
[21] A.F. Abdel-Magid, K.G. Carson, B.D. Harris, C.A. Maryanoff, R.D. Shah, J. Org.
Chem. 61 (1996) 3849e3862.
[22] M.B.-U. Surfraz, M. Akhtar, R.K. Allemann, Tetrahedron Lett. 45 (2004)
1223e1226.
[23] V. Liautard, V. Desvergnes, K. Itoh, H. Liu, O.R. Martin, J. Org. Chem. 73 (2008)
3103e3115.
[3] J. van Heijenoort, Nat. Prod. Rep. 8 (2001) 503e519.
[4] A. Bouhss, D. Mengin-Lecreulx, D. Le Beller, J. van Heijenoort, Mol. Microbiol.
34 (1999) 576e585.
[5] D.S. Boyle, W.D. Donachie, J. Bacteriol. 180 (1998) 6429e6432.
[6] A. Bouhss, M. Crouvoisier, D. Blanot, D. Mengin-Lecreulx, J. Biol. Chem. 279
(2004) 29974e29980.
[24] (a) J. Rokach, J. Adams, R. Perry, Tetrahedron Lett. 24 (1983) 5185e5188;
(b) Y. Le Merrer, C. Gravier-Pelletier, D. Micas-Languin, F. Mestre, A. . Duréault,
J.C. Depezay, J. Org. Chem. 54 (1989) 2409e2416.
[25] B. Al-Dabbagh, X. Henry, M. El Gachi, G. Auger, D. Blanot, C. Parquet,
D. Mengin-Lecreulx, A. Bouhss, Biochemistry 47 (2008) 8919e8928.
[26] G. Auger, J. van Heijenoort, D. Mengin-Lecreulx, D. Blanot, FEMS Microbiol.
Lett. 219 (2003) 115e119.
[7] T. Stachyra, C. Dini, P. Ferrari, A. Bouhss, J. van Heijenoort, D. Mengin-Lecreulx,
D. Blanot, J. Bitton, D. Le Beller, Antimicrob. Agents Chemother. 48 (2004) 897e902.
[8] K. Isono, M. Uramoto, H. Kusakabe, K. Kimura, K. Isaki, C.C. Nelson,
J.A. McCloskey, J. Antibiot. 38 (1985) 1617e1621.
[9] (a) T. Takeuchi, M. Igarashi, H. Naganawa, M. Hamada, PCT Int. Appl. (2001)
WO 2001012643;
(b) M. Igarashi, N. Nakagawa, N. Doi, S. Hattori, H. Naganawa, M. Hamada,
J. Antibiot. 56 (2003) 580e583.
[10] A. Takatsuki, K. Arima, G. Tamura, J. Antibiot. 24 (1971) 215e223.
[27] M. Crouvoisier, G. Auger, D. Blanot, D. Mengin-Lecreulx, Biochimie 89 (2007)
1498e1508.