Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1

Article abstract of DOI:10.1016/j.bmc.2010.03.012

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR₁₆₅ and molecules interfering with VEGF₁₆₅ binding to NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound, derived from d-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids arginine, and tryptophane or threonine. At 100 μM, all compounds exhibited a weak affinity for NRP-1, the most efficient being the bis-guanidinylated compound 32 (IC₅₀ = 92 μM) which could be considered as a new NRP-1 non-peptidic ligand.

Full text of DOI:10.1016/j.bmc.2010.03.012

Bioorganic & Medicinal Chemistry 18 (2010) 3285–3298
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Sugar-based peptidomimetics as potential inhibitors of the vascular
endothelium growth factor binding to neuropilin-1
Alexandre Novoa a, Nadia Pellegrini-Moïse a, Denise Bechet b, Muriel Barberi-Heyob b, Yves Chapleur a,
*
a Groupe SUCRES, UMR 7565, Nancy Université-CNRS, BP 70239, F-54506 Vandœuvre-lès-Nancy, France
b CRAN, UMR 7039, Nancy Université-CNRS, Centre Alexis Vautrin, Avenue de Bourgogne, F-54511 Vandœuvre-lès-Nancy, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuropilin-1 (NRP-1) is a co-receptor of VEGFR165 and molecules interfering with VEGF165 binding to
NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four
amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis
and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound,
Received 25 November 2009
Revised 5 March 2010
Accepted 8 March 2010
Available online 12 March 2010
derived from
arginine, and tryptophane or threonine. At 100
D
-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids
M, all compounds exhibited a weak affinity for NRP-1,
l
Keywords:
the most efficient being the bis-guanidinylated compound 32 (IC50 = 92 lM) which could be considered
as a new NRP-1 non-peptidic ligand.
Neuropilin-1
Peptidomimetics
Scaffold
Ó 2010 Elsevier Ltd. All rights reserved.
Carbohydrates
1. Introduction
genesis.5 For these reasons, molecules that interfere with VEGF165
binding to NRP-1 can modulate the behavior of cancer and endo-
thelial cells. NRP-1 seems to be a promising target in the develop-
ment of new angiogenic modulators and in targeting strategies.
Peptide inhibitors of NRP-1 currently in development for use as
antiangiogenic agents, are all basic in character and function and
incorporate an arginine residue.6 Recently, several peptides have
been reported to modulate VEGF-NRP-1 binding and studies have
revealed structural basis for ligand binding to NRP-1. A bicyclic
peptide antagonist (EG 3287) has been characterized and showed
the importance of the C-terminal six amino-acid domain encoded
by exon 8 for NRP-1 binding which corresponds to the CDKPRR se-
quence.7 These conclusions were further confirmed by the discov-
ery that the tuftsin peptide (TKPR) selectively binds to NRP-1 and
blocks VEGF binding to that receptor.8 The crystal structure of
NRP-1 in complex with tuftsin provided the first structural infor-
mation of NRP-1 in interaction with a ligand. The C-terminal argi-
nine of tuftsin mainly contributes to the interaction between
tuftsin and NRP-1.9 A cyclic peptide (CPQPRPLC sequence) isolated
from a random phage display library has been validated as a VEGF-
R ligand targeting NRP-1. NMR spectroscopy has shown that recep-
tor binding is mediated by the RPL pattern.10,11 Interestingly, a
heptapeptide ATWLPPR, selected by screening a phage display li-
brary, was described as an effective antagonist of VEGF binding
to NRP-1 and can be considered as a potent inhibitor of tumor
Angiogenesis or formation of new blood vessels is an important
biological process under healthy physiological conditions and in
variety of diseases including cancer.1 Vascular endothelial growth
factors (VEGFs) are key molecules in blood vessel formation and
represent the most important growth factors involved in tumor
angiogenesis.2 Among growth factors over-expressed during angi-
ogenesis, VEGF121 and VEGF165 are major isoforms, mediating their
biological effects through receptors located on the endothelial
cells, that is, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR). Neuropi-
lin-1 (NRP-1), identified as a co-receptor of VEGF165, the most
abundant isoform is a 140 kDa membrane protein, with a large
extracellular domain and a short cytoplasmic tail. NRP-1 was first
identified as a receptor for semaphorin, a family of axonal che-
morepellent proteins. In the nervous system, this receptor forms
a high affinity semaphorin-binding complex with a receptor tyro-
sine kinase, plexin that mediates semaphorin-induced growth cone
collapse. On the endothelium, NRP-1 is expressed together with
VEGFR-2. Therefore, NRP-1 directly binds VEGF165 and acts as a
co-receptor along with VEGR-2.3 It was also reported that NRP-1
forms a ternary complex between VEGF165, VEGFR-2 and NRP-1.4
NRP-1 potentiates KDR-mediated endothelial cells migration and
proliferation. Moreover, some tumor cells can express high levels
of NRP-1, which is typically their only VEGF165 receptor. Indeed,
the latter mediates effects on tumor progression and tumor angio-
angiogenesis (IC50 = 19 l
M).12 Structure-function analyses of
ATWLPPR based on alanine-scanning or amino acid deletion
showed the importance of the C-terminal sequence LPPR and par-
ticularly the key role of C-terminal arginine.13 Moreover, ATWLPPR
* Corresponding author. Tel.: +33 3 83 68 47 73; fax: +33 3 83 68 47 80.
E-mail address: yves.chapleur@srsmc.uhp-nancy.fr (Y. Chapleur).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.012

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A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
has recently been conjugated to a TriPhenylChlorin type (TPC) pho-
tosensitizer via a 6-aminohexanoic acid (Ahx) spacer. The coupling
compound TPC-Ahx-ATWLPPR binds to NRP-1 (IC50 = 171 lM), tar-
were easily separated by chromatography. Starting from the E-iso-
mer, the saturated ester 2 was obtained in excellent yield (98%) by
reductive hydrogenation in ethyl acetate in presence of 10 mol % of
Pd/C. Interestingly, hydrogenation of Z-isomer proceeded via prior
isomerization to E-isomer and thus needed 40 mol % of Pd/C to
provide the saturated compound 2 in 60% yield. Hydrogenation
of exo-glycal 1 proceeds via a high stereocontrol of double bond
reduction from the less crowded b face and led to compound 2
geting endothelial cells and tumor cells, and exhibits enhanced
efficiency in in vitro photodynamic therapy (PDT) as compared to
TPC alone.14 TPC-Ahx-ATWLPPR was stable in vitro in human and
mouse plasma for at least 24 h but following intra-venous injection
in glioma-bearing nude mice, was degraded in vivo at various
rates, depending on the organ considered. TPC-Ahx-A was identi-
fied as the main metabolic product, and biodistribution studies
suggested that its appearance in plasma mainly resulted from the
degradation of the peptidic moiety into organs of the reticuloendo-
thelial system. According to in vitro cell culture experiments, TPC-
Ahx-ATWLPPR was significantly degraded after incorporation in
human umbilical vein endothelial cells, mainly into TPC-Ahx-A.15
Given the poor bioavailability and instability of peptidic ligands,
the design of small molecules as peptidomimetics, targeting endo-
thelial cells is attractive.16 The use of carbohydrates as scaffolds to
construct new active compounds for mimicking bioactive peptides
has emerged in the last 10 years. The first example of the use of a
monosaccharide as template for the construction of peptidomimet-
ics was related to somatostatin.17 Since these works, the use of car-
bohydrates as scaffolds for the construction of bioactive
compounds has been reported.18
In connection with our ongoing program on the design of sugar-
based peptidomimetics18c,m,n we describe here the design, synthe-
sis and biological evaluation of series of sugar-based peptidomi-
metics inspired by known NRP-1 peptidic ligands, that is,
ATWLPPR. The choice of the carbohydrate scaffold was done by vi-
sual analogy with the heptapeptide. The LPP sequence was consid-
ered as a rigid scaffold bearing arginine and tryptophane or
threonine. Thus, we proposed the use of a lipophilic and rigid sugar
template to replace the LPP scaffold. The trioxabicyclo[3.3.0] sys-
with the methoxycarbonylmethyl moiety on the
a face as indi-
cated by the J3,4 = 4 Hz (ulosonic acid numbering) coupling con-
stant in favor of a cis arrangement of H-3 and H-4 protons.20
Saponification of 2 in a THF/H2O mixture in presence of KOH led
to the key acid 3 in 98% yield. Using carbonyldiimidazole as acti-
vating agent, amides 4 and 5 were obtained in 86% and 78% yield
by coupling 3 with tryptamine and 1-(R)-aminopropan-2-ol as mi-
metic groups of tryptophane and threonine side chain, respec-
tively. In view of further chemical manipulations, protection of
the hydroxyl function of amide 5 was achieved in quantitative
yield by treatment with acetic anhydride in pyridine. Selective re-
moval of the exocyclic isopropylidene acetal on compounds 4 and
6 under several acidic conditions has been investigated. The use of
acetic acid in a mixture of THF/H2O at 65 °C and PPTS (15 mol %) in
MeOH furnished the mono deprotected compound in modest
yields (40% and 30%, respectively). The most efficient method
was the use of aqueous HCl in MeOH (1:3) which furnished diols
7 and 8 in 93% and 78%, respectively. Oxidative cleavage of the diol
moiety by NaIO4 gave aldehydes 9 and 10 in good yields (92% and
78%, respectively). These aldehydes have been isolated as partial
hydrates without detectable epimerisation as confirmed by the
structure of the amines 11–22 prepared by reductive amination.
As known from the literature, in the RGD sequence mimics, the
guanidine function of the R residue may be replaced by guanidine
or amidine surrogates.21 Starting from the functionalized alde-
hydes 9 and 10, different amines commonly used as guanidine
mimetics were introduced by reductive amination.22 To this end,
various nucleophiles commercially available or prepared by rou-
tine chemistry from readily available starting materials were used
(Table 1).23 Reductive aminations have been carried out in MeOH
in the presence of acetic acid and NaBH3CN. The expected amines
11–22 were prepared in modest to good yields as pure isolated
compounds (Scheme 2, Table 1). Compounds 18 and 21 were diffi-
cult to purify and thus isolated in rather low yields. The acetyl
function of compounds 18–21 was removed using Zemplen meth-
od giving compounds 23–27 in excellent yields (Table 1).
tem readily accessible from sugar c-lactones was envisioned as a
good starting point and was decorated with mandatory guani-
dine/arginine residue, present on both peptides. The two other sig-
nificant residues tryptophane and threonine should be mimicked
by an appropriate side chain (Fig. 1).
2. Chemistry
On the basis of our experience and literature precedents, we
planned to construct a C-glycosyl-sugar amino acid suitable for
further functionalization at both ends. Our synthetic plan implied
on the one hand the introduction of tryptophane and threonine
side chains by amide bond formation. On the other hand, introduc-
tion of a guanidine function or a mimetic group thereof, should be
performed by reductive amination of a suitable aldehyde easily ob-
tained by diol cleavage.
For the introduction of a guanidine group, the C-glycosyl com-
pounds 17 and 22 bearing a diaminoethane spacer were prepared.
Removal of the Cbz protecting group of compounds 17 and 22 was
achieved by catalytic hydrogenation giving compounds 28 and 29
in good yield (Scheme 3). Treatment of the free amines 28 and
29 with N,N0-bis(benzyloxycarbonyl)-S-methylisothiourea in ex-
cess in DMF in the presence of HgCl2 gave guanidinylated deriva-
tives.24 However, careful examination of 1H and 13C NMR showed
that the isolated compounds were the bis-guanidinylated deriva-
Our first target was the carboxylic acid 3 (Scheme 1). The
known exo-glycal 1 was prepared from the corresponding pro-
tected D-gulonolactone by a Wittig reaction and obtained as a mix-
ture of stereoisomers (E/Z 1.2:1) in 80% yield.19 The two isomers
R1
=
O
CONH R1
R2 HN
N
W
T
Trp side chain
Thr side chain
R2 = amidine, guanidine mimetics
O
O
guanidine, arginine
R Arg or Arg side chain
Sugar amino-acid scaffold
OH
as mimetic of LPP sequence
Figure 1. Design of sugar-based peptidomimetics.

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3287
O
O
O
CONHR1
COOR
O
O
COOMe
a
O
O
O
c
O
O
O
O
O
O
O
4-6
1 (Z or E)
2 R = Me
3 R = H
b
4 R1 =
HN
2-(1H-indol-3-yl)ethyl
2-(hydroxy)propyl
5 R1 =
6 R1 =
OH
d
2-(acetoxy)propyl
OAc
O
O
CONHR1
f
CONHR1
HO
HO
OHC
e
O
O
O
O
7 R1 = 2-(1H-indol-3-yl)ethyl
8 R1 = 2-(acetoxy)propyl
9
R1 = 2-(1H-indol-3-yl)ethyl
10 R1 = 2-(acetoxy)propyl
Scheme 1. Reagents and conditions: (a) H2, Pd/C, EtOAc, 25 °C, 24 h, [98% for E-isomer; 60% for Z-isomer]; (b) KOH, THF/H2O (1:1), 25 °C, 24 h, 98%; (c) R1NH2, CDI, THF, 25 °C,
12 h, [86% for 4; 78% for 5]; (d) Ac2O, pyridine, 25 °C, 12 h, 99%; (e) HCl (1 N)/MeOH (1:3), 0–25 °C, [12 h, 93% for 7; 6 h, 78% for 8]; (f) NaIO4, MeOH, 25 °C, 14 h, [92% for 9;
78% for 10].
tives 30 or 31 resulting from the reaction of both primary and sec-
ondary amine groups of 28 and 29. Removal of Cbz protecting
group of 30 by catalytic hydrogenation yielded the expected guani-
dinoglycoside 32. On hydrogenation of compound 31 in MeOH,
concomitant removal of the acetyl group occurred giving the fully
deprotected compound 33. The presence of two guanidine func-
tions was unambiguously confirmed by mass spectrometry and
13C NMR spectra, two signals, characteristic of guanidine groups,
being observed around d 150 ppm.
Toovercomethisdifficulty, theintroductionofthe guanidinefunc-
tion underMitsunobu conditions was explored (Scheme 4).25 Starting
from the aldehyde 9, alcohol 34 was obtained in 45% yield by reduc-
tive aminationwith3-aminopropanol. Activationof theprimary alco-
hol with triphenylphosphine and diethylazodicarboxylate followed
by treatment with N,N0,N000-tri-(tert-butoxycarbonyl)guanidine gave
compound 35. This compound results from intramolecular nucleo-
philic attack of the secondary amine on the tert-butoxycarbonyl pro-
tectinggroupoftheguanidineresidueleadingtoafivememberedring
as confirmed by mass spectrometry. Removal of the two remaining
tert-butoxycarbonyl protecting groups was achieved by acidic treat-
ment (TFA) giving the cyclic urea 36 in 72% yield.
To solve the problem raised by the presence of the secondary
amine on compounds 17 and 22, introduction of the guanidine
function was planned using the previously used efficient reductive
amination of aldehydes 9 and 10. In this case the N-(2-amino-
ethyl)-N0,N000-bis-(tert-butoxycarbonyl)guanidine prepared accord-
ing to literature procedures was used as the amine component
(Scheme 5).23b This way, the protected guanidinoglycosides 37
and 38 were obtained in about 40% isolated yield. The removal of
tert-butyloxycarbonyl groups was achieved by acidic treatment
with TFA in CH2Cl2.
scaffold. Based on some literature precedents, N-alkylation of L-
arginine amino acid by reductive amination seemed reasonable.26
For easier chemical manipulation, the reaction was carried out
with L-arginine methyl ester hydrochloride on both aldehydes 9
and 10 (Scheme 6). Amines 41 and 42 were obtained in 75% and
68% yield, respectively, after purification on reverse phase column
chromatography. Hydrolysis of the methyl ester was performed by
treatment with LiOH in a THF/water mixture (3:1). The resulting
peptidomimetic compounds 43 and 44 have also been purified
by reverse phase chromatography and obtained in good yields
(68% and 87%, respectively).
3. Biological evaluation
The binding of peptidomimetic derivatives to recombinant NRP-
1 protein was determined using a competition assay previously de-
scribed by Tirand et al.14 Briefly, biotinylated VEGF165 was incu-
bated with recombinant chimeric NRP-1 protein in the presence
of the compound to be tested. The biotinylated VEGF165 remaining
after washing steps was detected by chemiluminescence thanks to
HRP-conjugated streptavidin. The binding of biotinylated VEGF
(negative control) to recombinant chimeric NRP-1 protein was
evaluated in the presence of VEGF165 and ATWLPPR as positive
controls. Affinities were estimated as IC50 values, that is, the con-
centration of competitor that displaced 50% of biotinylated
VEGF165 binding, using the medium effect method.
A
preliminary screening with inhibitor concentrations of
100
lM was performed. At this concentration, all the compounds
exhibited a modest affinity for NRP-1 as compared to the affinity
of the reference peptide ATWLPPR (Figs. 2–4).
In the tryptamine series, mimicking tryptophane side chain, the
use of aromatic or heteroaromatic guanidine surrogates as in com-
pounds 11–16 seems deleterious to the activity as compared to a
guanidinyl group which induces the activity of compounds 36–37
and 39. It seems important for the guanidyl group to be on a flexible
Taking into account the position, at the C-terminus, of the argi-
nine residue in all peptidic ligands of NRP-1 it was reasoned that
its carboxylic function may play a role in the recognition process.
Thus, we attempted to introduce a whole arginine residue on our

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A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
Table 1
Reductive amination of aldehydes 9 and 10
O
O
O
O
O
O
NH
R2HN
R2HN
R2HN
OH
OAc
N
H
N
H
N
H
O
O
O
O
O
O
11-17
23-27
18-22
Entry
R2NH2
2-(1H-Indol-3-yl)ethyl series
2-(Acetoxy)propyl series
2-(Hydroxy)propyl series
Product
Yield (%)
Product
Yield (%)
Product
Yield (%)
NH2
NH2
NH2
1
2
11
12
55
—
—
23
40 (two steps)
55
50
18
19
25
45
24
25
70
85
N
N
3
4
5
13
14
15
N
H
N
55
47
20
21
50
25
26
27
80
82
N
H
NH2
NH2
N
N
N
H
N
6
7
NH2
NH2
16
17
45
50
—
—
—
—
—
—
N
H
22
75
CbzNH
O
O
CONHR1
R2HN
CONHR1
dine function in compounds 31, 33, 38 and 40 failed to displace the
binding of biotinylated VEGF165 to NRP-1, compared to guanidine
surrogates for compounds 18–21 and 23–27.
OHC
a
O
O
O
O
The reported studies showing the key role of C-terminal argi-
nine in ATWLPPR led to the hypothesis of a possible role of the car-
boxylic function of the R residue when located at the C terminus as
in the model compound ATWLPPR.13 However, the modest activity
of compounds 43 and 44 bearing a N-linked arginine residue, is
somewhat disappointing and suggests that other important factors
are involved in the binding to NRP-1.
9
R1 = 2-(1H-indol-3-yl)ethyl
10 R1 = 2-(acetoxy)propyl
11-17 R1 = 2-(1H-indol-3-yl)ethyl
18-22 R1 = 2-(acetoxy)propyl
23-27 R1 = 2-(hydroxy)propyl
b
Scheme 2. Reagents and conditions: (a) R2NH2, NaBH3CN, MeOH/AcOH (15:1),
0–25 °C, 24 h; (b) Na°, MeOH, 25 °C, 20 min.
The binding to NRP-1 of the most affine compound 32 was deter-
minedin the range of concentrationsvarying from 10
lM to 500
lM.
arm like in the acyclic analogs 37 and 39 as deduced from the weaker
activity of compounds 35 and 36 in which the guanidyl group is
embedded in a cycle. Moreover, significant improvement of activity
is observed with guanidinyl residues as in compounds 30 and 32.
Theseresultsconfirmthataguanidylgroupisa primaryrequirement
for a significant activity and that surrogates of this group, often used
for example to mimic the RGD sequence,21 are not useful here. In this
series, the most potent compound is the fully deprotected glycoside
Indeed, the compound 32 showed affinity for NRP-1 (IC50 = 92
l
M,
Fig. 5). However, we previously evaluated VEGF plasma levels in
nude mice xenografted with U87 cells; plasma concentrations of
VEGF (165 and 121 isoforms) were around 25 pg/mL (0.5–0.7 pM)
lower than Kd values (Kd ꢀ10–700 pM), considering a molecular
weight of 35 and 45 kDa for VEGF121 and VEGF165 homodimers,
respectively.15c Hence, it is also interesting to note that the level of
VEGF165 in the blood compartment can not hinder the recognition
of the peptidomimetic 32 by NRP-1. A non-specific binding due to
the highly basic character of this compound, should be excluded tak-
ing into account the weaker affinity of bis-guanidine compound 33.
To sum up this study, it appears that a tryptophane mimic and a
genuine guanidine residue are required to obtain some activity, the
most potent compound 32 fulfill these requirements and exhibits
32 which exhibited 65% inhibition for NRP-1 at 100
which an IC50 = 92 M was determined (Fig. 2).
lM and for
l
Concerning the two other series, the compounds have been
tested with and without the hydroxyl protection, as a way to ex-
plore the significance of the OH group on the binding (Figs. 3 and
4). As depicted on Figs. 3 and 4, the peptidomimetics containing
a hydroxyl group are slightly more potent than the corresponding
acetylated compounds, suggesting that the hydroxyl group of the
threonine mimic does not play an essential role in the binding of
our compounds on NRP-1. In these series, the presence of a guani-
an IC50 of 92 lM. Mimics of threonine even with guanidine residue
led to less active compounds. The incorporation of a terminal argi-
nine residue in compounds 43 and 44 did not improve significantly
the inhibition in contrast with a recent report.27

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3289
CbzHN
NCbz
CbzHN
NCbz
SMe
H
N
O
O
CONHR1
CONHR1
N
R3HN
HN
CbzN
O
O
O
O
NHCbz
b
30 R1 = 2-(1H-indol-3-yl)ethyl
31 R1 = 2-(acetoxy)propyl
17 R1 = 2-(1H-indol-3-yl)ethyl, R3 = Cbz
22 R1 = 2-(acetoxy)propyl, R3 = Cbz
28 R1 = 2-(1H-indol-3-yl)ethyl, R3 = H
29 R1 = 2-(acetoxy)propyl, R3 =H
a
c
a
HN
NH2
O
CONHR1
N
HN
HN
O
O
NH2
32 R1 = 2-(1H-indol-3-yl)ethyl
33 R1 = 2-(hydroxy)propyl
Scheme 3. Reagents and conditions: (a) H2, Pd/C, MeOH, 25 °C, 24 h, [90% for 28, 98% for 29]; (b) HgCl2, DMF, NEt3, 25 °C, 14 h, [35% for 30, 25% for 31]; (c) H2, Pd/C, MeOH,
25 °C, 24 h, [90% for 32 and 33].
0
Scheme 4. Reagents and conditions: (a) (i) HO–(CH2)2–NH2, Et3N, Ti(OiPr)4, MeOH, MS 4 ÅA, 25 °C, 20 h; (ii) NaBH4, 0 °C, 2 h, 45%; (b) DEAD, PPh3, THF, 25 °C, 18 h, 35%; (c)
TFA, CH2Cl2, 0–25 °C, 72%.
H
N
O
O
CONHR1
NBoc
CONHR1
BocHN
HN
RHN
OHC
NH2
N
H
O
O
O
O
NR
a
37 R = Boc, R1 = 2-(1H-indol-3-yl)ethyl
38 R = Boc, R1 = 2-(acetoxy)propyl
39 R = H, R1 = 2-(1H-indol-3-yl)ethyl
40 R = H, R1 = 2-(acetoxy)propyl
9
R1 = 2-(1H-indol-3-yl)ethyl
10 R1 = 2-(acetoxy)propyl
b
b
Scheme 5. Reagents and conditions: (a) NaBH3CN, MeOH (15:1), AcOH, 25 °C, 24 h, [40% for 37 and 38]; (b) TFA, CH2Cl2, 0–25 °C [80% for 39; 38% for 40].
lead us to suggest that our scaffold does not efficiently replace
the LPP sequence present in the heptapeptide ATWLPPR and
must be tuned to arrange the crucial residues (guanidine and in-
dole) in the appropriate spatial directions. These results may also
suggest the need for another, yet to be discovered, interaction in
the receptor with a residue not present in our compounds. Fur-
ther work is in progress to improve the potency of this lead
structure.
4. Conclusion
In conclusion, in this pioneering study to design non-peptidic
ligands of NRP-1, new peptidomimetics based on known peptidic
ligands were prepared starting from a sugar bicyclic system as a
rigid scaffold. The synthesized compounds showed a modest
affinity for the recombinant chimeric NRP-1 protein. The most
potent compound 32 exhibiting an IC50 of 92 lM. These results

3290
A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
NH
O
O
CONHR1
CONHR1
HN
COOR
H2N
N
H
OHC
a
O
O
O
O
41 R = Me, R1 = 2-(1H-indol-3-yl)ethyl
42 R = Me, R1 = 2-(acetoxy)propyl
43 R = H, R1 = 2-(1H-indol-3-yl)ethyl
44 R = H, R1 = 2-(hydroxy)propyl
9
R1 = 2-(1H-indol-3-yl)ethyl
10 R1 = 2-(acetoxy)propyl
b
b
Scheme 6. Reagents and conditions: (a)
87% for 44].
L-ArgOMe.HCl, NaBH3CN, MeOH/AcOH (15:1), 25 °C, 24 h, [75% for 41; 68% for 42]; (b) LiOH, H2O/THF (1:3), 25 °C, 24 h, [68% for 43;
A
B
120
100
80
60
40
20
0
O
O
NH
R2HN
N
H
O
O
11-17, 28, 30, 32, 35,
36, 37, 39, 41, 43
Compounds
Figure 2. (A) Binding of biotinylated VEGF to NRP-1in the presence of VEGF165, ATWLPPR and new synthesized compounds at 100
experiments. (B) General structure of the [2-(1H-indol-3-yl)ethyl] series.
lM. Vertical bars represent S.D. for n = 3
A
B
120
100
80
60
40
20
0
O
O
R2HN
OAc
N
H
O
O
18-22, 29, 31, 38, 40, 42
Compounds
Figure 3. (A) Binding of biotinylated VEGF to NRP-1in the presence of VEGF165, ATWLPPR and new synthesized compounds at 100 lM. Vertical bars represent S.D. for n = 3
experiments. (B) General structure of the [2-(acetoxy)propyl] series.
A
B
120
100
80
60
40
20
0
O
O
R2HN
OH
N
H
O
O
23-27, 33, 44
Compounds
Figure 4. (A) Binding of biotinylated VEGF to NRP-1in the presence of VEGF165, ATWLPPR and new synthesized compounds at 100 lM. Vertical bars represent S.D. for n = 3
experiments. (B) General structure of the [2-(hydroxy)propyl] series.

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3291
100
90
80
70
60
50
40
30
20
10
0
to prevent non-specific interactions. Binding of the peptidomimet-
ic derivatives to NRP-1 was assessed using 5 ng/mL of biotinylated
VEGF165 (R&D Systems) in blocking buffer containing 2 lg/mL hep-
arin. Biotinylated VEGF165 was added to the coated wells, in com-
petition with an excess of derivatives, alone without any addition
(negative control) or unlabelled VEGF165 as positive control of
binding (0.5 lg/mL, R&D Systems). ATWLPPR (100 lM), 0.5%
Tween-20 was also used as positive control. After a 2 h-incubation
at room temperature, the plates were washed and the amount of
bound biotinylated VEGF165 stained with streptavidin horseradish
peroxidase conjugate (R&D Systems) and assayed. After 20 min at
room temperature, reaction was stopped by the addition of Stop
Solution (R&D Systems). Optical densities were measured at
450 nm. Results were expressed as relative absorbance to wells
containing only blocking buffer. Three wells per condition were
used for each compound. The assay was performed according to
Tirand et al. [14].
1
10
Concentration (µM)
100
5.3. Methyl 3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-
Figure 5. Binding of biotinylated VEGF (5 ng/mL; 110 pM) to recombinant NRP-1
protein in the presence of g/mL heparin was evaluated when increasing
D-gulo-L-glycero-octonic acid methyl ester (2)
2
l
concentrations of ATWLPPR (lozenge) and compound 32 (square). Data points
show the mean S.D.; n = 3.
To a solution of 1 (100 mg, 0.32 mmol) in ethyl acetate (10 mL)
was added Pd/C (10%) (10% w/w for 1E, 40% w/w for 1Z). The mix-
ture was then stirred under H2 atmosphere. After 24 h, the reaction
mixture was filtered through a pad of Celite and the solvent re-
moved in vacuo to give 2 as a white solid. The crude compound
was used without further purification. Yield 99% from 1E, 60% from
1Z as a white solid; Rf = 0.42 (hexane/EtOAc 1:1). Mp = 91–93 °C
5. Experimental part
5.1. General
Solvents and liquid reagents were purified and dried according
to recommended procedures. All commercially available chemicals
were obtained from Sigma–Aldrich. TLC analyses were performed
using standard procedures on Kieselgel 60 F254 plates or RP-18
F254 plates (Merck). Compounds were visualized using UV light
(254 nm) and (or) a solution of cerium sulfate tetrahydrate and
phosphomolybdic acid in 10% aqueous sulfuric acid as developing
agent. A specific revelator for guanidine function has been used
in several cases: successive spraying of 0.1% of 2-naphthol in 4%
NaOH aqueous and of 2% of Br2 in 4% NaOH aqueous. Column chro-
(recrystallized from hexane/CH2Cl2); ½a D20
ꢁ
= ꢂ11 (c 0.4, CHCl3); IR
(film)
m ;
2980, 2930, 2873, 1787, 1742 cmꢂ1 1H NMR (CDCl3,
250 MHz): d 1.29 (s, 3H), 1.39 (s, 3H), 1.45 (s, 6H), 2.86–2.91 (m,
2H), 3.52 (dd, 1H, J = 8.0 Hz, J = 4.0 Hz), 3.69 (s, 3H), 3.73 (m, 1H),
3.92 (ddd, 1H, J = 7.5 Hz, J = 6.0 Hz, J = 4 Hz), 4.21 (dd, 1H,
J = 8.0 Hz, J = 6.0 Hz), 4.38 (m, 1H), 4.62 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 4.80 (dd, 1H, J = 6.0 Hz, J3.4 = 4.0 Hz); 13C NMR (CDCl3,
62.9 MHz): d 24.7, 25.3, 25.7, 26.5, 33.3, 51.5, 65.2, 75.1, 77.3,
80.7, 80.8, 82.5, 109.1, 112.2, 168.7; HRMS (ESI) calcd for
C15H24NaO7: 339.1414, [M+Na]+; found 339.1409.
matography was performed on Silica Gel SI 60 (63–200
(Merck). Preparative reverse phase chromatography was per-
formed on LiChroprepÒRP-18 (40–63
m) (Merck). FTIR spectra
lm)
l
5.4. 3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-
glycero-octonic acid (3)
D-gulo-L-
were recorded on a Perkin–Elmer spectrum 1000 on NaCl windows
or KBr pellets. Melting points were determined with a Tottoli appa-
ratus and are uncorrected. Optical rotations were measured on a
Perkin–Elmer 141 polarimeter. 1H and 13C NMR spectra were re-
corded on a Bruker spectrometer AC250 (250 MHz and 62.9 MHz,
respectively) and DRX400 (400 MHz and 100.6 MHz, respectively).
For complete assignment of 1H and 13C signals, two-dimensional
1H, 1H COSY and 1H, 13C correlation spectra were recorded. Chem-
ical shifts (d) are given in ppm relative to the solvent residual peak.
The following abbreviations are used for multiplicity of NMR sig-
nals: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multi-
plet, br = broad signal. The J values given refer to apparent
multiplicities and do not represent the true coupling constants.
Mass spectra were obtained on a VG-Platform Micromass-Waters
(ESI+/quad) and a Shimadzu GCMS-QP2010 apparatus (CI). HRMS
spectra were recorded on a Bruker MicroTOFq apparatus.
To a stirred solution of 2 (200 mg, 0.63 mmol) in THF (3 mL) was
added 3 mL of an aqueous solution of KOH (70 mg, 1.26 mmol,
2 equiv) at room temperature. After 24 h HCl (1 N) was added until
pH 3. The solution was concentrated ad half in vacuo. The product
was extracted with CH2Cl2 (3 ꢃ 15 mL). The organic layer was dried
over MgSO4, filtered and concentrated under reduced pressure to
give 3 (190 mg, 98%) as as a colorless gum, which was used without
further purification. Rf = 0.46 (CH2Cl2/MeOH 9:1); ½a D20
= ꢂ13 (c 1.0,
ꢁ
CHCl3); IR (film)
m ;
3473, 3182, 2986, 2930, 2868, 1790, 1725 cmꢂ1
1H NMR (CDCl3, 250 MHz): d 1.29 (s, 3H), 1.39 (s, 3H), 1.45 (s, 6H),
2.86–3.02 (m, 2H), 3.54 (dd, 1H, J = 8.0 Hz, J = 4.0 Hz), 3.72 (m, 1H),
3.95 (ddd, 1H, J = 7.5 Hz, J = 6.0 Hz, J = 4.0 Hz), 4.21 (dd, 1H,
J = 8.0 Hz, J = 6.0 Hz), 4.35 (m, 1H), 4.64 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 4.79 (dd, 1H, J = 6.0 Hz, J = 4.0 Hz); 13C NMR (CDCl3,
62.9 MHz): d 25.0, 25.4, 25.9, 26.8, 33.2, 66.1, 75.5, 77.3, 81.1, 81.1,
83.6, 110.0, 113.1, 176.3; HRMS (ESI) calcd for C14H22NaO7:
325.1258, [M+Na]+; found 325.1245.
5.2. Binding of peptidomimetic derivatives to recombinant NRP-
1 protein
Neuropilin-1 was obtained from R&D Systems (Lille, France), as
recombinant chimeric protein. The surface of Maxisorp micro-
5.5. Representative procedure for amide synthesis
plates (Dutscher) was coated with NRP-1 (2
l
g/mL) in phosphate
To a stirred solution of 3 (570 mg, 1.9 mmol) in THF (2 mL) was
added under argon N,N-carbonyldiimidazole (306 mg, 1.9 mmol) at
room temperature. After 30 min, the appropriate amine was added
(1.9 mmol). After stirring for 12 h under an argon atmosphere, the
buffered saline (PBS), overnight at room temperature. The plates
were blocked with phosphate buffer saline (PBS) containing 0.5%
bovine serum albumin (BSA, blocking buffer) during 1 h at 37 °C,

3292
A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
solvent was removed under reduced pressure. The amide was puri-
fied by preparative high pressure column chromatography.
removed in vacuo. The product was extracted with CH2Cl2
(3 ꢃ 30 mL). The organic layer was dried over MgSO4, filtered and
the solvent was removed under reduced pressure to give the corre-
sponding diol used without further purification.
5.5.1. 3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene-N-[2-
(1H-indol-3-yl)ethyl]-D-gulo-L-glycero-octonoamide (4)
Yield 80% (670 mg) as a pale yellow solid; Rf = 0.57 (CH2Cl2/
5.7.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-N-[2-(1H-
MeOH 9:1); mp = 67–69 °C (recrystalized in hex/CH2Cl2);
indol-3-yl)ethyl]-D-gulo-L-glycero-octonoamide (7)
½
a 2D0
ꢁ
= ꢂ18 (c 1.0, CHCl3); IR (film)
m
3333, 2980, 2930, 2874, 1706,
Yield 80% as a beige solid; Rf = 0.42 (CH2Cl2/MeOH 9:1);
1650 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.24 (s, 3H), 1.40 (s, 3H),
;
mp = 103–104 °C (recrystalized in Hex/CH2Cl2); ½a D20
ꢁ
= ꢂ1 (c 1.0,
1.43 (s, 3H), 1.47 (s, 3H), 2.63 (d, 2H, J = 7.5 Hz), 2.99 (t, 2H,
J = 6.5 Hz), 3.48–3.56 (m, 2H), 3.63 (m, 1H), 3.73 (m, 1H), 3.95 (td,
1H, J = 7.5 Hz, J = 4.0 Hz), 4.21 (dd, 1H, J = 8.0 Hz, J = 6.0 Hz), 4.35
(m, 1H), 4.58 (dd, 1H, J = 6.0 Hz, J = 4.0 Hz), 4.63 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 5.93 (t, 1H, J = 5.0 Hz), 7.07–7.24 (m, 3H), 7.37 (d, 1H,
J = 7.0 Hz), 7.61 (d, 1H, J = 7.0 Hz), 8.38 (s, 1H); 13C NMR (CDCl3,
62.9 MHz): d 25.1, 25.3, 26.0, 26.2, 27.2, 36.4, 40.1, 66.5, 76.1, 79.0,
81.4, 81.7, 83.9, 110.2, 111.6, 113.0, 113.1, 119.0, 119.7, 122.4,
123.2, 127.8, 136.8, 170.6; MS (ESI) calcd for C24H32N2O6Na:
467.2168, [M+Na]+; found 467.4.
CHCl3); IR (film) m ;
3299, 2980, 2930, 2870, 1641 cmꢂ1 1H NMR
(acetone-d6, 400 MHz): d 1.27 (s, 3H), 1.39 (s, 3H), 2.57 (d, 2H,
J = 7.5 Hz), 2.96 (t, 2H, J = 6.5 Hz), 3.45–3.55 (m, 3H), 3.62 (m,
1H), 3.73 (m, 1H), 3.82 (td, 1H, J = 7.5 Hz, J = 4.0 Hz), 3.89 (m,
1H), 4.72 (dd, 1H, J = 6.0 Hz, J = 4.0 Hz), 4.76 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 7.02 (t, 1H, J = 7.5 Hz), 7.10 (t, 1H, J = 7.5 Hz), 7.20 (s,
1H), 7.40 (d, 1H, J = 7.5 Hz), 7.61 (d, 1H, J = 7.5 Hz), 10.02 (s, 1H);
13C NMR (acetone-d6, 100.6 MHz): d 24.5, 25.6, 25.8, 35.9, 40.0,
63.5, 71.5, 78.3, 81.4, 82.3, 82.4, 111.6, 111.7, 112.8, 118.8, 118.9,
121.5, 123.1, 128.1, 137.2, 170.1; MS calcd for C21H28N2NaO6:
427.18, [M+Na]+: found 427.27.
5.5.2. 3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene- N-[(2-
(R)-hydroxy)propyl]-
D
-gulo-
L
-glycero-octonoamide (5)
5.7.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-N-[(2-(R)-
Yield 75% (510 mg) as a colorless syrup; Rf = 0.55 (CH2Cl2/MeOH
acetoxy)propyl]-D-gulo-L-glycero-octonoamide (8)
9:1); ½a 2D0
ꢁ
= ꢂ9 (c 1.0, CHCl3); IR (film)
m
3428, 2981, 2927, 2873,
Yield 80% as a pale yellow amorphous solid; Rf = 0.42 (CH2Cl2/
1661 cmꢂ1
;
1H NMR (CDCl3, 400 MHz): d 1.17 (d, 3H, J = 6.0 Hz),
MeOH 9:1); ½a 2D0
ꢁ
= +10 (c 1.0, CHCl3); IR (film)
m 3369, 2986,
1.28 (s, 3H), 1.38 (s, 3H), 1.44 (s, 3H), 1.46 (s, 3H), 2.63 (dd, 1H,
J = 14.5 Hz, J = 7.0 Hz), 2.74 (dd, 1H, J = 14.5 Hz, J = 6.5 Hz), 3.02
(m, 1H), 3.47 (ddd, 1H, J = 14.0 Hz, J = 6.0 Hz, J = 3.0 Hz), 3.52 (dd,
1H, J = 8.0 Hz, J = 4.0 Hz), 3.72 (dd, 1H, J = 8.5 Hz, J = 7.0 Hz), 3.89
(ddd, 1H, J = 8.0 Hz, J = 6.0 Hz, J = 3.0 Hz), 3.96 (ddd, 1H, J = 7.0 Hz,
J = 6.5 Hz, J = 3.5 Hz), 4.21 (dd, 1H, J = 8.5 Hz, J = 7.0 Hz), 4.37 (dt,
1H, J = 8.0 Hz, J = 7.0 Hz, J = 7.0 Hz), 4.63 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 4.71 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.47 (t, 1H,
J = 6.0 Hz); 13C NMR (CDCl3, 62.9 MHz): d 20.9, 24.8, 25.5, 26.0,
26.9, 36.3, 47.3, 66.1, 67.2, 75.5, 78.6, 81.1, 81.6, 83.5, 110.0,
113.0, 171.5; HRMS (ESI) calcd for C17H29NO7Na: 382.1836,
[M+Na]+; found 382.1834.
2943, 2878, 1728, 1653 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.23
;
(d, 3H, J = 6.5 Hz), 1.29 (s, 3H), 1.45 (s, 3H), 2.06 (1s, 3H), 2.57
(dd, 1H, J = 15.0 Hz, J = 5.0 Hz), 2.66 (dd, 1H, J = 15.0 Hz,
J = 8.0 Hz), 3.32 (m, 1H), 3.43 (m, 1H), 3.58 (dd, 1H, J = 7.0 Hz,
J = 3.5 Hz), 3.70 (dd, 1H, J = 11.0 Hz, J = 5.0 Hz), 3.80 (dd, 1H,
0
J = 11.0 Hz, J7.8 = 4.0 Hz), 3.86 (m, 1H), 4.05 (m, 1H), 4.66 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz), 4.72 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 5.04 (m,
1H), 6.55 (br s, 1H); 13C NMR (CDCl3, 62.9 MHz): d 17.9, 21.4,
24.6, 25.9, 36.2, 44.4, 66.3, 70.1, 70.7, 77.9, 81.0, 81.8, 81.9,
112.6, 170.9, 171.7; HRMS (ESI) calcd for C16H27NO8Na:
384.1629, [M+Na]+: found 384.1635.
5.8. General procedure for aldehyde synthesis
5.6. 3,6-Anhydro-2-deoxy-4,5:7,8-di-O-isopropylidene- N-[(2-(R)-
acetoxy)propyl]-
D
-gulo-
L
-glycero-octonoamide (6)
To a stirred solution of diol 7 or 8 (1.51 mmol) in methanol
(32 mL), was added NaIO4 (646 mg, 3.02 mmol, 2 equiv) under ar-
gon. After stirring 14 h at room temperature, the solvent was re-
moved ad half in vacuo. The mixture was diluted with CH2Cl2
(80 mL). The organic layer was washed with water (3 ꢃ 10 mL),
dried over MgSO4, filtered and the solvent was removed under re-
duced pressure. Aldehydes 9 and 10 were used without further
purification.
To a stirred solution of 5 (206 mg, 0.57 mmol) in pyridine (6 mL)
was added under argon acetic anhydride (120 mg, 1.14 mmol,
2 equiv). After stirring 12 h at room temperature, the solvent was
co-evaporated with toluene and CH2Cl2 in vacuo. The crude product
was used without further purification. Yield 95% (230 mg) as color-
less liquid; Rf = 0.42 (CH2Cl2/MeOH 95:5); ½a D20
ꢁ
= +4 (c 1.0, CHCl3);
IR (film) m
3336, 2981, 2932, 2867, 1736, 1656 cmꢂ1; 1H NMR (CDCl3,
400 MHz): d 1.23 (d, 3H, J = 6.0 Hz), 1.28 (s, 3H), 1.38 (s, 3H), 1.43 (s,
3H), 1.45 (s, 3H), 2.06 (s, 3H), 2.66 (dd, 1H, J = 14.5 Hz, J = 7.0 Hz),
2.76 (dd, 1H, J = 14.5 Hz, J = 6.5 Hz), 3.37 (m, 1H), 3.45 (m, 1H),
3.53 (dd, 1H, J = 8.0 Hz, J = 4.0 Hz), 3.70 (dd, 1H, J = 8.0 Hz,
J = 7.5 Hz), 3.94 (ddd, 1H, J = 7.0 Hz, J = 6.5 Hz, J = 3.5 Hz), 4.21 (dd,
1H, J = 8.0 Hz, J = 6.5 Hz), 4.36 (m, 1H), 4.62 (dd, 1H, J = 6.0 Hz,
J = 4.0 Hz), 4.71 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 5.00 (m, 1H), 6.21 (t,
1H, J = 6.0 Hz); 13C NMR (CDCl3, 100.6 MHz): d 17.8, 21.7, 25.2,
25.8, 26.3, 27.1, 36.2, 44.1, 66.4, 70.2, 75.9, 78.8, 81.4, 81.7, 84.0,
110.2, 113.1, 170.7, 171.1; HRMS (ESI) calcd for C19H31NO8Na:
424.1942, [M+Na]+; found 424.1940.
5.9. General procedure for reductive amination
To a stirred solution of 9 or 10 (0.50 mmol, 1 equiv) in a mixture
of methanol (7 mL) and acetic acid (0.5 mL), was added at 0 °C un-
der argon the appropriate nucleophile (0.60 mmol, 1.2 equiv) and
NaBH3CN (1.1 equiv). After 24 h at room temperature, the solvent
was removed in vacuo and the mixture was diluted with CH2Cl2
(50 mL), washed with water (5 mL) and a satd aqueous solution
of NaHCO3 (2 ꢃ 5 mL) and water (5 mL). The organic layer was
dried over MgSO4, filtered and the solvent was removed under re-
duced pressure. The compound was purified by column
chromatography.
5.7. Representative procedure for diol synthesis
5.9.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(phenyl
To a stirred solution of 4 or 6 (1.0 mmol) in methanol (20 mL) at
0 °C, was added an aqueous solution of 1 N HCl (7 mL). After stir-
ring at room temperature (12 h for 5, 6 h for 6) was added a sat.
aqueous solution of NaHCO3 until pH 7. Half of the solvent was
methyl)amino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-heptono-
amide (11)
Yield 55% as a colorless gum; Rf = 0.37 (AcOEt/MeOH 9:1);
= +11 (c 1.0, CHCl3); IR (film) 3283, 3053, 2986, 2924,
½
a 2D0
ꢁ
m

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3293
2851, 1650 cmꢂ1; 1H NMR (CDCl3, 250 MHz): d 1.27 (s, 3H), 1.40 (s,
3H), 2.55 (d, 2H, J = 6.5 Hz), 2.88 (t, 2H, J = 6.0 Hz), 2.98 (t, 2H,
J = 6.5 Hz), 3.53–3.70 (m, 3H), 3.75 (m, 1H), 3.82 (s, 2H), 4.57 (dd,
1H, J = 6.0 Hz, J = 3.5 Hz), 4.63 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.11
(t, 1H, J = 5.0 Hz), 6.99 (d, 1H, J = 2.0 Hz), 7.15–7.25 (m, 2H), 7.28
(d, 1H, J = 7.0 Hz), 7.35–7.45 (m, 5H), 7.60 (d, 1H, J = 7.0 Hz), 8.04
(s, 1H); 13C NMR (CDCl3, 62.9 MHz): d 25.2, 25.5, 26.2, 36.7, 40.0,
48.0, 54.5, 78.4, 81.3, 81.4, 81.9, 111.6, 112.7, 113.3, 119.1, 119.3,
122.4, 122.5, 127.6, 127.8, 128.7 (2C), 128.9 (2C), 136.7, 140.4,
170.9; HRMS (ESI) calcd for C27H34N3O4: 464.2544, [M+H]+: found
464.2539.
1653 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.25 (s, 3H), 1.38 (s,
;
3H), 2.50–2.60 (m, 2H), 2.85–2.95 (m, 4H), 2.98 (t, 2H, J = 6.0 Hz),
3.41 (t, 2H, J = 5.5 Hz), 3.45–3.60 (m, 2H), 3.65–3.75 (m, 2H), 4.53
(dd, 1H, J4.5 = 6.0 Hz, J4.3 = 3.5 Hz), 4.58 (dd, 1H, J4.5 = 6.0 Hz,
J6.5 = 3.5 Hz), 5.20 (br s, 1H), 6.36 (t, 1H, J = 5.0 Hz), 6.43 (t, 1H,
J = 7.5 Hz), 6.58 (dd, 1H, J = 7.5 Hz, J = 4.0 Hz), 7.07 (s, 1H), 7.10 (t,
1H, J = 7.5 Hz), 7.17 (t, 1H, J = 7.5 Hz), 7.37 (d, 1H, J = 7.5 Hz), 7.42
(m, 1H), 7.60 (d, 1H, J = 7.5 Hz), 8.07 (d, 1H, J = 4.0 Hz), 9.58 (s,
1H); 13C NMR (CDCl3, 100.6 MHz): d 25.1, 25.4, 26.2, 36.7, 40.0,
41.6, 47.9, 49.2, 78.4, 80.9, 81.3, 81.9, 108.2, 111.8, 112.7, 113.1,
113.3, 119.1, 119.6, 122.3, 122.7, 127.9, 137.0, 138.1, 147.9,
159.0, 171.0; HRMS (ESI) calcd for C27H36N5O4: 494.2762,
+
**
5.9.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(pyridin-4-
[M+H] : found 494.2786.
ylmethyl)amino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-
heptonoamide (12)
5.9.6. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(pyrimi-
din-2-ylamino)ethylamino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-
heptonoamide (16)
Yield 55% as a colorless gum; Rf = 0.12 (AcOEt/MeOH 9:1);
½
a 2D0
ꢁ
= +13 (c 1.0, CHCl3); IR (film)
m
3283, 1650 cmꢂ1 1H NMR
;
(CDCl3, 400 MHz): d 1.27 (s, 3H), 1.41 (s, 3H), 2.58 (d, 2H,
J = 5.5 Hz), 2.86 (t, 2H, J = 6.0 Hz), 2.98 (t, 2H, J = 6.5 Hz), 3.60–3.70
(m, 3H), 3.80–3.95 (m, 3H), 4.60 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.64
(dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.10 (br s, 1H), 7.03 (s, 1H), 7.13 (t,
1H, J = 7.5 Hz), 7.20 (t, 1H, J = 7.5 Hz), 7.26 (d, 2H, J = 5.0 Hz), 7.35
(d, 1H, J = 7.5 Hz), 7.61 (d, 1H, J = 7.5 Hz), 8.54 (d, 2H, J = 5.0 Hz);
13C NMR (CDCl3, 100.6 MHz): d 25.1, 25.6, 26.2, 36.6, 40.2, 48.0,
53.1, 78.4, 81.1, 81.4, 81.8, 111.6, 112.7, 113.4, 119.1, 119.8, 122.5,
123.4 (2C), 127.8, 129.2, 136.8, 149.6, 150.1 (2C), 170.7; HRMS
(ESI) calcd for C26H33N4O4: 465.2596, [M+H]+: found 465.2532.
Yield 45% as a colorless gum; Rf = 0.08 (AcOEt/MeOH 9:1);
= +3 (c 1.0, CHCl3); IR (film) 3288, 3045, 2927, 2857,
1650 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.26 (s, 3H), 1.39 (s,
½
a 2D0
ꢁ
m
;
3H), 2.50–2.55 (m, 2H), 2.85–2.90 (m, 4H), 2.99 (t, 2H, J = 6.0 Hz),
3.50–3.65 (m, 4H), 3.59 (m, 1H), 3.68 (m, 1H), 4.56 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz), 4.60 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 5.66 (t,
1H, J = 5.0 Hz), 6.33 (t, 1H, J = 5.0 Hz), 6.55 (t, 1H, J = 5.0 Hz), 7.09
(s, 1H), 7.11 (t, 1H, J = 7.5 Hz), 7.19 (t, 1H, J = 7.5 Hz), 7.38 (d, 1H,
J = 7.5 Hz), 7.60 (d, 1H, J = 7.5 Hz) , 8.28 (d, 2H, J = 5.0 Hz), 9.18 (s,
1H); 13C NMR (CDCl3, 100.6 MHz): d 25.1, 25.4, 26.2, 36.8, 40.0,
41.1, 47.9, 49.2, 78.5, 81.1, 81.3, 81.9, 111.1, 111.7, 112.7, 113.3,
119.1, 119.7, 122.4, 122.6, 127.9, 136.9, 158.5 (2C), 162.7, 171.0;
HRMS (ESI) calcd for C26H35N6O4: 495.2720, [M+H]+: found
495.2722.
5.9.3. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(1H-
benzoimidazol-2-ylmethyl)amino]-N-[2-(1H-indol-3-yl)ethyl]-
L
-galacto-heptonoamide (13)
Yield 50% as colorless gum; Rf = 0.18 (AcOEt/MeOH 9:1);
½
a 2D0
ꢁ
= +12 (c 1.0, CHCl3); IR (film)
m 3255, 3059, 2980, 2924, 2851,
1647 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.28 (s, 3H), 1.35 (s, 3H),
;
5.9.7. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(N-benzy
2.54 (d, 2H, J = 5.5 Hz), 2.89 (t, 2H, J = 6.0 Hz), 2.98 (t, 2H,
J = 6.5 Hz), 3.43 (m, 1H), 3.52–3.70 (m, 2H), 3.75 (m, 1H), 4.07 (d,
1H, J = 15.5 Hz), 4.15 (d, 1H, J = 15.5 Hz), 4.52 (m, 1H), 4.57 (m, 1H),
6.28 (t, 1H, J = 5.0 Hz), 7.01 (s, 1H), 7.10 (t, 1H, J = 7.5 Hz), 7.16 (t,
1H, J = 7.5 Hz), 7.24 (m, 2H), 7.34 (d, 1H, J = 7.5 Hz), 7.59 (m, 3H),
9.47 (s, 1H); 13C NMR (CDCl3, 100.6 MHz): d 25.2, 25.4, 26.2, 36.7,
40.0, 47.7, 48.1, 78.4, 81.0, 81.4, 81.9, 111.8, 112.8, 112.9, 119.0,
119.6, 122.3 (2C), 122.8 (4C), 127.8, 136.9, 154.2 (3C), 171.1; HRMS
(ESI) calcd for C28H34N5O4: 504.2605, [M+H]+: found 504.2623.
loxycarbonylamino)ethylamino]-
indol-3-yl)ethyl]-amide (17)
L-galacto-heptono-N-[2-(1H-
Yield 50% as a colorless gum; Rf = 0.12 (AcOEt/MeOH 9:1);
= +7 (c 1.5, CHCl3); IR (film) ;
3315, 2927, 1704, 1650 cmꢂ1
½
a 2D0
ꢁ
m
1H NMR (CDCl3, 400 MHz): d 1.24 (s, 3H), 1.39 (s, 3H), 2.00 (br s,
1H), 2.56 (d, 2H, J = 6.5 Hz), 2.70–2.75 (m, 2H), 2.80 (d, 2H,
J = 6.0 Hz), 2.96 (t, 2H, J = 6.5 Hz), 3.20–3.25 (m, 2H), 3.44 (m,
1H), 3.50–3.65 (m, 2H), 3.76 (m, 1H), 4.55–4.60 (m, 2H), 5.11 (s,
2H), 5.40 (t, 1H, J = 5.0 Hz), 6.18 (t, 1H, J = 5.0 Hz), 7.02 (d, 1H,
J = 2.0 Hz), 7.10 (td, 1H, J = 7.5 Hz, J = 2.0 Hz), 7.18 (td, 1H,
J = 7.5 Hz, J = 2.0 Hz), 7.25–7.40 (m, 6H), 7.59 (d, 1H, J = 7.5 Hz),
8.82 (s, 1H); RMN 13C (CDCl3, 100.6 MHz): d 24.9, 25.3, 26.0,
36.4, 39.9, 40.6, 47.6, 49.1, 66.9, 78.1, 80.9, 81.1, 81.6, 111.5,
112.5, 113.0, 118.9, 119.5, 122.2, 122.4, 127.6, 128.3 (3C), 128.7
(2C), 136.6, 136.7, 156.9, 171.0; HRMS (ESI) calcd for
C30H39N4O6: 551.2864, [M+H]+: found 551.2859.
5.9.4. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(1H-
benzoimidazol-2-yl)ethylamino]-N-[2-(1H-indol-3-yl)ethyl]-
galacto-heptonoamide (14)
L-
Yield 55% as a colorless gum; Rf = 0.12 (AcOEt/MeOH 9:1);
D = +12 (c 1.0, CHCl3); IR (film) 3234, 3051, 2927, 2851,
1648 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.29 (s, 3H), 1.38 (s,
[
a]
m
;
3H), 2.55–2.55 (m, 2H), 2.95–3.00 (m, 4H), 3.05–3.15 (m, 4H),
3.50–3.55 (m, 2H), 3.66 (m, 1H), 3.82 (m, 1H), 4.58 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz), 4.64 (dd, 1H, J = 3.5 Hz, J = 6.0 Hz), 6.27 (t,
1H, J = 5.0 Hz), 7.02 (s, 1H), 7.11 (t, 1H, J = 7.5 Hz), 7.18 (t, 1H,
J = 7.5 Hz), 7.22 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz), 7.37 (d, 1H), 7.55–
7.60 (m, 3H), 9.07 (s, 1H); 13C NMR (CDCl3, 100.6 MHz): d 25.1,
25.5, 26.2, 28.4, 36.6, 40.1, 47.7 (2C), 78.4, 80.4, 81.4, 81.9, 111.7,
112.9, 113.1, 115.1 (2C), 119.1, 119.7, 122.4, 122.5, 122.6 (2C),
127.9, 136.9, 138.6 (2C), 154.4, 170.9; HRMS (ESI) calcd for
C29H36N5O4: 518.2767, [M+H]+: found 518.2758.
5.9.8. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(pyridin-4-
ylmethyl)amino]-N-[(2-(R)-acetoxy)propyl]-L-galacto-
heptonoamide (18)
Yield 25% as a colorless gum; Rf = 0.10 (AcOEt/MeOH 9:1);
½
a 2D0
ꢁ
= +13 (c 1.0, CHCl3); IR (film) ;
m
3294, 2932, 1734, 1656 cmꢂ1
1H NMR (CDCl3, 250 MHz): d 1.21 (d, 3H, J = 6.0 Hz), 1.30 (s, 3H),
1.43 (s, 3H), 2.01 (1s, 3H), 2.28 (br s, 1H), 2.61 (d, 2H, J = 5.0 Hz),
2.91 (d, 2H, J = 6.5 Hz), 3.35–3.50 (m, 2H), 3.68 (td, 1H, J = 6.5 Hz,
J = 3.0 Hz), 3.84 (d, 1H, J = 5.0 Hz), 3.90 (s, 2H), 4.63–4.70 (m, 2H),
4.97 (m, 1H), 6.31 (t, 1H, J = 6.0 Hz), 7.27 (d, 2H, J = 6 Hz), 8.52 (d,
2H, J = 6.0 Hz); 13C NMR (CDCl3, 62.9 MHz): d 17.8, 21.4, 25.0,
26.0, 36.4, 44.1, 47.9, 52.9, 70.1, 78.3, 81.0, 81.3, 81.6, 112.5,
123.2 (2C), 149.4, 149.9 (2C), 170.7, 170.9; HRMS (ESI) calcd for
C21H32N3O6: 422.2286, [M+H]+: found 422.2287.
5.9.5. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(pyridin-
2-ylamino)ethylamino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-
heptonoamide (15)
Yield 47% as a colorless gum; Rf = 0.08 (AcOEt/MeOH 9:1);
= +2 (c 1.0, CHCl3); IR (film) 3283, 3051, 2927, 2857, 1723,
½
a 2D0
ꢁ
m

3294
A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
5.9.9. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[1H-
benzoimidazol-2-ylmethyl)amino]-N-[(2-(R)-acetoxy)propyl-
galacto-heptonoamide (19)
69.9, 77.9, 80.8, 81.1, 81.4, 112.3, 127.7, 128.0 (2C), 128.5
(2C), 136.6, 156.5, 170.6, 170.8; HRMS (ESI) calcd for C25H38N3O8:
508.2653, [M+H]+: found 508.2641.
L
-
Yield 45% as a colorless gum; Rf = 0.12 (AcOEt/MeOH 9:1);
= +21 (c 1.0, CHCl3); IR (film) 3261, 3061, 2986, 2927,
1734, 1658 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.23 (d, 3H,
½
a 2D0
ꢁ
m
5.9.13. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(phenyl-
methyl)amino]-N-[(2-(R)-hydroxy)propyl]-L-galacto-heptono-
;
J = 6.0 Hz), 1.37 (s, 3H), 1.43 (s, 3H), 2.05 (1s, 3H), 2.61 (dd, 1H,
J = 15.0 Hz, J = 5.0 Hz), 2.66 (dd, 1H, J = 14.0 Hz, J = 7.0 Hz), 2.99
(dd, 1H, J = 12.5 Hz, J = 5.5 Hz), 3.08 (dd, 1H, J = 12.5 Hz,
J = 7.0 Hz), 3.37 (m, 1H), 3.46 (m, 1H), 3.67 (ddd, 1H, J = 7.0 Hz,
J = 5.5 Hz, J = 4.0 Hz), 3.93 (ddd, 1H, J = 7.0 Hz, J = 5.0 Hz,
J = 3.5 Hz), 4.14 (d, 1H, J = 16.0 Hz), 4.25 (d, 1H, J = 16.0 Hz), 4.69
(dd, 1H, J = 6.0 Hz, J = 4.0 Hz), 4.75 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz),
5.02 (m, 1H), 6.29 (t, 1H, J = 6.0 Hz), 7.24 (dd, 2H, J = 6.0 Hz,
J = 3.0 Hz), 7.58 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz); 13C NMR (CDCl3,
100.6 MHz): d 18.0, 21.6, 25.4, 26.3, 36.5, 44.4, 47.9, 48.2, 70.4,
78.4, 81.0, 81.6, 81.8, 112.9, 115.5 (2C), 122.6 (2C), 138.7, 154.4
(2C), 171.0, 171.4; HRMS (ESI) calcd for C23H33N4O6: 461.2395,
[M+H]+: found 461.2389.
amide (23)
Yield 40% (for two steps) as a colorless gum; Rf = 0.10 (AcOEt/
MeOH 9:1); ½a 2D0
ꢁ
= +5 (c 0.8, CHCl3); IR (film) m 3315, 2927, 2852,
1650 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.08 (d, 3H, J = 6.5 Hz),
;
1.25 (s, 3H), 1.40 (s, 3H), 2.52 (dd, 1H, J = 15.5 Hz, J = 6.0 Hz), 2.61
(dd, 1H, J = 15.5 Hz, J2 = 9.0 Hz), 2.87 (d, 2H, J = 6.0 Hz), 2.95 (m,
1H), 3.37 (ddd, 1H, J= 13.5 Hz, J = 6.5 Hz, J = 3.0 Hz), 3.62 (td, 1H,
J = 6.0 Hz, J = 3.0 Hz), 3.75 (s, 2H), 3.78–3.88 (m, 2H), 4.58–4.65
(m, 2H), 6.69 (t, 1H, J = 5.5 Hz), 7.25–7.30 (m, 5H); 13C NMR (CDCl3,
62.9 MHz): d 21.0, 24.9, 26.0, 36.4, 47.2, 47.8, 54.1, 66.7, 78.2, 80.7,
81.4, 81.6, 112.5, 127.3, 128.4 (2C), 128.6 (2C), 139.6, 171.7; HRMS
(ESI) calcd for C20H31N2O5: 379.2227, [M+H]+: found 379.2226.
5.10. General procedure for acetate deprotection
5.9.10. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(1H-
benzoimidazol-2-yl)ethylamino]-N-[(2-(R)-acetoxy)propyl]-
galacto-heptonoamide (20)
L
-
To a solution of amides 18–22 (0.15 mmol) in methanol (6 mL)
was added a catalytic amount of sodium. The mixture was stirred
for 20 min at room temperature. Amberlite IR-120 was added until
pH 4 and then filtered. The solvent was removed under reduced
pressure. When necessary, the compound was purified by column
chromatography.
Yield 50% as a colorless gum; Rf = 0.10 (AcOEt/MeOH 9:1);
= +15 (c 1.0, CHCl3). IR (film) 3256, 2986, 2943, 1734,
1650 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.20 (d, 3H, J = 6.0 Hz),
½
a 2D0
ꢁ
m
;
1.33 (s, 3H), 1.42 (s, 3H), 2.06 (s, 3H), 2.61 (d, 2H, J = 6.5 Hz), 2.66
(dd, 1H, J = 14.0 Hz, J = 7.0 Hz), 3.02 (d, 2H, J = 6.0 Hz), 3.05–3.15
(m, 4H), 3.35–3.45 (m, 2H), 3.67 (td, 1H, J = 6.0 Hz, J = 3.0 Hz),
3.93 (td, 1H, J = 6.5 Hz, J = 3.0 Hz), 4.65–4.60 (m, 2H), 4.95 (m,
1H), 6.37 (t, 1H, J = 6.0 Hz), 7.18 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz),
7.52 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz); 13C NMR (CDCl3, 62.9 MHz): d
17.8, 21.4, 25.0, 26.0, 28.6, 36.2, 44.1, 47.7 (2C), 70.1, 78.1, 80.6,
81.4, 81.6, 112.7, 114.9 (2C), 122.1 (2C), 138.5, 154.7 (2C), 170.7,
171.0; HRMS (ESI) calcd for C24H35N4O6: 475.2551, [M+H]+: found
475.2521.
5.10.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(pyridin-
4-ylmethyl)amino]-N-[(2-(R)-hydroxy)propyl]-L-galacto-
heptonoamide (24)
Yield 70% as a colorless gum; Rf = 0.24 (AcOEt/MeOH 2:1);
½
a 2D0
ꢁ
= ꢂ9 (c 1.0, CHCl3); IR (film)
m
3315, 1650 cmꢂ1 1H NMR
;
(CDCl3, 400 MHz): d 1.18 (d, 3H, J = 6.5 Hz), 1.32 (s, 3H), 1.46 (s,
3H), 2.56 (dd, 1H, J = 14.5 Hz, J = 5.0 Hz), 2.66 (dd, 1H, J = 14.5 Hz,
J = 8.5 Hz), 2.96 (d, 2H, J = 6.0 Hz), 3.05 (ddd, 1H, J = 13.5 Hz,
J = 8.0 Hz, J = 6.0 Hz), 3.43 (ddd, 1H, J = 13.5 Hz, J = 6.0 Hz,
J = 3.0 Hz), 3.76 (td, 1H, J = 6.5 Hz, J = 3.5 Hz), 3.88–3.96 (m, 4H),
4.64–4.69 (m, 2H), 6.72 (t, 1H, J = 6.0 Hz), 7.34 (d, 2H, J = 5.5 Hz),
8.57 (d, 2H, J = 5.5 Hz); 13C NMR (MeOH-d4, 62.9 MHz): d 21.0,
25.0, 26.3, 30.9, 36.5, 47.9, 52.7, 67.4, 79.6, 80.7, 82.7, 83.1,
113.6, 125.3 (2C), 149.4, 150.4 (2C), 173.6; HRMS (ESI) calcd for
C19H30N3O5: 380.2180, [M+H]+: found 380.2183.
5.9.11. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7[2-(pyridin-
2-ylamino)ethylamino]-N-[(2-(R)-acetoxy)propyl]-L-galacto-hep-
tonoamide (21)
Yield 15% as a colorless gum; Rf = 0.22 (AcOEt/MeOH 2:1);
= +15 (c 1.0, CHCl3); IR (film) 3321, 3067, 2927, 1731,
1658 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.22 (d, 3H, J = 6.0 Hz),
½
a 2D0
ꢁ
m
;
1.29 (s, 3H), 1.42 (s, 3H), 2.03 (s, 3H), 2.39 (br s, 1H), 2.57 (dd,
1H, J = 13.5 Hz, J = 4.5 Hz), 2.65 (dd, 1H, J = 13.5 Hz, J = 6.0 Hz),
2.94 (m, 4H), 3.30–3.45 (m, 4H), 3.66 (td, 1H, J = 6.0 Hz, J = 3 Hz),
3.93 (td, 1H, J = 7.0 Hz, J = 3.0 Hz), 4.60–4.70 (m, 2H), 4.98 (m,
1H), 5.08 (br s, 1H), 6.40 (d, 1H, J = 8.5 Hz), 6.45 (t, 1H, J = 6.0 Hz),
6.55 (dd, 1H, J = 6.5 Hz, J = 5.0 Hz), 7.39 (ddd, 1H, J = 8.5 Hz,
J = 6.5 Hz, J = 2.0 Hz), 7.58 (dd, 1H, J = 5.0 Hz, J = 2.0 Hz); 13C NMR
(CDCl3, 62.9 MHz): d 17.8, 21.4, 25.0, 26.0, 36.4, 41.6, 44.0, 47.8,
49.0, 70.1, 78.2, 81.0, 81.3, 81.6, 107.4, 112.5, 112.9, 137.5,
148.2, 159.0, 170.8, 171.0; HRMS (ESI) calcd for C22H35N4O6:
451.2551, [M+H]+: found 451.2525.
5.10.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[1H-benzo-
imidazol-2-ylmethyl)amino]-N-[(2-(R)-hydroxy)propyl]-L-galacto-
heptonoamide (25)
Yield : 85% as a colorless gum; Rf = 0.35 (AcOEt/MeOH 2:1);
½
a 2D0
ꢁ
= +7 (c 1.0, CHCl3); IR (film)
m
3288, 2981, 2932, 1648 cmꢂ1
;
1H NMR (CDCl3, 400 MHz): d 1.29 (d, 3H, J = 6.5 Hz), 1.30 (s, 3H),
1.39 (s, 3H), 2.51 (dd, 1H, J = 14.0 Hz, J = 3.5 Hz), 2.66 (dd, 1H,
J = 14.0 Hz, J = 10.0 Hz), 2.87 (m, 1H), 2.95–3.05 (m, 2H), 3.60–
3.71 (m, 2H), 3.88 (dt, 1H, J = 10.0 Hz, J = 3.0 Hz), 4.06 (m, 1H),
4.13 (s, 2H), 4.62 (dd, 1H, J = 6.0 Hz, J = 4.0 Hz), 4.68 (dd, 1H,
J = 6.0 Hz, J = 3.0 Hz), 6.74 (br s, 1H), 7.24 (dd, 2H, J = 6.0 Hz,
J = 3.0 Hz), 7.57 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz); 13C NMR (CDCl3,
100.6 MHz): d 21.0, 24.8, 26.0, 36.8, 47.4, 47.8, 48.6, 66.1, 78.6,
80.7, 81.5, 81.6, 112.8, 115.1 (2C), 122.6 (2C), 138.5, 153.1 (2C),
171.9; HRMS (ESI) calcd for C21H31N4O5: 419.2289, [M+H]+: found
419.2289.
5.9.12. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-(N-benzy-
loxycarbonylamino)ethylamino]-N-[(2-(R)-acetoxy)propyl]-
cto-heptonoamide (22)
Yield 75% as a colorless gum; Rf = 0.14 (AcOEt/MeOH 9:1);
= +10 (c 0.7, CHCl3); IR (film) 3315, 2976, 2933, 2868,
1715, 1656 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.21 (d, 3H,
L-gala-
½
a 2D0
ꢁ
m
;
J = 6 Hz), 1.29 (s, 3H), 1.44 (s, 3H), 2.04 (s, 3H), 2.56–2.65 (m,
2H), 2.75–2.95 (m, 4H), 3.23–3.45 (m, 4H), 3.62 (m, 1H), 3.86 (m,
1H), 4.61–4.70 (m, 2H), 4.96 (m, 1H), 5.10 (s, 2H), 5.38 (br s, 1H),
5.50 (br s, 1H), 6.27 (br s, 1H), 7.30–7.40 (m, 5H); 13C NMR (CDCl3,
62.9 MHz): d 17.5, 21.2, 24.7, 25.8, 36.1, 43.9, 47.5, 49.0, 50.5, 66.6,
5.10.3. 3,6-Anhydro-2-deoxy- -4,5-O-isopropylidene-7-[2-(1H-
benzoimidazol-2-yl)ethylamino]-N-[(2-(R)-hydroxy)propyl]-
L-
galacto-heptonoamide (26)
Yield 80% as a colorless gum; Rf = 0.20 (AcOEt/MeOH 3:2);
= 0° (c 1.0, CHCl3); IR (film)
3283, 2981, 2932, 1648 cmꢂ1
½
a 2D0
ꢁ
m
;

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3295
1H NMR (MeOH-d4, 250 MHz): d 1.15 (d, 3H, J = 6.5 Hz), 1.29 (s,
5.12. Generale procedure for guanidinylation
3H), 1.40 (s, 3H), 2.59 (dd, 1H, J = 14.0 Hz, J = 6.0 Hz), 2.67 (dd,
1H, J = 14.0 Hz, J = 7.0 Hz), 3.04 (d, 2H, J = 6.0 Hz), 3.10–3.25 (m,
6H), 3.74 (td, 1H, J6.7 = 6.0 Hz, J5.6 = 3.0 Hz), 3.78–3.87 (m, 1H),
3.88 (ddd, 1H, J = 7.0 Hz, J = 6.0 Hz, J = 3.0 Hz), 4.71 (dd, 1H,
J = 5.0 Hz, J = 3.0 Hz), 4.74 (dd, 1H, J = 5.0 Hz, J = 3.0 Hz), 7.22 (dd,
2H, J = 6.0 Hz, J = 3.0 Hz), 7.54 (dd, 2H, J = 6.0 Hz, J = 3.0 Hz); 13C
NMR (MeOH-d4, 62.9 MHz): d 21.0, 24.9, 26.2, 29.4, 36.6, 45.3,
47.8, 48.5, 67.4, 79.5, 81.1, 82.7, 83.0, 113.5, 115.6 (2C), 123.4
(2C), 139.6, 154.5 (2C), 173.6; HRMS (ESI) calcd for C22H33N4O5:
433.2445, [M+H]+: found 433.2445.
To a stirred solution of amine 28 or 29 (45 mg, 0.108 mmol) in
DMF (3 mL) was added N,N0-bis(dibenzyloxycarbonyl)-S-meth-
ylisothiourea 17 (77 mg, 0.216 mmol, 2 equiv), HgCl2 (29 mg,
0.108 mmol, 1 equiv) and Et3N (33 mg, 0.324 mmol, 3 equiv) under
argon at room temperature. After stirring for 14 h at room temper-
ature, the solvent was removed in vacuo and the residue was dis-
solved in EtOAc (40 mL). The organic layer was washed with water
(5 mL) and brine (5 mL) and then dried over MgSO4, filtered and
the solvent was removed under reduced pressure. The compounds
were purified by column chromatography.
5.10.4. 3,6-Anhydro-2-deoxy–4,5-O-isopropylidene-7[2-
(pyridin-2-ylamino)ethylamino]-N-[(2-(R)-hydroxy)propyl]-
galacto-heptonoamide (27)
5.12.1. 3,6-Anhydro-2-deoxy-N-[2-(1H-indol-3-yl)ethyl]-4,5-O-
isopropylidene-7-[[N-[bis[(phenylmethoxy) carbonyl]]amino-
iminomethyl]-N-[2-[bis[(phenylmethoxy)carbonyl]]guanidinoeth-
yl]amino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-heptonoamide
(30)
L-
Yield 82% as a colorless gum; Rf = 0.15 (AcOEt/MeOH 2:1);
½
a 2D0
ꢁ
= +10 (c 1.0, CHCl3); IR (film)
m
3310, 1650 cmꢂ1 1H NMR
;
(MeOH-d4, 400 MHz): d 1.16 (d, 3H, J = 6.5 Hz), 1.32 (s, 3H), 1.44
(s, 3H), 2.60–2.72 (m, 2H), 3.05 (dd, 2H, J = 11.0 Hz, J = 5.5 Hz),
3.13 (d, 2H, J = 6.0 Hz), 3.15 (dd, 1H, J = 13.5 Hz, J = 6.5 Hz), 3.21
(dd, 1H, J = 13.5 Hz, J = 5.0 Hz), 3.50 (t, 2H, J = 6.0 Hz), 3.78 (td,
1H, J = 6.0 Hz, J = 3.5 Hz), 3.84 (m, 1H), 3.99 (ddd, 1H, J = 7.5 Hz,
J = 6.0 Hz, J= 3.5 Hz), 4.74 (dd, 1H, J = 6.0 Hz, J= 3.5 Hz), 4.77 (dd,
1H, J = 6.0 Hz, J = 3.5 Hz), 6.60 (m, 2H), 7.47 (ddd, 1H, J = 8.5 Hz,
J = 7.0 Hz, J = 2.0 Hz), 7.98 (m, 1H); 13C NMR (MeOH-d4,
100.6 MHz): d 21.0, 24.9, 26.3, 36.5, 41.4, 47.8, 48.4, 50.4, 67.4,
79.6, 80.2, 82.6, 83.0, 110.5, 113.6, 113.7, 139.1, 147.9, 160.4,
173.5; HRMS (ESI) calcd for C20H33N4O5: 409.2445, [M+H]+: found
409.2444.
Yield 35% (40 mg) as
= +12 (c 1.0, CHCl3); IR (film)
1643 cmꢂ1 1H NMR (CDCl3, 250 MHz): d 1.24 (s, 3H), 1.27 (s,
a
colorless gum; Rf = 0.62 (AcOEt);
½
a 2D0
ꢁ
m
3349, 2924, 2853, 1734,
;
3H), 2.30–2.42 (m, 2H), 2.75–3.00 (m, 2H), 3.22–3.36 (m, 2H),
3.50–3.70 (m, 6H), 3.78 (m, 1H), 3.90 (m, 1H), 4.30–4.38 (m, 2H),
5.05 (s, 2H), 5.12 (s, 2H), 5.19 (s, 4H), 6.95 (d, 1H, J = 2.0 Hz),
7.04–7.18 (m, 2H), 7.26–7.38 (m, 20H), 7.42 (m, 1H), 7.55 (d, 1H,
J = 7.5 Hz), 8.45 (br s, 1H), 9.04 (br s, 1H), 9.50 (br s, 1H); 13C
NMR (CDCl3, 62.9 MHz): d 24.8, 26.0, 29.9, 36.4 (2C), 39.6 (3C),
67.4 (2C), 68.0, 68.5, 78.7, 79.2, 80.7, 82.0, 111.6, 112.2, 112.7,
118.7, 119.2, 121.9, 123.0, 127.7, 128.3 (4C), 128.6 (4C), 128.7
(4C), 128.9 (4C), 129.0 (4C), 134.8 (2C), 136.7, 136.8 (2C), 153.7
(2C), 156.5 (2C), 163.6 (2C), 170.1; HRMS (ESI) calcd for
C56H61N8O12: 1037.4392, [M+H]+: found 1037.4390.
5.11. General procedure for carbamate deprotection
5.12.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[[N-
[bis[(phenylmethoxy)carbonyl]]aminoiminomethyl]-N-[2-
[bis[(phenylmethoxy)carbonyl]]guanidinoethyl] amino]-N-[(2-
To a solution of 17 or 22 (80 mg, 0.145 mmol) in methanol
(6 mL) was added Pd/C (10%) (15% w/w). The mixture was stirred
under H2 atmosphere. After 24 h the reaction mixture was filtered
through a pad of Celite and the solvent removed in vacuo.
(R)-acetoxy)propyl]-
Yield 25% (30 mg) as
= ꢂ2 (c 0.7, CHCl3); IR (film)
1618 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 1.21 (d, 3H, J = 6.0 Hz),
L
-galacto-heptonoamide (31)
colorless gum; Rf = 0.51 (AcOEt);
3332, 2981, 2938, 1734, 1642,
a
½
a 2D0
ꢁ
m
5.11.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-
(amino)ethylamino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-
heptonoamide (28)
;
1.21 (s, 3H), 1.33 (s, 3H), 2.04 (1s, 3H), 2.50 (d, 2H, J = 6.0 Hz),
3.36 (m, 1H), 3.43 (m, 1H), 3.55–3.70 (m, 6H), 3.87 (m, 1H), 3.96
(m, 1H), 4.35–4.45 (m, 2H), 4.96 (m, 1H), 5.10 (s, 4H), 5.16 (s,
4H), 6.05 (br s, 1H), 7.35–7.40 (m, 20H), 8.49 (br s, 1H), 9.34 (br
s, 1H), 11.63 (br s, 1H); 13C NMR (CDCl3, 100.6 MHz): d 17.9,
21.6, 25.1, 26.1, 36.4, 39.1, 44.2, 48.0, 49.5, 67.6 (2C), 68.7 (2C),
70.2, 78.9, 79.2, 81.1, 81.8, 113.0, 128.5 (2C), 128.7 (4C), 128.9
(4C), 128.9 (4C), 129.1 (4C), 129.2 (2C), 135.0 (2C), 137.0 (2C),
153.9 (2C), 156.7 (2C), 163.9 (2C), 170.5, 171.0; HRMS (ESI) calcd
for C51H60N7O14: 994.4198, [M+H]+: found 994.4187.
Yield 90% as a colorless liquid; Rf = 0.08 (AcOEt/MeOH 2:1);
= +8 (c 1.0, CHCl3); IR (film) 3251, 2976, 2933, 2857,
1645 cmꢂ1 1H NMR (MeOH-d4, 250 MHz): d 1.30 (s, 3H), 1.44 (s,
½
a 2D0
ꢁ
m
;
3H), 2.59 (m, 2H), 2.85–3.00 (m, 8H), 3.51 (m, 2H), 3.61 (m, 1H),
3.86 (m, 1H), 4.62 (m, 1H), 4.69 (m, 1H), 7.00 (td, 1H, J = 7.5 Hz,
J = 1.0 Hz), 7.06 (td, 1H, J = 7.5 Hz, J = 1.0 Hz), 7.10 (s, 1H), 7.37
(br d, 1H, J = 7.5 Hz), 7.59 (br d, 1H, J = 7.5 Hz); 13C NMR (MeOH-
d4, 62.9 MHz): d 25.1, 26.3, 26.4, 36.7, 40.3, 41.6, 45.5, 47.2, 79.5,
81.7, 82.6, 83.0, 112.4 (2C), 113.4, 119.5, 119.7, 122.5, 123.7,
128.9, 138.2, 173.3; HRMS (ESI) calcd for C22H33N4O4: 417.2496,
[M+H]+: found 417.2523.
5.13. General procedure for carbamate deprotection
To a solution of 30 or 31 (30 mg, 0.04 mmol) in methanol (2 mL)
was added Pd/C (10%) (15% w/w). The mixture was stirred under
H2 atmosphere. After 24 h, the reaction mixture was filtered
through a pad of Celite and the solvent removed in vacuo. The mix-
ture was purified by column chromatography on C18 silica gel
(eluent H2O/CH3CN 7/3 with 0.1% of TFA) and the product was
lyophilized.
5.11.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[2-
(amino)ethylamino]-N-[(2-(R)-acetoxy)propyl]-L-galacto-
heptonoamide (29)
Yield 99% as a colorless gum; Rf = 0.08 (AcOEt/MeOH 2:1);
= ꢂ26 (c 1.0, CHCl3); IR (film) 3277, 2981, 2933, 2873,
1734, 1650 cmꢂ1 1H NMR (MeOH-d4, 250 MHz): d 1.20 (d, 3H,
½
a 2D0
ꢁ
m
;
J = 6.0 Hz), 1.31 (s, 3H), 1.44 (s, 3H), 2.03 (1s, 3H), 2.57–2.65 (m,
2H), 2.82–2.97 (m, 4H) , 3.23–3.45 (m, 4H), 3.66 (m, 1H), 3.91
(m, 1H), 4.67–4.77 (m, 2H), 4.96 (m, 1H); 13C NMR (MeOH-d4,
62.9 MHz): d 17.9, 21.3, 25.1, 26.4, 36.5, 41.4, 44.6, 49.0, 51.9,
71.0, 79.4, 81.6, 82.7, 83.1, 113.4, 172.5, 173.6; HRMS (ESI) calcd
for C17H32N3O6: 374.2286, [M+H]+: found 374.2285.
5.13.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[N-
(aminoiminomethyl)-N-(2-guanidinoethyl)amino]-N-[2-(1H-
indol-3-yl)ethyl]-L-galacto-heptonoamide (32)
Yield 90% (13 mg) as a white solid; Rf = 0.24 (H2O/CH3CN/TFA
6:4:0.1); ½a 2D0
ꢁ
= +9 (c 0.3, MeOH); IR (film) m 3363, 2927, 2851,

3296
A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
1672 cmꢂ1 1H NMR (MeOH-d4, 400 MHz): d 1.32 (s, 3H), 1.47 (s,
white solid; Rf = 0.34 (AcOEt); ½a D20
ꢁ
= +8 (c 0.4, CHCl3); IR (film)
m
;
3H), 2.52–2.66 (m, 2H), 2.96 (t, 2H, J = 7 Hz), 3.36–3.42 (m, 2H),
3.43–3.65 (m, 6H), 3.75 (m, 1H), 3.92 (ddd, 1H, J = 8.0 Hz,
J = 5.5 Hz, J = 3.5 Hz), 4.69 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.77 (dd,
1H, J = 6 Hz, J = 3.5 Hz), 7.02 (t, 1H, J = 7.5 Hz), 7.07–7.12 (m, 2H),
7.35 (d, 1H, J = 7.5 Hz), 7.58 (d, 1H, J = 7.5 Hz); 13C NMR (MeOH-
d4, 62.9 MHz): d 23.8, 23.9, 25.2, 35.5, 39.6, 40.5, 48.9, 49.1, 78.8,
79.4, 81.3, 81.9, 111.3, 112.2, 112.7, 118.3, 118.6, 121.3, 122.5,
127.8, 137.1, 158.3, 159.5, 172.0; HRMS (ESI) calcd for
C24H37N8O4: 501.2932, [M+H]+: found 501.2943.
3320, 2975, 2932, 2873, 1763, 1709, 1650 cmꢂ1 1H NMR (CDCl3,
;
400 MHz): d 1.24 (s, 3H), 1.38 (s, 3H), 1.50 (s, 18H), 2.50 (dd, 1H,
J = 13.5 Hz, J = 3.5 Hz), 2.58 (dd, 1H, J = 13.5 Hz, J = 5.5 Hz), 2.94 (t,
2H, J = 6.5 Hz), 3.20–3.53 (m, 6H), 3.60–3.80 (m, 4H), 4.50–4.60
(m, 2H), 6.24 (t, 1H, J = 5.5 Hz), 7.01 (d, 1H, J = 2.0 Hz), 7.05 (t,
1H, J = 7.5 Hz), 7.14 (t, 1H, J = 7.5 Hz), 7.35 (d, 1H, J = 7.5 Hz), 7.55
(d, 1H, J = 7.5 Hz), 9.12 (br s, 1H), 10.54 (br s, 1H); 13C NMR (CDCl3,
62.9 MHz): d 24.7, 25.2, 25.8, 28.10 (6C), 36.1, 40.1, 41.1, 42.3, 42.8,
78.0, 78.2 (2C), 79.3, 80.6, 81.4, 111.4, 112.6, 112.7, 118.6, 119.1,
121.8, 122.4, 127.4, 136.5, 144.7 (2C), 156.7 (2C), 170.2; HRMS
(ESI) calcd for C34H48N6NaO9: 707.3375, [M+Na]+: found 707.3411.
5.13.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[[N-(aminoi
minomethyl)-N-(2-guanidinoethyl)]amino]-N-[(2-(R)-hydroxy)
propyl]-
L
-galacto-heptonoamide (33)
5.16. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[3-[N-
(aminoiminomethyl)]-2-oxo-1-imidazolidinyl-N-[2-(1H-indol-
3-yl)ethyl]]-L-galacto-heptonoamide (36)
Yield 90% (11 mg) as white solid; Rf = 0.35 (H2O/CH3CN/TFA
6:4:0.1); ½a 2D0
ꢁ
= ꢂ5 (c 0.5, MeOH). IR (film)
m
3342, 2976, 2927,
1.17 (d, 3H,
1674 cmꢂ1
;
1H NMR (MeOH-d4, 400 MHz):
d
J = 6.0 Hz), 1.34 (s, 3H), 1.49 (s, 3H), 2.61–2.72 (m, 2H), 3.14 (dd,
1H, J = 13.5 Hz, J = 7.0 Hz), 3.25 (dd, 1H, J = 13.5 Hz, J = 5.0 Hz),
3.52 (t, 2H, J = 6.0 Hz), 3.60–3.71 (m, 4H), 3.79–3.87 (m, 2H), 4.00
(m, 1H), 4.76 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.82 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz); 13C NMR (MeOH-d4, 100.6 MHz): d 19.9,
23.7, 25.2, 35.3, 38.7, 46.7, 48.7, 49.6, 66.4, 78.9 (2C), 81.2, 81.9,
112.8, 157.8, 158.2, 172.4; HRMS (ESI) calcd for C17H34N7O5:
416.2621, [M+H]+: found 416.2622.
To a stirred solution of 35 (70 mg, 0.1 mmol) in CH2Cl2 (10 mL)
was added 1 mL of TFA at 0 °C. After stirring for 14 h at room tem-
perature, the solvent was removed in vacuo and coevaporated with
Et2O to remove residual TFA. The residue was chromatographed on
C18 silica gel (eluent H2O/CH3CN 7:3 with 0.1% of TFA) and the
product was lyophilized. Yield 72% (35 mg) as a white solid;
Rf = 0.26 (H2O/CH3CN/TFA 6:4:0.1); ½a D20
ꢁ
= +8 (c 0.6, MeOH); IR
(film)
m ;
3353, 2986, 2932, 1739, 1681 cmꢂ1 1H NMR (MeOH-d4,
250 MHz): d 1.27 (s, 3H), 1.43 (s, 3H), 2.51 (dd, 1H, J = 13.0 Hz,
J = 4.0 Hz), 2.59 (dd, 1H, J = 13.0 Hz, J = 6.0 Hz), 2.92 (t, 2H,
J = 7.0 Hz), 3.39–3.80 (m, 9H), 3.86 (ddd, 1H, J = 7.5 Hz, J = 5.5 Hz,
J = 3.5 Hz), 4.61 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.68 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz), 6.99 (ddd, 1H, J = 8 Hz, J = 7 Hz, J = 1 Hz),
7.06 (s, 1H), 7.07 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1 Hz), 7.32 (d,
1H, J = 7.0 Hz), 7.55 (d, 1H, J = 8.0 Hz); 13C NMR (MeOH-d4,
62.9 MHz): d 23.4, 24.8 (2C), 35.2, 40.0 (2C), 41.7, 42.7, 78.1,
78.6, 81.0, 81.5, 110.8, 111.9, 112.2, 117.9, 118.2, 120.9, 122.1,
127.4, 136.7, 154.9, 155.1, 171.7; HRMS (ESI) calcd for
C24H33N6O5: 485.2512, [M+H]+: found 485.2503.
5.14. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[(2-hydroxy)
ethylamino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-heptonoamide
(34)
To a stirred solution of 9 (400 mg, 1.075 mmol, 1 equiv) in
methanol (20 mL), was added under argon 3-aminopropanol
(197 mg, 3.225 mmol, 3 equiv), NEt3 (1.075 mmol, 1 equiv), Ti(i-
0
PrO)4 (catalytic amount) and 4 ÅA molecular sieves (80 mg). After
stirring at room temperature for 20 h, NaBH4 (1.1 equiv) was added
at 0 °C and stirred at room temperature for 2 h. The solvent was
then removed in vacuo and the mixture was diluted with CH2Cl2
(50 mL), washed with water (5 mL) and a sat. aqueous solution of
NaHCO3 (2 x 5 mL) and water (5 mL). The organic layer was dried
over MgSO4, filtered and the solvent was removed under reduced
pressure. The compound was purified by column chromatography.
Yield 45% (200 mg) as a colorless gum; Rf = 0.10 (AcOEt/MeOH
5.17. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[N-[2-[bis
[(tert-butyloxy)carbonyl]]guanidinoethyl]amino]-N-[2-(1H-
indol-3-yl)ethyl]-L-galacto-heptonoamide (37)
Prepared using the general procedure of reductive amination.
2:1); ½a 2D0
ꢁ
= +7 (c 0.5, CHCl3); IR (film)
;
m
3297, 2986, 2934, 2857,
Yield 40% as a white solid; Rf = 0.14 (AcOEt/MeOH 9:1); ½a D20
ꢁ
= +9
;
1652 cmꢂ1
1H NMR (CDCl3, 250 MHz): d 1.23 (s, 3H), 1.39 (s,
(c 0.4, CHCl3); IR (film)
m
3326, 2975, 2932, 1720, 1642 cmꢂ1
1H
3H), 2.52 (m, 2H), 2.70–2.98 (m, 8H), 3.42–3.68 (m, 3H), 3.73 (td,
1H, J = 6.5 Hz, J = 3.5 Hz), 4.49 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.54
(dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.25 (t, 1H, J = 5.5 Hz), 7.01 (s, 1H),
7.08 (ddd, 1H, J = 8.0 Hz, J = 1.0 Hz), 7.16 (ddd, 1H, J = 8.0 Hz,
J = 1.0 Hz), 7.36 (br d, 1H, J = 8.0 Hz), 7.56 (br d, 1H, J = 7.5 Hz),
9.10 (br s, 1H); 13C NMR (CDCl3, 62.9 MHz): d 24.7, 25.0, 25.8,
36.2, 39.7, 47.7, 51.3, 60.7, 77.9, 80.8, 81.0, 81.4, 111.3, 112.2,
112.6, 118.6, 119.2, 121.9, 122.3, 127.4, 136.5, 170.6; HRMS (ESI)
calcd for C22H31N3NaO5: 440.2156, [M+Na]+: found 440.2156 .
NMR (CDCl3, 400 MHz): d 1.24 (s, 3H), 1.37 (s, 3H), 1.45 (s, 9H),
1.50 (s, 9H), 2.48 (m, 1H), 2.60 (m, 1H), 2.60–2.80 (m, 4H), 2.97
(t, 2H, J = 6.5 Hz), 3.32–3.50 (m, 4H), 3.56–3.70 (m, 2H), 4.52 (dd,
1H, J = 6.0 Hz, J = 3.5 Hz), 4.57 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.32
(br t, 1H, J = 5.5 Hz), 7.03–7.16 (m, 3H), 7.36 (br d, 1H, J = 7.5 Hz),
7.58 (br d, 1H, J = 7.5 Hz), 8.61 (br t, 1H, J = 5.0 Hz), 9.20 (br s,
1H), 11.54 (br s, 1H); 13C NMR (CDCl3, 62.9 MHz): d 24.8, 25.2,
25.9, 28.2 (3C), 28.3 (3C), 36.4, 39.8, 40.5, 47.4, 48.3, 77.9, 79.5,
81.0, 81.2, 81.6, 83.3, 111.5, 112.3, 112.8, 118.7, 119.2, 121.9,
122.2, 127.6, 136.6, 153.3, 156.4, 163.6, 170.7; HRMS (ESI) calcd
for C33H51N6O8: 659.3763, [M+H]+: found 659.3724.
5.15. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[3-[N-[bis
[(tert-butyloxy)carbonyl]]aminoiminomethyl]-2-oxo-1-
imidazolidinyl]-N-[2-(1H-indol-3-yl)ethyl]-
heptonoamide (35)
L-galacto-
5.18. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[N-[2-[bis
[(tert-butyloxy)carbonyl]]guanidinoethyl]amino]-N-[(2-(R)-
acetoxy)propyl]-L-galacto-heptonoamide (38)
To a solution of C-glycoside 34 (160 mg, 0.384 mmol) N,N0,N00-
tri-(tert-butoxycarbonyl)guanidine (413 mg, 1.1 mmol) and PPh3
(151 mg, 0.58 mmol) in dry THF (10 mL) at 0 °C under argon was
Prepared using general procedure of reductive amination. Yield
40% as a white solid; Rf = 0.11 (AcOEt/MeOH 9:1); ½a D20
= ꢂ5 (c 0.3,
ꢁ
added dropwise via syringe diethyl azodicarboxylate (98
lL,
CHCl3); IR (film) m
3349, 2979, 2934, 1732, 1645, 1592 cmꢂ1 1H
;
0.58 mmol) over 5 min. The reaction was stirred at room tempera-
ture for 18 h. The solvent was evaporated in vacuo. The compound
was purified by column chromatography. Yield 35% (85 mg) as a
NMR (CDCl3, 250 MHz): d 1.24 (s, 3H), 1.30 (s, 3H), 1.46 (s, 9H),
1.49 (s, 9H), 2.05 (s, 3H), 2.52–2.66 (m, 2H), 2.96 (t, 2H,
J = 6.5 Hz), 3.30–3.70 (m, 6H), 3.80–4.00 (m, 2H), 4.60–4.75 (m,

A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
3297
2H), 4.98 (m, 1H), 6.29 (br t, 1H, J = 6.0 Hz), 7.91 (br s, 1H), 8.62 (br
s, 1H), 11.47 (br s, 1H); 13C NMR (CDCl3, 62.9 MHz): d 17.5, 21.2,
24.8, 25.8, 28.2 (3C), 28.4 (3C), 36.2, 40.3, 40.7, 47.3, 48.3, 69.9,
77.9, 78.1, 78.3, 81.0, 81.2, 81.4, 112.3, 153.0, 156.2, 163.5, 170.5,
171.1; MS (ESI) calcd for C28H49N5O10: 615.3479, [M+H]+: found
616.4.
1H, J = 8.0 Hz, J = 7.0 Hz, J = 1 Hz), 7.35 (d, 1H, J = 7.0 Hz), 7.59 (d,
1H, J = 8.0 Hz); 13C NMR (MeOH-d4, 62.9 MHz): d 23.3, 24.2, 24.7,
24.8, 26.2, 34.9, 40.1, 40.3, 45.5, 52.4, 59.3, 76.3, 78.6, 81.0, 81.6,
110.8, 111.8, 112.6, 117.8, 118.2, 120.9, 122.0, 127.4, 136.7,
157.3, 169.1, 171.7; HRMS (ESI) calcd for C27H41N6O6: 545.3082,
[M+H]+: found 545.3087.
5.19. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-[N-[2-
guanidinoethyl]amino]-N-[2-(1H-indol-3-yl)ethyl]-L-galacto-
heptonoamide (39)
5.22. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-(
L
-arginyl
methyl ester)-N-[(2-(R)-acetoxy)propyl]-L-galacto-heptonoamide
(42)
To a stirred solution of 37 (70 mg, 0.1 mmol) in CH2Cl2 (10 mL)
was added 1 mL of TFA at 0 °C. After stirring for 14 h at room tem-
perature, the solvent was removed in vacuo and coevaporated with
Et2O. The residue was chromatographed on C18 silica gel (eluant
H2O/CH3CN 7:3 with 0.1% of TFA) and the product was lyophilized.
Yield 80% (30 mg) as a white solid; Rf = 0.31 (H2O/CH3CN/TFA
Prepared using the general procedure of reductive amination
with L-arginine methyl ester. Yield 68% as a white solid; Rf = 0.37
(H2O/CH3CN/TFA 6:4:0.1); ½a D20
ꢁ
= +6 (c 0.2, MeOH); IR (film) m
3347, 3164, 2986, 2943, 1736, 1658 cmꢂ1 1H NMR (MeOH-
;
d4MeOH-d4, 250 MHz): d 1.21 (d, 3H, J = 6.5 Hz), 1.31 (s, 3H), 1.45
(s, 3H), 1.60–1.80 (m, 2H), 1.90–2.05 (m, 2H), 2.05 (s, 3H), 2.63
(d, 2H, J = 6.5 Hz), 3.24 (m, 4H), 3.35–3.45 (m, 2H), 3.84 (m, 1H),
3.87 (s, 3H), 3.99 (td, 1H, J = 6.5 Hz, J = 3.5 Hz), 4.13 (dd, 1H,
J = 7.0 Hz, J = 5.5 Hz), 4.75 (dd, 1H, J = 6 Hz, J = 3.5 Hz), 4.80 (m,
1H), 5.00 (m, 1H); 13C NMR (MeOH-d4, 62.9 MHz, 62.9 MHz): d
16.3, 19.8, 23.3, 24.2, 24.7, 26.4, 34.6, 40.3, 43.3, 45.6, 52.4, 59.4,
69.5, 76.3, 78.5, 81.0, 81.6, 112.6, 157.3, 169.2, 171.3, 172.0; HRMS
(ESI) calcd for C22H40N5O8: 502.2871, [M+H]+: found 502.2886.
6:4:0.1); ½a 2D0
ꢁ
= +10 (c 0.8, MeOH); IR (film)
m 3348, 2986, 2922,
2852, 1669 cmꢂ1
;
1H NMR (MeOH-d4, 250 MHz): d 1.29 (s, 3H),
1.44 (s, 3H), 2.60 (m, 2H), 2.95 (t, 2H, J = 7 Hz), 3.22–3.28 (m,
2H), 3.34–3.58 (m, 6H), 3.82–3.96 (m, 2H), 4.69 (dd, 1H,
J = 6.0 Hz, J = 3.5 Hz), 4.76 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 6.99
(ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.0 Hz), 7.05–7.12 (m, 2H), 7.33
(br d, 1H, J = 7.0 Hz), 7.56 (br d, 1H, J = 8.0 Hz); 13C NMR (MeOH-
d4, 62.9 MHz): d 23.2, 24.6, 24.7, 34.8, 37.4, 40.1, 46.2, 46.7, 76.1,
78.6, 80.8, 81.6, 110.8, 111.8, 112.6, 117.8, 118.2, 120.9, 122.0,
127.4, 136.7, 157.6, 171.6. HRMS (ESI) calcd for C23H35N6O4:
459.2714, [M+H]+: found 459.2762.
5.23. General procedure for saponification
To a stirred solution of 41 and 42 (0.11 mmol) in THF/H20
(6 mL/2 mL) was added LiOH (3 equiv for 41, 5 equiv for 42). After
stirring for 14 h at room temperature, a solution of HCl (3 N) was
added dropwise until pH 6–7. The solvent was removed in vacuo
and the residue was chromatographed on C18 silica gel (eluant
H2O/CH3CN 7:3 with 0.1% of TFA) and the product was freeze dried.
5.20. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7[N-[2-
guanidinoethyl]amino]-N-[(2-(R)-acetoxy)propyl]-L-galacto-
heptonoamide (40)
To a stirred solution of 38 (80 mg, 0.1 mmol) in CH2Cl2 (10 mL)
was added 1 mL of TFA at 0 °C. After stirring for 14 h at room tem-
perature, the solvent was removed in vacuo and coevaporated with
Et2O. The residue was chromatographed on C18 silica gel (eluant
H2O/CH3CN 7/3 with 0.1% of TFA) and the product was lyophilized.
Yield 38% (20 mg) as a white solid; Rf = 0.31 (H2O/CH3CN/TFA
5.23.1. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-(
N-[2-(1H-indol-3-yl)ethyl]- -galacto-heptonoamide (43)
Yield 68% as a white solid; Rf = 0.44 (H2O/CH3CN/TFA 6:4:0.1);
= +13 (c 1.0, MeOH); IR (film) 3353, 3185, 2986, 2932,
2852, 1696, 1664 cmꢂ1 1H NMR (MeOH-d4, 400 MHz): d 1.32 (s,
L-arginyl)-
L
½
a 2D0
ꢁ
m
6:4:0.1); ½a 2D0
ꢁ
= ꢂ3 (c 0.3, MeOH); IR (film)
m 3347, 3181, 2992,
;
2938, 1672 cmꢂ1
;
1H NMR (MeOH-d4, 250 MHz): d 1.20 (d, 3H,
3H), 1.47 (s, 3H), 1.68 (m, 1H), 1.82 (m, 1H), 1.92–2.09 (m, 2H),
2.62 (m, 2H), 2.97 (t, 2H, J = 7.5 Hz), 3.24 (t, 2H, J = 7.0 Hz), 3.36–
3.59 (m, 4H), 3.87 (dt, 1H, J = 8.0 Hz, J = 3.0 Hz), 3.96 (td, 1H,
J = 6.0 Hz, J = 3.5 Hz), 4.01 (dd, 1H, J = 7.0 Hz, J = 5.0 Hz), 4.73 (dd,
1H, J = 6.0 Hz, J = 3.5 Hz), 4.80 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 7.02
(t, 1H, J = 7.5 Hz), 7.09 (s, 1H), 7.10 (t, 1H, J = 7.5 Hz), 7.35 (d, 1H,
J = 7.5 Hz), 7.58 (d, 1H, J = 7.5 Hz); 13C NMR (MeOH-d4,
62.9 MHz): d 23.6, 24.7, 25.1, 25.2, 26.5, 35.2, 40.6, 40.7, 45.8,
60.1, 76.3, 79.0, 81.4, 82.1, 111.3, 112.2, 113.1, 118.2, 118.6,
121.3, 122.4, 127.8, 137.2, 157.7, 170.3, 172.1; HRMS (ESI) calcd
for C26H39N6O6: 531.2926, [M+H]+: found 531.2905.
J = 6.5 Hz), 1.31 (s, 3H), 1.45 (s, 3H), 2.04 (s, 3H), 2.63 (m, 2H),
3.16–3.26 (m, 2H), 3.32–3.39 (m, 2H), 3.41–3.49 (m, 2H), 3.60 (t,
2H, J = 6 Hz), 3.89 (dt, 1H, J = 9 Hz, J = 3 Hz), 4.00 (td, 1H,
J = 6.5 Hz, J = 3 Hz), 4.73–4.82 (m, 2H), 4.92–5.05 (m, 1H); 13C
NMR (MeOH-d4, 62.9 MHz): d 17.7, 21.2, 24.7, 26.1, 36.0, 38.9,
44.7, 47.6, 48.2, 70.9, 77.6, 80.0, 82.3, 83.0, 114.0, 159.0, 172.8,
173.4; HRMS (ESI) calcd for C18H34N5O6: 416.2504, [M+H]+: found
416.2470.
5.21. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-(
L-arginyl
methyl ester)-N-[2-(1H-indol-3-yl)ethyl]-
(41)
L-galacto-heptonoamide
5.23.2. 3,6-Anhydro-2-deoxy-4,5-O-isopropylidene-7-(
N-[(2-(R)-hydroxy)propyl]- -galacto-heptonoamide (44)
Yield 87% as a white solid; Rf = 0.60 (H2O/CH3CN/TFA 6:4:0.1);
= +5 (c 0.4, MeOH); IR (film) 3358, 3185, 2981, 2943, 2884,
1674 cmꢂ1 1H NMR (MeOH-d4, 400 MHz):
1.17 (d, 3H,
J = 6.5 Hz), 1.33 (s, 3H), 1.48 (s, 3H), 1.62 (m, 1H), 1.70–1.96 (m,
3H), 2.60–2.74 (m, 2H), 3.10–3.30 (m, 3H), 3.34–3.44 (m, 2H),
3.60 (m, 1H), 3.68 (m, 1H), 3.82–3.92 (m, 2H), 4.02 (m, 1H), 4.78
(dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 4.82 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz);
13C NMR (MeOH-d4, 62.9 MHz): d 19.6, 23.2, 24.5, 24.7, 26.6,
34.9, 40.4, 45.6, 46.4, 46.6, 65.9, 76.0, 78.5, 81.1, 81.7, 112.6,
157.3, 171.9, 172.1; HRMS (ESI) calcd for C19H36N5O7: 446.2609,
[M+H]+: found 446.2626.
L-arginyl)-
L
Prepared using the general procedure of reductive amination
½
a 2D0
ꢁ
m
with L-arginine methyl ester. Yield 75% as a white solid; Rf = 0.34
(H20/CH3CN/TFA 6:4:0.1); ½a D20
ꢁ
= +10 (c 0.9, CHCl3); IR (film)
m
;
d
3353, 3180, 2991, 2943, 2884, 1747, 1669 cmꢂ1
;
1H NMR
(MeOH-d4, 400 MHz): d 1.32 (s, 3H), 1.47 (s, 3H), 1.64 (m, 1H),
1.74 (m, 1H), 1.93–2.10 (m, 2H), 2.57–2.67 (m, 2H), 2.97 (t, 2H,
J = 7.0 Hz), 3.24 (t, 2H, J = 7.0 Hz), 3.36 (m, 1H), 3.43–3.58 (m,
3H), 3.86 (m, 1H), 3.87 (s, 3H), 3.96 (ddd, 1H, J = 7.5 Hz,
J = 6.0 Hz, J = 3.5 Hz), 4.19 (dd, 1H, J = 7.5 Hz, J = 5.0 Hz), 4.73 (dd,
1H, J = 6.0 Hz, J = 3.5 Hz), 4.79 (dd, 1H, J = 6.0 Hz, J = 3.5 Hz), 7.02
(ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1 Hz), 7.09 (s, 1H), 7.11 (ddd,

3298
A. Novoa et al. / Bioorg. Med. Chem. 18 (2010) 3285–3298
Bechet, D.; Becuwe, P.; Tirand, L.; Vanderesse, R.; Frochot, C.; Guillemin, F.;
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