Synthesis and Structure - Activity Relationship of Mannose-Based Peptidomimetics Selectively Blocking Integrin α4β7 Binding to Mucosal Addressin Cell Adhesion Molecule-1

Article abstract of DOI:10.1021/jm020487h

As part of our ongoing research in the development of α4β7 integrin antagonists, we are interested in peptidomimetics based on a rigid scaffold to allow the display of essential side chains in a suitable binding conformation while eliminating backbone ami

Full text of DOI:10.1021/jm020487h

5752
J . Med. Chem. 2003, 46, 5752-5762
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip of Ma n n ose-Ba sed
P ep tid om im etics Selectively Block in g In tegr in r4â7 Bin d in g to Mu cosa l
Ad d r essin Cell Ad h esion Molecu le-1
Elsa Locardi,† J u¨rgen Boer,† Armin Modlinger,† Anja Schuster,‡ Bernhard Holzmann,‡ and Horst Kessler*,†
Institut fu¨r Organische Chemie and Biochemie, Technische Universita¨t Mu¨nchen, Lichtenberg Strasse 4,
85747 Garching, Germany, and Chirurgische Klinik und Poliklinik, Technische Universita¨t Mu¨nchen,
Ismaninger Strasse 22, 81675 Mu¨nchen, Germany
Received October 29, 2002
As part of our ongoing research in the development of R4â7 integrin antagonists, we are
interested in peptidomimetics based on a rigid scaffold to allow the display of essential side
chains in a suitable binding conformation while eliminating backbone amide bonds and therefore
improving pharmacokinetic parameters of the drug. Except for a few examples, peptidomimetics
scaffolds have only been moderately successful and often yield molecules that lack the potency
of their peptide counterparts. However, we present herein a successful application of using a
rigid scaffold. Starting from a mannopyranoside analogue previously discovered in our
laboratory as an inhibitor of the R4â1/vascular cell adhesion molecule interaction, a biased
library of functionalized carbohydrates was developed. One compound emerged from this library
as an active and selective antagonist toward the R4â7/mucosal addressin cell adhesion molecule
interaction. Conformational implications and the relevance of different pharmacophoric patterns
will be discussed in order to explain the reverse selectivity and enhanced affinity.
In tr od u ction
cruitment of lymphocytes to inflamed colon and reduce
significantly the severity of colonic inflammatory dis-
ease.9 Moreover, antibodies against â7 integrins pro-
tected mice from the development of insulin-dependent
diabetes.10 Therefore, the R4â7/MAdCAM-1 adhesion
pathway represents a potent and organ-specific target
for therapeutic modulation of inflammatory diseases of
the gastrointestinal tract and autoimmune diabetes.
Our approach to developing a MAdCAM-1 proteino-
mimetic uses a carbohydrate moiety as rigid scaffold,
carrying the essential side chains in a suitable binding
conformation. There are only a few research groups who
succeeded in the transition from bioactive peptides to
small-molecule inhibitors using rigid scaffolds such as
cyclohexane.11-16 In a pioneering work, Hirschmann,
Nicolaou, and Smith developed somatostatin non-pep-
tide peptidomimetics utilizing â-D-glucose as novel
scaffolding.11 We present here another successful ap-
plication of this concept. On the basis of a D-mannose
core, we developed in a rational combinatorial approach
an active and selective inhibitor of the R4â7/MAdCAM
interaction.
A peptide-based structure-activity study was carried
out in our laboratory, leading to a number of potent and
selective R4â7 inhibitors.17 Cyclic hexapeptides were
designed and synthesized in an effort to mimic the
MAdCAM’s LDT binding motif by incorporation into a
rigid peptide core that adopts only specific conforma-
tions. These compounds were later used as templates
for the design of a non-peptidic R4â7 antagonist based
on â-D-mannose as the rigid scaffold, carrying the Leu,
Asp, and Thr mimetics at 6, 1, and 2 positions of the
pyranoside, respectively. The mannose core mimicked
the bioactive conformation of the constrained peptides,
displaying the aspartic acid at the i + 1 position of a
The infiltration of leukocytes to the site of inflamma-
tion contributes to the pathogenesis of a number of
human autoimmune diseases such as multiple sclerosis
and rheumatoid arthritis and results from a series of
adhesive and activating events involving multiple re-
ceptor/ligand interactions. The inflammatory processes
leading to tissue damage and disease are mediated in
part by the R4 integrins, R4â1 and R4â7, expressed on
the leukocyte cell surface. These glycoprotein receptors
modulate cell adhesion via interaction with their pri-
mary ligands, vascular cell adhesion molecule-1 (VCAM-
1) and mucosal addressin cell adhesion molecule-1
(MAdCAM-1), expressed in the affected tissue. Upon
binding, the combined integrin/CAM interacts at the cell
surface to result in firm adhesion of the leukocyte to
the vessel wall followed by extravasation into the
underlying tissue and subsequent activation and/or
proliferation of inflammatory cells.
In particular, the gut-specific trafficking of lympho-
cytes from the vascular to gastrointestinal mucosa and
lymphoid tissues is mediated by adhesive interactions
with MAdCAM-1 and the homing receptor R4â7.1-3
MAdCAM-1 specifically binds both human and mouse
lymphocytes that express the homing receptor R4â7 and
participates in the homing of these cells to the mucosal
endothelium.4,5 Organ-specific adhesion of normal lym-
phocytes and lymphoma cells to high endothelial venules
of Peyer’s patches is mediated by R4â7 integrins.6-8 In
mouse models it was demonstrated that antibodies
specific for â7 integrins and/or MAdCAM-1 block re-
* To whom correspondence should be addressed. Phone: ++49-89-
289-13301. Fax: +49 89-289-13210. E-mail: Horst.Kessler@ch.tum.de.
† Institut fu¨r Organische Chemie and Biochemie.
‡ Chirurgische Klinik und Poliklinik.
10.1021/jm020487h CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/22/2003

Mannose-Based Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5753
lar, the acid-catalyzed reaction of ethyl 1-thio-R-D-
mannopyranoside with cyclohexane-1,2-diones resulted
in selective protection of diequatorial, 3,4-diol function-
alities as a cyclohexane-1,2-diacetal (CDA).26
Rapid selective protection of the 2-position of CDA
mannoside 1, reported in Scheme 1, could be achieved
by benzylation with benzyl bromide using KOH in
DMSO, affording the 2-benzyl-protected sugar 2 in 75-
85% yield. The regioselectivity was unambiguously
assigned from the multiplicity of the hydroxyl proton
F igu r e 1. Comparison between a lead peptidic inhibitor of
the R4â7/MAdCAM interaction and the mannose-based pep-
tidomimetic.18
1
in H NMR and by the observation of a long-range 13C-
1H correlation between the benzylic protons and C2 of
the sugar ring in the heteronuclear multiple-bond
correlation (HMBC) spectrum. This reversal of selectiv-
ity (secondary versus primary hydroxyl group) was
already observed by Ley and co-workers who attributed
it to the higher basicity and reactivity of the oxyanion
at the 2-position.27 Direct protection of the 2-position
of PDA mannoside 13 did not work, but it was achieved
in two steps: regioselective protection of the primary
alcohol with tert-butyldiphenylsilyl chloride (TBDPSCl)
and subsequent benzylation of the secondary alcohol
(Scheme 2).
F igu r e 2. Development of a biased library of mannose
peptidomimetics.
To introduce the Ser and Leu side chains as well as
the benzyl and methyl groups, we used the Williamson
reaction, which is still the best general method for the
preparation of unsymmetrical ethers. We used solid
KOH in DMSO.28 In the case of secondary halides, an
excess of halide was needed because of elimination that
occurred as a side reaction. Many other functional
groups can be present in the molecule without interfer-
ence.
â-turn (Figure 1). Unfortunately, the designed man-
nopyranoside had no inhibitory activity against R4â7/
MAdCAM interaction, but one analogue where the Thr
side chain is replaced by the Ser side chain demon-
strated inhibition of VCAM-mediated binding of R4â1
J urkat cells (IC50 = 3.7 mM).18 Our interest in R4â7
integrin antagonists prompted us to investigate the
possibility of changing the selectivity and enhancing the
potency of this carbohydrate peptidomimetic, as well as
of shortening the synthetic strategy.
The Leu side chain was also introduced by Wittig
olefination of the sugar aldehyde with subsequent
double bond reduction at the 6-position. The primary
alcohol was oxidized to aldehyde according to known
procedures using DMSO as an oxidizing reagent and
sulfur trioxide pyridine complex as an alcohol activating
reagent. As reported in Scheme 3, the in situ prepared
ylide was then reacted with the crude aldehyde in cold
toluene. The resulting double bond was subsequently
reduced by p-toluenesulfonylhydrazide in dimethoxy-
ethane to give sugars 26 and 27. Another method we
used for selective reduction of a double bond in the
presence of a benzyl group was Pd/C catalyzed hydro-
genation with a nitrogen-containing base or amine as
poison.
Resu lts a n d Discu ssion
Design of a Bia sed Libr a r y of P ep tid om im etics.
In our strategy, we maintained the original D-mannose
core and the Leu and Asp pharmacophores at the 6 and
1 positions of the sugar ring, respectively (Figure 2). For
SAR studies, a spatial screening of the Asp pharma-
cophore was carried out by synthesizing and comparing
the biological effects of the â- and R-anomers. The Ser
side chain, which is known to be irrelevant for activity,
was replaced at position 2 by the Phe side chain. In fact,
a remarkable number of different research groups have
discovered phenylalanine-based R4 antagonists.19-24
The phenanthrene-9,10-diacetals (PDA) group was at-
tached to the D-mannose core as a hydrophobic source
at the 3 and 4 positions. The mannose scaffold was
rigidified by a trans ring fusion between a dioxane ring
and the pyranoside at the 3 and 4 positions. The Leu
mimetic was introduced either via ether linkage or via
a carbon-carbon bond-forming reaction to examine the
effects on activity of changes in polarity and in the
conformational space available for this pharmacophore.
Syn th etic Str a tegies. The synthetic protocol of the
desired functionalized mannopyranosides required an
orthogonal set of protecting groups. The orthogonal
protecting groups were successively cleaved, as speci-
fied, and replaced by pharmacophoric side chains.
We used the concept of 1,2-diacetals, developed by Ley
et al.,25 as a regioselective protecting group for the
diequatorial 1,2-diol units in carbohydrates. In particu-
The Asp side chain was introduced via O-glycosida-
tion. Attempts toward the synthesis of â-mannose
linkages have been well reviewed recently by Gridley
and Osborn.29 The methodology we used to overcome
the anomeric effect is a modified Koenigs-Knorr cou-
pling using insoluble silver salt promoters to block the
R-face of mannosyl halides and direct glycosidation to
give â-mannosides (Scheme 1).30 The â-anomeric con-
figuration was verified by determination of the inter-
proton distances between protons at positions 1, 3, and
5 derived from nuclear Overhauser effect spectrometry
(NOESY) spectra and by 13C NMR spectroscopy, based
on the general rules31,32 governing the stereochemical
1
dependence of the J (13C-1H) coupling constant at the
anomeric center in hexopyranoses, which for the â-ano-
mer was <160 Hz, while that for the R-anomer was
>170 Hz. For the glycosyl donor, we used predominantly

5754 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Locardi et al.
Sch em e 1a
a
i
i
Reagents: (a) BnBr, KOH in DMSO, 75-85%; (b) BuBr or PrI, KOH in DMSO, 90-95%; (c) 90% TFA in DCM, 80%; (d) MeI, KOH
in DMSO, 70%; (e) Br2 in DCM at 0 °C; (f) HOCH2CH2(OMe)2, Ag2CO3 in DCM at 0 °C, 75%, R/â 1:5; (g) HOCH2CH2(OMe)2, NIS, AgOTf
in DCM at 0 °C, 80%, R/â 6:1; (h) H2, Pd/C in MeOH, 98%; (i) BrCH2CH2OBn, KOH in DMSO, 95%; (l) HCl in H2O/THF; (m) NaClO2,
t
2-methyl-2-butene in BuOH; (n) H2, Pd/C in MeOH, 98%; (o) HOCH2COOMe, Ag2CO3 in DCM at 0 °C, 75%, R/â 1:5; (p) HOCH2COOMe,
NIS, AgOTf in DCM at 0 °C, 80%, R/â 6:1; (q) MeOH/NaOH 1 M.
Sch em e 2a
a
i
Reagents: (a) TBDPSCl, imidazole in DMF, 85%; (b) BnBr, KOH in DMSO, 95%; (c) TBAF in THF, 96%; (d) BuBr, KOH in DMSO,
95%; (e) Br2 in DCM at 0 °C; (f) HOCH2CH2OH, Ag2CO3 in DCM/THF at 0 °C, 90%, R/â 1:1; (g) TBDPSCl, imidazole in DMF, 85%; (h) H2,
Pd/C in MeOH, 98%; (i) BrCH2CH2OBn, KOH in DMSO, 95%; (l) TBAF in THF, 96%; (m) PySO3, TEA in DCM/DMSO 3:1; (n) NaClO2,
t
2-methyl-2-butene in BuOH; (o) H2, Pd/C in MeOH, 98%.
mannosyl bromide, which was prepared via direct
activation of the SEt group in ethyl 1-thio-R-D-manno-
sides with bromine in dichloromethane. In the case of
less reactive S-glycosides, as for 28 and 29 reported in
Scheme 3, we got the desired halide in two steps:
hydrolysis of the SEt group with NBS/H+ and subse-

Mannose-Based Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5755
Sch em e 3a
a
Reagents: (a) PySO3, TEA in DCM/DMSO 3:1; (b) toluene, ∆; (c) KN[Si(CH3)3]2 in toluene at 0 °C, 70% for 24, E/Z > 1:10; (d) TosNHNH2,
NaOAc in DME at 80 °C, 60-65%; (e) H2, Pd/C, TEA in MeOH, 80%; (f) 90% TFA in DCM, 80%; (g) MeI, KOH in DMSO, 90%; (h) NBS,
HCl in ACN/H2O, 90-95%; (i) SOCl2; (l) HOCH2COOMe, Ag2CO3 in DCM at 0 °C, 75%, R/â 1:5; (m) HOCH2COOMe, NIS, AgOTf in DCM
at 0 °C, 80%, R/â 6:1; (n) NaOH in MeOH/H2O, 99%.
quent reaction with thionyl chloride leading to the
mannosyl chloride almost quantitatively. The Asp side
chain (glycosyl acceptor) was masqueraded as a methyl
ester that was subsequently hydrolyzed to give the final
sugars 20-23 or acetal-protected aldehyde as for com-
pounds 7 and 8 (Scheme 1). The latter strategy was
chosen to avoid the problematic CR acidity of the
carboxyl acid derivatives that might result in undesired
byproducts during ether formation. After cleavage of the
acetal, the free aldehyde was immediately oxidized to
the corresponding carboxylic acid using sodium chlorite
and 2-methyl-2-butene as scavenger for the hypochlorite
that is produced during the oxidation.33 For the PDA-
protected sugar, the Asp side chain was introduced as
a primary alcohol via the Koenigs-Knorr reaction using
ethylene glycol as a glycosyl acceptor, leading to an R/â
ratio of 1:1 (Scheme 2). In fact, hydrolysis of the
dimethyl acetal, required by the previous protocol, in
this case caused byproducts because of a concurrent
aldol condensation. The alcohol functionality was finally
oxidized to carboxylic acid 19 in a two-step reaction.
commonly used. We chose the latter system to get the
R-mannose glycosides (Scheme 1).
Biologica l Activity. The inhibition of 38-â7 (R4â1-,
R4â7+) cell adhesion to MAdCAM-1 in the presence of
1 mg/mL of the sugar is reported in Table 1. In addition
to biological activity, selectivity is a major goal in drug
development. To verify selectivity (the ability of these
compounds to inhibit specific interactions of integrins
with their cognate ligands), binding of the structurally
related integrin R4â1 to VCAM-1 was investigated.
Adhesion mediated by R4â1 integrin was tested using
J urkat (R4â1+, R4â7-) lymphoma cells. To compare the
potencies of our antagonists with literature known
active compounds, the activity of a literature known
R4â7 antagonist (34)34 and of a cyclic peptide (35), which
was derived by Wang et al. from the crystal structure
of VCAM,35 was measured, and the IC50 curves were
reported in Figure 3.
The â-anomer of compound 20 showed inhibitory
activity toward the R4â7/MAdCAM interaction with an
IC50 of 420 µM (Figure 3C). This result is comparable
to the activity of the literature known tripeptide R4â7
antagonist (34), showing in our assay an IC50 of 280 µM
(Figure 3A). In a previous report,34 the IC50 of 34 was
found to be 3 µM. However, when the potencies of
different compounds are compared, it is important to
consider that the measured IC50 values are highly
dependent on reagent concentrations and assay condi-
tions. Therefore, only a comparison between the relative
potencies of antagonists measured in the same assay is
informative.
Furthermore, the tripeptide 34 did not show any
selectivity with respect to R4â1 binding to VCAM-1
(Table 1). Considering that the two receptors share a
common R4 chain, it is not surprising that many of the
potent R4â7/MAdCAM antagonists are inhibitors of
R4â1 as well. In contrast, the R4â1/VCAM-1 interaction
was not affected significantly by the â-anomer of 20,
Stereoselective R-O-glycosidation was carried out
using the thioglycoside methodology. This approach
employs a thioglycoside donor (e.g., having an alkylthio
aglycon), which is condensed directly with an alcohol
acceptor in the presence of a thiophilic agent, where the
latter is generated by a promoter system and transforms
the aglycon into a good leaving group. In this case, a
selectivity toward the more favorable R-anomer was
expected. In fact, R-mannosides are thermodynamically
and kinetically favored and are the major products when
the oxonium ion is an intermediate in the glycosidation
reaction. There are numerous promoter systems for
thioglycoside activation, of which sulfenyl halides/silver
trifluoromethanesulfonate, such as PhSCl/AgOTf and
MeSBr/AgOTf, or N-iodosuccinimide (NIS) combined
with either trifluoromethanesulfonic acid (TfOH) or
silver trifluoromethanesulfonate appear to be the most

5756 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Locardi et al.
Ta ble 1. Effect of Mannose-Based Peptidomimetics on 38-â7
Lymphoma and J urkat Cell Binding to Immobilized VCAM-1
and MAdCAM-1
F igu r e 3. IC50 curves measured for (A) 34 toward the R4â7/
MAdCAM interaction (IC50 ) 280 ( 51 µM),34 (B) 35 toward
the R4â1/VCAM interaction (IC50 ) 1443 ( 317 µM),35 and (C)
the â-anomer of 20 toward the R4â7/MAdCAM interaction (IC50
) 420 ( 84 µM). The standard deviations were determined
on the basis of three independent measurements for each point
in the curves.
instance, the R4â7 activity of the â-anomer of 20 is
i
almost completely lost as the Bu group is replaced by
i
the Pr group as for 21 (Table 1).
St r u ct u r a l St u d ies. Conformational analysis was
carried out for the â-anomer of 20 based on NMR data
and molecular modeling. The NOESY spectrum ac-
quired in CD3CN is reported in Figure 4. Long-range
connectivities involving aromatic protons of the Phe
pharmacophore and protons of the Leu side chain
indicated hydrophobic interactions between these resi-
dues. These proximities were also observed in a more
polar solution (D2O/CD3CN, 1:1). A simulating anneal-
ing protocol generated 50 structures that were classified
into five families based on the orientation of the Asp,
Phe, and Leu pharmacophores. Representative struc-
tures for each family were superimposed in Figure 5.
During the whole simulation, the sugar core remained
rigid. The five structures were classified based on the
NOE deviations, which of course have to be considered
as average values because of the mobility of the side
chains. However, the structure forming a hydrophobic
region between the aromatic ring of Phe and the Leu
side chain, which can be clearly seen in the side view of
Figure 5, is in agreement with the NOE data, and it is
by far the most populated conformation during simu-
lated annealing.
a
Cell adhesion is presented as percent of control medium in
the presence of 1 mg/mL antagonist. Each percent value represents
the average of at least four measurements ( the standard
deviation. Reference 34. c Reference 35. Reference 18.
b
d
demonstrating its high selectivity for integrin R4â7
(Table 1). The development of selective inhibitors of the
R4â7/MAdCAM interaction is important to in vivo
migration of leukocytes to the gastrointestinal tract,
where only MAdCAM expression has been linked to
human disease,36 without interfering with R4â1/VCAM-1
immune functions.
These present studies also reconfirmed the necessity
of â-orientation of the Asp side chain for activity, as
found for the â-anomer of 11 toward the R4â1/VCAM-1
interaction.18 In fact, the R-anomer of 20, as for the
R-anomer of 11, did not show any inhibitory activity.
Besides, the original chain length for the Leu pharma-
cophore present in 11 also turned out to be a winning
solution for blocking R4â7 binding to MAdCAM. For
Several possible mechanisms could explain the selec-
tivity of the â-anomer of 20 for R4â7. This sugar lacks

Mannose-Based Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5757
incorporation of the PDA group, as source of aromaticity,
at the 3 and 4 positions of 11-â causes a decrease of
R4â1 activity in 19-â as well. Besides, changes in
antagonist conformation, due to the constraints intro-
duced by the CDA group, can have a marked effect on
binding affinity. An additional possibility exists that the
activity and selectivity of 20-â can be dictated by the
hydrophobic region created by the Leu and Phe interac-
tions, as confirmed by NMR spectroscopy and molecular
modeling.
Con clu sion s
Interfering protein/protein interaction by small non-
peptidic molecules is one of the great challenges in
medicinal chemistry. On the basis of a D-mannose core,
we developed in a rational combinatorial approach a
selective inhibitor of the R4â7/MAdCAM interaction.
The biased library of mannose peptidomimetics origi-
nated from an inhibitor of the R4â1/VCAM interaction
previously developed in our laboratory. In our strategy,
we maintained the original mannose core and the Leu
and Asp pharmacophores at the 6 and 1 positions of the
sugar ring, respectively, occurring in the binding motif
of R4 natural ligands. A spatial screening for the Asp
pharmacophore was carried out by measuring the
activity for both R and â anomers. The Ser side chain
at position 2 was replaced by the Phe side chain.
Aromatic residues were introduced at the 3 and 4
positions to increase hydrophobicity of the mannose
peptidomimetics. The mannose scaffold was rigidified
by a trans ring fusion between a dioxane ring and the
pyranoside at the 3 and 4 positions. The length, polarity,
and conformational space available to the Leu side chain
were modified by carbon-carbon linkage.
F igu r e 4. Region of the NOESY spectrum acquired for the
â-anomer of 20 in CD3CN at 275 K. Long-chain connectivities
involving the aromatic protons of Phe are labeled.
a critical R4â1 binding interaction, like the Ser side
chain in 11. This could also explain the negative effect
on activity of the replacement of the ethylene glycol
moiety at the 2-position of the original R4â1 antagonist
11-â by the benzyl group as for the â-anomer of 22.
Otherwise, the relatively bulky CDA group sterically
hinders binding to R4â1 but not to R4â7. In fact, the
F igu r e 5. Stereoview of the superposition of representative structures of the â-anomer of 20 for each of the five cluster determined
by molecular modeling.

5758 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Locardi et al.
(IV) Gen er a l P r oced u r e for th e Syn th esis of Ma n n osyl
Br om id e. A solution of ethyl-1-thio-R-D-mannopyranoside
substrate (1 mmol) in 10 mL of dried dichloromethane was
treated with bromine (53 µL, 164 mg, 1 mmol) at 0 °C for 10-
15 min. The solution was successively washed with 10%
aqueous NaHSO3 and water, dried (Na2SO4), and concentrated.
The residue was used immediately as a glycosyl donor in a
Koenigs-Knorr type reaction.
(V) Gen er a l P r oced u r e for R-O-Glycosid ic Lin k a ge
F or m a tion . A solution of the ethyl-1-thio-R-D-mannopyrano-
side substrate (1 mmol) and alcohol (3 mmol), which was
previously rotoevaporated with toluene and dried under
vacuum, in dry dichloromethane (10 mL) containing freshly
activated 4 Å molecular sieves (2 g) was treated with N-
iodosuccinimide (1.2 mmol), followed by a solution of silver
trifluoromethanesulfonate (1.2 mmol) in 1 mL of dry toluene.
The reaction was essentially complete (as judged by TLC) after
the addition of the triflate. The mixture was then diluted with
CH2Cl2 and filtered. The filtrate was washed with 10% aqueous
sodium thiosulfate, saturated aqueous sodium bicarbonate,
and brine. The solution was dried (Na2SO4) and concentrated
to an oil that was flash-chromatographed on silica gel (gradient
elution: hexane/EtOAc 4:1 to hexane/EtOAc 1:1) to give the
title compounds in 75-85% yield as an R/â mixture 6:1.
(VI) Gen er a l P r oced u r e for â-O-Glycosid ic Lin k a ge
F or m a tion . Solid Ag2CO3 (6 mmol) was added at 0 °C to a
solution of mannosyl bromide (from step IV) or chloride (from
step III) (3 mmol), the specified glycosyl acceptor (10 mmol),
4 Å molecular sieves (2 g), and 10 mL of dichloromethane,
previously stirred at room temperature for 1 h. Stirring was
continued overnight in the dark. Successively, the mixture was
filtered through Celite and concentrated. The residue was
purified by flash column chromatography (gradient elution:
hexane/EtOAc 5:1 to hexane/EtOAc 2:1) to give the title
compounds in 70-75% yield as an R/â mixture 1:5.
(VII) Gen er a l P r oced u r e for P r im a r y Alcoh ol Oxid a -
tion to Ald eh yd e. A solution of 2,3,4-protected ethyl-1-thio-
R-D-mannopyranoside (0.6 mmol) and Et3N (380 µL, 2.72
mmol) in CH2Cl2/DMSO (3:1, 12 mL) was cooled to 0 °C and
treated with sulfur trioxide pyridine complex (344 mg, 2.16
mmol). After being stirred at 0 °C for 30 min, the mixture was
diluted with CH2Cl2 (100 mL), washed with H2O (2×) and
brine, dried (Na2SO4), and concentrated to give crude aldehyde,
which was used in the following reaction without purification.
(VIII) Gen er a l P r oced u r e for th e Syn th esis of th e
P h osp h on iu m Sa lt. A solution of triphenylphosphine (10
mmol) and the alkyl halide (10 mmol) in dry toluene (20 mL)
was heated under reflux for 24 h. After this time the mixture
was cooled with ice and filtrated. The white precipitate was
washed with cold toluene, dried under vacuum, and used
immediately.
(IX) Gen er a l P r oced u r e for Wittig Olefin a tion of
Su ga r Ald eh yd e. A mixture of the phosphonium salt (0.5
mmol) obtained from general procedure VIII, activated 4 Å
powdered molecular sieves (480 mg), and anhydrous toluene
(5 mL) was cooled to -10 °C. To this suspension was added
potassium bistrimethylsilylamide (1 mL of 0.5 M solution in
toluene, 0.5 mmol) as base, followed by the aldehyde (0.5
mmol), obtained in step VII, dissolved in anhydrous toluene
(2 mL). While being stirred, the suspension was allowed to
slowly warm to room temperature (about 3 h), maintained for
an additional 30 min at room temperature, and then filtered
through Celite. The solvent was evaporated, and the residue
was dissolved in CH2Cl2. The organic layer was washed with
water (10 mL), dried (Na2SO4), and concentrated. Flash
chromatography (12:1 hexane/EtOAc) of the residue furnished
the olefin in 65-70% yield.
SAR studies reconfirmed the â-orientation of the Asp
side chain to be necessary for activity. The basis for
affinity and selectivity toward the R4â7/MAdCAM in-
teraction is likely to be the association of the Leu and
Phe pharmacophores, which might be accommodated in
a hydrophobic pocket of the receptor, together with the
conformational restraints introduced by the cyclohex-
ane-1,2-diacetal group at the 3 and 4 positions. Besides,
the mannose-based antagonist mimics the active con-
formation of R4â7 selective cyclic peptides, previously
developed in our group. Furthermore, compound 20
exhibits a -1 < log P < 5 (log P ) 3.35), which is one
requirement for orally availability according to Lipinski
et al.37 In conclusion, this class of peptidomimetics
overcomes the limitations of peptidic drugs generally
associated with mediocre absorption and poor metabolic
stability, among other factors, and represents a promis-
ing candidate for drug development.
Exp er im en ta l Section
Gen er a l Meth od s. All chemicals were used as supplied
without further purification. All organic solvents were distilled
before use. Pd/C was donated by Degussa (Frankfurt/M.,
Germany). RP-HPLC analysis and semiscale preparations
were carried out on a Waters (high-pressure pump 510,
multiwavelength detector 490E, chromatography workstation
Maxima 820), Beckman (high-pressure pump 110B, gradient
mixer, controller 420, UV detector Uvicord from Knauer), and
Amersham Pharmacia Biotech (A¨ kta Basic 10/100, autosam-
pler A-900) instruments. RP-HPLC preparative separations
were carried out on Beckman System Gold (high-pressure
pump module 126, UV detector 166). C18 columns were used.
Solvents were the following: (A) H2O + 0.1% CF3COOH and
(B) CH3CN + 0.1% CF3COOH with UV detection at 220 and
254 nm. HPLC-ESI mass spectra were recorded on a Finnigan
NCQ-ESI with HPLC conjunction LCQ (HPLC system Hewlett-
Packard HP 1100, Nucleosil 100 5C18). Degrees of purity given
for final products refer to the latter HPLC system.
(I) Gen er a l P r oced u r e for Willia m son Eth er Syn th e-
sis. To DMSO (15 mL) was added powdered KOH (20 mmol
per replaceable hydrogen of substrate). After the mixture was
stirred for 10 min, the substrate alcohol (1 mmol) was added,
followed immediately by the alkyl halide (10 mmol per
replaceable hydrogen of substrate). Stirring was continued
until completion of the reaction, monitored by TLC, after which
the mixture was poured into water (20 mL) and extracted with
diethyl ether or dichloromethane (3 × 20 mL). The combined
organic extracts were washed with water (5 × 10 mL) and
dried with Na2SO4, and the solvents were removed under
reduced pressure. The residue was purified by flash column
chromatography (gradient elution: hexane/EtOAc 9:1 to hex-
ane/EtOAc 4:1) to give the title compounds in 70-95% yield.
(II) Gen er a l P r oced u r e for th e SEt Clea va ge of Eth yl-
1-t h io-r-D-m a n n op yr a n osid e. The ethyl-1-thio-R-D-man-
nopyranoside substrate (4 mmol) was dissolved in 60 mL of
CH3CN/H2O 2:1 solution. At room temperature, N-bromosuc-
cinimide (8 mmol) and 1 mL of concentrated HCl were added.
The solution was stirred for 3 h until completion of the
reaction, monitored by TLC, after which CH3CN was evapo-
rated and the residue was extracted with dichloromethane (3
× 20 mL). The combined organic extracts were dried with Na2-
SO4 and evaporated and the residue was purified by flash
column chromatography (gradient elution: hexane/EtOAc 5:1
to hexane/EtOAc 2:1) to give the title compounds in 90-95%
yield.
(III) Gen er a l P r oced u r e for th e Syn th esis of Ma n n osyl
Ch lor id e. The R,â-D-mannopyranoside mixture (4 mmol) from
general procedure II was dissolved at room temperature in
pure thionyl chloride (10 mL), and the mixture was stirred
for 2 h. After this time, the solution was evaporated and the
(X) Gen er al P r ocedu r e for CDA Depr otection . A sample
of 20 mmol of 3,4-CDA protected 1-thiomannopyranoside was
stirred at room temperature for 1 h in 90% TFA solution in
CH2Cl2. At completion of the reaction, the solvents were
evaporated and the residue was purified by flash chromatog-
raphy (2:1 hexane/EtOAc) to give a colorless oil in 75-85%
yield.
residue was used immediately as a glycosyl donor in
a
Koenigs-Knorr type reaction.

Mannose-Based Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5759
(XI) Gen er a l P r oced u r e for Alk en e R ed u ct ion w it h
p-Tolu en esu lfon ylh yd r a zid e. To a warmed (85 °C), stirred
solution of alkene substrate (0.5 mmol) and freshly recrystal-
lized p-toluenesulfonylhydrazide (285 mg, 1.53 mmol) in
dimethoxyethane (10 mL) was added 1 M aqueous sodium
acetate (1.53 mL) in six portions during 3 h. After an additional
3 h at 85 °C, the mixture was diluted with H2O (5 mL) and
extracted with CH2Cl2 (2 × 30 mL). The organic phase was
dried (Na2SO4) and concentrated. The residue was eluted from
a column of silica gel with hexane/AcOAc 12:1 to give the
reduced product in 60-65% yield.
(XII) Gen er a l P r oced u r e for Selective Hyd r ogen a tion
of Alk en e in th e P r esen ce of th e Ben zyl Gr ou p . A solution
of the alkene substrate (0.5 mmol), 30 mg of 10% Pd/C (50%
water), and ethylenediamine as poison (0.25 mmol) in MeOH
(10 mL) was stirred at room temperature under hydrogen
atmosphere (50 mbar) for 12 h until completion of the reaction,
monitored by TLC. The solution was then filtered through
Celite, the solvent was evaporated, and the residue was used
in the following reaction without purification (75-80% yield).
(XIII) Gen er a l P r oced u r e for Ben zyl Gr ou p Clea va ge.
A solution of the benzyl-protected alcohol (0.5 mmol) and 60
mg of 10% Pd/C (50% water) in MeOH (15 mL) was stirred at
room temperature under a hydrogen atmosphere (balloon) for
1 h until completion of the reaction, monitored by TLC. The
solution was then filtered through Celite, the solvent was
evaporated, and the residue was used in the following reaction
without purification (90-98% yield).
preferentially the R-anomer. The benzyl group was then
cleaved following general procedure XIII, and the residue was
coupled with bromoethyl-2-benzyl ether under Williamson
conditions as described in step I (95% yield). A solution of [2′,2′-
dimethoxyethyl]-2-O-[2′-benzyloxyethyl]-6-O-isobutyl-3,4-di-O-
methyl-D-mannopyranoside 9 (0.2 mmol, R and â anomers were
kept in different flasks) in a mixture of 10 mL of 2 N aqueous
HCl and 10 mL of THF was stirred at room temperature for 1
h until completion of the reaction (TLC). The solution was
neutralized with NaHCO3, the aldehyde was extracted with
ether, and the organic extract was concentrated. The residue
was then dissolved in 8 mL of tert-BuOH and 3 mL of 2-methyl-
2-butene. An equivalent of NaClO2 (18.0 mg, 0.2 mmol) and
0.3 g (2.52 mmol) of sodium dihydrogen phosphate were
dissolved in 3 mL of H2O, and the mixture was added dropwise
to the previous solution. At completion of the reaction (3 h),
the solution was treated with an aqueous Na2SO3 to destroy
the excess NaClO2. The solvent was evaporated, and the
aqueous solution was extracted with EtOAc. The organic
extract was dried (Na2SO4) and concentrated, and the residue
was used in the following reaction without purification. The
benzyl group was then cleaved as outlined in general procedure
XIII, and both R and â anomers were purified by HPLC (80%
yield).
[Car boxylm eth yl]-2-O-[2′-h ydr oxyeth yl]-6-O-isopr opyl-
3,4-d i-O-m eth yl-R,â-D-m a n n op yr a n osid e.12 Ethyl-2-O-ben-
zyl-6-O-isopropyl-3,4-di-O-methyl-1-thio-R-D-mannopyrano-
side 6 was reacted with Br2, as outlined in general procedure
IV, and the freshly prepared mannosyl bromide as glycosyl
donor was successively coupled to anhydrous glycol aldehyde
dimethyl acetal (step VI) to give preferentially the â-anomer,
or 6 was reacted directly with glycol aldehyde dimethyl acetal
as described in general procedure V to give preferentially the
R-anomer. The benzyl group was then cleaved following
general procedure XIII, and the residue was coupled with
bromoethyl-2-benzyl ether under Williamson conditions de-
scribed in step I (95% yield). A solution of [2′,2′-dimethoxy-
ethyl]-2-O-[2′-benzyloxyethyl]-6-O-isopropyl-3,4-di-O-methyl-
D-mannopyranoside 10 (0.2 mmol, R and â anomers were kept
in different flasks) in a mixture of 10 mL of 2 N aqueous HCl
and 10 mL of THF was stirred at room temperature for 1 h
until completion of the reaction (TLC). The solution was
neutralized with NaHCO3, the aldehyde was extracted with
ether, and the organic extract was concentrated. The residue
was then dissolved in 8 mL of tert-BuOH and 3 mL of 2-methyl-
2-butene. An equivalent of NaClO2 (18.0 mg, 0.2 mmol) and
0.3 g (2.52 mmol) of sodium dihydrogen phosphate were
dissolved in 3 mL of H2O, and the mixture was added dropwise
to the previous solution. At completion of the reaction (3 h),
the solution was treated with an aqueous Na2SO3 to destroy
the excess NaClO2. The solvent was evaporated, and the
aqueous solution was extracted with EtOAc. The organic
extract was dried (Na2SO4) and concentrated, and the residue
was used in the following reaction without purification. The
benzyl group was then cleaved as outlined in step XIII, and
both R and â anomers were purified by HPLC (80% yield).
Eh yl-2-O-ben zyl-3,4-O-[9′,10′-d im eth oxyp h en a n th r en e-
9′,10′-d iyl]-6-O-isob u t yl-1-t h io-r-D-m a n n op yr a n osid e.15
Ehyl-3,4-O-[9′,10′-dimethoxyphenanthrene-9′,10′-diyl]-1-thio-
R-D-mannopyranoside26 13 (13 mmol) and imidazole (39 mmol)
were dissolved in 50 mL of DMF, and the solution was stirred
at room temperature. Successively, tert-butyldiphenylsilyl
chloride (TBDPSCl, 17 mmol) was added dropwise. Stirring
was continued for 3 h until completion of the reaction, followed
by TLC. The solvent was evaporated under vacuum, and the
residue was purified by flash column chromatography (gradi-
ent elution: hexane/EtOAc 9:1 to hexane/EtOAc 4:1) to give
the title compound as a colorless oil (11.0 mmol, 85% yield).
The TBDPS-protected mannoside was then benzylated at
position 2 following the general procedure I using benzyl
bromide (95% yield). TBDPS removal was carried out over-
night under stirring in 20 mL of THF using 2 equiv of
tetrabutylammonium fluoride (TBAF) of a 1 M TBAF solution
in THF. The solvent was evaporated, and the residue was
Eth yl-2-O-ben zyl-3,4-O-[1′,2′-dim eth oxycycloh exan -1′,2′-
diyl]-1-th io-r-D-m an n opyr an oside.2 Ethyl-3,4-O-[1′,2′-dimeth-
oxycyclohexan-1′,2′-diyl]-1-thio-R-D-mannopyranoside26 1 was
benzylated at position 2 as outlined in general procedure I
using 1.2 equiv of benzyl bromide (75-85% yield).
Eth yl-2-O-ben zyl-3,4-O-[1′,2′-dim eth oxycycloh exan -1′,2′-
d iyl]-6-O-isobu tyl-1-th io-r-D-m a n n op yr a n osid e.3 Ethyl-2-
O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-1-thio-R-
D-mannopyranoside 2 was alkylated at position 6 as outlined
in general procedure I using isobutyl bromide (95% yield).
Eth yl-2-O-ben zyl-3,4-O-[1′,2′-dim eth oxycycloh exan -1′,2′-
d iyl]-6-O-isop r op yl-1-th io-r-D-m a n n op yr a n osid e.4 Ethyl-
2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-1-thio-
R-D-mannopyranoside 2 was alkylated at position 6 as outlined
in general procedure I using isopropyl iodide (90% yield).
Eth yl-2-O-ben zyl-6-O-isobu tyl-3,4-d i-O-m eth yl-1-th io-
r-D-m a n n op yr a n osid e.5 CDA deprotection of ethyl-2-O-ben-
zyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-6-O-isobutyl-1-
thio-R-D-mannopyranoside 3 was carried out as outlined in step
X. Subsequent methylation at the 3 and 4 positions following
general procedure I using methyl iodide afforded 5 in 70%
yield. Activation of the SEt group in the presence of MeI caused
the formation of methylmannopyranoside as a byproduct.
Before purification by flash column chromatography, the
excess of toxic alkyl halide was destroyed with a 15% solution
of ammonium hydroxide.
Eth yl-2-O-ben zyl-6-O-isop r op yl-3,4-d i-O-m eth yl-1-th io-
r-D-m a n n op yr a n osid e.6 CDA deprotection of ethyl-2-O-ben-
zyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-6-O-isopropyl-
1-thio-R-D-mannopyranoside 4 was carried out as outlined in
step X. Subsequent methylation at the 3 and 4 positions
following general procedure I using methyl iodide afforded 6
in 70% yield. Activation of the SEt group in the presence of
MeI caused the formation of methylmannopyranoside as a
byproduct. Before purification by flash column chromatogra-
phy, the excess of toxic alkyl halide was destroyed with a 15%
solution of ammonium hydroxide.
[Ca r boxylm eth yl]-2-O-[2′-h yd r oxyeth yl]-6-O-isobu tyl-
3,4-d i-O-m eth yl-R,â-D-m a n n op yr a n osid e.11 Ethyl-2-O-ben-
zyl-6-O-isobutyl-3,4-di-O-methyl-1-thio-R-D-mannopyrano-
side 5 was reacted with Br2, as outlined in general procedure
IV, and the freshly prepared mannosyl bromide as glycosyl
donor was successively coupled to anhydrous glycol aldehyde
dimethyl acetal (from step VI) to give preferentially the
â-anomer, or 5 was reacted directly with glycol aldehyde
dimethyl acetal as described in general procedure V to give

5760 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Locardi et al.
purified by flash column chromatography (gradient elution:
hexane/EtOAc 6:1 to hexane/EtOAc 4:1) (96% yield). The 6
position was then alkylated following the general procedure I
using isobutyl bromide (95% yield).
successively coupled to anhydrous glycolic acid methyl ester
(step VI) to give preferentially the â-anomer, or 4 was reacted
directly with glycolic acid methyl ester as described in general
procedure V to give preferentially the R-anomer. Hydrolysis
of the methyl ester in 10 mL of a 1:1 solution of 1 M NaOH
and MeOH for 1 h afforded quantitatively R- and â-21, which
were purified by HPLC.
[Ca r b oxylm et h yl]-2-O-b en zyl-6-O-isob u t yl-3,4-d i-O-
m et h yl-R,â-D-m a n n op yr a n osid e.22 Ethyl-2-O-benzyl-6-O-
isobutyl-3,4-di-O-methyl-1-thio-R-D-mannopyranoside 5 was
reacted with Br2, as outlined in general procedure IV, and the
freshly prepared mannosyl bromide as glycosyl donor was
successively coupled to anhydrous glycolic acid methyl ester
(step VI) to give preferentially the â-anomer, or 5 was reacted
directly with glycolic acid methyl ester as described in general
procedure V to give preferentially the R-anomer. Hydrolysis
of the methyl ester in 10 mL of a 1:1 solution of 1 M NaOH
and MeOH for 1 h afforded quantitatively R- and â-22, which
were purified by HPLC.
[Ca r b oxylm et h yl]-2-O-b en zyl-6-O-isop r op yl-3,4-d i-O-
m et h yl-r,â-D-m a n n op yr a n osid e.23 Ethyl-2-O-benzyl-6-O-
isopropyl-3,4-di-O-methyl-1-thio-R-D-mannopyranoside 6 was
reacted with Br2, as outlined in general procedure IV, and the
freshly prepared mannosyl bromide as glycosyl donor was
successively coupled to anhydrous glycolic acid methyl ester
(step VI) to give preferentially the â-anomer, or 6 was reacted
directly with glycolic acid methyl ester as described in general
procedure IV to give preferentially the R-anomer. Hydrolysis
of the methyl ester in 10 mL of a 1:1 solution of 1 M NaOH
and MeOH for 1 h afforded quantitatively R- and â-23, which
were purified by HPLC.
r-D-Ma n n o-n on -6-en op yr a n osid e, E t h yl-6,7,8,9-t et r a -
deoxy-2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-
8-m eth yl-1-th io.24 The free primary alcohol in ethyl-2-O-
benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-diyl]-1-thio-R-D-
mannopyranoside 2 was oxidized to aldehyde following the
general procedure VII. The aldehyde was then reacted with
isobutyltriphenylphosphonium chloride salt (step VIII) under
conditions described in step IX with good Z/E selectivity (>10:
1).
[2′-H yd r oxyet h yl]-2-O-b en zyl-3,4-O-[9′,10′-d im et h ox-
yp h en a n th r en e-9′,10′-d iyl]-6-O-isobu tyl-r,â-D-m a n n op y-
r a n osid e.16 Ehyl-2-O-benzyl-3,4-O-[9′,10′-dimethoxyphenan-
threne-9′,10′-diyl]-6-O-isobutyl-1-thio-R-D-mannopyranoside 15
was reacted with Br2 as outlined in general procedure IV.
Successively, the freshly prepared mannosyl bromide as gly-
cosyl donor was coupled to anhydrous ethylene glycol in 5: 1
DCM/THF solution (step VI). The addition of THF was needed
to dissolve the ethylene glycol and to create a homogeneous
system. Purification by flash column chromatography afforded
16 in 90% yield with a 1:1 R/â ratio. The two conformers were
collected together on purpose.
[Ca r b oxylm et h yl]-2-O-[2′-h yd r oxyet h yl]-3,4-O-[9′,10′-
d im et h oxyp h en a n t h r en e-9′,10′-d iyl]-6-O-isob u t yl-r,â-D-
m a n n op yr a n osid e.19 [2′-Hydroxyethyl]-2-O-benzyl-3,4-O-[9′,-
10′-dimethoxyphenanthrene-9′,10′-diyl]-6-O-isobutyl-R,â-D-
mannopyranoside 16 (6.5 mmol) and imidazole (19.5 mmol)
were dissolved in 25 mL of DMF, and the solution was stirred
at room temperature. Successively, tert-butyldiphenylsilyl
chloride (TBDPSCl, 8.5 mmol) was added dropwise. Stirring
was continued for 3 h until completion of the reaction, followed
by TLC. The solvent was evaporated under vacuum and the
residue was purified by flash column chromatography (gradi-
ent elution: hexane/EtOAc 9:1 to hexane/EtOAc 4:1) to give
the title compound as a colorless oil (5.5 mmol, 85% yield).
The benzyl group at position 2 was then cleaved as outlined
in general procedure XIII, and the residue was coupled with
bromoethyl-2-benzyl ether under Williamson conditions de-
scribed in step I (95% yield). TBDPS removal was carried out
overnight under stirring in 20 mL of THF using 2 equiv of
tetrabutylammonium fluoride (TBAF) of a 1 M TBAF solution
in THF. The solvent was evaporated, and the residue was
purified by flash column chromatography (gradient elution:
hexane/EtOAc 6:1 to hexane/EtOAc 4:1) (96% yield). The free
primary alcohol present in [2′-hydroxyethyl]-2-O-[2′-benzy-
loxyethyl]-6-O-isopropyl-3,4-di-O-methyl-D-mannopyrano-
side 18 (0.2 mmol, R and â anomers were kept in different
flasks) was then oxidized to aldehyde as described in general
procedure VII. The residue was then dissolved in 8 mL of tert-
BuOH and 3 mL of 2-methyl-2-butene. An equivalent of
NaClO2 (18.0 mg, 0.2 mmol) and 0.3 g (2.52 mmol) of sodium
dihydrogen phosphate were dissolved in 3 mL of H2O, and the
mixture was added dropwise to the previous solution. At
completion of the reaction (3 h), the solution was treated with
an aqueous Na2SO3 to destroy the excess NaClO2. The solvent
was evaporated, and the aqueous solution was extracted with
EtOAc. The organic extract was dried (Na2SO4) and concen-
trated, and the residue was used in the following reaction
without purification. The benzyl group was then cleaved
following the general procedure XIII, and both R and â
anomers were purified by HPLC (80% yield).
r-D-Ma n n o-oct-6-en op yr a n osid e, Eth yl-6,7,8-tr id eoxy-
2-O-ben zyl-3,4-O-[1′,2′-d im eth oxycycloh exa n -1′,2′-d iyl]-7-
m eth yl-1-th io.25 The free primary alcohol in ethyl-2-O-benzyl-
3,4-O-[1′,2′-dim et h oxycycloh exa n -1′,2′-diyl]-1-t h io-R-D-
mannopyranoside 2 was oxidized to aldehyde following the
general procedure VII. The aldehyde was then reacted with
isopropyltriphenylphosphonium iodide salt (step VIII) under
conditions described in step IX.
r,â-D-Man n o-n on -pyr an oside, [Car boxylm eth yl]-6,7,8,9-
t et r a d eoxy-2-O-b en zyl-3,4-d i-O-m et h yl-8-m et h yl.32 The
double bond present in R-D-manno-non-6-enopyranoside, ethyl-
6,7,8,9-tetradeoxy-2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-
1′,2′-diyl]-8-methyl-1-thio 24 was reduced with p-toluenesulfo-
nylhydrazide following general procedure XI or by hydrogen-
ation with poisoned Pd/C following general procedure XII. CDA
deprotection was carried out as outlined in X. Subsequent
methylation at the 3 and 4 positions as described in general
procedure I using methyl iodide afforded the desired R-D-
manno-non-pyranoside, ethyl-6,7,8,9-tetradeoxy-2-O-benzyl-
3,4-di-O-methyl-8-methyl-1-thio 28 in 90% yield. Before puri-
fication by flash column chromatography, the excess of toxic
alkyl halide was destroyed with a 15% solution of ammonium
hydroxide. To obtain preferentially the â-anomer, 28 was
reacted first with N-bromosuccinimide (step II) and second
with thionyl chloride (step III), and the freshly prepared
mannosyl chloride as glycosyl donor was successively coupled
to anhydrous glycolic acid methyl ester (step VI). To obtain
preferentially the R-anomer, 28 was reacted directly with
glycolic acid methyl ester following general procedure V.
Hydrolysis of the methyl ester in 10 mL of a 1:1 solution of 1
M NaOH and MeOH for 1 h afforded quantitatively R- and
â-32, which were purified by HPLC.
[Ca r boxylm eth yl]-2-O-ben zyl-3,4-O-[1′,2′-d im eth oxycy-
cloh e xa n -1′,2′-d iyl]-6-O-isob u t yl-r,â-D-m a n n op yr a n o-
sid e.20 Ethyl-2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-
1′,2′-diyl]-6-O-isobutyl-1-thio-R-D-mannopyranoside
3 was
reacted with Br2, as outlined in general procedure IV, and the
freshly prepared mannosyl bromide as glycosyl donor was
successively coupled to anhydrous glycolic acid methyl ester
(step VI) to give preferentially the â-anomer, or 3 was reacted
directly with glycolic acid methyl ester as described in general
procedure V to give preferentially the R-anomer. Hydrolysis
of the methyl ester in 10 mL of 1:1 solution of 1 M NaOH and
MeOH for 1 h afforded quantitatively R- and â-20, which were
purified by HPLC.
[Ca r boxylm eth yl]-2-O-ben zyl-3,4-O-[1′,2′-d im eth oxycy-
clo-h exa n -1′,2′-d iyl]-6-O-isop r op yl-r,â-D-m a n n op yr a n o-
sid e.21 Ethyl-2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-
1′,2′-diyl]-6-O-isopropyl-1-thio-R-D-mannopyranoside 4 was
reacted with Br2, as outlined in general procedure IV, and the
freshly prepared mannosyl bromide as glycosyl donor was
r,â-D-Ma n n o-oct-p yr a n osid e, [Ca r boxylm eth yl]-6,7,8-
tr ideoxy-2-O-ben zyl-3,4-di-O-m eth yl-7-m eth yl.33 The double

Mannose-Based Peptidomimetics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5761
bond present in R-D-manno-oct-6-enopyranoside, ethyl-6,7,8-
trideoxy-2-O-benzyl-3,4-O-[1′,2′-dimethoxycyclohexan-1′,2′-
diyl]-7-methyl-1-thio 25 was reduced with p-toluenesulfonyl-
hydrazide following the procedure described in general
procedure XI or by hydrogenation with poisoned Pd/C as
outlined in step XII. CDA deprotection was carried out
following general procedure X. Subsequent methylation at the
3 and 4 positions as described in general procedure I using
methyl iodide afforded the desired R-D-manno-oct-pyranoside,
ethyl-6,7,8-trideoxy-2-O-benzyl-3,4-di-O-methyl-7-methyl-1-
thio 29 in 90% yield. Before purification by flash column
chromatography, the excess of toxic alkyl halide was in this
case reacted with a 15% solution of ammonium hydroxide. To
obtain preferentially the â-anomer, 29 was reacted first with
N-bromosuccinimide (step II) and second with thionyl chloride
(step III) and the freshly prepared mannosyl chloride as
glycosyl donor was successively coupled to anhydrous glycolic
acid methyl ester (step VI). To obtain preferentially the
R-anomer, 29 was reacted directly with glycolic acid methyl
ester following general procedure V. Hydrolysis of the methyl
ester in 10 mL of a 1:1 solution of 1 M NaOH and MeOH for
1 h afforded quantitatively R- and â-33, which were purified
by HPLC.
Com p u ter Sim u la tion s. The structure calculations were
performed on Silicon Graphics computers. Energy minimiza-
tion (EM) and molecular dynamic (MD) calculations were
carried out with the program DISCOVER using the CVFF
force field.
50 After EM using steepest descent and the conjugate
gradient, the system was gradually heated from 300 to 800 K
and subsequently cooled to 300 K using 5 ps steps at every
temperature, each by direct scaling of velocities. Configura-
tions were saved every 25 ps for another 1250 ps. All the
structures coming from MD simulations were minimized using
again steepest descent and conjugate gradient algorithms.
During molecular modeling simulations, no restraints were
taken into account. The structures were evaluated at the end
on the basis of the NOE data.
Oth er P h ysica l Da ta . The log P values were calculated
on the basis of Crippen’s fragmentation.51
Ack n ow led gm en t. The authors gratefully acknowl-
edge Wilex AG and the Fonds der Chemischen Industrie
for financial support. We also thank Dr. Gerd Fischer
(Department of Chemistry, University of Munich) for
the HRMS data.
Biologica l Eva lu a tion . For MAdCAM-1 and VCAM su-
pernatants, 293T cells (embryonic fibroblasts from kidney) are
transfected with pCDNA3.1 IgG MAdCAM-1 and pCDNA3.1
IgG VCAM, respectively, and the supernatants are collected
after 2-3 days. For cell adhesion assay, 96-well plates were
coated with donkey antihuman IgG (J ackson ImmunoResearch
Laboratories Inc.) (0.5 µg in 100 µL of PBS/well) overnight at
4 °C. The plates were washed with adhesion buffer (Click’s
RPMI, 1% BSA, 1.0 mM Ca2+, 1.0 mM Mg2+) and incubated
for 30 min at room temperature with 25-150 µL/well MAd-
CAM or VCAM supernatant (depending on the concentration).
After being washed with adhesion buffer, the plates were
blocked with adhesion buffer for 1 h at 37 °C. The 38C13â7
lymphoma cells or J urkat cells were labeled for 30 min at 37
°C with 6 µg/mL H33342 dye (Calbiochem, La J olla, CA) in
cell adhesion buffer and washed with PBS containing 1 mM
EDTA and with PBS. The cells (8 × 105 cells/mL) were
resuspended in cell adhesion buffer. The compounds were
preincubated with the cells at the final concentration of 1 mg/
mL for 10 min at 37 °C. Subsequently, cells were allowed to
adhere for 30 min at 37°C and nonadherent cells were removed
by inverse centrifugation for 10 min at 50 g. Adhesion assays
were quantified by fluorimetry using a Cytofluor 2300 (Mil-
lipore, Bedford, MA).
Su p p or tin g In for m a tion Ava ila ble: ESI mass spectra
1
and H and 13C chemical shifts of compounds 2-6, 11, 12, 15,
16, 19-25, 32, and 33. This material is available free of charge
via the Internet at http://pubs.acs.org.
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4
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conformation. Homonuclear coupling constants were deter-
mined from one-dimensional spectra.

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