Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition

Article abstract of DOI:10.1016/j.bmcl.2010.01.141

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH₂-glycopeptides already reported to act as sLe^x mimics. The α and β anom

Full text of DOI:10.1016/j.bmcl.2010.01.141

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1957–1960
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and
P-selectin inhibition
Vanessa Gouge-Ibert a, Camille Pierry a, Florent Poulain a, Anne-Lise Serre a, Céline Largeau b,c,d,e
,
Virginie Escriou b,c,d,e, , Daniel Scherman b,c,d,e, Philippe Jubault a, Jean-Charles Quirion a, Eric Leclerc a,
*
*
a Université et INSA de Rouen, CNRS, UMR6014 & FR3038–IRCOF, 1 rue Tesnière, 76821 Mont Saint-Aignan Cedex, France
b Inserm, U640, Paris F-75006, France
c CNRS, UMR8151, Paris F-75006, France
d Université Paris Descartes, Faculté de Pharmacie, Chemical and Genetic Pharmacology Laboratory, Paris F-75270, France
e Ecole Nationale Supérieure de Chimie de Paris, Paris F-75005, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is
described. These molecules are difluorinated analogues of CH2-glycopeptides already reported to act as
Received 3 December 2009
Revised 21 January 2010
Accepted 23 January 2010
Available online 2 February 2010
sLex mimics. The
a
and b anomers of these CF2-glycopeptides have been prepared, as well as their
1-hydroxy analogues which were present in solution as an equilibrium mixture of - and b-pyranose
and - and b-furanose forms. These molecules showed inhibitory activities comparable to their CH2 coun-
terparts with a moderate influence of the pseudo-anomeric center configuration.
a
a
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Selectin inhibitors
sLex Mimics
C-Glycosides
Fluorine
Selectins are transmembrane glycoproteins which play an
important role in the recruitment of leukocytes during the early
stages of the inflammatory process.1,2 The release of signaling cyto-
kines by the damaged tissues stimulates the expression of these
calcium-dependent, cell-adhesion molecules on the surface of
endothelial cells. The weak, but effective affinity between selectins
and certain glycoproteins expressed by circulating leukocytes al-
lows them to slow down and roll along the endothelial wall. A
strong adhesion of the leukocytes on the vascular wall occurs after-
wards, thanks to the interaction between leukocytes integrins and
cell-adhesion molecules, allowing their transendothelial migra-
tion. The elucidation of this mechanism provided a new therapeu-
tic target since inappropriate or non-regulated recruitment of
leukocytes may cause severe damage to tissues and contribute to
inflammatory diseases (rheumatoid arthritis, asthma, lupus, etc.).
Furthermore, it has also been demonstrated that selectins played
a similar role in the metastasis of tumor cells.3
give rise to new inflammatory drugs.4,5 Due to the complex struc-
ture and tedious synthesis of sLex, the elaboration of smaller and
easily synthesized molecules, but still ensuring a binding to selec-
tins, has led to the discovery of many interesting inhibitors.6 For
example, Wong and Kaila rationally designed and prepared
a- or
b-C-mannosylpeptides as efficient inhibitors.7 The fucose unit of
sLex could indeed be replaced by a mannose moiety, thanks to a
close topology of the hydroxy groups interacting with selectins be-
tween these two sugars. The peptide moiety of these inhibitors fea-
tured the acidic function which is required for the binding to
selectins and which was provided by the sialic acid unit in sLex.2,7
Moreover, the enzymatic stability of these C-glycosidic structures
was a considerable advantage compared to the poor bioavailability
of O-glycosidic compounds.8
Our group has a long-standing interest in the synthesis of fluori-
nated C-glycosides, for which the replacement of the anomeric
oxygen by a CF2 group is expected to provide better sugar surrogates
than standard C-glycosides.9,10 Although in that case the methylene
group does not act as an oxygen mimic, we wished to prepare the
fluorinated analogues of Wong’s and Kaila’s inhibitors.11 It would
indeed allow us to assess the influence of a pseudo-anomeric CF2
group on the binding to selectins and therefore bring information
on the conformational and electronic changes that it may induce.
With the mannosyldifluoroacetates 4, 5 and 6 in hands, the
preparation of the corresponding mannopeptides 1, 2, and 3 was
The carbohydrate epitope of leukocyte glycoproteins involved
in the binding to selectins, known as PSGL-1 for P-selectin and
ESL-1 for E-selectin, is the tetrasaccharide sialyl Lewisx (sLex).2
The development of sLex derivatives, analogues or mimics as selec-
tin inhibitors has thus attracted considerable interest as it may
* Corresponding authors.
E-mail address: eric.leclerc@insa-rouen.fr (E. Leclerc).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.141

1958
V. Gouge-Ibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1957–1960
OH
OH
The 19F NMR spectra of these compounds indeed displayed four sets
OH
OH
OH
OH
of signals corresponding to four different compounds (two doublets
for each compound due to the diasterotopicity of the fluorine atoms).
The description of the 19FNMRspectrumof1ainD2Oanditsevolution
(Fig. 1) might bring some explanations (a similar trend was observed
for 1b). After dissolving the product obtained from hydrogenolysis in
D2O, afirst19FNMRspectrumwasreadilyrecordedwhich showedthe
presence of a major compound I, along with small but detectable
amounts of three other compounds II, III, and IV. After 30 min, the
compositionofthismixtureevolvedtoa44:41:10:5ratioforI:II:III:IV
with no further variation, even after a few days. The most reasonable
explanation for this was that an equilibration between the pyranose
F
F
F
F
O
O
O
HO
HO
HO
HO
HO
HO
O
R
O
R
O
OH
HN
F
HN
F
1
2
3
HN
R
OBn
OBn
O
OTBDPS
OBn
O
OBn
OBn
O
F
F
F
F
BnO
BnO
BnO
BnO
BnO
BnO
O
O
OH
O
OEt
F
OEt
and furanose forms and their a- or b-diastereomers occurred when
F
4
5
6
OEt
thedeprotected mannopeptides weredissolvedina proticpolar med-
ium. Unfortunately, the complexity of the 1H and 19F NMR spectra of
this mixture did not allow us to unambiguously determine the struc-
ture of each compound. However, one can reasonably postulate that
the two major compounds I and II would be the b-hexopyranose
and b-hexofuranose forms (i.e., the compounds for which the peptidic
chain is in b position) as they should be thermodynamically favored.
This structural flexibility could of course have an influence on
the binding to selectins. The preparation of mannopeptides 2 and
CO2H
CO2H
a : R =
b : R =
CO2H
Scheme 1. Mannosyl difluoroacetates as selectin inhibitors.
straightforward (Scheme 1).9b,d,e Type 1 compounds feature an addi-
tional hydroxy group at C-1 allowing them to exist as an equilibrium
mixture between what is supposed to be their
a
and b-pyranose and
3, which are locked in their a- or b-hexopyranose forms, might
a
and b-furanose forms. The synthesis of 1–3 and their evaluation as
then provide complementary information on the structural
E- and P-selectin inhibitors, described herein, would thus allow us to
estimate the importance of the C-1 configuration and the relevance
of using structurally flexible compounds.
requirements for such inhibitors. Our group recently reported a
synthesis of
a-mannosyldifluoroacetate 5, via an addition of a dif-
luoroketene acetal onto
D
-glucal 9, and its conversion to 2a and 2b
Mannoside 4 was obtained using a Reformatsky addition of
ethyl bromodifluoroacetate onto lactone 7.9e The reaction yielded
the most stable diastereomer, that is, the one placing the difluoro-
acetate group in equatorial position, with a 99:1 selectivity
through the same sequence of reactions which was used for the
synthesis of 1a and 1b (Scheme 3).9b A synthesis of b-mannosyldi-
fluoroacetate 6 was also described by our group, involving a Refor-
matsky addition onto the mannose-derived ketoaldehyde 10, and
allowed the preparation of 3a and 3b (Scheme 3).9d
(Scheme 2). Since lactone 7 is easily prepared from O-methyl-D-
mannose, this straightforward preparation of a mannosyldifluoro-
acetate appeared as a nice entry to modified glycopeptides similar
to the ones used by Wong and Kaila. The additional hydroxy group
at C-1, which could not be easily reduced, did not appear to us as a
drawback on first instance. The ester functionality was thus sapon-
ified and the acid was subjected to a peptidic coupling with diben-
zylated glutamate or ethyl 3-aminobenzoate using classical EDC/
HOBt conditions. Compounds 8a and 8b were obtained in moder-
ate yield and were subsequently deprotected using a simple
hydrogenolysis for 8a and a saponification/hydrogenolysis se-
quence for 8b (Scheme 2).12
Despite the hemi-ketal functionality, mannoside 4 and all its pro-
tected derivatives were perfectly stable and no equilibration towards
their C-1 epimers was ever observed even under basic conditions in
protic solvents. Debenzylated pseudo-glycopeptides 1a and 1b
behaved differently since the NMR spectra of these compounds in
D2O were much more complex than anticipated whereas mass spec-
trometry and elemental analysis confirmed the expected structure.
The targeted pseudo-mannopeptides were then tested in a
competition assay for human recombinant E- and P-selectins
coated to wells and using a cell-based assay (Table 1). The manno-
peptides were added together with HL-60 cells expressing sLex,
which therefore bind to the coated selectins as in the natural inter-
action between PSGL-1 and ESL-1 with P- and E-selectins.5c,13 The
ability of our compounds to displace the binding of HL-60 cells to
selectins was monitored so that a percentage of inhibition and IC50
could be assessed. Two sets of competition assays were performed.
Due to their straightforward synthesis, inhibitors 1a and 1b were
first prepared and tested. Their inhibitory activity prompted us to
synthesize mannopeptides 2 and 3 which were evaluated in a
second set of experiments. A control experiment was performed
each time by evaluating sLex as an inhibitor.
Compounds 1b, 2a, 2b, and 3b showed encouraging inhibitory
activities on E-selectins, with IC50 in the 2.5–5 mM range, but still
inferior to sLex (IC50 <1 mM, entry 1). The inhibitory activities of
thesecompoundsonP-selectinwereinthesamerangebut, thistime,
1- LiOH, 91%
OBn
OBn
OBn
OBn
OBn
OBn
2- HGlu(OBn)2
EDCI, HOBt, 53%
BrCF2CO2Et
Zn, THF, rfx
F
F
F
F
O
O
H
O
BnO
BnO
BnO
BnO
BnO
BnO
N
CO2Bn
CO2Et
70%
O
OH
OH
O
CO2Bn
8a
4
7
1- LiOH, 91%
2- Ethyl 3-aminobenzoate
EDCI, HOBt, 45%
H2, Pd/C, quant.
1a
OBn
OBn
O
F
F
1- LiOH, 38%
2- H2, Pd/C, quant.
H
N
BnO
BnO
CO2Et
1b
OH
O
8b
Scheme 2. Synthesis of 1-hydroxymannosides 1a and 1b.

V. Gouge-Ibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1957–1960
1959
Figure 1. 19F NMR spectrum of compound 1a.
OTBDPS
OBn
O
OAc
O
F
F
OZnEt
OEt
OBn
F
F
OSiMe3
OEt
OBn
OBn
3a
and
3b
O
2a
and
2b
O
F
F
BnO
BnO
AcO
AcO
O
BnO
BnO
CO2Et
Ref 9d
Ref 9b
CO2Et
Ref 9d
Ref 9b
BnO
OBn
9
6
F
OBn
F
10
5
Scheme 3. Synthesis of a- and b-mannosides 2a, 2b, 3a, and 3b.
Table 1
E- and P-selectin inhibition with mannosides 1–3
The synthesis of the difluorinated counterparts of the C-manno-
peptides described by Wong and Kaila gave rise to a new class of
selectin inhibitors which showed comparable activities. Taken as
a whole, the difluoromethylene group showed no striking influence
on the inhibitory activities of the compounds, except for an unex-
pected discrepancy with Kaila’s results regarding the glutamate
and 3-aminobenzoate side-chains. Since the greater size of the
pseudo-anomeric difluoromethylene group did not compromise
binding to selectins, other applications for these glycomimetics
are thus conceivable. A beneficial effect of the fluorine atom might
indeed be expected for compounds where the CF2 group acts as an
oxygen mimic.
Entry Compound E-Selectin
Inhibition at
E-Selectina P-Selectin
IC50 (mM) inhibition at
P-Selectina
IC50 (mM)
5 mMa (%)
5 mMa (%)
1
2
3
4
5
6
7
sLex
1a
1b
2a
2b
3a
3b
86
13
50
61
49
16
70
0.3
11
5
3
5
45
19
43
59
50
42
67
>5
11
6.5
3
5
11
2.5
15
2.5
a
All assays were performed in duplicate.
Acknowledgments
close to sLex (IC50 >5 mM, entry 1). The aminobenzoate residue (en-
tries 3 and 7) appeared to be a better sialic acid surrogate than the
We thank the Ministère de l’Enseignement Supérieur et de la
Recherche for a Ph.D Grant to V.G.-I. and Perigene Inc. for a Ph.D
Grant to F.P. and the Association pour la Recherche contre le Cancer
for financial support.
glutamate (entries 2 and 6), but the
a
configuration seemed benefi-
-manno-
cial to both compounds (entries 4 and 5). More generally,
a
sides were the most efficient compounds whereas the behaviour of
the structurally flexible 1-hydroxymannosides 1 (entries 2 and 3)
was highly similar to the behaviour of b-mannosides 3. The C-1 con-
figuration (compounds 1, 2, 3) and/or the pyranose/furanose form
involved in the binding (compounds 1) appeared to have a certain
influence on the inhibitory activities of the glutamate derivatives
(entries 2, 4, and 6). In contrast, the 3-aminobenzoate derivatives
showed similar activities for all compounds (entries 3, 5, and 7). This
last consideration is in disagreement with the results reported by
Kaila. Indeed, the b-mannosylglutamate prepared by this group led
to an appreciable E-selectin inhibition (IC50 = 6 mM) whereas a
3-aminobenzoate derivative displayed no activity.7b Finally, an ana-
logue of compound 1a, which featured a OC16H33 hydrophobic group
on C-6 instead of the free OH group, was also prepared and tested but
led to cell lysis during the competition assays. This modification,
which was efficient in the case of Wong’s inhibitors, was therefore
not further explored.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.141.
References and notes
1. Larsky, L. A. Annu. Rev. Biochem. 1995, 64, 113.
2. Bertozzi, C. Chem. Biol. 1995, 703.
3. Chen, S.; Fukuda, M. Methods Enzymol. 2006, 416, 371. and references cited
therein.
4. For two exhaustive reviews, see: (a) Kaila, N.; Thomas, B. E. Med. Chem. Rev.
2002, 22, 566; (b) Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.; Wong, C.-H.
Chem. Rev. 1998, 98, 833.
5. For more recent examples, see: (a) Moreno-Vargas, A. J.; Molina, L.; Carmona, A.
T.; Ferrali, A.; Lambelet, M.; Spertini, O.; Robina, I. Eur. J. Org. Chem. 2008, 2973;
(b) Ulbrich, H. K.; Luxenburger, A.; Prech, P.; Eriksson, E. E.; Soehnlein, O.;
Rotzius, P.; Lindbom, L.; Dannhardt, G. J. Med. Chem. 2006, 49, 5988; (c) Girard,

1960
V. Gouge-Ibert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1957–1960
C.; Dourlat, J.; Savarin, A.; Surcin, C.; Leue, S.; Escriou, V.; Largeau, C.;
F.; Gouge, V.; Castelot-Deliencourt, G.; Oulyadi, H.; Leclerc, E.; Jubault, P.;
Pannecoucke, X.; Quirion, J.-C. Synlett 2005, 2627; (f) Marcotte, S.; D’Hooge, F.;
Ramadas, S.; Pannecoucke, X.; Feasson, C.; Quirion, J.-C. Tetrahedron Lett. 2001,
42, 5879.
Herscovici, J.; Scherman, D. Bioorg. Med. Chem. Lett. 2005, 15, 3224; (d) Kaila,
N.; Somers, W. S.; Thomas, B. E.; Thakker, P.; Janz, K.; DeBernardo, S.; Tam, S.;
Moore, W. J.; Yang, R.; Wrona, W.; Bedard, P. W.; Crommie, D.; Keith, J. C.; Tsao,
D. H. H.; Alvarez, J. C.; Ni, H.; Merchese, E.; Patton, J. T.; Magnani, J. L.;
Camphausen, R. T. J. Med. Chem. 2005, 48, 4346.
10. (a) Castelot-Deliencourt-Godefroy, G.; Quirion, J.-C. EP 2009/2027138A2.; (b)
Quirion, J.-C.; Pannecoucke, X.; D’Hooge, F.; Marcotte, S.; Castelot-Deliencourt,
G.; Jubault, P.; Gouge, V. WO 2004/014928A2.
11. For CF2-linked disaccharide mimics as P-selectin inhibitors, see: (a) Pérez-
Castells, J.; Hernández-Gay, J. J.; Denton, R. W.; Tony, K. A.; Mootoo, D. R.;
Jiménez-Barbero, J. Org. Biomol. Chem. 2007, 5, 1087; (b) Denton, R. W.; Tony, K.
A.; Hernández-Gay, J. J.; Cañada, F. J.; Jiménez-Barbero, J.; Mootoo, D. R.
Carbohydr. Res. 2007, 342, 1624.
6. For a total synthesis of glycopeptides PSGL-1, ESL-1 and derivatives, see: (a)
Baumann, K.; Kowalczyk, D.; Kunz, H. Angew. Chem., Int. Ed. 2008, 47, 3445; (b)
Filser, C.; Kowalczyk, D.; Jones, C.; Wild, M. K.; Ipe, U.; Vestweber, D.; Kunz, H.
Angew. Chem., Int. Ed. 2007, 46, 2108.
7. (a) Kaila, N.; Chen, L.; Thomas, B. E.; Tsao, D.; Tam, S.; Bedard, P. W.;
Camphausen, R. T.; Alvarez, J. C.; Ullas, G. J. Med. Chem. 2002, 45, 1563; (b)
Kaila, N.; Thomas, B. E.; Thakker, P.; Alvarez, J. C.; Camphausen, R. T.; Crommie,
D. Bioorg. Med. Chem. Lett. 2001, 11, 151; (c) Tsai, C.-Y.; Park, W. C. K.; Weitz-
Schmidt, G.; Ernst, B.; Wong, C.-H. Bioorg. Med. Chem. Lett. 1998, 8, 2333; (d)
Wong, C.-H.; Moris-Varas, F.; Hung, S.-C.; Marron, T. G.; Lin, C.-C.; Gong, K. W.;
Weitz-Schmidt, G. J. Am. Chem. Soc. 1997, 119, 8152.
12. The experimental procedures for the synthesis of compounds 1 are similar to
the preparation of 2 reported in Ref. 9b. 1a: [a]D: +9.7 (c 0.06, H20, k = 436 nm).
1H NMR (300 MHz, D2O) d 4.58 (d, 1H, J = 4.9 Hz), 4.37–4.30 (m, 1H), 4.22–4.15
(m, 2H), 4.05–3.98 (m, 1H), 3.95–3.55 (m, 9H), 2.41–2.28 (m, 4H), 2.20–1.85
(m, 4H). 19F NMR (282.5 MHz, D2O) d À115.7 (d, J = 252.4 Hz, 1F), À116.8 (d, J =
252.4 Hz, 1F), À117.9 (d, J = 248.9 Hz, 1F), À118.8 (d, J = 262.3 Hz, 1F), À119.1
(d, J = 248.9 Hz, 1F), À119.7 (d, J = 262.3 Hz, 1F), À119.8 (d, J = 258.1 Hz, 1F),
À122.1 (d, J = 258.1 Hz, 1F). IR (neat) mmax 3368, 1694, 1682 cmÀ1. MS (ESI+):
m/z = 425 ([M+Na]+). Anal. Calcd for C13H19F2NO11: C, 38.72; H, 4.75; N 3.47.
Found C, 38.54; H, 4.61; N, 3.13. 1b: 1H NMR (300 MHz, D2O) d 8.20 (br s, 1H),
7.91–7.70 (m, 2H), 7.36 (br s, 1H), 3.97–3.38 (m, 6H). 19F NMR (282.5 MHz,
D2O) d À115.7 (d, J = 253.6 Hz, 1F), À116.7 (d, J = 253.6 Hz, 1F), À117.8 (d, J =
247.6 Hz, 1F), À118.7 (d, J = 262.2 Hz, 1F), À118.9 (d, J = 247.6 Hz, 1F), À120.3
(d, J = 262.2 Hz, 1F), À121.0 (d, J = 254.7 Hz, 1F), À122.4 (d, J = 254.7 Hz, 1F). IR
(neat) mmax 3328, 1702 cmÀ1. MS (FAB-): m/z = 392 ([MÀH]À). Anal. Calcd. for
C15H17F2NO9: C, 45.81; H, 4.36; N 3.56. Found C, 45.55; H, 4.24; N, 3.42.
13. DeFrees, S. A.; Kosch, W.; Way, W.; Paulson, J. C.; Sabesan, S.; Halcomb, R. L.;
Huang, D.-H.; Ichikawa, Y.; Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 66.
8. C-Glycosylation: (a) Nishikawa, T.; Adachi, M.; Isobe, M., 2nd ed.. In
Glycoscience: Chemistry and Chemical Biology; Fraser Reid, B., Tatstuta, K.,
Thiem, J., Coté, G. L., Flitsch, S., Ito, Y., Kondo, H., Nishimura, S., Yu, B., Eds.;
Springer: Berlin, Heidelberg, 2008; Vol. 3,
p 755; C-Glycosyl analogs of
oligosaccharide: (b) Vauzeilles, B.; Urban, D.; Doisneau, G.; Beau, J. M., 2nd
ed.. In Glycoscience: Chemistry and Chemical Biology; Fraser Reid, B., Tatstuta, K.,
Thiem, J., Coté, G. L., Flitsch, S., Ito, Y., Kondo, H., Nishimura, S., Yu, B., Eds.;
Springer: Berlin, Heidelberg, 2008; Vol. 9, p 2021.
9. (a) Poulain, F.; Leclerc, E.; Quirion, J.-C. Tetrahedron Lett. 2009, 50, 1803; (b)
Poulain, F.; Serre, A.-L.; Lalot, J.; Leclerc, E.; Quirion, J.-C. J. Org. Chem. 2008, 73,
2435; (c) Moreno, B.; Quehen, C.; Rose-Hélène, M.; Leclerc, E.; Quirion, J.-C. Org.
Lett. 2007, 9, 2477; (d) Karche, N. P.; Pierry, C.; Poulain, F.; Oulyadi, H.; Leclerc,
E.; Pannecoucke, X.; Quirion, J.-C. Synlett 2007, 123; (e) Cuenca, A. B.; D’Hooge,