Design and synthesis of cyclic sialyl Lewis X mimetics: A remarkable enhancement of inhibition by pre-organizing all essential functional groups

Article abstract of DOI:10.1016/S0040-4039(00)01653-1

Two rigid macrocyclic glycopeptides 1 and 2 were designed to mimic the tetrasaccharide SLe(X) as inhibitors of P-selectin. While compound 1 was found to be 1000-fold more potent than SLe(X) with IC₅₀ = 1 μM, compound 2 was not soluble in water for evaluation of its activity. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01653-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9499–9503
Design and synthesis of cyclic sialyl Lewis X mimetics: a
remarkable enhancement of inhibition by pre-organizing all
essential functional groups†
Chung-Ying Tsai, Xuefei Huang and Chi-Huey Wong*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 15 August 2000; revised 20 September 2000; accepted 21 September 2000
Abstract
Two rigid macrocyclic glycopeptides 1 and 2 were designed to mimic the tetrasaccharide SLeX as
inhibitors of P-selectin. While compound 1 was found to be 1000-fold more potent than SLeX with
IC50=1 mM, compound 2 was not soluble in water for evaluation of its activity. © 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: sialyl Lewis X; mimetics; inhibition enhancement.
Sialyl Lewis X (SLeX) is a tetrasaccharide often found at the non-reducing terminal of certain
glycoproteins and glycolipids as a ligand for selectins (Fig. 1(a)).1–6 The interaction between
SLeX and selectins plays important roles in neutrophil extravasation, lymphocyte recirculation,
Figure 1. (a) The structure of SLeX and its crucial functional groups (marked with a ) for binding with P-selectin.
(b) The structures of designed cyclic compounds 1 and 2. (c) The structures of previously prepared open form SLeX
mimetics 3 and 4
* Corresponding author. Tel: +1-858-784-2487; fax: +1-858-784-2409; e-mail: wong@scripps.edu
†
Dedicated to Professor Harry H. Wasserman on the occasion of his 80th birthday.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01653-1

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platelet adhesion as well as cancer metastasis. Development of SLeX mimetics as potential
therapeutics has been a subject of current interest.1 It has been demonstrated that the 3-hydroxyl
group of the fucose, the 4- and 6-hydroxyl groups of the galactose, and the negatively charged
carboxylate of the sialic acid are the crucial functional groups for the SLeX epitope binding to
P-selectin,7–9 the first selectin expressed when tissue injury occurs. Due to the weak affinity of
SLeX (IC50=1.3–3.0 mM) for P-selectin,10 efforts have been directed toward the design of SLeX
mimetics containing minimal functional groups to increase affinity.8,9,11 One of the approaches
is based on combinatorial chemistry using the Ugi reaction, from which compounds 3 and 4
(Fig. 1(c)) were found to be more active than SLeX.10 Compound 3 is about 15 times more
potent than SLeX, while introduction of a hydrophobic chain (such as that in compound 4) has
led to a further 20-fold increase in its potency. This result together with those from compounds
related to 4 with different hydrophobic groups indicates the possible presence of a hydrophobic
pocket in P-selectin near the binding site. In order to further improve the potency of the SLeX
mimics, we have decided to synthesize cyclic SLeX mimics 1 and 2 (Fig. 1(b)) with all the
recognition elements pre-organized in a defined manner to further lower the entropic energy
upon binding with selectin.
The cyclic glycopeptide 1 was synthesized by coupling the monosaccharide 5 and the
tripeptide 6 (Scheme 1). To prepare compound 5,
L
-galactose was converted to the fully
protected -galactoside 7 in 55% yield (Scheme 1-1). Upon treatment with 3-TMS 1-propyne
L
and TMSOTf, the anomeric methoxy group of 7 was displaced with allene (compound 8) in 71%
yield with concomitant removal of the benzyl group.11,12 Subsequent protection of the free
hydroxy group of compound 8 with acetic anhydride followed by ozonolysis, Jones oxidation
Scheme 1. Reagents and conditions: (a) (i) IRA-120, MeOH, 12 h; (ii) NaH (5 equiv.), BnBr (6 equiv.), Bu4NI (0.2
equiv.), 4 h, 55%; (b) TMSOTf (0.5 equiv.), 3-TMS-1-propyne (2 equiv.), CH3CN, 12 h, 71%; (c) (i) Ac2O, pyridine,
2 h; (ii) O3, then Me2S, 8 h; (iii) Jones’ oxidation, 0.5 h; (iv) cat. NaOMe, MeOH, 1 h, 65%; (d) (i) N-Boc-b-allyl-
aspartic acid 10 (1.1 equiv.), EDC (1.1 equiv.), HOBt (1.1 equiv.), NMM (2.0 equiv.), 2 h; (ii) TFA, 1 h, 90%; (e) (i)
N-Boc-O-benzyl- -serine 12 (1.1 equiv.), EDC (1.1 equiv.), HOBt (1.1 equiv.), NMM (2 equiv.); (ii) TFA, 1 h, 90%;
L-
L
(f) (i) 2 (1.1 equiv.), EDC (1.1 equiv.), HOBt (1.1 equiv.), NMM (2 equiv.), 2 h; (ii) Rh(PPh3)3Cl (0.1 equiv.), EtOH,
H2O, 2 h, 70%; (g) PPh3 (1.3 equiv.), DEAD (1.3 equiv.), THF, 12 h, 27%; (h) Pd/C, H2, AcOH/THF/H2O, 12 h, 83%

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and deacetylation gave the monosaccharide 5 in 65% yield in four steps. The tripeptide 6 was
prepared from the benzyl protected glutamic acid 9 with the allyl protected aspartic acid 10 and
benzyl protected serine 12 (Scheme 1-2). Coupling of the tripeptide 6 and monosaccharide 5 with
EDC followed by removal of the allyl protective group gave the open form glycopeptide
carboxylic acid 13 in 70% yield. Macrolactonization of 13 was accomplished in 27% yield under
Mitsunobu conditions to give the fully protected cyclic glycopeptide 14. Removal of all benzyl
groups by catalytic hydrogenation yielded the desired cyclic glycopeptide 1.13
Compound 2 was synthesized by coupling monosaccharide 5 with tripeptide 15, which was in
turn prepared from dipeptide 11 and the serine derivative 16. Attempts to directly synthesize 16
from serine esters failed as either Mitsunobu reaction on serine 17 or displacement of the
bromide from 18 led to the formation of dehydroserine 19, presumably through a-deprotonation
(Scheme 2-1). The serine derivative 16 was successfully synthesized in 45% yield by first reducing
D
-serine 20 to serinol 21 followed by Mitsunobu reaction to introduce the hydrophobic chain
(Scheme 2-2). Coupling of dipeptide 11 with serine derivative 16 and monosaccharide 5 followed
by deprotection gave the open form glycopeptide carboxylic acid 22 (Scheme 2-3) in 68% yield
in four steps. The macrolactonization of 22 was achieved using EDC, DMAP and DMAP·HCl14
Scheme 2. Reagents and conditions: (a) PPh3 (1.3 equiv.), DEAD (1.3 equiv.), 4-(n-heptyloxy)-phenol (1 equiv.), 12
h; (b) sodium 4-(n-heptyloxy)-phenoxide (1 equiv.), DMF, 12 h; (c) LiBH4 (1 equiv.), THF, 1 h, 97%; (d) (i) PPh3 (1.3
equiv.), DEAD (1.3 equiv.), 4-(n-heptyloxy)-phenol (1 equiv.); (ii) Pd/C, CH2Cl2; (iii) PDC, DMF, 48%; (e) (i) 16 (1.0
equiv.), EDC (1.1 equiv.), HOBt (1.1 equiv.), NMM (2.0 equiv.), 2 h; (ii) TFA, 1 h, 90%; (f) 5 (1.1 equiv.), EDC (1.1
equiv.), HOBt (1.1 equiv.), NMM (2.0 equiv.), 2 h; (ii) Pd(PPh3)4 (0.1 equiv.), morpholine (1.2 equiv.), 1 h, 75%; (g)
EDC (3.0 equiv.), DMAP (1.0 equiv.), DMAP·HCl (1.0 equiv.), 0°C, 40–50%; (h) Pd/C, H2, AcOH/THF/H2O

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at 0°C in 40–50% yield. Using other coupling reagents such as DEAD/PPh3, 2,4,6-trichloroben-
zoyl chloride,15,16 and 2,2%-dipyridyl disulfide17,18 either led to a much lower yield or completely
failed. Unfortunately, subsequent deprotection of all the benzyl groups of macrolactone 2319
under catalytic hydrogenation conditions gave a white solid product insoluble in the solvent
system such as water, DMSO, methanol, methanol/CH2Cl2/H2O, acetic acid, THF or acetoni-
trile, although mass spectra indicated a complete removal of all benzyl groups; this is most likely
due to the amphiphilic nature of compound 2. No racemization during peptide coupling was
observed in the preparation of 1 and 2.
Glycopeptide 1 was assayed11 and its IC50 value in inhibiting P-selectin was determined to be
1 mM as compared to 118 mM for compound 3.10 Therefore, pre-organization of the necessary
point of contacts by introducing a macrolactone ring indeed greatly increased the potency of the
mimics, to an extent similar to the hydrophobic effect exhibited in 4. Although compound 2 is
insoluble, changing the structure of the hydrophobic group may increase its solubility.
Acknowledgements
This research was supported by the NSF.
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13. 1: H NMR (600 MHz, D2O) l 4.55 (d, J=6.6 Hz, 1H), 4.51–4.54 (m, 2H), 4.43 (dd, J=1.8, 11.9 Hz, 1H), 4.30
(dd, J=4.9, 9.2 Hz, 1H), 4.11 (dd, J=3.5, 10.1 Hz, 1H), 3.99 (dd, J=9.7, 11.9 Hz, 1H), 3.91 (d, J=3.5 Hz, 1H),
3.90 (dd, J=6.6, 10.1 Hz, 1H), 3.81 (dd, J=5.7, 11.8 Hz, 1H), 3.75 (dd, J=4.4, 11.8 Hz, 1H), 3.66 (dd, J=1.8,
9.7 Hz, 1H), 2.87 (dd, J=4.4, 15.8 Hz, 1H), 2.81 (dd, J=4.9, 15.8 Hz, 1H), 2.30–2.35 (m, 2H), 2.06–2.11 (m, 1H),
1.83–1.88 (m, 1H); 13C NMR (150 MHz, D2O) l 177.86, 175.53, 172.53, 171.94, 171.48, 171.39, 75.66, 75.50,
70.79, 69.84, 67.96, 66.60, 61.48, 55.32, 52.96, 51.26, 36.38, 30.66, 26.70; HRMS calcd for C19H27N3NaO14 (FAB,
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M. J. Chem. Eur. J. 1996, 7, 847.

9503
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19. 23: 1H NMR (500 MHz, CDCl3) l 7.69 (d, J=9.5 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.22–7.36 (m, 12H),
7.17–7.22 (m, 4H), 7.08–7.16 (m, 4H), 7.03–7.06 (m, 2H), 6.93–6.98 (m, 2H), 6.53–6.62 (m, 4H), 5.41–5.48 (m,
1H), 5.16 (d, J=12.0 Hz, 1H), 5.03–5.08 (m, 1H), 4.96 (d, J=12.5 Hz, 1H), 4.92 (d, J=12.5 Hz, 1H), 4.86–4.92
(m, 1H), 4.81 (bs, 1H), 4.68 (d, J=12.1 Hz, 1H), 4.59 (d, J=12.5 Hz, 1H), 4.54 (d, J=12.5 Hz, 1H), 4.51 (d,
J=12.5 Hz, 1H), 4.49 (d, J=12.5 Hz, 1H), 4.41 (d, J=12.5 Hz, 1H), 4.32–4.39 (m, 2H), 4.24–4.29 (m, 1H),
4.13–4.18 (m, 1H), 3.95–4.03 (m, 2H), 3.68–3.75 (m, 2H), 3.17 (dd, J=11.0, 15.5 Hz, 1H), 2.75 (dd, J=5.2, 15.5
Hz, 1H), 1.94–2.15 (m, 2H), 1.72–1.88 (m, 2H), 1.60–1.72 (m, 2H), 1.30–1.45 (m, 2H), 1.16–1.36 (m, 6H), 0.89
(t, J=7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) l 171.93, 171.50, 170.60, 170.37, 170.22, 170.07, 153.94, 151.92,
137.94, 137.87, 137.84, 135.79, 135.41, 128.44, 128.38, 128.30, 128.08, 127.98, 127.73, 127.69, 127.56, 116.29,
115.24, 73.66, 72.41, 72.07, 69.41, 68.42, 67.10, 66.22, 31.79, 29.69, 29.40, 29.11, 26.03, 22.61, 14.01; MS calcd for
C67H75N3NaO15 (ESI, M+Na): 1185; found: 1185.
.