Synthesis and biological evaluation of PEG-tirofiban conjugates

Article abstract of DOI:10.1016/j.bmcl.2008.01.118

We have conjugated tirofiban, an antagonist of the GPIIb/IIIa integrin receptor, to PEG, and shown that these polymers effectively inhibit platelet aggregation. This inhibition decreased with the size of the polymer. Our goal was to develop new cryoprotec

Full text of DOI:10.1016/j.bmcl.2008.01.118

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2028–2031
Synthesis and biological evaluation of PEG-tirofiban conjugates
a,
a
b
b
*
´
´
´
Laurent Desaubry, Stephanie Riche, Patricia Laeuffer and Jean-Pierre Cazenave
aUMR 7175 LC1, Universite´ Louis Pasteur, CNRS, Faculte´ de Pharmacie, 67401 Illkirch, France
bINSERM U.311, EFS-Alsace, 10, rue Spielmann, BP 36, 67065 Strasbourg, France
Received 16 November 2007; revised 29 January 2008; accepted 29 January 2008
Available online 2 February 2008
Abstract—We have conjugated tirofiban, an antagonist of the GPIIb/IIIa integrin receptor, to PEG, and shown that these polymers
effectively inhibit platelet aggregation. This inhibition decreased with the size of the polymer. Our goal was to develop new cryo-
protective agents to store frozen platelets. Surprisingly, tirofiban-conjugated PEG did not exhibit any protection.
Ó 2008 Elsevier Ltd. All rights reserved.
Platelets cannot be conserved more than five days,
which causes regularly some shortage in the stocks of
blood banks. Consequently, there is an ongoing need
to develop a method to store platelets for a longer time.
Their storage in the frozen state has been limited by the
toxicity and the lack of efficiency of cryoprotective
agents.1 DMSO protects platelets from the stress of
freezing and thawing, however, residual DMSO in plate-
let concentrate induces nausea, vomiting, local vaso-
spasm and garlic-like taste, which makes it unsuitable
for clinical purposes.1,2 On the other hand, polyethylene
glycol (PEG), which has been used for many years as
a cryoprotectant for several tissues and cells, is well tol-
erated by the organism. However, its cryoprotective effi-
ciency is not sufficient for platelets.3 The main damages
occur at the level of the plasmic membrane that loses its
integrity due to formation of ice crystals during freezing
and thawing.
PEG
platelet
GPIIb/IIIa antagonist
GPIIb/IIIa receptor
Figure 1. Platelet wrapping by PEG conjugated to a GPIIb/IIIa
antagonist.
The first task of our research program was to design and
synthesize a GPIIb/IIIa antagonist that could be easily
conjugated to PEG. Tirofiban (1) is an antithrombotic
drug developed by Merck scientists that binds with a
high affinity to GPIIb/IIIa receptor even when platelets
are not activated (Kd = 13 nM).5 On the basis of SAR
studies that showed that the butylsulfonamide moiety
could be replaced by various arylsulfonamides,6 we envi-
sioned compound 2 to be a suitable ligand for conjuga-
tion to PEG (Fig. 2).
We aimed at improving this cryoprotection by conjugat-
ing PEG to the ligand of a membrane receptor concen-
trate the polymer close to the membrane where it could
display its cryoprotective effects (Fig. 1). We expected
this layer of cryoprotective polymer to maintain mem-
brane integrity during freezing and thawing. The
membrane receptor that we selected is the GPIIb/IIIa
integrin receptor because it is the most abundant one
at the platelet surface (more than 40,000 copies per
platelet).4 The ligand had to be an antagonist to prevent
it from triggering the aggregation of platelets.
The general approach to prepare 2 was based on the
synthesis of tirofiban developed by Merck scientists
(Scheme 1).7 Deprotonation and alkylation of picoline
3 with bromide 4 followed by deprotection afforded
pyridinylbutanol 5 (71% from 3). Hydrogenation over
Adams’ catalyst and N-Boc protection gave 6 (57%, 2
steps). This alcohol was used to alkylate protected tyro-
sine 7 by a Mitsunobu reaction (76%). Removal of the
Cbz and sulfonylation of the free amine completed the
synthesis of the GpIIb/IIIA-binding moiety of the
ligand.
Keywords: Poly(ethylene glycol), PEG; Cryopreservation, Platelet ag-
gregation; Antithrombotic; GPIIb/IIIa receptor.
Completion of the synthesis required the introduction
of the linker moiety by a Sonogashira’s reaction with
*
Corresponding author. E-mail: desaubry@chimie.u-strasbg.fr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.118

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L. Desaubry et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2028–2031
2029
O
O
N
HN
S O
COOH
N
HN
COOH
H
H
O
O
S O
1
2
H
O
N
O
H2N
O
O
Figure 2. Tirofiban (1) and its analogue to be conjugated (2).
OH
OH
NBoc
1) nBuLi, THF
(CH2)4
1) H2, PtO2, AcOH,
60 psi, 4 days
2) Boc2O, NaOH,
THF, H2O
Br
O
O
2)
(4), -78°C - r.t.
O
CBZ-TyrOMe (7),
Ph3P, DEAD, CH2Cl2
3) HCL 4N, 12 h
76%
57%
N
3
N
N
71%
CBZ-NH
COOMe
5
Boc
6
8
1) H2, Pd/C, EtOH
2) pBrPhSO2Cl,
Et3N, DMAP,
CH2Cl2, 0°C-r.t.,
15h
87 %
1) pNO2PhOCOCl, Et3N, CH2Cl2, 0°C-r.t., 12h
NH
O
2)
2 (11) (6 eq),
H2N
O
CH2Cl2, r.t., 12 h
3) Boc2O, THF, 24 h
O
BocN
67%
OH
10
HN
COOMe
H
9
O
N
O
O
S
O
BocHN
O
12
O
Pd(OAc)2, Bu4NHSO4 , Ph3P, Et3N, CH3CN, H2O
74%
Br
PEG
O
O
O
DMAP,
1) LiOH
90%
dioxane,
r.t.
2) AcOH
N
O
O N
95%
13
2
O
O
O
O
n ~ 45: PEG 2K
O
n ~ 230: PEG 10K
O
O
NHS
NHS
NHS
n
or
O
O
n ~ 110: PEG 5K-OMe
MeO
O
n
Na2CO3, NaHCO3, H2O, pH 9, 3 days
65-78%
NH
O
O
H
O
S
O
O
N
PEG
O
N
O
NH
H
O
O
HOOC
14: PEG 2K (ligand 2)2
15: PEG 10K (ligand 2)2
16: PEG 5K-OMe ligand 2
Scheme 1. Synthesis of ligand 2 and its conjugation to PEG.

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L. Desaubry et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2028–2031
2030
alkyne 12. This reagent was easily prepared in 3 steps
from butynol 10. Reaction of this alcohol with an excess
of 4-nitrophenylchloroformate cleanly provided an acti-
vated carbonate, whose reaction with an excess of dia-
mine 11 and subsequent protection as a Boc efficiently
afforded 12 (67%, 3 steps).
Next, we examined the cryoprotective properties of 14,
16 and regular PEG using Dayan’s method for freezing
platelets.14 Briefly, a solution of the polymer was added
to a suspension of human platelets in a glycerol–glucose
medium. This preparation was frozen in liquid nitrogen
using Dayan’s statically controlled cooling rate device,
carefully thawed in a bath at 37 °C, and washed by
centrifugation. We examined the swirling and the aggre-
gating properties and compared them to a non-frozen
sample. In all frozen samples, swirling was slightly
reduced, which indicates that a portion of the platelets
changed their shape and were not able to aggregate any-
more. Platelet frozen in the presence of regular PEG 2K
and PEG 10K retained partially their ability to aggre-
gate in response to ADP (Table 1).
Optimization of the Sonogashira coupling between 9
and 12 led to a satisfying yield of 74% under Jeffery’s
conditions.8 Saponification of the ester and deprotection
of the N-Boc afforded the ligand 2 (90%, 2 steps).
Initial attempts to conjugate 2 to PEG using 4-nitrophe-
nylchloroformate according to Schiavon’s procedure
gave low yields.9 However, the use of di-succinimidyle
carbonate 1310 in presence of DMAP in dioxane affor-
ded the corresponding activated PEG in 95% yields.
First attempts to conjugate these activated carbonates
with ligand 2 in chloroform or pyridine gave poor yields.
Finally, we found that performing this conjugation with
ligand 2 in an aqueous buffer at pH 9 efficiently afforded
the expected adducts 14–16 (65–78% yields).11,12
Surprisingly, this ability was totally lost with 14 and 16,
indicating that conjugating tirofiban analogue 2 to PEG
abolishes cryoprotective properties of PEG. This might
be explained by a destabilization of the membrane
bilayer by PEG due to a depletion interaction.15
None of the platelet samples responded to collagen,
which shows that the activation by collagen is more sen-
sitive to freezing stress, than the one by ADP.
Before examining the cryoprotective properties of
14–16, we tested whether PEG conjugates retain the
binding and pharmacological properties of tirofiban.
We checked that these conjugates inhibit the aggrega-
tion of platelets activated by ADP, collagen or TRAP
(a thrombin receptor agonist) according to established
procedures (Fig. 3).13 We showed that better inhibition
of platelet aggregation is achieved with a shorter poly-
mer conjugate: PEK 2K conjugate 14 abolished totally
the aggregation at a 10 lM concentration, while PEG
5K and 10K conjugates 15 and 16 required a 10-fold
higher concentration to achieve the same effect. This
could be due to the repulsive effect of PEG that tends
to repel these conjugated polymers from the membrane
and its receptors.
Several drugs have been conjugated to PEG in order to
modify their bioavailability.16 None of them were
designed for cryopreservation. We provided therein the
first experimental evidence that such an approach is
unlikely to be successful. We also demonstrated that
PEG-tirofiban conjugates effectively inhibit platelet
aggregation, and that this inhibition decreases with the
size of the polymer.
Table 1. Aggregation properties of platelet samples that were frozen in
the presence of 0.2 mM PEG or PEG-2 conjugates (expressed as a
percentage relative to a non-frozen sample)
% Aggregation % Aggregation
induced by ADP induced by collagen
We observed that the aggregation induced by collagen
and TRAP was less sensitive to the inhibition by PEG
conjugates than the one induced by ADP, however,
the activity profile remained the same. Overall, these
data confirmed that PEG conjugates 14–16 efficiently
bind to the GPIIb/IIIa receptor and do not exhibit del-
eterious effects on platelet function.
Control (no polymers)
PEG 2K
14
0
0
0
0
0
0
40
0
PEG 10K
16
32
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
ADP
collagen
TRAP
* *
*
*
* *
*
*
*
10 100 1 10 100 μM
0
0
1
10 100
1
10 100 1 10 100 μM
0
1
10 100
1
10 100 1 10 100 μM
0
1
10 100
1
Control (no polymer)
PEG 2K conjugate 14
PEG 5K conjugate 15
PEG 10K conjugate 16
Figure 3. Effect of PEG conjugates 14–16 on human platelet aggregation induced by ADP (5 lM), collagen (2.5 mg/ml) or TRAP (1 lM). The
concentrations of polymers are based on the quantity of conjugated tirofiban ( 0% aggregation).
*

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2031
7. Chung, J. Y. L.; Zhao, D.; Hughes, D. L.; Grabowski, E.
J. J. Tetrahedron 1993, 49, 5767.
Acknowledgment
8. Nguefack, J.-F.; Bolitt, V.; Sinou, D. Tetrahedron Lett.
1996, 37, 5527.
9. Veronese, F. M.; Largajolli, R.; Boccu, E.; Benassi, C.
A.; Schiavon, O. Appl. Biochem. Biotechnol. 1985, 11,
141.
´ ´
´ ´
Financial support from ‘Delegation Generale pour
l’Armement’ (Grant No. 0334037) is gratefully
acknowledged.
10. Zalipsky, S.; Seltzer, R.; Menon-Rudolph, S. Biotechnol.
Appl. Biochem. 1992, 15, 100.
References and notes
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jugate Chem. 2003, 14, 1038.
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B. J., Benson, E., Lane, N., Eds.; CRC Press: Boca Raton,
2004; pp 483–504.
2. Santos, N. C.; Figueira-Coelho, J.; Martins-Silva, J.;
Saldanha, C. Biochem. Pharmacol. 2003, 65, 1035.
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12. Compound 2 (2 equiv) reacted with succinimidyl car-
bonate PEG in an aqueous buffer at pH 9 according to
Ref. 11. After 72 h, the solution was filtered, dialyzed
three times and lyophilized to afford the adduct as a
white solid, which was analyzed by 1H NMR in
DMSO-d6.
13. Cazenave, J. P.; Ohlmann, P.; Cassel, D.; Eckly, A.;
Hechler, B.; Gachet, C. Methods Mol. Biol. 2004, 272, 13.
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Leckband, D.; Israelachvili, J.; Hui, S. W. Langmuir 1996,
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