Title of Invention

ANTITHROMBOTIC COMPOUND

Abstract The present invention relates compounds of the formula (A) oligosaccharide-spacer-GpIIb/IIIa antagonist (A), wherein the oligosaccharide is a negatively charged oligosaccharide residue comprising four to twenty five monosaccharide units, the charge being compensated by positively charged counterions, and wherein the oligosaccharide residue is derived from an oligosaccharide which has (AT-III mediated) anti-Xa activity per se; the spacer is a bond or an essentially pharmacologically inactive linking residue; the GpIIb/IIIa antagonist is a residue mimicking the RGD and/or K(QA)GD fragment of fibrinogen, typically comprising an optionally esterified carboxylate moiety and a basic moiety located within the residue at a distance of 10-20 Å from each other; or a pharmaceutically acceptable salt thereof or a prodrug or a solvate thereof. The compounds of the invention have antithrombotic activity and can be used in treating or preventing thrombotic diseases.
Full Text The invention relates to a new antithrombotic compound, a pharmaceutical composition
containing the compound as an active ingredient, as well as the use of said compound for the
manufacture of medicaments.
Acute myocardial infarction, ischemia and stroke are caused by the formation of an occlusive
thrombus in an atherosclerotic coronary artery. The arterial thrombus is formed by blood
platelets (thrombocytes) aggregating with (increased levels of) fibrinogen. This process is
associated with an excited state and imbalance of the coagulation system in which fibrinogen is
cleaved into fibrin clots. Intervention in one of these primary and secondary haemostatic
pathways is essential in the treatment of (arterial) thrombosis.
Serine proteases are enzymes which play an important role in the blood coagulation cascade.
Members of this group of proteases are for example thrombin, trypsin, factors VIIa, IXa, Xa,
XIa, XIIa, and protein C. Thrombin is the final serine protease enzyme in the coagulation
cascade. The prime function of thrombin is the cleavage of fibrinogen to generate fibrin
monomers, which are cross-linked to form an insoluble gel. In addition, thrombin regulates its
own production by activation of factors V and VIII earlier in the cascade. It also has important
actions at cellular level, where it acts on specific receptors to cause platelet aggregation,
endothelial cell activation and fibroblast proliferation. Thus thrombin has a central regulatory
role in haernostasis and thrombus formation. Factor Xa catalyzes the conversion of prothrombin
into thrombin. Inhibition of factor Xa effectively results in inhibition of the coagulation of
blood.
Platelet aggregation is triggered by several activators, not only by thrombin, but also by ADP,
collagen and epinephrin. In all cases, the final common pathway leading to platelet aggregation
is binding of fibrinogen to its receptor, the key membrane glycoprotein complex GPIIb/IIIa.
Therefore, inhibition of fibrinogen binding to this protein is. considered, a very effective way of
inhibiting .platelet aggregation for the prevention of (arterial) thrombus formation and the
treatment of thrombotic disorders.
GPIIb/IIIa (αIIbβ3) is a surface receptor belonging to the integrin family. Integrins are composed
of two chains, an α subvrnit and a β subunit, which are held together by noncovalent bonds in a

calcium dependent manner. GPIIb constitutes the a subunit (αIIb) with divalent cation binding
domains, whereas GPIIIa is a protypical β subunit (β3). Integrins have been isolated from cells
throughout the body and axe mediators of cell-cell and cell-substrate adhesion and signalling.
There are three binding sites on GPIIb/IIIa, one that recognises the amino sequence Arg-Gly-
Asp (RGD binding site), another that recognises Lys-Gln-Ala-Gly-Asp (KQAGD binding site)
and one that recognises Lys-Gly-Asp (KGD binding site).
On each circulating platelet, there are 35,000 to 100,000 GPIIb/IIIa complexes; most are
distributed on the platelet surface, with a smaller pool in an internal reserve. The GPIIb/IIIa
complex does not interact with its plasma ligands until platelets have been activated by
exogenous agonists such as ADP or thrombin. When this occurs, an inside-out signal is
generated that results in a conformational change in the extracellular portion of the complex that
renders the molecule capable of binding fibrinogen and other ligands.
Compounds mimicking the α-chain (RGD) and γ-chain (KQAGDV) fragments of fibrinogen
may act as antagonists. Numerous potent GPIIb/IIIa antagonists based on peptidomimetic
structures have been described previously.
Some (very) potent examples are Ro 435054, xemilofiban, RWJ 50042, tirofiban and lamifiban.
However, a significant number of GPIIb/IIIa antagonists showing excellent potencies and
pharmacological profiles in vitro, are not further developed or on hold after having reached late
phase clinical trials, due to a lack of consistent control of platelet aggregation and ambiguous
pharmacological behaviour, (partly) caused by the short half-lives of the compounds. The short
half-lives lead to large variations in plasma levels of the free drug and may contribute to
interindividual variability in dose response (monitoring therapy is required).
It was further reported by H. Darius in Thromb Res. 2001, 103, S117-S124 that in all large
clinical trials with GPIIb/IIIa antagonists the therapeutic effect was only minor and, moreover,
even an increased mortality in the glycoprotein Ilb/IIIa receptor-antagonist-treated group of
patients (orally, treated) had been observed. The narrow therapeutic window and limited
bioavailability of the drugs, together with the still very limited knowledge about the regulation
of the platelet fibrinogen receptor, were considered to be responsible for this therapeutic failure.
In conclusion, there is a need for GPIIb/IIIa antagonists having a predictable antithrombotic
effect, preferably with a longer half-life (to achieve consistent levels of inhibition of platelet
aggregation).

According to the present invention new compounds have now been found which are inhibitors
preferably having a mixed pharmacological profile by inhibiting two key targets in both the
coagulation cascade (factor Xa) and the platelet aggregation pathway (GpIIb/IIIa).
The compounds of this invention have the formula A
oligosaccharide-spacer-GpIIb/IIIa antagonist (A),
wherein
the oligosaccharide is a negatively charged oligosaccharide residue comprising four to twenty
five monosaccharide units, the charge being, compensated by positively charged counterions,'
and wherein the oligosaccharide residue is derived from an oligosaccharide which has (AT-IH
mediated) anti-Xa activity per se;
the spacer is a bond or an essentially pharmacologically inactive linking residue;
the GpIIb/IIIa antagonist is a residue mimicking the RGD and/or K(QA)GD fragment of
fibrinogen, typically comprising an optionally esterified carboxylate moiety and a basic moiety
located within the residue at a distance of 10-20 Å from each other;
or a pharmaceutically acceptable salt thereof or a prodrug or a solvate thereof.
The compounds of the invention are effective antithrombotic agents by both ATIII-mediated
inhibition of coagulation factor Xa and inhibition of platelet aggregation by antagonizing the
binding of fibrinogen to its receptor. When compared to the combination therapies known in the
art, wherein GpIIb/IIIa inhibitors are combined with anticoagulant therapies (such as described
in Expert Opin.Investig.Drugs (2003) 12(9), 1567, and in US 2003/0199457 Al), the
pharmacokinetic and pharmacodynamic profiles of the compounds of the present invention lead
to more consistent control of platelet aggregation and to less interindividual variability in dose
reponse. A further advantage of the compounds of the present invention is that the
pharmacological profile can be tuned; (a) by changing the type of oligosaccharide the binding to
ATIII is affected, resulting in an increase or decrease of the Xa inhibitory activity, and longer or
shorter halflives, respectively, (b) by changing the type' of GpIIb/IIIa antagonist the inhibition of
the platelet aggregation can be increased or decreased, (c) by changing the spacer length, further
tuning of the individual pharmacological activities of each compound is possible.

Other conjugates comprising an oligosaccharide have been reported, being synthetic conjugates
of a pentasaccharide and a direct thrombin inhibitor (Bioorg. Med. Chem. Lett. 1999, 9(14),
2013-8; WO 99/65934; WO 01/42262) or conjugates of two oligosaccharides, wherein one of
the oligosaccharides displays indirect AT-III mediated anti-thrombin activity, in addition to AT-
III mediated anti-Xa activity of the other oligosaccharide (EP 0,649,854). Though also
antithrombotic compounds, those compounds only act on factors of the coagulation cascade.
The compounds of the present invention, on the other hand, also act on the GPIIb/IIIa receptor
present on the surface of circulating platelets. The mechanism of action is therefore significantly
different from each of the other types of antithrombotic conjugates. With the compounds of the
present invention two complementary antithrombotic therapies are available within one single
drug substance.
The compounds of the present invention are useful for treating and (possibly) preventing
thrombotic diseases. This includes a number of thrombotic and prothrombotic states in which
the coagulation cascade is activated which include, but are not limited to, deep vein thrombosis,
pulmonary embolism, thrombophlebitis, arterial occlusion from thrombosis or embolism,
arterial reocclusion during or after angioplasty, restenosis following arterial injury or invasive
cardiological procedures, postoperative venous thrombosis or embolism, stroke and myocardial
infarction.
Any negatively charged oligosaccharide residue of four to twenty five monosaccharide units is
useable in the compounds of the present invention. Suitable compounds of the invention are
compounds wherein the oligosaccharide is a sulfated or phosphorylated oligosaccharide residue.
Preferably, the oligosaccharide residue is derived from an oligosaccharide which has (AT-III
mediated) anti-Xa activity per se, such as the oligosaccharides disclosed in EP 0,454,220, EP
0,529,715, WO 97/47659, WO 98/03554 and WO 99/36443. Preferred compounds according to
the invention are compounds wherein the oligosaccharide residue has four to sixteen
monosaccharide units, and most preferably is a sulfated pentasaccharide residue.' Preferred
pentasaccharide residues have the structure B


wherein Rl is independently OSO3- or (1-8C)alkoxy and the charge being compensated by
positively charged counterions.
Particularly preferred pentasaccharides have the structure C

wherein Rl is OCH3 or OSO3-.
the charge being compensated by positively charged counterions. In the most preferred
pentasaccharide of the structure C R1 is OCH3.
The spacer is a bond or an essentially pharmacologically inactive linking residue. In preferred
embodiments the spacer is an essentially pharmacologically inactive linking residue, preferably
having 1-50 atoms, the oxygen of the oligosaccharide residue not included. The chemical nature
of the spacer is of minor importance for the anti-thrombotic activity of the compounds of the
invention. However, the spacer of the compounds of the invention is preferably flexible.
Suitable spacers may easily be designed by a person skilled in the art. A more preferred length
of the spacer is 10-35 atoms, in particular 10-28. For synthetic reasons longer spacers are
considered less suitable, however, longer spacers may still succesfully be applied in the
compounds of the present invention. Preferred spacers contain at least one -(CH2CH2O)-
element. More preferred spacers contain more, preferably six -(CH2CH2O)- elements. The most
preferred spacer is *-(CH2CH2O)3-(CH2)2-NH-C(O)-CH2O-(CH2CH2O)3-(CH2)2-, the end
indicated with * being attached to the oxygen of the oligosaccharide residue.

The attachment site of the spacer to the GpIIb/IIIa antagonist residue may be chosen essentially
arbitrarily, provided that the GpIIb/IIIa antagonist activity is not abolished. Thus, the typically
present carboxylate moiety (optionally esterified) and basic moiety must remain unaffected.
In preferred compounds according to this invention, the GpIIb/IIIa antagonist residue is selected
from residues derived from Ro 435054, SC 54701 (xemilofiban) , RWJ 50042, sibrafiban (Ro
44 3888), lamifiban (Ro 449883), GPI562, FK 633, tirofiban (MK 383), orbofiban (SC 57101),
eptifibatide (C68 22), roxifiban (XV 459), elarofiban (RWJ 53308), SR 121787, lefradafiban
(BJSU 52), lotrafiban (SB 214857), gantofiban (YM 028), T-250, EF 5077, ZD 2486, TAK 029,
TP 9201, L 703014, SR 121566 (active form of SR 121787). Derivatives of said residues also
include chemically modified residues, wherein the part comprising the (optionally esterified)
carboxylate moiety and a basic moiety (or protected basic moiety) is retained.
Preferred GpIIb/IHa antagonist residues have the structure D
Y-N(H)-C(O)-X (D),
wherein Y is N(H)-C(O)-C(R2)(C(R2)2COOH) or
N(H)-C(O)-C(R2)(CH2aryl)-N(H)-C(O)-C(R2)(C(R2)2COOH),
O-phenylene-C(R2)2-C(R2)(COOH)-N(H)-C(O)-C(R2)(C(R2)2COOH),
O-phenylene-C(R2)2-C(R2)(C(O)-R3-O-C(R2)2COOH),
wherein R2 is independently H or (1-4C)alkyl; and wherein aryl is phenyl, hydroxyphenyl,
thiophenyl or pyridinyl and R3 is piperidinyl;
and X is benzamidine, (CH2)2-N(H)-C(O)-benzamidine, (CH2)2-C(O)-N(H)-benzamidine or

The most preferred compounds of the present invention are the compounds II, V, VIII, X, XI,
XII, XIII, XIV, XV and XVI as decribed in the examples, of which compound XIII has the
highest preference.

A positively charged counterfoil means H+, Na+, K+, Ca2+, and the like. Preferably the
compounds of formula (A) are in the form of their sodium salt.
The term basic moiety means any well known basic moiety, such as an amine, amidine
guanidine, piperidine, and the like.
With the phrase "at a distance of 10-20 Å from each other" the spatial orientation of the two
groups with respect to another is meant, not only measured along the bonds. Well known
modelling techniques are available to the person skilled in the art for the determination of the
distance. (See for example J.Med.Chem. 1994, 37,2537-2551).
The term (l-8C)alkyl means a branched or unbranched alkyl group having 1-8 carbon atoms, for
example methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, hexyl and octyl. Methyl
and ethyl are preferred alkyl groups.
The term "prodrug" means a compound which is metabolized in the body into the active
compound, e.g. a compound in which the basic moiety (such as an amino or benzamidrno

group) in the GpIIb/IIIa antagonist residue of the compound of formula A is protected, e.g. by a
hydroxy, (1-6C)alkoxy or (1-6C)alkoxycarbonyl group.
Solvates according to the invention include hydrates.
The compounds of the present invention can be prepared by optionally modifying earlier
described GPIIb/IIIa antagonists which are e.g. derived from Ro 435054, RWJ 50042 or SC
54701 (the pharmacologically active form of xemilofiban), tirofiban, lamifiban, or analogues
thereof, with amino acids, peptidomimetics or additional functional groups (e.g. -COOH, -NH2,
-SH, -OH or the like) using methods generally known in the art. An example of the synthesis of
such a modified RGD-analog is described in Bioorganic Chemistry 29, 357-379 (2001), where
the compound is suggested as a potential vector for targeted drug delivery. According to the
invention, the optionally modified GPIIb/IIIa antagonist part (a) is coupled directly to an
oligosaccharide or (b) is coupled to an oligosaccharide-spacer residue or (c) is coupled to a
spacer, which is subsequently is coupled to an oligosaccharide-spacer-residue (e.g. by methods
known from WO 99/65934; WO 01/42262). Any suitable oligosaccharide may be used for this
purpose, for example oligosaccharides known in literature (e.g. from EP 0,454,220 and EP
0,529,715, but not limited to these sources) or commercially available oligosaccharides. The
oligosaccharides may be phosphorylated at an appropriate point in time by methods known in
the' art, e.g. as described by Buijsman, R. et al. (Bioorg. Med. Chem. Lett. 1999,9, 2013-2018).

The coupling of the spacer to the oligosaccharide can for instance be performed by using the
methods described in EP 0,649,854.
Other examples of known GpIIb/IIIa antagonists which may serve as the (basis for the)
GpIIb/IIIa antagonist part of the compounds of the present invention (but not limited to these
examples): the compounds Ro 43 8857 (J. Med. Chem. 35, 4393 (1992)), Ro 48 3657, B1BL
12, FK 633, GR 144053, EMD 76 334, SR 121566, SB 208651, SC 54684, SC 52012, DMP
754, FR 158999, GR 200976, XV 788, MK 383 (tirofiban), RWJ 53308, ZD 2486, L 709780,
RGD 891, T 250, C 6822, BIBU 104, SB 214857, SC 57101, G 7453, TAK 029, XV 454, XV
459, L 734 217, DMP 802, SR 121787, TP 9201, DMP 757, SC 52012, RPR 109891, YM
68128, ME 3229, ME 3230, CT 50352, MK 852, S 1197, DMP 728, SC 57345, L 738 167, GR
233548, RO 438857, TA 993, YM 337, BIBW 194, BIBU 129, BIBW 98, tetrafibricin, L 703
014, BIBU 251, GR 91669, RG 13965, G 7446, PS 028, XR 300, NSL 9403, L 756568, S 1762,
L 746 223, L 767685, NSL 95301, G 4120, SB 207043, GR 83895, P246, L 739 758, XR 299,
SV 873, RWJ 50228, XQ 870, EF 5154, AR 0510, G 7570, G 7442, G 7464, RWJ 52656, TAK
024, MS 180, MS 28168, XU 063, XU 065, L 734115, SM 20302, TS 943, NSL 96184, UR
12947, XU 057, L 750034, UR 3216, UR 2922, CP 4632, AR 0598, SC 79992, SC 4992, RGD
039, ME 3277, T 250, SC 57099B, SKF 106760, SKF 107260, RWJ 52654, PSA 0613, CGH
400, NSL 95317, XT 111, RWJ 27755, L 736622, SC 46749, SM 20302, YM 570029, CY
311176 and compounds described in EP 0,529,858, WO 96/20172, EP 0,496,378, EP
0,530,505, Bioorg. & Med. Chem. 3, 539 (1995), WO 93/08174, J.Am.Chem.Soc. U5, 8861
(1993), J. Med. Chem. 43, 3453 (2000), Bioorg. Med. Chem. 3,337 (1995), US 5,239,113, US
5,344,957, US 5,973,003, US 5,703,125, WO 96/37464, WO 93/07867, US 5,378,712, EP
445,796, US 5,273,982, US 5,770,575,. WO 01/602813, EP 656,348, US 5,726,185, EP
505,868, EP 560,730, US 5,561,112, EP 513,675, US 5,574,016, WO 94/09030, EP 478,363,
US 5,292,756, US 5,206,373, WO 93/16994, US 5,312,923, EP 743,302, US 5,658,929, US
5,880,136, US 5,814,643 and US 6,040,317.
Also included into the present invention are compounds comprising newly designed GpIIb/IIIa
antagonist residues mimicking the RGD and/or K(QA)GD fragment of fibrinogen, typically
comprising an optionally esterified carboxylate moiety and a basic moiety located within the
residue at a distance of 10-20 A from each other.

The peptide coupling, a procedural step in the above described method to prepare the
compounds of the invention, can be carried out by methods commonly known in the art for the
coupling - or condensation - of peptide fragments such as by the azide method, mixed anhydride
method, activated ester method, the carbodiimide method, or, preferably, under the influence of
ammonium/uronium salts like TBTU, especially with the addition of catalytic and racemisation
suppressing compounds like N-hydroxysuccinimide and N-hydroxybenzotriazole. An overview
is given in The Peptides, Analysis, Synthesis, Biology, Vol 3, E. Gross and J. Meienhofer, eds.
(Academic Press, New York, 1981).
Amine functions present in the compounds may be protected during the synthetic procedure by
an N-protecting group, which means a group commonly used in peptide chemistry for the
protection of an a-amino group, like the tert-butyloxycarbonyl (Boc) group, the
benzyloxycarbonyl (Z) group, the 9-fluorenylmethyloxycarbonyl (Fmoc) group or the phthaloyl
(Phth) group, or may be introduced by demasking of an azide moiety. An overview of amino
protecting groups and methods for their removal is given in the above mentioned The Peptides,
Analysis, Synthesis, Biology, Vol 3.
Amidine functions, if present, can be left unprotected in the coupling step, or can be protected
using carbamate such as allyloxycarbonyl or benzyloxycarbonyl. The amidine function is
preferably introduced under mild conditions by using the l,2,4-oxadiazolin-5-one moiety as the
precursor.
Carboxylic acid groups may be protected by a group commonly used in peptide chemistry for
the protection of an α-carboxylic acid group, such as a tert-butyl ester. The carboxylic acid
group of the modified GPIIb/IIIa antagonist is preferably protected as a benzyl ester. Removal
of the protecting groups can take place in different ways, depending on the nature of those
protecting groups. Usually deprotection takes place under acidic conditions and in the presence
of scavengers or reductive conditions such as catalytic hydrogenation.
A prerequisite for conjugation of the GPIIb/IIIa antagonist to an oligosaccharide is the presence
of an orthogonally reactive anchoring group, such as a carboxylate group, which can be coupled
directly to an oligosaccharide residue or to an oligosaccharide-spacer derivative or via a spacer

to an oligosaccharide-spacer derivative. To allow such conjugation in most cases additional
modification of the GPIIb/IIIa antagonist is necessary.
Construction of the spacer-derived building blocks en route to compounds of the formula I can
be achieved in various ways using methods known in the art, either in a linear fashion by the
step-wise introduction of amino acids, their derivatives or peptidomimetics, or in convergent
manner by block-coupling of intermediate constructs.
The compounds of the invention, which can occur in the form of a free base, may be isolated
from the reaction mixture in the form of a pharmaceutically acceptable salt. The
pharmaceutically acceptable salts may also be obtained by treating the free base of formula (I)
with an organic or inorganic acid such as hydrogen chloride, hydrogen bromide, hydrogen
iodide, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, maleic acid,
malonic acid, methanesulphonic acid, fumaric acid, succinic acid, tartaric acid, citric acid,
benzoic acid, ascorbic acid and the like.
The compounds of this invention may possess chiral carbon atoms, and may therefore be
obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing
diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g.
crystallization of salts which are obtained from optically active acids and the racemic mixture,
or chromatography using chiral columns. For diastereomers straight phase or reversed phase
columns may be used.
The compounds of the invention may be administered enterally or parenterally. The exact dose
and regimen of these compounds and compositions thereof will necessarily be dependent upon
the needs of the individual subject to whom the medicament is being administered, the degree
of affliction or need and the judgement of the medical practitioner. In general parenteral
administration requires lower dosages than other methods of administration which are more
dependent upon absorption. However, the daily dosages are for humans preferably 0.0001-10
mg per kg body weight, more preferably 0.001-1 mg per kg body weight.
The medicament manufactured with the compounds of this invention may also be used as
adjuvant in (acute) anticoagulant therapy. In such a case, the medicament is administered with
other compounds useful in treating such disease states, such as aspirin, clopidogrel or statins.

Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the standard reference,
Gennaro et al., Remington's Pharmaceutical Sciences, (18th ed, Mack Publishing Company,
1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture) the
compounds may be compressed into solid dosage units, such as pills, tablets, or be processed
into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds
can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection
preparation, or as a spray, e.g. for use as a nasal spray.
For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants,
polymeric binders and the like is contemplated. In general any pharmaceutically acceptable
additive which does not interfere with the function of the active compounds can be used.
Suitable carriers with which the compositions can be administered include lactose, starch,
cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.
The invention is further illustrated by the following examples.
EXAMPLES
Abbreviations used
Aq. .. = aqueous
Ala = alanine
Alloc = allyloxycarbonyl
Asp = (L)-aspartate
ATIII = antithrombin III
Bn = benzyl
Boc = tert-butyloxycarbonyl
Bt = benzotriazole
r-Bu = tert-butyl
DCM = dichloromethane
DiPEA= N,N-diisopropylethylarnine
DMAP= N,N-dimethylaminopyridine
DMF = N,N-dimethylformarnide
EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride




Scheme 1
Ethyl N-(4-Cyano-benzoyI)-β-alanine (2)
A suspension of ethyl β-alanine.HCl (2.81 g, 18.3 mmol) and 4-cyano-benzoyl chloride (3.03 g,
18.0 mmol) in DCM (30 mL) was stirred overnight at RT. The mixture was diluted with EtOAc,
washed with aq. citric acid (3%), sat. aq. NaHCO3 and H2O. Drying (MgSO4) and evaporation
of the solvent gave compound 2 (2.52 g, 56%).
N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoyl)-β-alanine ethyl ester (3)

Compound 2 (2.50 g, 10.2 mmol) was dissolved in EtOH (70 mL). Hydroxylamine.HCl ( 0.99
g, 14.3 mmol) and Et3N (2.49 mL, 17.3 mmol) were added and the mixture was stirred
overnight at RT. The reaction was complete after refluxing for-1.5 h. The product precipitated
by cooling and concentration (~10 mL) and was filtered, rinsed with EtOH and dried under
vacuum.
Traces of H2O were removed from the crude hydroxybenzamidine (2.8 g, 10 mmol) by
coevaporation with pyridine (3 x 15 mL) after which it was dissolved in pyridine (100 mL).
Ethyl chloroformate (1.43 mL, 15.0 mmol) was added and the mixture was stirred at reflux
temperature for 18h. The reaction was quenched by pouring into H2O (200 mL), which was
followed by extraction with EtOAc (3 x 75 mL). The organic layer was dried (MgSO4) and
concentrated. Residual pyridine was removed by evaporation with toluene. Recrystallization
(EtOAc/hexane) afforded the product as a slightly pink-colored solid (2.48 g, 81%).
N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoyl)-β-alanine(4)
Compound 3 (0.60 g, 2.0 mmol) was dissolved in a mixture of l,4-dioxane/H2O (10 mL, 1/1,
v/v). Aq. NaOH (2.0 mL, 2N) was added and the mixture was stirred for 3h at RT. The reaction
mixture was neutralized with Dowex H+ and filtered. The filtrate was concentrated under
reduced pressure and the resulting white solid was dried under vacuum. Yield 0.51 g (93%).
1-Azido-1-deoxy-tetraethylene glycol (5)
Tetraethylene glycol (90 g, 0.46 mol) was cooled to 0°C and a solution of sodium hydroxide
(3.0 g, 75 mmol) in 10 mL of H2O was added, followed by the addition of 100 mL of THE The
mixture was vigourously stirred for 30 rnin. p-Toluenesulfonylchloride (8.8 g, 46 mmol) in. 50
mL of THF was slowly added at 0°C under vigourous stirring to keep a homogeneous mixture.
After stirring for 5h, the mixture was poured into 400 mL of H2O. The solution was extracted
with DCM (4 x 20 mL). The combined organic layers were washed with H2O (20 mL) and brine
(20 mL) and dried (MgSO4). After filtration the solvents were removed under reduced pressure
to afford the monotosylate as an oil.
The crude compound was dissolved in a mixture of EtOH (120 mL) and H2O (18 mL). Sodium
azide (3.25 g, 50 mmol) was added and the mixture was stirred at 60°C overnight. The EtOH

was evaporated in vacuo (50 mbar, 50°C) and 150 mL of EtOAc was added. The solution was
washed with brine (20 mL) and dried over MgSO4. After filtration the solvent was removed
under reduced pressure (50 mbar, 50°C), to give the azide 5 as an oil (6.25 g, 62% yield over
the two steps).
tert-Butyl 15-aza-3,6,9,12-tetraoxa-pentadecanoate (6)
Compound 5 (1.15 g, 5.25 mmol) was dissolved in toluene (10 mL). To the solution was added
a 50% aq. solution of sodium hydroxide (12.5N, 5 mL), tetrabutylammonium hydrogensulfate
(0.34 g, 1.05 mmol) and tert-butyl bromoacetate (4.27 mL, 26.3 mmol). The suspension was
vigourously stirred at RT. After 3h the mixture was diluted with EtOAc (10 mL), washed with
brine (10 mL), hydrochloric acid (2N, 5 mL) and H2O (5 mL). The aq. layers were extracted
with EtOAc after which the combined organic layers were dried (MgSO4). After concentration,
the crude product was purified by silica gel (20 g) column chromatography (eluent: heptane →
EtOAc, 1/0→0/1). Concentration of the appropriate fractions afforded the azide as a pale yellow
oil (1.25 g, 71%). Rf 0.8 (EtOAc/MeOH, 95/5, v/v).
Reduction of the azide was effected by hydrogenation over 10% Pd/C (100 mg) in EtOH (30
mL) containing acetic acid (0.2 mL). After 4h the catalyst was removed by filtration over two
layers of Whatman GF/A filter. The filtrate was concentrated, dissolved in DCM (25 mL) and
treated with Argonaut MP carbonate resin to remove the acetic acid. The resin was removed by
filtration and evaporation of the solvent gave compound 6 in a yield of 1.17 g (100%).
tert-Butyl(N-benzyloxycarbonyi-L-phenylalanyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate
(7)
Compound 6 (3.60 g, 11.7 mmol) and Z-Phe-OH (3.51 g, 11.7 mmol) were dissolved in DCM
(30 mL). HOBt (1.39 g,. 11.7 mmol), EDC (2.83 g, 14.0 mmol) and TEA (3.38 mL, 23.4 mmol)
were subsequently added and the solution was- allowed to stir overnight. The reaction mixture
was diluted with EtOAc (30 mL) and rinsed with sat. aq. NaHCO3 (2 x 10 mL), 5% aq. citric
acid (10 mL) and H2O (10 mL). The organic phase was dried (MgSO4) and concentrated under
reduced pressure. Purification was effected by silica gel (75g) column chromatography (eluent:

EtOAc→ EtOAc/MeOH, 95/5, v/v), to give compound 7 as a colorless oil (4.95 g, 72%). Rf 0.5
(EtOAc/MeOH, 95/5, v/v).
tert-Butyl(L-phenylalanyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(8)
Compound 7 (2.42 g, 4.11 mmol) was dissolved in EtOH (100 mL) containing acetic acid (0.4
mL) and stirred under an atmosphere of H2 in the presence of 10% Pd/C (0.1 g). After 16h, the
mixture was filtered and concentrated. Acetic acid was removed by repeated concentration of
the product in toluene and treatment with Argonaut MP carbonate resin (1.3 g) in DCM (20
mL). Filtration of the resin and evaporation of the solvent afforded compound 8 as a colorless
oil (1.69 g, 90%). Pvf 0.05 (EtOAc/MeOH, 95/5, v/v).
tert-Butyl(N-tert-butyloxycarbonyl-O-benzyl-L-aspartyI-L-phenylalanyl)-15-aza-3,6,9,12-
tetraoxa-pentadecanoate (9)
Compound 8 (1.69 g, 3.71 mmol) was coupled to Boc-AspBn-OH (1.20 g, 3.71 mmol) as
described earlier for the synthesis of compound 7, using NMM (0.84 mL, 7.4 mmol at pH 8.3)
instead of TEA. After work-up, a yellow oil was obtained which was purified by silica gel (50g)
column chromatography (eluent: EtOAc/heptane, 2/1→ EtOAc/MeOH, 95/5, v/v), to give
compound 9 as a colorless oil (2.30 g, 82%). Rf 0.6 (DCM/MeOH, 9/1, v/v).
tert-Butyl(O-benzyl-L-aspartyl-L-phenylalanyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate
(10)
Boc-protected compound 9 (1.09 g, 1.43 mmol) was treated with a 1.5 N solution of
hydrochloric acid in EtOAc (10 mL). After stirring for 100 rnin. at RT the reaction mixture was
cooled (0°C) and neutralized by pouring into a cooled solution of sat. aq. NaHCO3 (15 mL).
The aq. layer was extracted with EtOAc (2 x 10 mL), the combined organic layers were washed
with H2O (10 mL) and dried (MgSO4). Concentration afforded crude compound 10 which was
used without purification in the next reaction. Rf 0.2 (EtOAc/MeOH, 95/5, v/v).

tert-Butyl ({N-(4-[1,2,4-oxadiazol-5-onyl]-benzoyl)-β-alanyl}-O-benzyI-L-aspartyl-L-
phenylalanyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(11)
Carboxylic acid 4 (0.12 g, 0.42 mmol) and amine 10 (crude, max. 0.40 mmol) were dissolved in
DMF (5 mL). HOBt (50 mg, 0.42 mmol), EDC (94 mg, 0.50 mmol) and NMM (88 µL, 0.61
mmol) were subsequently added and the solution was allowed to stir overnight. The brown
solution was diluted with a mixture of EtOAc and 2-butanol (10 mL, 1/1, v/v) and extracted
with 3 % citric acid (5 mL), sat. NaHCO3 (5 mL) and brine (5 mL). The aq. phases were
consecutively extracted with EtOAc and the combined organic layers dried over MgSC>4.
Removal of the solvent gave crude compound 11 (0.27 g, 78%), which was used without
purification in the next reaction. Rf 0.2 (DCM/MeOH, 9/1, v/v).
({N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoyl)-β-alanyl}-O-benzyl-L-aspartyl-L-phenylalanyl)-
15-aza-3,6,9,12-tetraoxa-pentadecanoic acid (12)
Compound 11 (0.2 g, crude, max. 0.22 mmol) was stirred in a mixture of DCM (5 mL) and
TFA (3 mL). After 2h the solution was concentrated and remains of TFA were removed by
repeated concentration in toluene (3x5 mL). The product was purified by preparative HPLC-
MS to give compound 12 in pure form (0.11 g, 56% over the three steps from 9).
tert-Butyl({N-(4-amidinobenzoyl)-β-alanyl}-L-aspartyl-L-phenylaIanyl)-15-aza-3,6,9,12-
tetraoxa-pentadecanoate (I)

Compound 11 (65 mg, crude, max. 71 µmol) was dissolved in a mixture of EtOH (10 mL), H2O
(2 mL) and acetic acid (0.1 mL). 10% Pd/C (50 mg) was added and the mixture was stirred
overnight under an atmosphere of H2 gas. The catalyst was removed by filtration over two
layers of Whatman GF/A filter and the filtrate was concentrated. The product was obtained in
pure form by applying preparative HPLC-MS and lyophilization. Yield 7.7 mg (14%. ESI-MS:
787 [M+H]+.

General procedure for conjugation of spacer-derived GPIIb/IIIa antagonists 12, 23, 31,
33,43,50,52,54,62 to pentasaccharide 63, see Scheme 8 and 9
The spacer-derived carboxylic acid (33 umol) (i.e. compound 12, 23, 31, 33, 43, 50, 52, 54 or
62) was dried by coevaporation with DMF (2x2 mL), dissolved in DMF (1 mL) and stirred in
the presence of TBTU (10.5 mg, 33 umol) and DiPEA (5.7 µL, 33 µmol), under, an atmosphere
of N2. After 1h, the aminospacer-containing pentasaccharide 63 (56 mg, 31 µmol) was added.
The reaction mixture was stirred overnight at RT and analyzed by ion exchange (Mono-Q) and
reversed phase (Luna C18) chromatography. After complete consumption of the
pentasaccharide, the reaction mixture was concentrated ( The (crude) product (10 mg/mL in H2O) was deprotected by hydrogenation (H2) over 10% Pd/C
(an equal amount in weight was added with respect to the crude product). After 16h the solution
was degassed, filtered over a 0.45 µM HPLC filter and concentrated under reduced pressure
( buffer: H2O → 2M NaCl), followed by desalting with a Sephadex G25-column (H2O) and
lyophilization.
Methyl O-2,3-di-O-methyl-4-O-{({N-(4-amidinobenzoyl)-β-alanyl}-L-aspartyl-L-
phenylalanyl)-(15-aza-3,6,9,12-tetraoxa-pentadecanoyI)-(1-aza-4,7,10-trioxadodecyl)}-6-O-
sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1-
>4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-
idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-aIpha-D-glucopyranoside octakis
sodium salt (II)


The product was obtained by conjugation of 12 (21.4 mg, 24.8 µmol) with pentasaccharide 63
(42.4 mg, 23.6 µmol), purification and deprotection according, to the general procedure. White
solid, yield 34 mg (42%, 2 steps). 1H-NMR (D2O, 600 MHz, HH-COSY): δ 3.38-3.32 (8 x s,
34H, 8x OMe); ring D: 5.36 (d, 1H, H1), 4.18 (m, 1H, H6a), 4.04 (d, 1H, H6b), 3.80 (m, 1H,
H5), 3.47 (m, 1H, H3), 3.27 (dd, 1H, H2); ringE: 4.59 (d, 1H, H1), 3.82 (m, 1H, H4), 3.62 (m,
1H, H5), 3.45 (m, 1H, H3), 3.15 (m, 1H, H2); ring F: 5.27 (d, 1H, H1), 4.49 (t, 1H, H3), 4.33
(d, 1H, H6a), 4.20 (m, 1H, H2), 4.08 (m, 1H, H6b), 3.86 (t, 1H, H4); ring G: 4.92 (bs, 1H, H1),
4.54 (d, 1H, H5), 4.07 (m, 1H, H4), 3.74 (1H, dd, H3), 3.37 (m, 1H, H2); ring H: 4.99 (d, 1H,
HI), 4.25 (m, 1H, H2), 4.22 (m, 1H, H6a), 4.17 (dd, 1H, H6b), 3.92 (ddd, 1H, H5), 3.66 (t, 1H,
H4), 3.58 (m, 1H, H3); spacer: 3.96 (s, 2H, C(O)CH2O), 3.62-3.51 (m, 26H, 13 x CH2O), 3.33-
3.30 (m, 4H, OCH2CH2NHCH(O)CH2, OCH2CH2NHC(O)-Phe), 3.25 (m, 1H,
OCH2aCH2NHC(O)-Phe), 3.17 (m, 1H, OCH2bCH2NHC(O)-Phe); peptide: 7.84 (d, 2H, Harom
benzamidine), 7.79 (d, 2H, Harom benzamidine), 7.25 (t, 2H, Harom Phe), 7.20 (t, 1H, Harom Phe),
7.09 (d, 2H, Harom Phe), 4.51 (dd, 1H, CH Asp), 4.38 (t, 1H, CH Phe), 3.60 (m, 2H, CH2N β-
Ala), 2.83 (dAB, 2H, CH2Phe), 2.52 (t, 2H, C(O)CH2 (3-Ala), 2.44 (dAB, 2H, CH2 Asp).
ESI-MS: Calculated: 2511.4; found: m/z 1267.9 [M+H+Na]2+, 1256.9 [M+2H]2+, 1245.9
[M+2H-Na]2+, 852.9 rM+H+2Na]3+, 845.5 [M+2H+Na]3+, 838.3 [M+SH]3+, 639.9
[M+2H+2Na]4+, 1232.8 [M-2Na]2-, 1221.7 [M-3Na+H]2-, 1210.8 [M-4Na+2H]2-, 1199.8 [M-
5Na+3H]2-, 814.1 [M-3Na]3-.



Scheme 2
4-(tert-Butyloxycarbonylamino)-benzonitriIe(14)
4-Aminobenzonitrile (13, 2.95 g, 25 mmol) and di-tert-butyl dicarboriate (8.0 mL, 35 mmol)
were stirred in a mixture of THF (50 mL) and pyridine (4 mL) at 75°C. After 16h the red
coloured reaction mixture was cooled, diluted with EtOAc (30 mL) and washed with
hydrochloric acid (1N, 15 mL), sat. aq. NaHCO3 (15 mL) and brine (15 mL). The organic layer
was dried (MgSO4) and concentrated. The dark brown oil was purified by silica gel (75g)
column chromatography (eluent: EtOAc/heptane, 1/5→1/2, v/v) which afforded compound 14
as an orange oil (3.25 g, 60%), containing -30% bis-Boc protected product. The latter mixture
was used without further purification in the next reaction.
4-(1,2,4-Oxadiazol-5-onyI)-aniline (16)
Compound 14 (3.25 g, 14.9 mmol) was converted into oxadiazolone 15 as described earlier for
the preparation of compound 3. The resulting crude light orange solid was stirred for 5h in a
mixture of DCM (15 mL) and TFA (8 mL). Concentration under reduced pressure and
evaporation with toluene (2 x 10 mL) gave a brown solid which was purified by silica gel (40g)
column chromatography (eluent: EtOAc) to yield compound 16 (1.81 g, 69% over the three
steps). Rf 0.5 (EtOAc).
N-[4-(1,2,4-Oxadiazol-5-onyl)-phenyl]-succinamic acid (17)
Aniline derivative 16 (0.89 g, 5.0 mmol) was dissolved in pyridine (25 mL). Succinic anhydride
(0.75 g, 7.5 mmol) was added together with 4-iV,iV,-dimethyIaminopyridine (63 mg, 0.5 mmol)
and the mixture was stirred overnight at 100°C. The solution was concentrated under reduced
pressure and the pale brown solid was recrystallized from a mixture of EtOH/MeOH/H2O (60
mL, 5/35/1, v/v/v). After filtration, the mother liquid was concentrated to -10 mL to give
carboxylic acid 17 as a solid which was dried under vacuum at 50°C for 16h. Yield 1.28 g
(92%). Rf 0.3 (DCM/MeOH/AcOH, 100/10/1, v/v/v).

tert-Butyl(N-tert-butyloxycarbonyl-O-benzyl-L-aspartyl)-15-aza-3,6,9,12-tetraoxa-
pentadecanoate (18)
Compound 6 (0.94 g, 3.1 mmol) and Boc-AspBn-OH (0.99 g, 3.1 mmol) were dissolved in DCM
(8 mL). HOBt (0.36 g, 3.1 mmol), EDC (0.74 g, 3.8 mmol) and NMM (0.34 mL, 6.2 mmol)
were subsequently added and the solution was allowed to stir overnight. The reaction mixture
was diluted with EtOAc (10 mL) and rinsed with sat. aq. NaHCO3 (2x5 mL), 5% aq. citric acid
(5 mL) and H2O (5 mL). The organic phase was dried (MgSO4) and concentrated under reduced
pressure. Purification was established by silica gel (20g) column chromatography (eluent:
EtOAc/heptane, 3/1 →1/0, v/v), to give compound 18 as a colorless oil (1.15 g, 61%). Rf 0.7
(EtOAc/MeOH, 95/5, v/v).
tert-Butyl(O-benzyl-L-aspartyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(19)
Compound 18 (0.51 g, 0.83 mmol) was treated during 45 min according to the same reaction
conditions and work-up procedure as described earlier for the synthesis of compound 10.
Product 19 was used in the next reaction without further purification.
tert-Butyl N-{[4-(1,2,4-oxadiazol-5-onyl)-phenyl]-succinamyl-(O-benzyl-L-aspartyl)}-15-
aza-3,6,9,12-tetraoxa-pentadecanoate(20)
Compound 17 (0.30 mmol) and 19 (0.30 mmol) were coupled as described earlier for the
synthesis of 11. Crude compound 20 (0.17 g, 75%) was used without further purification in the
next reaction. Rf 0.3 (DCM/MeOH, 9/1, v/v).
tert-Butyl N-{(4-benzarnidino)-succinamyl}-L-aspartyl-15-aza-3,6,9,12-tetraoxa-
pentadecanoate (III)


Catalytic hydrogenation of 20 (32 mg, 41 µmol) was effected in a mixture of EtOH/H2O/AcOH.
Filtration, concentration and purification by preparative HPLC-MS gave spacer derivative III as
a white solid. Yield 20 mg (77%). ESI-MS: 640 [M+H]+.
tert-Butyl N-[4-(1,2,4-oxadiazol-5-onyI)-phenyl]-succinamyl-(O-benzyl-L-aspartyl-L-
phenylalanyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate (22)
Oxadiazolone 17 (24 mg, 88 µmol) and crude amine 10 (max. 58 mg, 88 µmol) were coupled as
described earlier for the synthesis of compound 11. Crude product 22 was used in the next
reaction without further purification. Rf 0.6 (DCM/MeOH, 9/1, v/v).
N-[4-(1,2,4-Oxadiazol-5-onyl)-phenyI]-succinamyl-(O-benzyl-L-aspartyl-L-phenylalanyI)-
15-aza-3,6,9,12-tetraoxa-pentadecanoic acid (23)
Crude compound 22 (max. 88 µmol) was stirred in a mixture of DCM (1.0 mL) and TFA (1.0
rnL). After 2h the solution was concentrated and the product was purified by preparative HPLC.
Concentration of the appropriate fractions afforded compound 23 in a yield of 31 mg (41% over
the last three steps).
tert-Butyl N-{(4-benzarmdino)-succinamyl}-L-aspartyl-L-phenylalanyl-15-aza-3,6,9,12-
tetraoxa-pentadecanoate (IV)


Crude compound 22 (max. 80 mg, 87 µmol) was deprotected by catalytic hydrogenation over
10% Pd/C (50 mg) in a mixture of EtOH (7 mL) and H2O (1 mL). After 24h, the mixture was
filtered and concentrated and the crude product was purified by preparative HPLC. Compound
IV was isolated as a white foam after lyophilization in a yield of 10.5 mg (15%). ESI-MS: 787
[M+H]+.
Methyl O-2,3-di-O-methyl-4-O-{[N-(4-benzamidyl)-succiriamyl]-L-aspartyl-L-
phenylalanyl)-(15-aza-3,6,9,12-tetraoxa-pentadecanoyl)-(1-amino-4,7,10-trioxadodecyl)}-
6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-
(1->4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-
idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranoside octakis
sodium salt (V)

Conjugation of carboxylic acid 23 (30.0 mg, 34.8 µmol) to pentasaccharide 63 (59.4 mg, 56.4
µmol), followed by purification arid deprotection was effected according to the general
procedure. Conjugate V was obtained as a white solid, yield 37.5 mg (48%, 2 steps).
1H-NMR (D2O, 600 MHz, HH-COSY): δ 3.43-3.32 (8 x s, 34H, 8x OMe); ringD: 5.36 (d, 1H,
H1), 4.21 (m, 1H, H6a), 4.05 (d, 1H, H6b), 3.79 (m, 1H, H5), 3.48 (m, 1H, H3), 3.33 (m, 1H,

H4), 3.20 (dd, 1H, H2); ring E: 4.58 (d, 1H, H1), 3.79 (m, 1H, H4), 3.65 (m, 1H, H5), 3.48 (m,
1H, H3), 3.15 (m, 1H, H2); ring F: 5.26 (d, 1H, H1), 4.48 (t, 1H, H3), 4.32 (d, 1H, H6a), 4.22
(m, 1H, H6b), 4.15 (dd, 1H, H2), 4.08 (m, 1H, H5), 3.84 (t, 1H, H4); ring G: 4.98 (bs, 1H, H1),
4.55 (d, 1H, H5), 4.07 (m, 1H, H4), 3.73 (1H, dd, H3), 3.37 (m, 1H, H2); ring H: 4.98 (d, 1H,
H1), 4.25 (dd, 1H, H2), 4.21 (m, 1H, H6a), 4.17 (m, 1H, H6b), 3.92 (ddd, 1H, H5), 3.66 (t, 1H,
H4), 3.58 (m, 1H, H3); spacer: 3.96 (s, 2H, C(O)CH2O), 3.61-3.51 (m, 26H, 13 x CH2O), 3.38-
3.30 (m, 4H, OCH2CH2NHCH(O)CH2, OCH2CH2NHC(O)-Phe), 3.19 (m, 1H,
OCH2aCH2NHC(O)-Phe), 3.12 (m, 1H, OCH2bCH2NHC(O)-Phe); peptide: 7.69 (d, 2H, Harom
benzamidine), 7.63 (d, 2H, Harom benzamidine), 7.25 (t, 2H, Harom Phe), 7.20 (t, 1H, Harom Phe),
7.09 (d, 2H, Harom Phe), 4.44 (dd, 1H, CH Asp), 4.38 (t, 1H, CH Phe), 2.90 (dAB, 2H, CH2
Phe), 2.75 (m, 2H, CH2 succinyl), 2.55 (m, 2H, CH2 succinyl), 2.43 (dAB, 2H, CH2 Asp).
ESI-MS: Calculated: 2511.4; found: m/z 1267.8 [M+H+Na]2+, 1256.8 [M+2H]2+, 1245.8
[M+2H-Na]2+, 852.8 [M+H+2Na]3+, 845.5 [M+2H+Na]3+, 838.2 [M+3H]3+, 639.9
[M+2H+2Na]4+, 1232.7 [M-2Na]2-, 1221.7 [M-3Na+H]2-, 1210.7 [M-4Na+2H]2-, 1199.7 [M-
5Na+3H]2-, 814.1 [M-3Na]3-.



Scheme 3
Ethyl N-3-[4-(N-tert-butyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotate (26)
A mixture of 3-(N-Boc-piperidyl)-propionic acid (24,1.00 g, 3.89 mmol), TBTU (1.38 g, 4.28
mmol) and DiPEA (2.70 mL, 15.6 mmol) in DCM (20 mL) was stirred for 5 min. Next, ethyl
(R)-nipecotate (L)-tartrate salt (25, 0.61 g, 3.89 mmol) was added and the solution was stirred
for 2h. The reaction was quenched with 5% aq. NaHCO3 (20 mL), washed with 3% aqeous
citric acid (20 mL) and H2O (20 mL). The aq. phases were extracted with DCM (3 x 10 mL) and
the combined organic layers were dried (MgSO4). Compound 26 (2.34 g) was obtained as a
brown-yellowish oil, which was used without further purification in the next reaction. Rf 0.7
(EtOAc/MeOH, 93/7, v/v).
Ethyl N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotate (28)
Crude compound 26 (max. 3.89 mmol) was stirred in a mixture of DCM (10 mL) and TFA (10
mL). After 1h the solution was concentrated under reduced pressure. Traces of TFA were
removed by repeated concentration in toluene (3 x 10 mL). Subsequently, crude piperidrne
derivative 27 was dissolved in DCM (10 mL), followed by the addition of DiPEA (2.02 mL,
11.7 mmol) and (iV)-benzyloxycarbonyloxy-succinidmide (1.94 g, 7.79 mmol). After stirring
overnight the reaction mixture was diluted with EtOAc (20 mL), rinsed with sat. aq. NaHCO3
(10 mL), 3% aq. citric acid and H2O (10 mL). The organic phase was dried over MgSO4 and
concentrated furnishing Z-protected compound 28 as a colorless oil. (1.37 g, 82% over the three
steps). Rf 0.5 (EtOAc).
N-3-[4-(N-beuzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotic acid(29)
A solution of compound 28 (0.35 g, 3.14 mmol) in 1,4-dioxane (20 mL) and 0.5M NaOH (20
mL) was stirred for 1h. Neutralization by Dowex 50 WX4-H+ ion-exchange, filtration and
concentration afforded carboxylic acid derivative 29.as a slightly yellowish colored oil (1.27g,
100%). Rf 0.05 (EtOAc).


tert-Butyl {N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotyl}-(O-
benzyl-L-aspartyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(30)
A mixture of compound 29 (0.24 g, 0.60 mmol) and crude 19 (max. 0.60 mmol), together with
EDC (0.14 g, 0.72 mmol) and NMM (90µL, 0.78 mmol) in DCM (5 mL) was stirred for 5h. The
solution was diluted with DCM (10 mL) and washed with sat. aq. NaHCO3 (10 mL), 3% aq.
citric acid and H2O (10 mL). The organic phase was dried over MgSO4 and concentrated. Crude
30 was purified by silica gel (5g) column chromatography (eluent: EtOAc/heptane/MeOH,
2/1/0→9/0/1, v/v/v). Yield 0.29 g (53%). Rf 0.6 (EtOAc/MeOH, 7/1, v/v).
{N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotyl}-(O-benzyl-L-
aspartyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoic acid (31)
Compound 30 (0.29 g, 0.32 mmol) was stirred in a mixture of DCM (5 mL) and TFA (5 mL).
After 3h the mixture was concentrated and repeatedly evaporated on toluene (3 x 10 mL). The
product was purified by preparative HPLC, to give 31 in a yield of 0.23 g (86%).
tert-Butyl{N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotyl}-(O-
benzyl-L-aspartyl)-L-phenyIalanyl-15-aza-3,6,9,12-tetraoxa-pentadecanoate(32)
Compound 29 (0.17g, 0.42 mmol) and crude 10 (0.25g, max. 0.38 mmol) were dissolved in
DCM (5 mL). HOBt (59 mg, 0.46 mmol), EDC (94 mg, 0.49 mmol) and NMM (88 µL, 0.63
mmol) were subsequently added and the solution was allowed to stir overnight. The reaction
mixture was diluted with EtOAc (10 mL) and rinsed with sat. aq. NaHCO3 (2x5 mL), 5% aq.
citric acid (5 mL) and H2O (5 mL). The organic phase was dried (MgSO4) and concentrated
under reduced pressure. Purification was effected by silica gel (5g) column chromatography
(eluent: DCM/MeOH, 1/0→ 95/5, v/v), to give compound 32 as a colorless oil (0.24 g, 60%).
Rf 0.3 (DCM/MeOH, 95/5, v/v).

{N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotyl}-(O-benzyl-L-
aspartyl)-L-phenylalanyl-15-aza-3,6,9,12-tetraoxa-pentadecanoic acid(33)
Compound 32 (0.20 g, 0.19 mmol) was deprotected and purified as described earlier for the
synthesis of compound 31. Yield 50 mg (30%).
tert-Butyl{N-3-[4-(N-benzyloxycarbonyl)-piperidinepropionyl]-(R)-(-)-nipecotyl}-(O-
benzyl-L-aspartate (34)
To a stirred mixture of compound 29 (0.69 g, 1.72 mmol) and AspBn-O-t-Bu ester (0.54 g, 1.72
mmol) in DCM (5 mL) was added TBTU (0.58 g, 1.80 mmol) and DiPEA (0.90 mL, 5.4 mmol).
After 2h the reaction mixture v/as diluted with EtOAc (10 mL) and washed with sat. aq.
NaHCO3 (2x5 mL), 5% aq. citric acid (5 mL) and H2O (5 mL). The organic phase was dried
over MgSO4 and concentrated under reduced pressure. Silica gel (25g) column chromatography
(eluent: EtOAc/heptane, 1/2→ 1/0, v/v) afforded pure compound 34 as a light yellow oil (0.84
g,73%).Rf 0.6(EtOAc).
tert-Butyl N-{[N-3-(4-piperidmepropionyl)-(R)-(-)nipecotyl]-L-aspartyl}-15-aza-3,6,9,12-
tetraoxa-pentadecanoate (VII)

Compound 31 (35 mg, 39 µmol) was deprotected by catalytic hydrogenation over 10% Pd/C (30
mg) in a mixture of EtOH (5 mL) and H2O (1 mL). After 16h, the mixture was filtered over two
layers of Whatman GF/C filter and concentrated. Compound VII was isolated as a white foam
after lyophilization in a yield of 24 mg (92%). ESI-MS: 673 [M+H]+.
tert-Butyl N-{[N-3-(4-piperidinepropionyl)-(R)-(-)-nipecotyl]-L-aspartyl-L-phenylalanyl}-
15-aza-3,6,9,12-tetraoxa-pentadecanoate(IX)


Compound 32 (45 mg, 43 µmol) was deprotected as described earlier for the synthesis of
compound VII. Preparative HPLC and lyophilization afforded pure IX in a yield of 8.8 mg
(26%). ESI-MS: 820 [M+H]+.
1{N-3-[4-piperidinepropionyl]-(R)-(-)-nipecotyl}-L-aspartate (VI)

Compound 34 (47 mg, 71 µmol) was deprotected by stirring in a mixture of DCM (5 mL) and
TFA (5 mL). After 5h the mixture was concentrated and repeatedly evaporated on toluene (3x5
mL). The crude intermediate exposed to catalytic hydrogenation over 10% Pd/C (30 mg) in a
mixture of 1,4-dioxane (5 mL) and H2O (5 mL). After 3h, the mixture was filtered over two
layers of Whatman GF/C filter, which were subsequently rinsed with MeOH. The filtrate was
concentrated and purified by preparative HPLC to give pure compound VI as a white foam after
lyophilization in a total yield of 9.8 mg (39%). ESI-MS: 384 [M+H]+.
Methyl O-2,3-di-O-meth}1-4-O-{[N-3-(4-piperidmepropionyl)-(R)-(-)-nipecotyl-(L)-
asparryl]-(15-aza-3,6,9,12-tetraoxa-pentadecanoyl)-(1-aza-4,7,10-trioxadodecyI)}-6-O-
sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1-
>4)-O-2,3,6-tri-O-sulfo-aIpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-
idopyranuronpsyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranoside octakis
sodium salt (VIII)


Conjugation of carboxylic acid derivative 31 (51 mg, 59 µmol) to pentasaccharide derivative 63
(105 mg, 56 µmol), subsequent deprotection of the crude product and purification was executed
as described in the general procedure. Compound VIII was obtained in pure form after
lyophilization. Yield 101 mg (76% over the two steps). 1H-NMR (DA 600 MHz, HH-COSY,
mixture of rotamers): 5 5.39 (d, 1H, H-1 ring D), 5.31 (bs, 1H, H-1 ring F), 5.00 (d, 1H), 4.95
(bs, 1H), 4.61 (bs, 1H), 4.59 (d, 1H), 4.50 (m, 1H), 4,34 (d, 1H), 4.27-4.20 (m, 7H), 4.19-4.15
(m, 2H), 4.10-4.03 (m, 3H), 4.02 (s, 2H CH2 Ac spacer), 3.92 (ddd, 1H), 3.88 (ddd, 1H), 3.86-
3.75 (m, 7H), 3.71 (dd, 1H), 3.69-3.52 (m, 38H), 3.49-3.43 (m, 14H), 3.40-3.30 (m, 12H), 3.26-
3.11 (m, 4H), 2.96-2.89 (m, 3H), 2.77 (m, 1H), 2.68 (m, 1H).
ESI-MS: Calculated: 2397.4; found: m/z 1221.6 [M+2Na]2+, 1210.6 [M+H+Na]2+, 1199.6
[M+2H]2+, 822.1 [M+3Na]3+, 814.8 [M+2H+Na]3+, 807.4 [M+H+2Na]3+, 1175.7 [M-2Na]2-
1164.7 [M-3Na+H]2-, 1153.8 [M-4Na+2H]2-, 776.2 [M-3Na]3-, 768.9 [M-4Na+H]3-, 761.5 [M-
3Na+H]3-, 570.9 [M-5Na+H]4-, 576.4 [M-4Na]2-, 456.5 [M-5Na]5-.
Methyl O-2,3-di-O-methyl-4-O-{[N-3-(4-piperidinepropionyl)-(R)-(-)-nipecotyl-(L)-
aspartyl-(L)-phenylalanyl]-(15-aza-3,6,9,12-tetraoxa-pentadecanoyl)-(1-aza-4,7,10-
trioxadodecyl)}-6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-
glucopyranuronosyl-(1->4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-
methyl-alpha-L-idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-
glucopyranoside octakis sodium salt (X)


Compound 33 (32.4 mg, 32.8 uxnol) was conjugated to pentasaccharide 63 (56.0 mg, 31.2
µmol), deprotected and purified according to the general procedure to give product X as a white
powder. Yield 49 mg (62% over the two steps).
1H-NMR (D2O, 600 MHz, HH-COSY, 1:1 mixture of retainers): 5 7.32-7.19 (m, 5H, H-arom
Phe), 5.39 (d, 1H, H-1 ring D), 5.29 (d, 1H, H-l ring F), 4.99 (d, 1H), 4.93 (bs, 1H), 4.59 (d,
1H), 4.53 (s, 1H), 4.50-4.43 (m, 3H), 4.33 (d, 1H), 4.26-4.15 (m, 8H), 4.13-4.08 (m, 3H), 4.06-
4.02 (d, 1H), 3.98 (d, 1H CH2 Ac spacer), 3.94-3.78 (m, 9H), 3.74 (bs, 1H), 3.68-3.52 (m, 38H),
3.50-3.42 (m, 14H), 3.38-3.30 (m, 12H), 3.27-2.85 (m, 6H), 2.80-2.70 (m, 2H).
ESI-MS: Calculated: 2546.4; found: m/z 1273.2 [M+2H]2+, 1284.2 [M+H+Na]2+, 1295.2
[M+2Na]2+, 856.5 (M+2H+Na]3+, 863.8 [M+H+2Na]3+, 871.1 [M+3Na]3+, 1238.3 [M+H-
3Na]2-, 1227.3 [M+2H-4Na]2-, 1216.3 [M+3H-5Na]2-, 825.2 [M-3Na]3-, .817.9 [M-4Na+H]3-,
810.5 [M-5Na+2H]3-, 803.2 [M-6Na+3H]3-, 613.1 [M-4Na]4-, 607.7 [M-5Na+H]4-, 485.9 [M-
5Na]5-, 401.1 [M-6Na]6-.



Scheme 4
N-(4-[1,2,4-Oxadiazol-5-onyl]-phenyl-succinamyl-O-benzyl-L-aspartate(36)
To a stirred solution of compound 17 (1.0 g) in DMF (35 mL), under a N2 atmosphere, NMM
(351 µL) was added followed by isobutylchloroformate (440 µL). After 5 minutes Asp(Bn)(t-
Bu) (1.12 g) was added followed by DiPEA (586 µl) and DMAP (7.8 mg). After 1h H2O was
added and the mixture was extracted with EtOAc (twice). The organic layer was washed with a
sat. Nad-solution, dried on MgSO4 and concentrated under reduced pressure. Toluene was
added, after which product 35 precipitated. Heptane was added and the product was filltered of
in a 55 % yield. Rf= 0.5 (DCM / MeOH / AcOH 9:1:0,1)
To 35 (843 mg) a mixture of DCM and TFA (40 mL, 1:1, v/v) was added and stirred for 3
hours. Then the mixture was concentrated after addition of toluene to give 36 in quantitative
yield. Rf 0.2 (Tol/EtOH 8:2, v/v)
N-Allyloxycarbonyl-L-tyrosine (38)
To a stirred solution of tyrosine (10 g) in 4N aq. sodium hydroxide (30 mL) cooled in an ice
bath was slowly added allyl chloroformate (6.4 mL) in a peiiod of 20 minutes. Ten minutes
after completion of the addition the ice bath was removed and 4N aq. sodium hydroxide (30
mL) and H2O (5mL) were added. After two hours at RT MeOH (60 mL) was added. After an
additional two hours the reaction mixture was extracted with Et2O. The aq. mixture was cooled
in an ice bath, acidified to pH 2-3 using 36-38% hydrochloric acid and extracted with EtOAc.
De organic extract was dried (Na2SO4) and concentrated under reduced pressure to give Alloc-
Tyr-OH (38) as an oil (14.5 g, 88%). Rf 0.35 (DCM/ MeOH/AcOH, 89/10/1, v/v/v).
N-Allyloxycarbonyl-4-O-benzyl-L-tyrosine(39)
Alloc-Tyr-OH (38, 5.5 g) was dissolved in DMF (40 mL) and half the volume of DMF was
removed under reduced pressure. The residue was cooled at 0°C under a N2 atmosphere and
sodium hydride (60% disperse, 1.9 g) was added in small portions. The suspension was stirred

for one hour at 0°C. Then benzyl bromide (1.88 mL) in DMF (5 mL) was added. After one hour
at RT 2N hydrochloric acid (7 mL) was added and the mixture concentrated under reduced
presure. To the residue H2O (50 mL) was added, the pH was adjusted to nine using 2N aq.
sodium hydroxide and washed with a mixture of toluene and EtOAc. EtOAc was added and the
mixture acidified to pH 3 using 2N hydrochloric acid. The organic layer was separated, dried
(Na2SO4) and concentrated. The residu was purified using silica gel column chromatography
(DCM/MeOH/AcOH, 89/10/1, v/v/v). The product from the column was dissolved in EtOAc,
washed with H2O, dried (Na2SO4) and concentrated to give compound 39 (4.66 g, 77%). Rf 0.5
(DCM/MeOH/AcOH, 89/10/1, v/v/v).
tert-Butyl (N-allyloxycarbonyl-4-O-benzyl-L-tyrosyl)-15-aza-3,6,9,12-tetraoxa-
pentadecanoate (40)
To a stirred solution of alloc-Tyr(Bn)-OH (39, 0.61 g) and tert-butyl 15-amino-3,6,9,12-
tetraoxa-tetradecanoate (6) (0.54 g) in THF (2 mL) was added TBTU (0.7 g) and NMM (0.38
mL). After 16 hours tert-butyl 15-amino-3,6,9,12-tetraoxa-pentadecanoate (6) (0.18 g), TBTU
(0.1 g) were added and the pH adjusted to 7 (wet pH-paper) using NMM. After 3 days the
reaction mixture was filtered and the filtrate concentrated under reduced pressure. The residue
was purified using silica gel column chromatography (EtOAc/heptane/EtOH, 65/33/2 to 50/0/1,
v/v/v) to give the title compound 40 (0.7 g, 67%). Rf 0.25 (EtOAc/heptane/EtOH, 66/33/1,
v/v/v).
tert-Butyl 15-N-(4-O-benzyl-L-yrosyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate (41)
To a stirred solution of compound 40 (0.43 g) in DCM (15 mL) was added H2O (75 µL),
tributyltin hydride (0.4 mL) and PdCl2(PPh3)2 (12 mg). The reaction was monitred by TLC
(EtOAc/ ethanol, 25/1, v/v). After completion of the reaction the reaction mixture was cooled at
. 0°C, H2O (10 mL) added and acidified to pH 4 using 2 N hydrochloric acid. The organic layer
separated, dried (Na2SO4) and concentrated under reduced presure. The residue was purified
using silica gel column chromatography (gradient of DCM/ MeOH, 95/5 to DCM/MeOH/N-
methyl morpholine, 450/50/3) to give the title compound 41 (0.36 g, 78%). Rf 0.4 (DCM/
MeOH, 10/1, v/v).

15-N-{4-[1,2,4-Oxadiazol-5-onyl]-phenyl-succinamyl-(O-benzyl-L-aspartyl)-(4-O-benzyl-L-
tyrosyl)}-15-aza-3,6,9,12-tetraoxa-pentadecanoate(43)
Compound 41 (360 mg, 0.49 mmol) and 36 (241 mg, 0.5 mmol) were dissolved in THF (5 mL).
To this mixture, NMM (108 µl, 0.98 mmol) was added followed by TBTU. The mixture was
allowed to stir overnight. The solution was filtered on a PVDF (0.45 µm) filter. Purification was
effected by silica gel column chromatography (eluent: DCM→ DCM/MeOH, 95/5, v/v), to give
compound 42 as an oil (560 mg, 100%). Rf 0.5 (DCM/MeOH, 9/1, v/v). Hydrolysis of the
compound was carried out as described for the preparation of compound 12. Purification was
carried out by silica gel column chromatography (DCM/MeOH/AcOH, 90/10/3, v/v/v), to give
compound 43 (185 mg, 39%). 1H-NMR (MeOD, 400 MHz): 7.70 (AB, 4H, H benzamidine),
7.32 (m, 10H, Bn), 7.12 (d, 2H, Tyr), 6.88 (d, 2H, Tyr), 5.10 (d, 2H, CH, Bn), 5.00 (s, 2H, CH2
Benzyl), 4.67 (m, 1H), 4.48 (m, 1H), 4.06 (s, 2H), 3.61 (m, 10H), 3.52 (m, 2H), 3.41 (m, 2H),
3.32 (m, 2H), 3.10 - 2.40 (m, 8H).
Methyl O-2,3-di-O-methyl-4-O- tyrosyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoyl}]-12-aza-3,6,9-trioxa-dodecyl>-6-O-sulfo-
alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(l->4)-O-
2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-
idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranoside octakis
sodium salt (XI)
The conjugation of compound 43 (62.2 mg) with pentasaccharide 63 (110 mg), followed by
deprotection and purification was carried out as outlined in the general procedure. Yield 22 mg
(14% over the 2 steps). 1H-NMR (D2O, 600 MHz, HH-COSY): 5 3.54-3.30 (8 x s, 34H, 8x
OMe); ring D: 5.28 (d, 1H, H1), 4.14 (m, 1H, H6a), 3.97 (m, 1H, H6b), 3.72 (m, 1H, H5), 3.37
(m, 1H, H3), 3.24 (m, 1H, H4), 3.12 (m, 1H, H2); ring E:.4.50 (d, 1H, H1), 3.73 (m, 3H,
H3,4,5), 3.16 (m, 1H, H2); ring F: 5.16 (d, 1H, H1), 4.39 (m, 2H, H3,4), 4.24 (d, 1H, H6a),
4.14 (m, 1H, H6b), 4.00 (m, 1H, H2), 3.97 (m, 1H, H5); ring G: 4.84 (bs, 1H, H1), 3.97 (m, 1H,
H4), 3.66 (m, 2H, H2,3); ring H: 4.91 (d, 1H, H1), 4.14 (m, 2H, H2,6a), 4.08 (m, 1H, H6b),

Scheme 5
tert-Butyl 15-hydroxy-3,6,9,12-tetraoxa-tetradecanoate (44)
To a stirred mixture of tetraethylene glycol (40 mL) and THF (15 mL) was added-potasium tert-
butoxide (2.8 g). The reaction mixture was heated at 40 to 50°C until a clear solution was
obtained. This solution was cooled to 0°C and tert-butyl bromoacetate ( 4 mL, 246 mmol) was
added in one portion. The cooling bath was removed and the reaction mixture was stirred at RT
for two hours. It was diluted with EtOAc and washed with brine twice. Both brine layers were
extracted with EtOAc four times. The combined organic layers were dried (Na2SO4) and
concentrated under reduced presure to give compound 44 (4.66 g). 1H-NMR (CDCl3, 400
MHz): δ 4.03 (s, 2H), 3.60-3.76 (m, 16H), 1.47 (s, 9H).
tert-Butyl 14-(toluene-4-sulfonyloxy)-3,6,9,12-tetraoxa-tetradecanoate(45)
To a stirred mixture of compound 44 (4.66 g) in DCM (30 mL) at 0°C were added NMM (2
mL) and p-toluenesulfonyl chloride (3.2 g). The cooling was removed and the reaction mixture
was stirred at RT for 18 hours. Brine was added and the mixture was extracted three times with
DCM. The combined organic layers were dried (Na2SO4) and concentrated under reduced
pressure to give an oil (7.4 g) that still contained starting material. This oil was again dissolved
in DCM (30 mL) and NMM (2 mL) and p-toluenesulfonyl chloride (3.2 g) were added to the
stirred solution at 0°C. After 2 days at RT brine was added and the mixture was extracted three
times with DCM. The combined organic layers were dried (Na2SO4) and concentrated under
reduced pressure. The crude product was purified using silica gel column chromatography
(EtOAc/heptanes, 1/1, v/v) to give compound 45 (4.6 g, 66%). Rf 0.15 (EtOAc/heptanes 1/1,
v/v).
tert-Butyl 14-[4-O-(N-tert-butyloxycarbonyl-L-tyrosine benzyl ester)]-3,6,9,12-tetraoxa-
tetradecanoate (47)

A stirred mixture of Boc-Tyr-OBn (46, 0.6 g), compound 45 (1.0 g), cesium carbonate (0.82 g)
and sodium iodide (0.12 g) in DMF (30 mL) was heated at 70°C under a N2 atmosphere. After
20 hours the reaction mixture was allowed to cool to RT and brine was added. The mixture was
extracted with EtOAc (four times). The combined organic layers were dried (Na2SO4) and
concentrated under reduced presure. The crude product was purified using silica gel column
chromatography (EtOAc/ toluene, gradient 1/2 to 1/1, v/v) to give compound 47 (0.92 g, 80%).
Rf 0.15 (EtOAc/heptanes, 1/1, v/v).
tert-Butyl 14- L-tyrosine benzyl ester}>-3,6,9,12-tetraoxa-tetradecanoate (49)
To a stirred mixture of compound 47 (0.42 g) in tert-butyl acetate (4 mL) at 0°C was added a
solution of 4 N hydrogen chloride in dioxane (2 mL). After 2 hours sulfuric acid (0.04 mL) was
added and after half an hour an additional portion sulfuric acid (0.04 mL) was added. After
another half an hour a sat. aq. solution of sodium hydrogencarbonate was added and the mixture
was extracted four times with EtOAc. The combined organic layers were dried (Na2SO4) and
concentrated under reduced presure to give compound 48 (0.14 g). This compound was
dissolved in THF (2 mL) and to this stirred solution was added compound 36 (0.11g), TBTU
(84 mg) and 30 µL NMM. After two hours the reaction mixture was concentrated under reduced
pressure. The residue was purified using silica gel column chromatography (EtOAc/EtOH,
gradient 1/0 to 9/1, v/v) to give compound 49 (90 mg, 36%). Rf 0.5 (EtOAc/EtOH 9/1, v/v).
14- L-tyrosyl}-3,6,9,12-tetraoxa-tetradecanoate (50)
Compound 49 (90 mg) was dissolved in dioxane and concentrated under reduced pressure. The
residue was dissolved in DCM (1 mL) and TFA (1 mL) was added. After two hours dioxane (5
mL) was added and the mixture was concentrated under reduced pressure. The residue was
dissolved in acetonitrile and concentrated under reduced pressure. The residue was purified
using HPLC:column LUNA 10u C18(2) 250x50mm, flow 50 mL/min, gradient 3% 0.1 N TFA
in H2O, 47 % acetonitrile and 50% H2O/acetonitril (10/1, v/v) to 3% 0.1 N TFA in H2O, 90 %

acetonitrile and 7 % H2O/acetonitri] (10/1 v/v) in 30 minutes to give compound 50 (73 rng,
85%). MS (EI): m/z 970 [M+H]+.
Methyl O-2,3-di-O-methyl-4-O- O-L-tyrosyl)]-3,6,9,12-tetraoxa-tetradecanoyl}-12-aza-3,6,9-trioxa-dodecyl>-6-O-sulfo-
alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1->4)-O-
2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-aIpha-L-
idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranoside octakis
sodium salt (XII)
The product was obtained by conjugation of compound 50 (40.3 mg, 41.5 umol) to
pentasaccharide 63 (71.1 mg, 39.5 umol), followed by purification and deprotection, according
to the general procedure. The product was obtained as a white fluffy solid, yield 63.5 mg (60%,
2 steps). 1H-NMR (D2O, 600 MHz, HH-COSY): δ-3.43-3.32 (8 x s, 34H, 8x OMe); ring D:
5.36 (d, 1H, H1), 4.21 (m, 1H, H6a), 4.05 (d, 1H, H6b), 3.77 (m, 1H, H5), 3.44 (m, 1H, H3),
3.31 (m, 1H, H4), 3.19 (dd, 1H, H2); ring E: 4.58 (d, 1H, H1), 3.79 (m, 1H, H4), 3.67 (m, 1H,
H5), 3.44 (m, 1H, H3), 3.16 (m, 1H, H2); ring F: 5.28 (d, 1H, H1), 4.48 (m, 1H, H3), 4.32 (d,
1H, H6a), 4.18 (m, 1H, H2), 4.15 (m, 1H, H6b), 4.07 (m, 1H, H5), 3.86 (t, 1H, H4); ring G:
4.92 (bs,-1H, H1), 4.07 (m, 1H, H4), 3.91 (dd, 1H, H3), 3.31 (m, 1H, H2), 3.18 (m, 1H, H5);
ring H: 4.98 (d, 1H, H1), 4.23 (dd, 1H, H2), 4.22 (m, 1H, H6a), 4.17 (m, 1H, H6b), 3.93 (ddd,
1H, H5), 3.59 (m, 1H, H4), 3.57 (m, 1H, H3); spacer: 3.96 (s, 2H, C(O)CH2O), 3.61-3.51 (m,
26H, 13 x CH2O), 3.38-3.30 (m, 4H, OCH2CH2NHCH(O)CH2, OCH2CH2NHC(O)-Phe), 3.19
(m, 1H, OCH2aCH2NHC(O)-Phe), 3.12 (m, 1H, OCH2bCH2NHC(O)-Phe);peptide: 7.70 (d, 2H,
Harom benzamidine), 7.62 (d, 2H, Harom benzamidine), 7.01 (d, 2H, Harom Tyr), 6.77 (d, 2H, Harom
Tyr), 4.57 (dd, 1H, CH Asp), 4.28 (dd, 1H, CH Tyr), 2.87 (dAB, 2H, CH2 Tyr), 2.50 (t, 2H, CH2
succinyl), 2.48 (t, 2H, CH2 succinyl), 2.48 (dAB, 2H, CH2 Asp).



Scheme 6
Benzyl N-(3-Carboxybenzenesulfonyl)-4-O-{4-(N-benzyloxycarbonyl-4-piperidinyl)-
butyl}-L-tyrosine (52)
Compound 51 (123 mg, prepared as described in Bioorg. Med. Chem. 2001, 29, 357-379) was
dissolved in a mixture of acetonitrile and H2O (10 mL, 6/4, v/v). Potassium carbonate (381 mg)
was added and the solution was cooled to 0oC. 3-Carboxybenzenesulfonyl chloride was added
in portions, during a period of 15 min. and the mixture was stirred for 30 min. at 0°C and 1h at
RT. The solution was cooled to 0°C again and another portion of 3-carboxybenzenesulfonyl
chloride (102 mg) was added. After 30 min. the reaction mixture was allowed to warm to RT
and stirred overnight. The mixture was acidified to pH 1 with 1N hydrochloric acid,
concentrated under reduced pressure and the crude product was dissolved in EtOAc. The
organic layer was washed with 1N hydrochloric acid, sat. solution of NaCl, dried (MgSO4) and
concentrated to give compound 52 (0.11 g, 73%). Rf 0.5 (DCM/MeOH, 9/1, v/v). MS (ESI)
M+=729.
Benzyl N- benzenesulfonyl>-4-O-{4-(N-benzyloxycarbonyl-4-piperidinyl)-butyl}-L-tyrosine(54)
Crude compound 52 (216 mg) and compound 6 (49 mg) were dissolved in DCM (5 mL). TBTU
(145 mg) and NMM (82 uL) were added and the mixture was stirred overnight at RT. The
mixture was diluted with EtOAc, washed with sat. NaHCO3, citric acid and sat. NaCl. The
combined aq. phases were extracted with ethyl actetate after which the combined organic phases
were dried with MgSO4 and concentrated to give 0.31 g of 68 as a brown oil. Next, crude
compound 53 was stirred in a mixture of DCM (5 mL) and TFA (2.5 mL). After 4h the mixture
was concentrated. The product was purified by preparative LC/MS (C18, acetonitrile/H2O,
0.01% TFA) to give 84 mg of compound 54.
Methyl O-2,3-di-O-methyl-4-O- tetradecyl)]-keto}-benzenesulfonyl>-4-O-{4-(4-piperidinyl)-butyl}-L-tyrosine>-12-aza-
3,6,9-trioxa-dodecyl>>-6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-
D-glucopyranuronosyl-(1->4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-

O-methyl-alpha-L-idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-
glucopyranoside octakis sodium salt (XIII)
Conjugation of carboxylic acid 54 (53 mg) to pentasaccharide 63 (95 mg), followed by
purification and deprotection was effected according to the general procedure. Conjugate XIH
was obtained as a white foam. Yield 77 mg (58% over the two steps).
1H-NMR (D2O, 600 MHz, HH-COSY): δ 3.43-3.32 (8x s, 34H, 8x OMe); ring D: 5.39 (d, 1H,
H1), 4.22 (m, 1H, H6a), 4.04 (d, 1H, H6b), 3.79 (m, 1H, H5), 3.46 (m, 1H, H3), 3.34 (m, 1H,
H4), 3.24 (dd, 1H, H2); ring E: 4.59 (d, 1H, H1), 3.82 (m, 1H, H4), 3.66 (m.. 1H, H5), 3.52 (m,
1H, H3), 3.20 (m, 1H, H2); ring F: 5.28 (d, 1H, H1), 4.48 (t, 1H, H3), 4.34 (d, 1H, H6a), 4.22
(m, 1H, H2), 4.09 (m, 1H, H5), 3.85 (m, 1H, H4); ring G: 4.92 (bs, 1H, H1), 4.52 (bs, 1H, H5),
4.09 (m, 1H, H4), 3.75 (1H, m, H3), 3.35 (dd, 1H, H2); ring H: 4.99 (d, 1H, H1), 4.22 (m, 2H,
H2, H6a), 4.16 (m, 1H, H6b), 3.91 (ddd, 1H, H5), 3.68 (m, 1H, H4), 3.60 (m, 1H, H3); spacer:
3.91 (s, 2H, C(O)CH2O), 3.66-3.32 (m, 32H, 16 x CH2). peptide: 7.85 (dt, 1H, Harom), 7.75 (t,
1H, Harom), 7.62 (dt, 1H, Harom), 7.43 (t, 1H, Harom), 6.83 (d, 2H, Harom Tyr), 6.48 (d, 2H, Harom
Tyr), 3.86 (m, 2H, CH2O), 3.65 (m, 1H, H-1 Tyr), 3.36 (m, 2H, CH2N), 2.91 (m, 2H, CH2N),
1.91 (m, 2H, CH2piperidyl), 1.72 (m, 2H, CH2 butyl), 1.59 (m, 1H, CH piperidyl), 1.44 (m, 2H,
CH2 butyl), 1.36 (m,4H).
ESI-MS: m/z 1272.3 [M+2TEA-2H]2-, 814.5 [M+TEA-3H]3-, 799.1 [M-3H]3-, 585.0 [M-4H]4-.
Methyl O-2,3-di-O-methyl-4-O- piperidinyl)-butyl}-L-tyrosine>-12-aza-3,6,9-trioxa-dodecyl>>-6-O-sulfo-alpha-D-
glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1->4)-O-2,3,6-tri-
O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1-
>4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranoside octakis sodium salt (XIV)
Conjugation of carboxylic acid 52 (22.5 mg) to pentasaccharide 63 (52.6 mg), followed by
purification and deprotection, was effected according to the general procedure. Conjugate XIV
was obtained as a white foam. Yield 38.4 mg (58% over the two steps).
1H-NMR (D2O, 600 MHz, HH-COSY): δ 3.43-3.32 (8x s, 34H, 8x OMe); ring D: 5.39 (d, 1H,
H1), 4.22 (m, 1H, H6a), 4.04 (d, 1H, H6b), 3.79 (m, 1H, H5), 3.46 (m, 1H, H3), 3.34 (m, 1H,

H4), 3.20 (dd, 1H, H2); ring E: 4.58 (d, 1H, H1), 3.82 (m, 1H, H4), 3.66 (m, 1H, H5), 3.52 (m,
1H, H3), 3.18 (m, 1H, H2); ring F: 5.28 (d, 1H, H1), 4.49 (t, 1H, H3), 4,34 (d, 1H, H6a), 4.20
(m, 1H, H2), 4.08 (m, 1H, H5), 3.85 (m, 1H, H4); ring G: 4.94 (bs, 1H, H1), 4.54 (bs, 1H, H5),
4.08 (m, 1H, H4), 3.75 (1H, m, H3), 3.35 (dd, 1H, H2); ring H: 4.98 (d, 1H, H1), 4.22 (m, 2H,
H2, H6a), 4.17 (m, 1H, H6b), 3.92 (ddd, 1H, H5), 3.68 (m, 1H, H4), 3.60 (m, 1H, H3); spacer:
3.62-3.38 (m, 16H, 8 x CH2). peptide: 7.83 (dt, 1H, Harom), 7.71 (t, 1H, Harom), 7.62 (dt, 1H,
Harom), 7.42 (t, 1H, Harom), 6.81 (d, 2H, HaromTyr), 6.46 (d, 2H, Harom Tyr), 3.84 (m, 2H, CH2O),
3.66 (m, 1H, H-1 Tyr), 3.33 (m, 2H, CH2N), 2.90 (m, 2H, CH2N, 1.93 (m, 2H, CH2 piperidyl),
1.72 (m, 2H, CH2 butyl), 1.59 (m, 1H, CH piperidyl), 1.44 (m, 2H, CH2 butyl), 1.34 (m, 4H).
ESI-MS: m/z 1155.2 [M+2TEA-2H]2-, 736.3 [M+TEA-3H]3-, 702.6 [M-3H]3-.



Scheme 7
tert-ButyI 14-[4-O-(N-allyloxycarbonyl-L-tyrosyl)]-3,6,9,12-tetraoxa-tetradecanoate
(55)
Compound 38 (1.0 g) was dissolved in DMF (50 mL) and 10 mL of DMF was removed under
reduced pressure. The residue was cooled -at 0°C under an N2 atmosphere and sodium hydride
(60% disperse, 0.34 g) was added in small portions. The suspension was stirred for one hour at
0°C. Then compound 45 (1.3 g) in DMF (4 mL) was added. After 24 hours at RT the reaction
mixture was concentrated under reduced pressure. To the residue H2O (50 mL) was added and
the mixture was washed with EtOAc. EtOAc and solid sodium chloride was added and the
mixture was acidified to pH 3 using 2N aq. hydrochloric acid. The organic layer was separated,
dried (Na2SO4) and concentrated. The residue was purified using silica gel column
chromatography (DCM/ MeOH/AcOH, 949/50/1, v/v/v). The product from the column was
dissolved in EtOAc, washed with H2O, dried (Na2SO4) and concentrated to give compound 55
(0.6 g, 37%). Rf 0.4 (EtOAc/pyridine/AcOH/H2O 270/16/9/4, v/v/v/v).
Benzyl 2-[(4-piperidinyl)oxy]acetate (57)
tert-Butyl 2-[(4-piperidinyl)oxy] acetate (56) was prepared as previously described in /. Med.
Chem. 1992, 35, 4393-4407. To compound 56 (4.3 g 20 mmol) 50 mL of DCM/TFA (1:1) was
added and the solution was allowed to stir for about 30 minutes. The mixture was concentrated
under reduced pressure. The crude product was dissolved in a mixture of EtOH (40 mL) and
NMM (7 mL) to which was added di-tert-butyl-dicarbonate (4.8 g, 22 mmol). The mixture was
allowed to stir for about 55 h. Next, the reaction mixture was concentrated under reduced
pressure. To the residue 4 M NaOH (250 mL) and Et2O were added and the layers were
separated after extraction. The H2O layer was acidified to pH 3 using 2N hydrochloric acid and
was subsequently extracted 3 times with EtOAc. The. combined organic layers were dried
(magnesium sulfate) and concentrated to give 2-[4-(N-Boc-piperidinyl)oxy]acetate (2.1 g, 40%).
The latter crude product was dissolved in acetone (25 mL) and triethylamine (3.4 mL). Benzyl
bromide (965 µL, 8.1 mmol) was added and the reaction mixture was allowed to stir overnight.



Ice water (200 mL) and EtOAc were added. The mixture was acidified to pH 3 with 2N
hydrochloric acid. The organic layer was washed with sat. NaHCO3 and brine, dried (Na2SO4)
and concentrated to give 760 mg of the crude product. The product was purified using silica gel
column chromatography (heptane/EtOAc, 1/1, v/v) to give 418 mg (15%) of pure product. Rf
0.47 (heptane/EtOAc, 1/1, v/v). The latter product was dissolved in DCM/TFA (10 mL, 1/1,
v/v) and stirred for 1 h. The reaction mixture was concentrated under reduced pressure and
toluene (5 mL) was added. Dissolved in EtOAc (50 mL) and washed with sat. NaHCO3. The aq.
layer was extracted twice with EtOAc. The combined organic layers were dried (Na2SO4) and
concentrated to give 331 mg (100%) of the title compound 57. 1H-NMR (MeOD, 400 MHz,
HH): 5 7.37 (m, 5H, Ar), 6.00 (s, 2H), 4.17 (s, 2H), 3.56 (m,- 1H), 3.16 (m, 1H), 2.76 (m, 1H),
1.98 (m, 1H) 1.63 (m, 1H).
tert-Butyl 14-{[4-O-(N-allyloxycarbonyI-L-tyrosyl)]-2-[(4-piperidinyl)oxy]acetate benzyl
ester}-3,6,9,12-tetraoxa-tetradecanoate (58)
Coupling of compound (55) (400 mg, 0.72 mmol) and (57) (215 mg, 0.86 mmol) was performed
as described earlier for compound 64. The product was purified by silica gel column
chromatography (DCM → DCM/MeOH, 95/5, v/v) to give 587 mg (91 %) of the title
compound 58. Rf 0.65 (DCM/MeOH/AcOH, 95/5/0.3, v/v/v).
tert-Butyl 14-{4-O-L-tyrosyl-2-[(4-piperidinyl)oxy]acetate benzyl ester}-3,6,9,12-tetraoxa-
tetradecanoate (59)
To a solution of compound 58 (587 mg, 0.66 mmol) in DCM (15 mL) was subsequently added
H2O (75 µL), morpholine (115 µL, 1.32 mmol) and PdCl2(PPh3)2. After 3h the reaction mixture
was pourred in a H2O/brine (1/1, v/v) mixture which was extracted with DCM. The organic
layer was dried (Na2SO4) and concentrated. The residue was purified using silica gel column
chromatography (DCM/ MeOH, 9/1, v/v) to give 433 mg (89 %) of the title compound 59. Rf
0.66 (DCM/MeOH, 9/1, v/v).
N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoic acid (60)

p-Cyanobenoic acid methyl ester (5.0 g, 31 mmol) was converted into the corresponding
oxadiazolinone as described for the synthesis of compound 3. The crude product (4.6 g, 20.9
mmol) was dissolved in a mixture of THF (50 mL) and MeOH (50mL). H2O (50 mL) was
added, which gave a suspension, followed by 4 N aq. NaOH (10 mL). After 6 h the organic
solvents were removed by distillation under reduced pressure. The aq. layer was extracted with
EtOAc/toluene (1/2, v/v), acidified with 2 N hydrochloric acid and the precipitate was filtered
off. The crude product was dried at 40 °C under reduced pressure to give 4.2 g (97 %) of
compound 60. ESI-MS: 207 [M+H]+.
tert-Butyl 14-{{N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoyl}-{4-O-L-tyrosyl-2-[(4-
piperidinyl)oxy]acetate benzyl ester}}-3,6,9,12-tetraoxa-tetradecanoate (61)
To a solution of compound 59 (433 mg, 0.59 mmol) and 60 (134 mg, 0.65 mmol) in DMF (6
mL), TBTU (227 mg, 0.71 mmol) and NMM (130 µl, 1.18 mmol) were added. After 16 h of
stirring at RT the reaction mixture was concentrated under reduced pressure. EtOAc was added
and the organic layer was washed with brine, dried (Na2SO4) and concentrated. The product was
purified by HPLC (C18, ACN/H2O) to give 337 mg (64 %) of compound 61. ESI-MS: 891
[M+H]+, 913 [M+Na]+, 835 [M-tBu+H]+
14-{{N-(4-[1,2,4-Oxadiazol-5-onyl]-benzoyl}-{4-O-L-tyrosyl-2-[(4-piperidinyl)oxy]acetate
benzyl ester}}-3,6,9,12-tetraoxa-tetradecanoate (62)
Hydrolysis of the t-Bu ester was carried out as previously described for the preparation of
compound 12. The product was purified by HPLC (C18, ACN/H2O/3% 0.1 N TFA(aq)) to give
181 mg (57 %) of the desired compound 62. 1H-NMR (MeOD, 400 MHz): 7.91 (AB, 4H, Harom
benzamidine), 7.35 (m, 5H, Bn), 7.19 (d, 2H, Tyr), 6.88 (dd, 2 H, Tyr), 5.24 (m, 1H, CH Tyr),
5.18 (d, 2H, CH, Bn), 4.17 (d, 2H), 4.10 (s, 2H), 4.08 (m, 2H), 3.80 (m, 2H), 3.65 (m, 14H),
3.58 (m, 1H), 3.32 (m, 2 H), 3.07 (m, 2H), 1.82 -1.01 (m, 4H). ESI-MS: 835 [M+H]+.

3.84 (m, 1H, H5), 3.51 (m, 1H, H3); spacer: 3.88 (s, 2H, O(O)CH2,O), 3.54-3.24 (m,32H)
peptide: 7.59 (d, 2H, Harom benzamidine), 7.53 (d, 2H, Harom benzamidine), 6.84 (d, 2H, Harom
Tyr), 6.63 (d, 2H, Harom Tyr), 4.38 (dd, 1H, CH Asp), 4.21 (t, 1H, CH Tyr), 2.70 (m, 2H, CH,
Tyr), 2.47(m, 1H, CH2 Asp), 2.30 (dd, 1H, CH, Asp), 2.74-2.40 (m, 4H, succinyl).


Methyl O-2,3-di-O-methyl-4-O- oxadiazol-5-onyl]-benzoyl}-{4-O-L-tyrosyl-2-[(4-piperidinyl)oxy]acetate benzyl ester}}-
3,6,9,12-tetraoxa-tetradecanoyl>-6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-
methyl-beta-D-glucopyranuronosyl-(1->4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1-
>4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-
D-glucopyranoside octakis sodium salt (XV)
The conjugation of compound 62 (51.7 mg) with pentasaccharide 63 (106 mg), followed by
deprotection and purification was carried out as described in the general procedure. Yield 127
mg (87% over the 2 steps).









Pharmacological Data
La. In vitro test for inhibition of human platelet aggregation induced by ADP
Introduction:
Addition of adenosine diphosphate (ADP) to human platelet rich plasma (PRP) in vitro induces
platelet aggregation. This aggregation can be assessed by measuring the optical density (OD) of
the PRP. The in vitro test described here was used to determine the inhibitory activity of a test
compound on the ADP-mduced aggregation of human platelets. A microplare reader is used to
measure the activity of several compounds simultaneously.
Test medium: Platelets from healthy human volunteers who have not taken any drug during the
preceding 10 days.
Ref.compound: In this assay, tirofiban (AGGRASTAT® (MSD) purchased as 0.25 mg/mL
concentrate for i.v. infusion) inhibits human platelet aggregation induced by 5 uM ADP by 50%
at a concentration of 30 - 60 nM (IC5O).
Vehicle: Test compounds should preferably be dissolved in MQ H2O at a concentration of 1
mM. An alternative vehicle is 0.9% NaCl in MQ H2O. The compound solution (either in MQ
H2O or 0.9% NaCl in MQ H2O) is further diluted in 0.9% NaCl in MQ H2O.
Technique:
Reagents
1. Platelet rich plasma (PRP):
Free-flowing blood (at least 100 mL) is taken from a healthy volunteer and collected in 0.1
volume 3.8% sodium citrate. 2H2O in distilled H2O (w/v). The final concentration is 0.38%
sodium citrate.
The citrated blood is centrifuged at 1,600 N/kg (160 g) at RT. After 15 min, centrifugation is
discontinued with the brake turned off and the supernatant (=PRP) is collected.
2. ADP (Kordia/ Chrono-par # 384). Before use, a solution of 50 uM in 0.9% NaCl is
prepared.
Equipment
1. Sysmex blood cell counter model KX-21.
2. Labsystems iEMS reader MF with a 620 run filter, an orbital shaker set at
1,000 rpm and a constant temperature of 37°C. Absorption is measured with the

Labsystems iEMS program.
3. Blood collection system 600 mL with needle, art P4203 (NPBI).
4. 96 wells flat bottom microplates (Greiner Labortechnik).
Procedure
The platelets in the supernatant (PRP) are counted using a Sysmex blood cell counter and the
supernatant is diluted with PPP to obtain a PRP containing 400,000 ± 50,000 plt/µL. PRP
should stabilize at RT for at least 20 min but not longer than 3 h.
ADP-induced aggregation
150 µL PRP is pipetted into a well of the microplate. 30 uL test compound in a range of
concentrations (7 concentrations per compound) or vehicle is added and the microplate is placed
in the Labsystems iEMS reader MF at 37 °C. The optical density (OD62O) is then measured at
620 nm. After shaking for 2 min in the reader (1,000 rpm),'the OD is measured for the second
time. This is to verify the stability of the platelets (absence of spontaneous platelet aggregation).
Then, 20 µL of 50 µM ADP solution is added and the OD is kinetically measured every min for
14 min at 620 nm (Fig. 1). Between two measurements, the plate is shaking for 40 seconds at
1,000 rpm. Each test compound is investigated in at least 2 experiments using PRP from
different volunteers. Usually, the highest final concentration of the test compound is between
1E-6 and 1E-7M and the compound concentrations are decreased by a factor 2 until a platelet
aggregation inhibition of less than 25% is obtained.
Evaluation of response ADP-induced aggregation
The mean OD at each compound concentration (including vehicle) is calculated at t= 0 min and
t = 10 min. The percentage inhibition at each concentration is calculated using the formula:
100 % - (ODcompound at t=0 min - ODcompound at t=10 min) x 100 %
(ODvehicle at t=0 min - ODvehicle at t=10 min)
The IC50 of the test compound is the concentration of the test compound at which the ADP-
induced platelet aggregation is reduced by 50%. For this, the percentage inhibition values are

plotted against compound concentration. Then, IC50, nH (Hill slope) and efficacy of the test
compound is calculated using Graphpad Prism 3.0 (with variable slope).
Quantities required: 2 mg.
Reference: Salmon, DM. (1996) Thrombosis Research 84,213-216.


I.b. In vitro test for inhibition of human platelet aggregation induced by TRAP
Introduction: Addition of trombm receptor agonist peptide (TRAP) to washed human platelets
(WPL) in vitro induces platelet aggregation. This aggregation can be determined by measuring
the optical density of the WPL. The in vitro test described here is used to analyze the activity of
a test compound to inhibit TRAP-induced aggregation of human platelets. A microplate reader
is used to measure the activity of several compounds simultaneously.
Test medium: Platelets from healthy human volunteers who have not taken any drug during the
preceding 10 days.
Ref. compound: In this assay, tirofiban (AGGRASTAT® (MSD)) purchased as 0.25 mg/mL
concentrate for i.v. infusion) inhibits the human platelet aggregation induced by 5 uM TRAP by
50% at a final concentration of 30 - 60 nM (IC50).
Vehicle: Test compounds should preferably be dissolved in MQ H2O at a concentration of 1
mM. An alternative vehicle is 0.9% NaCl in MQ H2O. The compound solution (either in MQ
H2O or 0.9% NaCl in MQ H2O) is further diluted in 0.9% NaCl in MQ H2O.
Technique:
Reagents .
1. Platelet rich plasma (PRP):
Free-flowing blood (at least 100 mL) is taken from a healthy volunteer and collected in 0.1
volume 3.8% sodium citrate. 2H2O in MQ H2O (w/v). The final concentration is 0.38% sodium
citrate. The citrated blood is centrifuged at 1,600 N/kg (160 g) in Hettich Rotanta/AP centrifuge
(750 rpm) at RT. After 15 min, centrifugation is discontinued with the brake turned off and the
supernatant (=PRP) is collected.
2. Washed platelets (WPL):
After adding freshly made 1 µL PGI2 solution per ml PRP, the suspension is centrifuged at
13,500 N/kg (1,350 g) for 10 min in a Hettich Rotanta/AP centrifuge (i.e., 2,800 rpm) at RT.
After gentle resuspension of the pellet in the same volume of fresh Watson buffer containing 5

ng/mL PGI2, the suspension is centrifuged at 1,350 g (2,800 rpm) for 10 min at RT again. The
final platelet pellet is resuspended in Watson buffer to give approx. 400,000 + 50,000
platelets/mL.
3. Watson buffer:
134 mM NaCl, 2.9 mM KC1, 12 mM NaHCO3,0.34 mM Na2HPO4.2H2O, 1 mM MgCl2.6H2O,
5 mM Glucose and 5 mM HEPES in MQ H2O. The pH is adjusted to 7.4 with 1 M NaOH.
4. Prostaglandin I2 (Sigma, P6188 powder):
The PGI2 stock solution of 1 mg/mL in 1 M KOH is stored in aliquots of 100 µL at -20°C.
Immediately before use, a solution of 5 µg/mL in ice-cold 0.9% NaCl in MQ H2O is prepared.
5. Human Fibrinogen (Kordia/ERL, art nr: FIB 2 powder):
0.5 g fibrinogen powder is dissolved in 50 mL MQ H2O under vacuum. This stock solution is
stored in aliquots of 1 mL at -20°C. Before use, a solution of 0.5 mg/mL in saline is prepared.
6. TRAP
Before use, a solution of 50 µM TRAP in 0.9% NaCl in MQ H2O is prepared. For each test, a
fresh solution is made.
Equipment
1. Sysmex cell counter model KX-21
2. Labsystems iEMS reader MF with a 405 nm filter, an orbital, shaker set at
1,000 rpm and a constant temperature of 37°C. Absorptions are measured with
the Labsystems iEMS program.
3. Blood collection system 600 mL with needle, art nr P4203 (NPBI)
4. 96 wells microplates (Greiner Labortechnik) flat-bottom
Procedure: The WPL concentration is counted in a Sysmex blood cell counter and the
suspension is diluted with Watson buffer to obtain a concentration of 400,000 ± 50,000 plt/µL.
Before use, WPL is allowed to stabilize at room temperature for at least 20 min but not longer
than 3-4 hours.
TRAP-induced aggregation: 150 µL WPL is pipetted into a well of a microplate. 15 uL test
compound or vehicle and 15 µL fibrinogen solution is added and the microplate is placed in the
microplate reader at 37 °C. Then, the optical density (OD) is measured at 405 nm and after

shaking for 2 min in the reader, the OD405 is measured again to verify the stability of the
platelets (absence of spontaneous platelet aggregation). 20 µL of 50 µM TRAP is added and the
OD405 is kinetically measured every min for 14 min at 405 nm. Between two measurements,
the plate is shaking for 40 seconds at 1,000 rpm (Fig. 1). For determination of the IC50 of a test
compound, each test compound is investigated in at least 2 experiments using WPL from
different volunteers. The usual starting concentration of the compound is between 1E-6M and
1E-7M in the incubation medium and the compound concentration is decreased by a factor 2
until a platelet aggregation inhibition of less than 25% is obtained.
Evaluation of response: The mean OD of each concentration (including vehicle) is calculated at
t= 0 min and t = 10 min. The percentage inhibition at each concentration is calculated using the
formula:
100 % - fODcompound at t=0 min - ODcompound at t=10 min) x 100%
(ODvehicle at t=0 min - ODvehicle at t=10 min)
The concentrations of the compound are plotted against the percentage inhibition. The IC50, nH
(Hill slope) and efficacy of the test compound are calculated using Graphpad Prism 3.0 (with
variable slope). The IC50 of the test compound is the concentration at which the TRAP-induced
platelet aggregation is reduced by 50%.
Quantities required: 2mg.
Reference: Salmon D.M. (1996) Thrombosis Research 84, 213-216.



II. Determination of anti-Factor Xa activity in buffer
(Microtiter plate method)
Introduction:
Activated factor X (Xa) is a factor in the coagulation cascade; its activity is slightly inhibited by
antithrombin III (AT-III). Anticoagulants can inhibit Xa directly or, like heparin, by potentiating
the inhibitory activity of AT-III. Anti-Xa activity can be assessed by determination of the rate of
hydrolysis of the chromogenic substrate S-2222 in the presence of AT-III. This assay is used to
determine anti-Xa activity of heparinoid preparations in Kabi buffer.
Test medium: Kabi buffer.
Ref. compound: International standard heparin (11.4 anti-Xa U/mg).
Technique:
Reagents
1. Kabi buffer
Composition of the buffer: NaCl 10.17 g (174 mmol); edetate disodium dihydrate 3.26 g (9.6
rnmol); tromethamine (Tris) 6.11 g (50.4 mrnol); made up to 1 L with ultrapure H2O *. The pH
of the solution is adjusted to 7.4 with hydrochloric acid (0.10 mol/L)
* For all aq. solutions ultrapure H2O (Milli-Q quality) is used.
2. Xa solution
Bovine factor Xa (Kabi Diagnostica, Stockholm, Sweden) is dissolved in Kabi buffer to give a
solution containing 1.5 U/mL (0.75 nKat/mL).
3. S-2222 solution
S-2222 (Kabi Diagnostica) is dissolved in ultrapure H2O to give a solution containing 0.375
mg/mL (0.5 mmol/L).
4. AT-III solution
Human AT-III (Kabi Diagnostica) is dissolved in Kabi buffer to give a solution containing 0.25
U/mL. A fresh solution has to be prepared daily.
5. Standard solution of the calibration sample
Standard heparin is dissolved in Kabi buffer to give a standard solution containing approx. 0.25
anti-Xa U/mL.

Procedure:
Preparation of test samples
Each preparation is dissolved in ultrapure H2O and diluted with Kabi buffer to the required
concentrations in the range of 0.02 - 0.2 anti-Xa U/mL
Determination of Xa activity
Each test sample (0.05 mL) is pipetted into a well of a microtiter plate at RT. AT-III solution
(0.05 mL) is added to each sample and the plate is shaken using a Vari-shaker.
An aliquot of Xa solution (0.05 mL) is pipetted into each well 10 min following addition of
AT-III solution and the plate is shaken again. Exactly 2 min following addition of Xa solution,
0.1 mL S-2222 solution is pipetted into each well and the plate is shaken again. For all additions
a 12-channel pipette is used.
The remaining amount of Xa catalyses the hydrolysis of S-2222,' the rate of which is measured
photometrically following incubation periods of 2 and 22 min respectively at RT. The
absorbance of each sample is measured at 405 nm using a Reader Microelisa, model 310C
(Organon Teknika, Oss, The Netherlands) and the increase in absorbance (AOD) is calculated.
Each test sample is determined in duplicate. With every 10 samples, a blank (0.05 mL Kabi
buffer) is included.
Calibration curve
From an aliquot of the standard solution of the calibration sample a range of dilutions is made
(dilution factor 1,3 for heparin samples). The resulting standard samples (approx. 15 samples)
should contain between 0,01 - 0,25 anti-Xa U/mL. Within each run, 0,05 mL of each standard
sample is tested at least 3 times as described under Determination of Xa activity. A calibration
curve is obtained by fitting a straight line to
log mean AOD (blank) - mean AOD (standard sample)
mean AOD (standard sample)
against log anti-Xa U/mL values, using the method of least squares.
Evaluation of responses:
For each sample the mean anti-Xa activityin U/mL is determined using the calibration curve.

For comparison, the anti-Xa activity data of the free pentasaccharide XXII, the "parent" of the
oigosaccharide part of the conjugates, are also presented.

Pharmacokinetics
Guinea pigs (DH) were administered 0.5 µmol/kg (i.v.) of anticoagulant. The half-life of the
pentasaccharide XXII, the "parent" of the oligosaccharide part of the conjugates, and conjugate
VIII were indirectly assigned by ex-vivo determination of plasma anti-Xa activity at specific
time-intervals as described above. The half-lives of the reference GPIIb/IIIa antagonists XVII,
XIX and XX (see Scheme 10) were determined by measuring the plasma concentration at
specific time-intervals using LC-MS/MS.


Conclusion:
The halflife of the conjugate compound VIII of this invention is significantly prolonged when
compared to the reference GpIIb/IIIa antagonists.

We claim:
1. A compound of the formula (A)
oligosaccharide-spacer-GpIIb/IIIa antagonist (A),
wherein
the oligosaccharide is a negatively charged oligosaccharide residue comprising four to twenty
five monosaccharide units, the charge being compensated by positively charged counterions, and
wherein the oligosaccharide residue is derived from an oligosaccharide which has (AT-III mediated)
anti-Xa activity per se ; where said oligosaccharide is a sulfated pentasaccharide of formula B

wherein R1 is-independently OSO3- or (1-8C)alkoxy and the charge being compensated by
positively charged counterions such as herein described
the spacer is an essentially pharmacologically inactive linking residue:
the GpIIb/IIIa antagonist is a residue mimicking the RGD and/or K(QA)GD fragment of fibrinogen,
typically comprising an optionally esterified carboxylate moiety and a basic moiety located within the
residue at a distance of 10-20 Å from each other, where said GpIIb/IIIa antagonist residue is
selected from residues derived from Ro 435054, SC 54701 (xemilofiban), RWJ 50042, sibrafiban
(Ro 44 3888), lamifiban (Ro 449883) GPI 562, FK 633, tirofiban (MK 383), orbofiban (SC 57101),
eptifibatide (C68 22), roxifiban (XV 459), elarofiban (RWJ 53308), SR 121566 (active form of SR 121787),
lefradafiban (BIBU 52), lotrafiban (SB 214857), gantofiban (YM 028), T-250, EF 5077, 2D 2486, TAK
029, TP 9201 or L 703014; or the GpIIb/IIIa antagonist residue has the structure D
-Y-N(H)-C(O)-X (D),
wherein Y is N(H)-C(O)-C(R2)(C(R2)2COOH) or
N(H)-C(O)C(R2)(CH2aryl)N(H)C(O)-C(R2)(C(R2)2COOH),
O-phenylene-C(R2)2-C(R2)(COOH)-N(H)-(O)-C(R2)(C(R2)2COOH),
O-phenylene-C(R2)2-C(R2)(C(O)-R3-O-C(R2)2COOH),
wherein R2 is independently H or (1-4C)alkyl; and wherein aryl is phenyl,
hydroxyphenyl, thiophenyl or pyridinyl and R3 is piperidinyl;
and X is benzamidine, (CH2)2-N(H)-C(O)benzamidine, (CH2)2-C(O)N(H)-
benzamidine or

or a pharmaceutically acceptable salt thereof or a solvate thereof.

2. A compound as claimed in claim 1, wherein the sulfated pentasaccharide of formula B has the
formula C

wherein R1 is OCH3 or OSO3-,
the charge being compensated by positively charged counterions,
3. The compound as claimed in claim 1, wherein the linking residue has a length of 1-50 atoms.
4. The compound as claimed in any one of claims 1 to 3, wherein the linking residue comprises at
least one -(CH2CH2O)- element.
5 A compound as claimed in claim 1, selected from the following compounds:




6. A process for the preparation of the compound of formula (A) as claimed in anyone of claims
1 to 5, comprising a step wherein optionally modified GPIIb/IIIa antagonist residue (a) is coupled
directly to an oligosaccharide or (b) is coupled to an oligosaccharide-spacer residue or (c) is coupled
to a spacer, which is subsequently is coupled to an oligosaccharide-spacer-residue.
7. A pharmaceutical composition comprising the compound as claimed in any one cf claims 1 to
5 and pharmaceutically suitable auxiliaries.
8. The compound as claimed in any one of claims 1 to 5, treating or preventing thrombotic diseases.


The present invention relates compounds of the formula (A) oligosaccharide-spacer-GpIIb/IIIa antagonist (A),
wherein the oligosaccharide is a negatively charged oligosaccharide residue comprising four to twenty five monosaccharide units,
the charge being compensated by positively charged counterions, and wherein the oligosaccharide residue is derived from an
oligosaccharide which has (AT-III mediated) anti-Xa activity per se; the spacer is a bond or an essentially pharmacologically
inactive linking residue; the GpIIb/IIIa antagonist is a residue mimicking the RGD and/or K(QA)GD fragment of fibrinogen,
typically comprising an optionally esterified carboxylate moiety and a basic moiety located within the residue at a distance of 10-20
Å from each other; or a pharmaceutically acceptable salt thereof or a prodrug or a solvate thereof. The compounds of the invention
have antithrombotic activity and can be used in treating or preventing thrombotic diseases.

Documents:


Patent Number 248843
Indian Patent Application Number 2403/KOLNP/2006
PG Journal Number 35/2011
Publication Date 02-Sep-2011
Grant Date 30-Aug-2011
Date of Filing 24-Aug-2006
Name of Patentee SANOFI-AVENTIS
Applicant Address 174 AVENUE DE FRANCE FR-75013 PARIS
Inventors:
# Inventor's Name Inventor's Address
1 BUIJSMAN ROGIER CHRISTIAN ST.ANTHONIUSSTRAAT 26 NL-5341 LD OSS PAYS BAS
2 MEULEMAN DIRK GERRIT C/O.N.V. ORGANON WETH VAN ESCHSTRAAT 1 P.O.BOX 20 NL-5340 BH OSS
3 VAN BOECKEL CONSTANT MERCURIUSSTRAAT 32 NL-5345 LX OSS PAYS BAS
4 DE KORT MARTIN C/O.N.V. ORGANON WETH VAN ESCHSTRAAT 1 P.O.BOX 20 NL-5340 BH OSS
PCT International Classification Number C07H 11/00
PCT International Application Number PCT/EP2005/002881
PCT International Filing date 2005-03-03
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04005343.1 2004-03-05 EUROPEAN UNION