Synthesis and evaluation of a small library of graftable thrombin inhibitors derived from (l)-arginine

Article abstract of DOI:10.1016/j.ejmech.2006.07.010

Novel piperazinyl-amide derivatives of N-α-(aryl-sulfonyl)-l-arginine were synthesized as graftable thrombin inhibitors, in the context of biomaterials' design. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position and the introduction of a trifluoromethyl group as XPS (X-ray Photoelectron Spectroscopy) tag on the sulfonamide moiety were evaluated in vitro against human α-thrombin. All the compounds of the library were found to be active at the micromolar level, as the reference TAME (N-tosyl-l-arginine methyl ester). The blood compatibilization improvement of poly(ethylene terephthalate) (PET) membrane, coated or grafted by wet chemistry treatment with one representative inhibitor of the library, was also evaluated, showing interesting decrease in blood clot formation.

Full text of DOI:10.1016/j.ejmech.2006.07.010

European Journal of Medicinal Chemistry 42 (2007) 37e53
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of a small library of graftable
thrombin inhibitors derived from (L)-arginine
Claudio Salvagnini a, Sonia Gharbi a, Thierry Boxus b, Jacqueline Marchand-Brynaert a,
*
a Unite´ de Chimie Organique et Me´dicinale, Universite´ catholique de Louvain, Baˆtiment Lavoisier,
Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium
b Mettler-Toledo GMbH, AutoChem, Sonnenbergstrasse 74, CH-8603 Schwerzenbach, Switzerland
Received 21 March 2006; received in revised form 17 July 2006; accepted 20 July 2006
Available online 28 September 2006
Abstract
Novel piperazinyl-amide derivatives of N-a-(aryl-sulfonyl)-L-arginine were synthesized as graftable thrombin inhibitors, in the context of
biomaterials’ design. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position and
the introduction of a trifluoromethyl group as XPS (X-ray Photoelectron Spectroscopy) tag on the sulfonamide moiety were evaluated in vitro
against human a-thrombin. All the compounds of the library were found to be active at the micromolar level, as the reference TAME (N-tosyl-L-
arginine methyl ester). The blood compatibilization improvement of poly(ethylene terephthalate) (PET) membrane, coated or grafted by wet
chemistry treatment with one representative inhibitor of the library, was also evaluated, showing interesting decrease in blood clot formation.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Thrombin inhibitors; Arginine derivatives; Graftable inhibitors; Blood-compatible materials
1. Introduction
Many strategies for preventing and treating thromboem-
bolic events have focused on the inhibition of thrombin gener-
ation, as well as on the inhibition of the thrombin action [5].
Heparin was in clinical use for more than 50 years. This nat-
ural compound (linear polysaccharide with alternating uronic
acid and glucosamino units bearing carboxyl, sulfonic acid
and sulfonamide groups) acts as an anticoagulant by activating
antithrombin, which then inhibits thrombin, but its therapeutic
use suffer severe limitations [6]. The quest for the ideal anti-
coagulant has produced several synthetic agents (molecules
of low molecular weight) acting as direct thrombin inhibitors
[1]. The covalently bound inhibitors (binding to Ser-195) are
electrophilic compounds derived from the tripeptide (D)-Phe-
Pro-Arg (fibrinogen recognition sequence), with the peptide
arginal Efegatran and the chloromethylene ketone PPACK as
leads [7]. High affinity inhibitors (non-covalently bound, inter-
acting with Asp-189), called steric inhibitors, were also de-
signed to block thrombin activity. In this family, the lead
compounds are N-tosyl-(L)-arginine methyl ester (TAME)
and benzamidine. Argatroban [8] and Ximelagatran [9] belong
Thrombin is a key-enzyme in the blood coagulation system
and represents a main target in medicinal chemistry [1]. In-
deed, this enzyme is the final common mediator of both the in-
trinsic and extrinsic coagulation pathways; it triggers platelet
activation, production of factors V, VIII, IX, and mediates
the proteolytic cleavage of fibrinogen to fibrin [2]. Thrombin
is formed by an A chain of 36 amino acids and a B chain of
259 amino acids connected by a disulfur bridge. This enzyme
contains the catalytic triad (Asp-102, His-57, Ser-195) charac-
teristic of the chymotrypsin family. From X-ray diffraction
data, three important binding site pockets have been identified:
the specificity pocket S1 with Asp-189, the hydrophobic prox-
imal pocket S2 (also called P-pocket) and a larger hydropho-
bic distal pocket S3 (also called D-pocket) [3,4].
* Corresponding author. Tel.: þ32 10 472740; fax: þ32 10 474168.
E-mail address: marchand@chim.ucl.ac.be (J. Marchand-Brynaert).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.07.010

38
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
to this class of reversible thrombin inhibitors; they are pres-
ently the only synthetic inhibitors approved and available for
medical purpose.
CH3
[aromatic motif]
SO2
HN
O
NH
In our laboratory, we use active molecules stemming from
medicinal chemistry approaches as tools for the biocompatibili-
zationofpolymermaterials. Ourstrategyreliesuponthecovalent
grafting of biologically active molecules, via a spacer-arm, on
the surface of polymer devices. In this way, we confer specific
properties selectively to the material surface, while the phys-
ico-chemical and mechanical properties of the bulk remain un-
changed [10]. This methodology has been successfully applied
to the preparation of novel supports for the in vitro mammalian
cells’ cultivation: the grafting of peptidomimetic molecules of
the Arg-Gly-Asp (RGD) sequence, designed to interact with
aVb3 integrin receptor, promoted Caco-2 cell adhesion on
poly(ethylene terephthalate) track-etched membranes [11].
Now we are interested in blood-compatible materials. Poly-
mer devices preventing clot formation are generally heparin-
ized materials. Heparin can be attached to blood-contacting
devices according to material surface available functional
groups and suitable treatments: activation of heparin carbox-
ylic groups with carbodiimides [12,13] or deaminative cleav-
age [14] and grafting on aminated surfaces; activation of
surface amino groups with isocyanates [15] or glutaraldehyde
[16e18]. Heparin-like materials were also produced by sur-
face chemical modification. For example, unsaturated
polymers were modified with N-chlorosulfonyl isocyanate
[19e22]; isoprene with fuming sulfuric acid [23]. Sulfonated
forms of polystyrene [24], dextran [25], chitosans [26] and
more recently polyrotaxanes [27] were also prepared.
Thrombomodulin, an anticoagulant transmembrane protein
found on endothelial cells’ surface [28], was also investigated
for the incorporation on biomaterials’ surface in order to ob-
tain non-thrombogenic devices [29e34]. Recently, nitric oxide
releasing/generating substances were incorporated into poly-
mer materials in order to avoid thrombosis and stenosis on bio-
medical implants [35e37]. Nitric oxide is in fact a well-known
potent anti-platelet agent, preventing adhesion and activation
of platelets, and a smooth cell proliferation inhibitor [38].
Instead, little attention has been devoted, so far, to the use
of synthetic drugs for preventing blood coagulation on mate-
rials. We found only two representative examples: (i) an acryl-
amide derivative of Argatroban was graft-polymerized on the
surface of polyurethanes [39], and (ii) a p-amino-benzamidine
derivative was immobilized on maleic anhydride copolymer
films via a spacer [40].
N
NH2
SO2
HN
O
COOH
N
[spacer]
N
NH
CH3
NH
H2N
NH
NH
H2N
A
B
Fig. 1. Structure of the model compound Argatroban (A) and proposed struc-
tures of graftable thrombin inhibitors (B).
(iii) the piperazinyl amide moiety bearing a spacer-arm for
surface grafting. Comparatively to Argatroban A, we sup-
pressed the chiral centres on both the N- and C-substituents
of the arginine template, and replaced the piperidine ring by
a piperazine ring with the objective of using the N-4 atom
as anchoring point of various spacer-arms. The validity of
our design had to be established (theoretically and experimen-
tally) before to use molecules B in any biomaterial applica-
tion. A molecular modelling and docking study of the possible
positioning of one representative of target molecules B (aromatic
motif ¼ m-trifluoromethyl-phenyl; spacer ¼ propyl) into the
thrombin active site, showed two modes of binding of similar
energies, one of them allowing the spacer to point towards
the surface of the enzyme and come out from the cavity [41].
In this article, we describe the synthesis of a small library
of graftable thrombin inhibitors (B) and the evaluation of their
activity against human thrombin in view to select good candi-
dates for materials’ hemocompatibilization.
2. Results and discussion
Compounds B (Fig. 1) were readily accessible according to
Scheme 1: N,N0-protected arginine (Boc-Arg(NO2)) was trans-
formed into mixed anhydride and then coupled with an azacy-
clohexane derivative to furnish an amide (Boc-Arg(NO2)-Ai,
step i). After selective deprotection of the Boc group (H2N-
Arg(NO2)-Ai, step ii), the arylsulfonyl motif was introduced
by reaction with a chlorosulfonyl derivative (Sj-Arg(NO2)-
Ai, step iii). The final step was the deprotection of all masked
functions (N-nitro-Arg and chain substituent X on the pipera-
zine motif) by catalytic hydrogenation (AiSj, step iv) [42e44].
The experimental conditions of this four-step synthesis
have been manually set up and exemplified with a few rep-
resentatives (Aryl ¼ m-trifluoromethyl-phenyl; Y ¼ CH2,
NH, N-(CH2)3-NH2). However, the optimization of our de-
sign of graftable inhibitors required the (parallel) synthesis
of another series of compounds in order to address the fol-
lowing questions, when comparing Argatroban (A) and the
Our objective was to prepare hemocompatible polymer
membranes by the surface grafting of thrombin inhibitors. In
this context of biomaterials, the surface bound inhibitors are
not allowed to be processed by the enzyme. We thus selected
a simplified structure (B) of Argatroban (A) (steric inhibitor),
equipped with a spacer-arm, as starting point of our research
(Fig. 1).
Molecules B were derived from (L)-arginine and feature the
following characteristics: (i) the guanidyl function for ionic in-
teraction with Asp-189 in the S1-pocket; (ii) the lipophilic sul-
fonamide moiety for interaction with the S2eS3 binding sites;

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
39
NH2
NH2
NH2
NO2
NO2
N
H
N
N
H
N
NO2
H
N
N
H
N
H2N
O
H
N
Boc
Boc
i
ii
O
N
N
O
OH
X
X
Boc-Arg(NO2)
Boc-Arg(NO2)-Ai
H2N-Arg(NO2)-Ai
iii
NH2
NH2
O
O
O
Ar
Ar
NO2
O
S
N
H
N
S
N
H
NH
HN
HN
iv
O
O
N
N
Y
X
AiSj
Sj-Arg(NO2)-Ai
Scheme 1. Synthetic sequence adapted for parallel synthesis. Reagents and conditions: (i) NMM, ClCO2-iBu (IBC), THF then azacyclohexane HN(C2H4)2X, and
NMM; (ii) TFA/CH2Cl2 (1:1), then aq. NaOH; (iii) ArylSO2Cl, Et3N, CH2Cl2; (iv) H2, Pd/C, EtOH, HOAc; Y ¼ X with deprotected terminal amino group.
target molecules (B): what could be the effect of (a) the re-
placement of the piperidine motif by a piperazine motif, (b)
the presence of a spacer-arm on this motif, (c) the simplifi-
cation of the aryl group structure, but with the introduction
of a spectroscopic tag (CF3) for XPS analysis of the
biomaterial?
For this purpose, we selected nine building blocks for the
synthesis of the carboxamide moiety (A1 to A9, Fig. 2) and
(-NH)
O
N
O
HN
HN
O
N
N
HN
HN
HN
O
A1
A2
A3
A4
A5
(-NH2)
HN
PNB =
HN
O
N
(-NH2)
H
N
N
O
NO2
H
N
( )
PNB
12
O
( )
O
PNB
10
O
A6
A7
(-NH2)
HN
O
HN
H
N
N
O
PNB
N
O
O
N
H
O
( )
PNB
3
O
(-NH2)
A8
A9
CF3
CH3
NH
N
SO2Cl
SO2Cl
SO2Cl
SO2Cl
S1
S2
S3
S4
Fig. 2. Building blocks considered in parallel (and manual) synthesis. Residues after deprotection by hydrogenation are indicated in brackets.

40
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
four building blocks for the synthesis of the sulfonamide moi-
ety (S1 to S4, Fig. 2). S1 to S4, and A1 to A5 are commer-
cially available, while A6 to A9 equipped with a spacer-arm
have been prepared by conventional chemistry. A6 and A9
were previously described [41]. A7 was obtained in three steps
from 12-amino-dodecanoic acid (Scheme 2, part 1): after
N-protection with p-nitrobenzyl chloroformate, the carboxylic
acid was coupled to 1-Boc-piperazine using the mixed anhy-
dride method of activation; Boc deprotection with trifluoroace-
tic acid (TFA) and neutralization gave the block A7 in 50%
overall yield. A8 resulted from the N-alkylation of 1-Boc-pi-
perazine with 2-[2-[2-( p-nitro-benzyloxycarbonylamino]-
ethoxy]-ethoxy]-ethyl mesylate followed by Boc cleavage
(Scheme 2, part 2). The mesylate was obtained from 2-[2-[2-
chloroethoxy]-ethoxy]-ethanol: after chloride substitution
with azide, the azide was reduced by hydrogenation into amine
which was protected as usual; the terminal alcohol was then
activated by mesylation. The block A8 was recovered in
50% overall yield for six steps.
Our strategy for the introduction of a spacer-arm on the po-
tential inhibitors B generates a novel basic centre, the N-4
atom of piperazine. The possible disturbance due to this func-
tion towards the enzyme recognition was evaluated, thanks to
the blocks A1 to A5. The effect of the nature (hydrophobic or
hydrophilic) and the length (3 to 12 atoms) of the spacer-arm
was controlled with blocks A6 to A9; in all cases the terminal
function for surface anchorage was a primary amine because
the activated polymer surfaces usually display carboxylic or
hydroxyl functions.
After surface grafting, the polymers were routinely ana-
lysed by X-ray photoelectron spectroscopy (XPS) for the
quantification of fixed molecules. This requires a fluorine tag
(CF3) on the molecules of interest. The lipophilic sulfon-
amides moiety was used for that purpose (block S1); the effect
of this structural modification on the biological activity was
estimated thanks to blocks S2 to S4, S4 being close to Arga-
troban (Fig. 1, A).
The facilities of parallel automated synthesis (AutoChem
from Mettler-Toledo) were used to prepare the library AiSj.
(Scheme 1). The manual protocols used previously to synthe-
size A1S1, A3S1, A6S1 and A9S1 [41] had to be adapted: in-
troduction of solutions of reagents instead of solids; reduction
of volumes; suppression of concentration steps, when possible;
suppression of intermediate chromatographic purifications;
purifications by liquideliquid extractions; NMR controls re-
placed by mass spectrometry (MS) and thin layer chromatog-
raphy (TLC). Protocols are summarized in Table 1. Step i
(C-functionalization of protected arginine; Scheme 1) was re-
alized individually, with chromatographic purifications of
Boc-Arg-(NO2)-Ai, or in parallel synthesis, without other pu-
rifications than the liquideliquid extractions.
After first deceiving results in performing step ii (Boc de-
protection) with the automated system, due to pure TFA han-
dling problems, we did not further investigate deprotection
step in the parallel automated workstations and this operation
was manually achieved. Step iii (N-functionalization with sul-
fonyl chlorides) used the crude TFA salts of amines [41] or
preferably the free amines H2N-Arg(NO2)-Ai obtained by
O
H
H
ii
A7
iii
i
O
N
O
COOH
11
N
PNB
N
H2N
COOH
11
(1)
(2)
PNB
11
95
O
N
O
Boc
O
75
70
O
OH
iv
97
O
OH
N3
Cl
O
v
95
O
vi
69
O
OH
PNB
O
OH
H2N
O
O
N
H
O
vii
90
Boc
O
N
O
O
viii
92
O
N
PNB
OSO2Me
PNB
O
O
N
O
N
H
O
H
iii
95
A8
Scheme 2. Synthesis of the piperazine linked spacer-arms. Reagents and conditions: (i) PNB-OCOCl, THF, NaOH$H2O, 0 ꢀC then H3Oþ; (ii) ClCO2-iBu, Et3N,
THF, then 1-Boc-piperazine; (iii) TFA/CH2Cl2 (1:1), 20 ꢀC then NaOH$H2O; (iv) NaN3, NaI, H2O, 50 ꢀC; (v) H2, Pd/C, MeOH; (vi) PNB-OCOCl, Et3N, CH2Cl2,
0 ꢀC; (vii) MeSO2Cl, pyridine, CH2Cl2, 0 ꢀC; (viii) 1-Boc-piperazine, NaI, DIPEA, CH3CN.

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
41
Table 1
Compared protocols
Step Manual (optimized bench protocol)
Automated
i
Boc-Arg(NO2) (1 equiv) and NMM (2 equiv) in THF (conc: 2%)
Addition of IBC (1 equiv) at ꢂ20 ꢀC; 30 min at ꢂ20 ꢀC
Addition of azacyclohexane (1 equiv) and NMM (1 equiv) at ꢂ20 ꢀC;
20 min at ꢂ20 ꢀC; 1 h at RT
Boc-Arg(NO2) (1 equiv) and NMM (2 equiv) in THF (conc: 5%)
Addition of IBC (1 equiv) at 5 ꢀC; 50 min at 5 ꢀC
Addition of azacyclohexane (1 equiv) and NMM (1 equiv) at 5 ꢀC; 50 min at
5 ꢀC; 1 h at RT
Concentration under vacuum (Rotavapor, 40 ꢀC)
Extraction EtOAc/brine; drying over MgSO4; evaporation
Column chromatography on SiO2
Filtration (TLC analysis); overnight evaporation under fume hood
Extraction EtOAc/NaOH 1 N; evaporation of organic phase under fume hood
MS control of the crude
ii
Dissolution in TFA/CH2Cl2, 1:1 (conc: 3e5%); 2e3 h at RT
Concentration under vacuum (Rotavapor, RT)
Addition of TFA/CH2Cl2, 1:1 (conc: 10%), 2 h at RT
Evaporation under fume hood
Addition of ether; formation of a viscous oil
Trituration in ether; solid washing with ether (three times)
Dissolution in NaOH 1 N and extraction with CH2Cl2 (or EtOAc)
Drying over MgSO4; evaporation
iii
(Free) amine (1 equiv) and Et3N (2e4 equiv) in CH2Cl2 (conc: 3%)
Addition of sulfonyl chloride (1.2 equiv) at 0 ꢀC
TFA amine (1 equiv) and Et3N (4e6 equiv) in CH2Cl2 (conc: 5e10%)
Addition of sulfonyl chloride (1.2 equiv), at 5 ꢀC, in solution (S1 and S2 in
CH2Cl2; S3 in CH2Cl2 þ 2% DMF; S4 in pyridine)
Shaking 1 h at 5 ꢀC and 2 h at RT
Stirring for 30 min at 0 ꢀC and 2 h at RT
Washing with brine, drying over MgSO4, concentration
Column chromatography on SiO2
Sulfonamide dissolved in EtOH/AcOH, 1:1 (conc: 5%)
Washing with brine
MS control of the crude. Purification by SFC
Sulfonamide dissolved in EtOH/AcOH, 1:1 (conc 10%). MS control of the
crude
iv
Pd 10% on C, H2 (1 atm), 50 ꢀC, 12 h
Filtration, concentration
Crystallization from H2O/MeOH, 1:1 and washing with ether
NMM ¼ N-Methyl-morpholine; THF ¼ tetrahydrofuran; IBC ¼ isobutyl chloroformate; RT ¼ room temperature; TFA ¼ trifluoroacetic acid; MS ¼ mass spectrom-
etry; SFC ¼ supercritical fluid chromatography; TLC ¼ thin layer chromatography.
dissolution of the corresponding TFA salt in aqueous NaOH
and extraction with CH2Cl2 or EtOAc [45,46] (manually or
with the automate). According to the nature of the amine part-
ner (TFA salt or free base), the amount of triethylamine used
for the coupling with arylsulfonyl chlorides was adjusted (see
Table 1). Similar yields were obtained following both proto-
cols. These coupling reactions were readily performed in
parallel synthesis. In all cases, the expected products Sj-
Arg-(NO2)-Ai were identified in the crude mixtures by MS
and TLC, with two exceptions. In the A4 series, we recovered
practically no materials after liquideliquid extractions, and in
the S4 series, the coupling products were identified in only two
crudes (A5 and A8). Some products Sj-Arg-(NO2)-Ai of the
series A5, A6 and A8 have been purified by supercritical fluid
chromatography (SFC). Finally, step iv (hydrogenation) was
performed on chromatographed products (S1 series, manual
synthesis) or on crude products (S2eS4 series, parallel synthe-
sis) with similar results. The yields obtained from steps i to iv
are collected in Table 2. Identifications of final products AiSj
and intermediates by HRMS or elemental analysis are pre-
sented in Table 3.
The arginine derivatives AiSj were evaluated for their activ-
ity against human thrombin. The activities were measured by
adapting the protocol of Lottenberg et al. [47]. A chromogenic
substrate, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroani-
lide dihydrochloride, was used as competitor. A mixture of sub-
strate and tested compound at different concentrations was
treated with the enzyme; the appearance of the substrate hydro-
lysis product ( p-nitroaniline) was spectrophotometrically mea-
sured at 405 nm as a function of time. Increasing the inhibitor
concentration resulted in a decrease of p-nitroaniline detection.
The slope of the curve (absorbance versus time) gave the reac-
tion rate and the velocity ratio V0/Vi was calculated for each con-
centration of inhibitor (Graph 1). These values were plotted
against the inhibitor concentration and the points were fitted
with a linear curve (Graph 2). The inhibition constant Ki was cal-
culated from the slope of the curve (m), knowing Km and the sub-
strate initial concentration [S] according to the equation:
Km
1
Ki ¼
m ð½Sꢁ þ KmÞ
The results are collected in Table 4. All the tested com-
pounds were found to be active in the micromolar range,
from 1 to 15 mM. In view to validate these rather low activi-
ties, a reference compound structurally close to the AiSj com-
pounds and commercially available was tested. In our hands,
the measured Ki of TAME (N-tosyl-L-arginine methyl ester)
was 6e10 mM. The reported Ki of TAME [48] and Argatroban
(structure A in Fig. 1) [49] are 40 mM and 20 nM, respectively.
Thus, our compounds are definitively less active than Argatro-
ban: the nanomolar activity of the drug has been lost with our
structural modifications. Our biological results showed that the
nature of the aryl group of the sulfonamide moiety has no
great influence on the activity (compare series S1 to S2, S3,
S4); hence, the m-trifluoromethyl-phenyl substituent remains
a good choice, combining the requirements of thrombin inhi-
bition and spectroscopic detection (XPS tag). On the other
hand, the nature of the atom in position 4 of the azacyclohex-
ane moiety (compare series A1, A2, A5), also poorly influ-
enced the activity (Y ¼ CH2, O, N-Ac in Table 4). However,
the presence of a basic motif (series A3; Y ¼ NH) seemed

42
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
Table 2
Reaction yields
the entry into the enzymic cavity, after inhibitor immobiliza-
tion, a long spacer-arm could be more appropriate.
Blocks
Step
i
The micromolar activity of compounds AiSj should be suf-
ficient to induce the expected biological response, namely the
inhibition of blood coagulation, on the surface devices. In-
deed, it has been previously reported that the surface grafting
of simple benzamidines (which are millimolar inhibitors)
could improve the hemocompatibility of polymer films [40].
After deposition of inhibitor A6S1 on the surface of a poly
(ethylene terephthalate) (PET) track-etched membrane (coat-
ing at 10 nmol/cm2), the blood clotting was reduced by 22%
(Table 5); this effect corresponds to 38% of the activity of hep-
arin coated at high concentration. The first experiments of co-
valent grafting of A6S1 via the hydroxyl chain-ends of the
polymer furnished a material displaying about 54 pmol/cm2
of inhibitor (calculated from the F/C atomic ratio determined
by XPS). These surface modified PET membranes showed
8% inhibition of blood clotting (Table 6). Thus a coated or
grafted inhibitor retains its activity and this activity is still de-
tectable at surface concentrations in the picomolar range.
ii
iii
iv
A1
S1
S2
S3
S4
A2
S1
S2
S3
S4
A3
S1
S2
S3
S4
A4
S1
S2
S3
S4
A5
S1
S2
S3
S4
A6
S1
S2
S3
S4
A7
S1
S2
S3
S4
A8
S1
S2
S3
S4
A9
S1
69
85
75/(>100)*
(>100)*
(>100)*
(>100)*c
90
58*
61*
e
70a
65
40
46
62
74
58
83
100
90
71/(86)*
(>100)*
(>100)*
(>100)*c
80/60*
55*
62*
e
70/(90)*
(>100)*
(98)*
(>100)*c
90
81*
74*
e
80
(<10)*c
(<10)*c
(<10)*c
nd
nd
nd
e
(0)*c
˜
100
100
100
90
3. Conclusion
(39)*; 18*b
(65)*; 33*b
(67)*; 30*b
(91)*; 28*b
81*
85*
34*
70*
We have prepared a small library of thrombin inhibitors
featuring the general structure B (Fig. 1). Despite the presence
of a piperazinyl moiety equipped with various spacer-arms, all
the compounds retained the activity of the parent structures,
i.e. the corresponding piperidine, morpholine, piperazine and
4-acetyl-piperazine derivatives. This experimentally validates
our design of graftable inhibitors for improving blood compat-
ibility of polymeric materials.
First attempts to improve the blood compatibility of a poly-
ester membrane by surface immobilization of thrombin inhib-
itors were encouraging as we observed a sensible decrease in
clot formation. Further experiments are on the way to increase
the level of surface grafting of active molecules on PET and
other materials, in view to reduce more significantly the coag-
ulation phenomenon when such devices are placed in contact
with blood.
61
75
(>100)*; 36*b
(>100)*; 49*b
(>100)*c
88*
81*
e
67
68
(>100)*
(>100)*
(>100)*c
28*
51*
e
60
75
(>100)*; 29*b
(>100)*; <5*b
(89)*; 8*b
88*
nd
nd
77
82
90
Yields in brackets are of the crude products; yields with * correspond to results
of parallel synthesis.
4. Experimental
a
PYBOP was used as coupling agent.
Purification by SFC (supercritical fluid chromatography).
Coupling product not present in the crude from MS analysis.
b
4.1. Chemistry (see Tables 2 and 3)
c
Reagents and solvents were purchased from Acros chimica,
Aldrich or Fluka. Tetrahydrofuran was dried over sodium/ben-
zophenone, then distilled. Column chromatographies were car-
ried out with silica gel 60 (70e230 mesh ASTM) supplied by
Merck. The IR spectra were recorded with PerkineElmer
1710 and Shimadzu Benelux FTIR-8400S instruments, only
the most significant adsorption bands being reported. The
mass spectra were obtained with a Finnigan MAT TSQ-70 in-
strument. The high resolution mass spectra (HRMS) were per-
formed at the University of Mons, Belgium (Prof.
R. Flammang). The microanalysis was performed at the Chris-
topher Ingold Laboratories of the University College, London
(Dr. A. Stones). The melting points were determined with an
to slightly diminish the activity. In the series AiS1 the pres-
ence of a long alkyl spacer-arm on the piperazine motif
(Y ¼ N-(CH2)12-NH2, N-(C2H4O)2-C2H4-NH2) reduced also
the activity, but less than in the case of the spacer-arm fixed
via an amide linkage (Y ¼ N-CO-(CH2)11-NH2). Lastly, a hy-
drophilic spacer of PEG type (compound A8S1) was not more
appropriate than a simple alkyl chain such as aminopropyl
(A9S1). Accordingly, inhibitors A9S1 and A7S1 provide
good candidates for surface binding, but inhibitors A8S1 and
A6S1 can also be considered. Indeed, the differences between
the tested inhibitors AiSj are not so relevant to allow a deep
insight on structureeactivity relationship. Moreover, to favour

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
43
Table 3
EA/MS analysis and TLC profile of intermediates and final tested compounds (in bold)
Compound
Elemental analysis
(%) or HRMS; found (calcd.)
Formula
TLC (Rf; solv.)
Boc-Arg(NO2)-A1
H2N-Arg(NO2)-A1
S1-Arg(NO2)-A1
S2-Arg(NO2)-A1
S3-Arg(NO2)-A1
A1S1
425.1912 (425.1915)
309.1643 (309.1651)
C16H30N6O5 (þK)
C11H22N6O3 (þNa)
C18H25N6O5SF3 (þ0.5H2O)
C18H29N6O5S
0.5; CH2Cl2
0; CH2Cl2
C, 42.78 (42.94); H, 4.97 (5.40); N, 16.23 (16.69); S, 6.77 (6.37)
441.0 (54%)a
477.1 (67%)a
450.1762 (450.1787)
0.8; MeOH/CH2Cl2 1:9
0.8; MeOH/CH2Cl2 1:9
0.8; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0.5; CH2Cl2
C21H29N6O5S
C18H27N5O3SF3
C18H30N5O3S
C21H30N5O3S
A1S2
A1S3
396.2062 (396.2069)
432.2059 (432.2069)
Boc-Arg(NO2)-A2
H2N-Arg(NO2)-A2
S1-Arg(NO2)-A2
S2-Arg(NO2)-A2
S3-Arg(NO2)-A2
A2S1
411.1970 (411.1968)
289.1628 (289.1624)
C15H28N6O6 (þNa)
C10H21N6O4
0; CH2Cl2
519.1238 (519.1250)
465.1544 (465.1532)
501.1516 (501.1532)
C17H23N6O6SF3 (þNa)
C17H26N6O6S (þNa)
C20H26N6O6S (þNa)
C17H24N5O4SF3 (þAcOH$1.5H2O)
C17H28N5O4S
0.5; MeOH/CH2Cl2 1:9
0.5; MeOH/CH2Cl2 1:9
0.6; MeOH/CH2Cl2 1:9
0.6; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0.6; CH2Cl2
C, 42.30 (42.38); H, 5.58 (5.80); N, 12.70 (13.00)
398.1874 (398.1862)
434.1854 (434.1862)
A2S2
A2S3
C20H27N5O4S
Boc-Arg(NO2)-A3
H2N-Arg(NO2)-A3
S1-Arg(NO2)-A3
S2-Arg(NO2)-A3
S3-Arg(NO2)-A3
A3S1
C, 52.32 (52.18); H, 6.85 (6.63); N, 18.04 (17.98)
422.2172 (422.2152)
C23H35N7O7 (þ0.5H2O)
C18H28N7O5
C25H30N7O7SF3
0; CH2Cl2
C, 47.52 (47.69); H, 4.85 (4.80); N, 15.13 (15.57); S, 5.13 (5.09)
576.0 (88%)a
612.0 (90%)a
0.5; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0.6; IsopOH/CH2Cl2 1:9
0.6; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0.4; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0.4; MeOH/CH2Cl2 1:9
0.4; MeOH/CH2Cl2 1:9
0.4; MeOH/CH2Cl2 1:9
0.5; Cyhex/EtOAc 5:4
0; Cyhex/EtOAc 5:4
0.7; MeOH/CH2Cl2 1:9
0.7; MeOH/CH2Cl2 1:9
0.7; MeOH/CH2Cl2 1:9
0.7; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0; MeOH/CH2Cl2 1:9
0.5; EtOAc/isopOH 1:1
0; EtOAc/isopOH 1:1
0.4; IsopOH/CH2Cl2 1:9
0.4; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0.8; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0.8; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0.3; IsopOH/CH3CN 2:8
0; IsopOH/CH3CN 2:8
0.5; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
C25H34N7O7S
C28H34N7O7S
C, 38.85 (39.18); H, 6.12 (6.40); N, 13.97(14.4)
397.2035 (397.2022)
433.2008 (433.2022)
C17H25N6O3SF3 (þAcOH$4H2O)
A3S2
A3S3
C17H29N6O3S
C20H29N6O3S
C17H33N7O5(þ0.5H2O)
C12H26N7O3
Boc-Arg(NO2)-A4
H2N-Arg(NO2)-A4
S1-Arg(NO2)-A4
S2-Arg(NO2)-A4
S3-Arg(NO2)-A4
Boc-Arg(NO2)-A5
H2N-Arg(NO2)-A5
S1-Arg(NO2)-A5
S2-Arg(NO2)-A5
S3-Arg(NO2)-A5
S4-Arg(NO2)-A5
A5S1
C, 48.33 (48.10); H, 8.21 (8.07); N, 23.15 (23.10)
316.2101 (316.2097)
524.1893 (524.1903)
C19H29N7O5SF3
C19H32N7O5S
C22H32N7O5S
C17H31N7O6Na
C12H23N7O4
470.2183 (470.2186)
506.2170 (506.2185)
452.2234 (452.2230)
330.1894 (330.1890)
538.0 (100%)a
C19H28N7O6SF3
C19H30N7O6S
C22H30N7O6S
C21H29N8O6S
C19H28N6O4SF3
484.1 (100%)a
520.1 (90%)a
521.1 (100%)a
493.1841 (493.1845)
A5S2
A5S3
A5S4
439.2131 (439.2128)
475.2121 (475.2128)
480.2386 (480.2393)
C
C
19H31N6O4S
22H31N6O4S
C21H34N7O4S
C35H59N9O9 (þ0.5H2O)
C30H52N9O7
Boc-Arg(NO2)-A6
H2N-Arg(NO2)-A6
S1-Arg(NO2)-A6
S2-Arg(NO2)-A6
S3-Arg(NO2)-A6
A6S1
C, 55.70 (55.39); H, 8.00 (7.97); N, 16.14 (16.61)
650.4001 (650.3990)
880.3596 (880.3615)
C37H54N9O9SF3 (þNa)
C37H58N9O9SF3
C40H58N9O9S
804.2 (5%)
840.2 (5%)
634.3737 (634.3726)
580.4003 (580.4009)
C29H51N7O3SF3
C29H54N7O3S
C32H54N7O3S
A6S2
A6S3
616.3994 (616.4009)
786.4150 (786.4126)
Boc-Arg(NO2)-A7
H2N-Arg(NO2)-A7
S1-Arg(NO2)-A7
S2-Arg(NO2)-A7
S3-Arg(NO2)-A7
A7S1
C35H57N9O10 (þNa)
C30H50N9O8
C37H52N9O10SF3
664.3768 (664.3782)
C, 50.17 (50.97); H, 5.94 (6.01); N, 14.0 (14.46)
818.1 (5%)a
854.1 (33%)a
C37H56N9O10
C40H56N9O10
S
S
C, 50.00 (50.43); H, 7.36 (7.44); N, 12.32 (12.48)
594.3793 (594.3802)
630.3795 (630.3802)
C
29H48N7O4SF3 (þ2AcOH$H2O)
A7S2
A7S3
C29H52N7O4S
C32H51N7O4S
C29H47N9O11
C24H40N9O9
Boc-Arg(NO2)-A8
H2N-Arg(NO2)-A8
S1-Arg(NO2)-A8
S2-Arg(NO2)-A8
S3-Arg(NO2)-A8
S4-Arg(NO2)-A8
A8S1
C, 49.75 (49.92); H, 6.94 (6.79); N, 17.43 (18.01)
598.2949 (598.2949)
806.2734 (806.2755)
C31H43N9O11SF3
752.2 (5%)
788.1 (10%)
C31H46N9O11
C34H46N9O11
S
S
789.1 (5%)
582.2709 (582.2685)
C33H45N10O11S
C23H39N7O5SF3
(continued on next page)

44
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
Table 3 (continued)
Compound
Elemental analysis
(%) or HRMS; found (calcd.)
Formula
TLC (Rf; solv.)
A8S2
528.2935 (528.2968)
C
23H42N7O5S
0; IsopOH/CH2Cl2 1:9
0.1; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
0.5; IsopOH/CH2Cl2 1:9
0; IsopOH/CH2Cl2 1:9
Boc-Arg(NO2)-A9
H2N-Arg(NO2)-A9
S1-Arg(NO2)-A9
A9S1
C, 48.62 (48.66); H, 6.16 (6.76); N, 19.05 (19.65)
C, 48.14 (48.18); H, 6.43 (6.35); N, 24.15 (24.08)
C, 46.06 (45.96); H, 5.09 (4.96); N, 16.98 (17.23)
C, 44.37 (44.03); H, 6.32 (6.62); N, 14.68 (14.98)
C26H41N9O9 (þH2O)
C21H33N9O7
C28H36N9O9SF3
C20H32N7O3SF3 (þ2AcOH$1.5H2O)
a
MS (APCI) m/z (rel. ab %).
1
Electrothermal microscope and are uncorrected. The H and
13C NMR spectra were recorded on Varian Gemini 300 (at
300 MHz for proton and 75 MHz for carbon) or Bruker AM-
500 spectrometers (at 500 MHz for proton and 125 MHz for
carbon); the chemical shifts were reported in ppm (d) down-
field from tetramethylsilane (TMS).
(1H, m), 5.20 (2H, s), 7.52 (2H, d, J ¼ 8.6 Hz), 8.22 (2H, d,
J ¼ 8.6 Hz); 13C NMR (75 MHz, CDCl3) d (ppm): 24.7,
26.7, 29.0, 29.1, 29.2, 29.3, 29.4, 29.9, 33.8, 41.3, 65.1,
123.7, 128.0, 144.1, 150.2, 155.7, 178.2; MS (APCI) m/z
(ass, rel. ab. %): 395.0 (M þ 1, 100); Anal. calcd. for
C20H30N2O6: C, 60.90; H, 7.65; N, 7.10%. Found: C, 60.55;
H, 7.45; N, 6.76%.
4.1.1. Preparation of the spacer-arms (A7 and A8)
The preparation of the spacer-arms A6 and A9 has been de-
scribed elsewhere [41].
4.1.1.2. 4-[12-(4-Nitro-benzyloxycarbonylamino)-dodecanoyl]-
piperazine-1-carboxylic acid tert-butyl ester (Scheme 2, part 1).
12-(4-Nitro-benzyloxycarbonylamino)-dodecanoic acid (3.0 g,
7.8 mmol, 1 equiv) was dissolved in dry THF (120 mL) and
NEt3 (1.095 g, 7.8 mmol, 1 equiv). The solution was cooled
down to ꢂ25/ꢂ20 ꢀC and then isobutyl chloroformate (1 mL,
7.8 mmol, 1 equiv) was added dropwise. The solution was
stirred for 15 min at ꢂ20/ꢂ25 ꢀC and then tert-butyl-pipera-
zine-1-carboxylate (1.432 g, 7.8 mmol, 1 equiv) was added
dropwise to the reaction mixture that was stirred for additional
20 min at low temperature and then 2 h at room temperature.
The solution was concentrated under reduced pressure and the
solid residue was extracted and partitioned between EtOAc
and brine. The organic layer was then washed with water, dried
over MgSO4 and evaporated under reduced pressure. The pure
product was isolated by column chromatography purification
(3.0 g, 70% yield). Mp ¼ 71.9e72.3 ꢀC; Rf ¼ 0.5 (SiO2,
CH2Cl2/isopropanol 99:1, UV); IR (CH2Cl2 liquid film on
NaCl) nmax (cmꢂ1): 3300, 2916, 2848, 1713, 1698, 1633,
1528, 1418, 1351, 1265, 1232, 1160, 1132; 1H NMR
(300 MHz, CDCl3) d (ppm): 1.26 (m, 14H), 1.47 (s, 9H), 1.61
(m, 4H), 2.32 (m, 2H), 3.19 (q, 2H, J ¼ 6.8 Hz), 3.43 (m,
6H), 3.58 (m, 2H), 4.83 (m, 1H), 5.18 (s, 2H), 7.51 (d, 2H,
4.1.1.1. 12-(4-Nitro-benzyloxycarbonylamino)-dodecanoic acid
(Scheme 2, part 1). 12-Aminododecanoic acid (250 mg,
1.16 mmol, 1 equiv) was dissolved in THF (3 mL), water
(3 mL) and NaOH 1 N (1.14 mL, 1 equiv). 4-Nitrobenzyl
chloroformate (246 mg, 1.16 mmol, 1 equiv) in THF (1 mL)
and NaOH 1 N (1.3 mL, 1.1 equiv) were added simultaneously
to the reaction mixture at 0 ꢀC. The solution was stirred at
0 ꢀC for 10 min and then for 2 h at room temperature, check-
ing pH to be above 10. HCl 1 N was added to the reaction mix-
ture to reach pH 1, and the solution was then extracted twice
with EtOAc. The organic layers were combined, washed with
HCl 1 N and water, dried over MgSO4, and evaporated under
reduced pressure to give a yellow solid that was purified by
column chromatography on SiO2 to obtain pure carbamate
(0.345 g) as a white solid (75% yield). Mp ¼ 107.9e
108.9 ꢀC; Rf ¼ 0.3 (SiO2, EtOAc/cyclohexane 3:5, UV); IR
(CH2Cl2 liquid film on NaCl) nmax (cmꢂ1): 3367, 2920,
1
2851, 1725, 1689, 1613, 1527, 1351, 1256, 1246; H NMR
(300 MHz, CDCl3) d (ppm): 1.27 (14H, m), 1.52 (2H, m),
1.64 (2H, m), 2.36 (2H, t, J ¼ 7.1 Hz), 3.21 (2H, m), 4.84
Graph 1. p-Nitroaniline release versus time monitored at 405 nm. Effect of inhibitor (compound A1S1) concentration on substrate S-2238 hydrolysis.

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
45
Graph 2. Velocity ratio versus inhibitor concentration. Linear fitting for Ki calculation (R2 ¼ 0.9984).
J ¼ 8.7 Hz), 8.21 (d, 2H, J ¼ 8.7 Hz); 13C NMR (75 MHz,
CDCl3) d (ppm): 25.3, 26.7, 28.4, 29.2, 29.4, 29.9, 33.3, 41.3,
43.6, 45.4, 65.0, 80.2, 123.7, 128.1, 144.2, 147.3, 154.6,
155.8, 171.8; MS (APCI) m/z (ass, rel. ab. %): 563.0 (M þ 1,
75), 507.1 (M ꢂ 55, 100); Anal. calcd. for C29H46N5O7: C,
61.90; H, 8.24; N, 9.96%. Found: C, 61.68; H, 8.35; N, 9.81%.
29.4, 29.9, 32.9, 43.4, 65.1, 123.7, 128.1, 144.1, 147.2,
155.8, 171.7; MS (APCI), m/z (ass, rel. ab. %): 925
(M$M þ 1, 12), 463.3 (M þ 1, 100); Anal. calcd. for
C26H39N4F3O7: C, 54.16; H, 6.82; N, 9.72%. Found: C,
53.77; H, 6.84; N, 9.54%.
4.1.1.3. (12-Oxo-12-piperazin-1-yl-dodecyl)-carbamic acid 4-
nitro-benzyl ester (A7). 4-[12-(4-Nitro-benzyloxycarbonyla-
mino)-dodecanoyl]-piperazine-1-carboxylic acid tert-butyl
ester (3 g, 5.33 mmol, 1 equiv) was dissolved in CH2Cl2
(100 mL) and TFA (0.912 g, 8.0 mmol, 1.5 equiv). The reac-
tion mixture was stirred at room temperature for 4 h. The sol-
vent was then removed by rotary evaporation at reduced
pressure and the solid residue was triturated and suspended
in Et2O to obtain the desired product as trifluoroacetate salt
(3.06 g, quantitative yield). Rf ¼ 0 (SiO2, CH2Cl2/isopropanol
99:1, UV); IR (CH2Cl2 liquid film on NaCl), nmax (cmꢂ1):
4.1.1.4. 2-[2-(2-Azido-ethoxy)-ethoxy]-ethanol (Scheme 2, part
2). 2-[2-(2-Chloroethoxy)-ethoxy]-ethanol (5.0 g, 29.7 mmol,
1 equiv) was dissolved in water (30 mL). NaI (0.9 g,
6 mmol, 0.2 equiv) and NaN3 (20 g, 0.3 mol, 10.4 equiv)
were added in one portion and the solution was stirred at
50 ꢀC for 54 h. The mixture was filtered and extracted four
times with EtOAc. The aqueous phase was saturated with
NaCl and extracted two times with EtOAc. The organic layers
were combined, evaporated under reduced pressure to give the
pure azide (5.016 g) as a yellow liquid (97% yield). Rf ¼ 0.5
(SiO2, AcOEt, UV); IR (CH2Cl2 liquid film on NaCl) nmax
(cmꢂ1) 3415, 2873, 2359, 2338, 2109, 1653, 1458, 1300,
1
3328, 2926, 2854, 1677, 1523, 1347, 1252, 1202, 1178; H
1
NMR (300 MHz, CDCl3) d (ppm): 1.25 (m, 14H), 1.49 (m,
2H), 1.60 (m, 2H), 2.32 (m, 2H), 3.17 (m, 6H), 3.77 (m,
2H), 3.88 (m, 2H), 4.86 (br, 1H), 5.17 (s, 2H), 7.51 (d, 2H,
J ¼ 8.7 Hz), 8.20 (d, 2H, J ¼ 8.7 Hz), 10.0 (br, 2H); 13C
NMR (50 MHz, CDCl3) d (ppm): 25.0, 26.7, 29.2, 29.3,
1119, 1070, 933; H NMR (300 MHz, CDCl3) d (ppm): 2.60
(br, 1H), 3.36 (t, 2H, J ¼ 5.2 Hz), 3.59 (m, 2H), 3.65 (m,
6H), 3.72 (m, 2H); 13C NMR (50 MHz, CDCl3) d (ppm):
51.0, 62.3, 70.6, 70.9, 71.9, 73.1; MS (APCI), m/z (ass, rel.
ab. %): 148.1 ((M ꢂ N2) þ 1, 100).
Table 4
Inhibition constants Ki in mM (compound code in brackets)
NH2
NH
F3C
H3C
S
i
SO2
N
H
N
H
HN
O
N
A
i
CH2
O
2.3e2.6 (A1S1)
0.9e1.8 (A2S1)
5.6e5.7 (A3S1)
2.5e2.8 (A5S1)
7.3e8.2 (A6S1)
2.5e5.7 (A7S1)
11.5e15.3 (A8S1)
1.9e5.7 (A9S1)
0.9e3.2 (A1S2)
1.5e2.6 (A2S2)
5.4e6.0 (A3S2)
4.4e5.3 (A5S2)
1.0e2.2 (A6S2)
3.7e4.5 (A7S2)
e
0.7e1.8 (A1S3)
0.4e0.9 (A2S3)
6.4e6.7 (A3S3)
1.0e1.2 (A5S3)
5.3e5.8 (A6S3)
8.0e8.3 (A7S3)
e
e
e
e
NH
N-CO-CH3
N-(CH2)12-NH2
1.2e2.0 (A5S4)
e
e
e
e
N-CO-(CH2)11-NH2
N-(C2H4-O)2-C2H4-NH2
N-(CH2)3-NH2
e
e

46
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
Table 5
Inhibitor coating effect on blood clot formation
The product was crystallized from a solution of pentane/
CH2Cl2 1:1, filtered and washed with cold pentane to give the
pure carbamic ester (5.447 g, 69% yield). Mp: 81e84 ꢀC;
Rf ¼ 0.1 (SiO2, EtOAc, UV); IR (KBr) nmax (cmꢂ1): 3428,
3334, 2933, 2873, 1721, 1607, 1521, 1453, 1347, 1256, 1110,
Surfacea
Inhibitor
concentration (mg)
Clot weightb % Inhibition Relative
activity (%)
PET, native
membrane
PET, coated
with heparin
PET, coated
with A6S1
a
e
27.5 ꢃ 1.5
e
1
1059, 910, 735; H NMR (500 MHz, CDCl3) d (ppm): 3.41
500 Uc/cm2
11.4 ꢃ 0.5
58
22
100
38
(q, 2H, J ¼ 5.3 Hz), 3.58 (q, 2H, J ¼ 5.3 Hz), 3.60 (m, 2H),
3.65 (m, 4H), 3.73 (m, 2H), 5.19 (s, 2H), 5.53 (t, 1H), 7.51
(d, 2H, J ¼ 8.8 Hz), 8.21 (d, 2H, J ¼ 8.8 Hz); 13C NMR
(125 MHz, CDCl3) d (ppm): 40.84, 61.61, 65.01, 69.85,
70.25, 72.38, 123.6, 128.00, 143.97, 147.45, 155.81; MS
(APCI), m/z (ass, rel. ab. %): 329.0 (M þ 1, 37); Anal. calcd.
for C14H20N2O7: C, 51.22; H, 6.14; N, 8.53%. Found: C,
51.36; H, 6.24; N, 8.39%.
10 nmol/cm2 21.6 ꢃ 3.0
PET samples are disks of 2.6 cm of diameter, placed in contact with human
blood as described in Section 4.
b
Results are the mean of three independent experiments ꢃ standard
deviation.
c
USP heparin units.
4.1.1.5. 2-[2-(2-Amino-ethoxy)-ethoxy]-ethanol (Scheme 2,
part 2). 2-[2-(2-Azido-ethoxy)-ethoxy]-ethanol (5.246 g,
29.9 mmol, 1 equiv) was dissolved in MeOH (60 mL). Pd/C
(0.9 g, 0.15 mmol, 0.005 equiv) was added, then the flask
was purged with argon and filled with hydrogen. The solution
was stirred 24 h at room temperature. The mixture was filtered
and the solvent evaporated at reduced pressure to obtain the
pure amino alcohol (0.4266 g, 95% yield). Rf ¼ 0 (SiO2,
AcOEt, UV); IR (KBr) nmax (cmꢂ1): 3383, 3053, 2873,
2305, 2253, 1603, 1458, 1422, 1350, 1265, 1116, 1072, 909,
735; 1H NMR (CD3OD/300 MHz) d (ppm): 2.92 (t, 2H,
J ¼ 5.2 Hz), 3.59 (t, 2H, J ¼ 5.2 Hz), 3.61 (m, 6H), 3.66 (m,
2H) 3.73 (t, 2H, J ¼ 4.5 Hz); 13C NMR (50 MHz, CDCl3)
d (ppm): 40.9, 61.0, 70.0, 70.1, 71.6, 72.6; MS (APCI), m/z
(ass, rel. ab. %): 150.2 (M þ 1, 100).
4.1.1.7. Methanesulfonic acid 2-{2-[2-(4-nitro-benzyloxycar-
bonylamino)-ethoxy]-ethoxy}-ethyl ester (Scheme 2, part
2). The N-protected alcohol (5.375 g, 16.38 mmol, 1 equiv)
was dissolved in distilled CH2Cl2 (50 mL) and the solution
was cooled down to 0 ꢀC. Pyridine (2.589 g, 32.76 mmol,
2 equiv) and methanesulfonyl chloride (2.800 g, 24.57 mmol,
1.5 equiv), both diluted in CH2Cl2 (5 mL) were added drop-
wise to the reaction mixture. After stirring at room tempera-
ture for 24 h, the solution was washed with HCl 1 N, then
a saturated solution of NaHCO3 and brine. The organic phase
was dried over MgSO4, concentrated under reduced pressure
and the liquid residue was purified by flash chromatography
to obtain the mesylate (5.990 g) as yellow gel (90% yield).
Rf ¼ 0.3 (SiO2, CH2Cl2/EtOAc 7:3, UV); IR (KBr) nmax
(cmꢂ1): 3393, 2946, 2872, 1733, 1717, 1700, 1607, 1521,
1540, 1507, 1347, 1248, 1172, 1108, 1014, 973, 920, 854,
1
4.1.1.6. {2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethyl}-carbamic acid
4-nitro-benzyl ester (Scheme 2, part 2). 2-[2-(2-Amino-eth-
oxy)-ethoxy]-ethanol (3.601, 24.1 mmol, 1 equiv) was dis-
solved in distilled CH2Cl2 (100 mL) and the mixture was
cooled down to 0 ꢀC. A solution of p-nitrobenzyl chloroformate
(5.363 g, 24.1 mmol, 1 equiv) dissolved in CH2Cl2 (20 mL) and
NEt3 (2.699 g, 24.1 mmol, 1 equiv) was added dropwise and si-
multaneously to the reaction mixture. The solution was stirred
15 min at 0 ꢀC and additional 2 h at room temperature. The
mixture was then washed twice with HCl 1 N, once with water
and NaOH 1 N, and finally with brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure.
801, 775, 738; H NMR (300 MHz, CDCl3) d (ppm): 2.02
(s, 3H), 3.39 (dt, 2H, J ¼ 4.8, 5.7 Hz), 3.55 (q, 2H,
J ¼ 4.8 Hz), 3.62 (m, 2H), 3.64 (m, 2H), 3.75 (tt, 2H,
J ¼ 3.8, 4.8 Hz), 4.36 (tt, 2H, J ¼ 3.8, 4.8 Hz), 5.18 (s, 2H),
5.43 (t, 1H, J ¼ 5.7 Hz), 7.50 (d, 2H, J ¼ 8.7 Hz), 8.19 (d,
2H, J ¼ 8.7 Hz); 13C NMR (75 MHz, CDCl3) d (ppm): 38.1,
41.4, 65.5, 69.4, 69.5, 70.4, 70.6, 71.0, 124.2, 128.5, 144.5,
147.8, 156.3; MS (APCI), m/z (ass, rel. ab. %): 407.0
(M þ 1, 100); Anal. calcd. for C15H22N2O9S: C, 44.33; H,
5.46; N, 6.89%. Found: C, 44.09; H, 5.54; N, 6.47%.
4.1.1.8. 4-(2-{2-[2-(4-Nitro-benzyloxycarbonylamino)-ethoxy]-
ethoxy}-ethyl)-piperazine-1-carboxylic acid tert-butyl ester
(Scheme 2, part 2). The mesylate (5.90 g, 14.52 mmol,
1 equiv) was dissolved in CH3CN (50 mL). NaI (2.18 mmol,
14.52 mmol, 1 equiv) was dissolved in H2O (1.5 mL) and added
to the mesylate solution that was stirred for 30 min at 85 ꢀC.
Boc-piperazine (2.71 g, 14.52 mmol, 1 equiv) dissolved in DI-
PEA (1.87 g, 14.52 mmol, 1 equiv) and CH3CN (5 mL) was
added in one portion and the solution was stirred for 24 h at
room temperature. Then the solution was concentrated, the solid
residue dissolved in AcOEt and washed with brine. The organic
phase was dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by flash chromatography to
give the desired product as yellow gel (7.21 g, 92% yield).
Table 6
Inhibitor grafting effect on blood clot formation
Surfacea
Inhibitor
concentration
(by XPS)
Clot weightc
(mg)
% Inhibition
PET, blankb
PET, grafted
with A6S1
<0.5% (adsorption)
1.9% or 54 pmol/cm2
29.7 ꢃ 0.4
27.5 ꢃ 0.5
e
8
a
See Table 5.
b
This sample has been exposed to all the chemical treatments, but with the
omission of the surface activating agent, i.e. tosyl chloride.
c
See Table 5.

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
47
Rf ¼ 0.5 (SiO2, CH2Cl2/isopropanol 9:1, UV); IR (CH2Cl2 liq-
uid film on NaCl) nmax (cmꢂ1): 3310, 2936, 2868, 1725, 1691,
1606, 1523, 1458, 1422, 1365, 1347, 1247, 1170, 1130, 1005,
937, 859, 803, 771, 738; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.44 (s, 9H), 2.49 (m, 4H), 2.64 (m, 2H), 3.40 (dt,
2H, J ¼ 5.0, 5.3 Hz), 3.45 (m, 4H), 3.56 (q, 2H, J ¼ 5.3 Hz),
3.60 (m, 4H), 3.65 (m, 2H), 5.19 (s, 2H), 5.54 (t, 1H,
J ¼ 5.0 Hz), 7.51 (d, 2H, J ¼ 8.8 Hz), 8.21 (d, 2H,
J ¼ 8.8 Hz), 13C NMR (75 MHz, CDCl3) d (ppm): 28.3, 40.9,
42.6, 43.6, 53.2, 57.6, 65.0, 68.4, 69.8, 70.1, 70.2, 79.7, 123.6,
128.0, 144.0, 147.5, 154.5, 155.9; MS (APCI), m/z (ass, rel.
ab. %): 497.2 (M þ 1, 100); Anal. calcd. for
C23H36N4O8$0.5H2O: C, 54.64; H, 7.38; N, 11.08%. Found:
C, 54.53; H, 7.40; N, 10.93%.
4.1.2.1.
[(S )-4-(N0-Nitro-guanidino)-1-(morpholine-4-car-
bonyl)-butyl]-carbamic acid tert-butyl ester (Boc-Arg(NO2)-
A2). N-a-Boc-N-a-nitro-L-arginine (0.500 g, 1.56 mmol,
1 equiv) and PyBop (0.817 g, 1.56 mmol, 1 equiv) were dis-
solved in dry CH2Cl2 (8 mL). Morpholine (0.545 g,
6.26 mmol, 4 equiv) was dissolved in dry CH2Cl2 (2 mL)
and added to the reaction mixture with stirring at room tem-
perature. After 1 h 30 min the solution was filtered to elimi-
nate the white precipitate, and then washed once with NaOH
1 N. The organic layer was concentrated at reduced pressure
to give a brown gel (0.9 g) containing 56% of desired product
(84% raw yield, by NMR). The solid residue can be submitted
directly to the next reaction or purified by column chromatog-
raphy (0.452 g, 70% yield). 1H NMR (300 MHz, CDCl3)
d (ppm): 1.42 (s, 9H), 1.64 (m, 2H), 1.73 (m, 2H), 3.22 (m,
1H), 3.47 (m, 2H), 3.59 (m, 2H), 3.67 (m, 5H), 4.55 (m,
1H), 5.80 (m, 1H), 7.80 (s, 2H), 8.95 (s, 1H); IR (CH2Cl2 liq-
uid film on NaCl) nmax (cmꢂ1): 3310, 2961, 2922, 2847, 1701,
1626, 2529, 1446, 1371, 1270, 1164, 1114, 847.
4.1.1.9. {2-[2-(2-Piperazin-1-yl-ethoxy)-ethoxy]-ethyl}-carba-
mic acid 4-nitro-benzyl ester (A8). The N-protected compound
(4.39 g, 8.84 mmol, 1 equiv) was dissolved in a 1:9 mixture of
TFA/CH2Cl2 (7 mL) and the solution was stirred at room tem-
perature for 4 h. The solution was concentrated under reduced
pressure to give a brown gel that was triturated and suspended
in Et2O until precipitation of a brown solid occurred. The solid
residue was dissolved in water and basified to pH 12 with
a 1 N NaOH solution that was saturated with NaCl, and ex-
tracted four times with CH2Cl2. The organic layers were com-
bined, dried over MgSO4 and concentrated at reduced pressure
to give the pure deprotected product (3.339 g) as a brown gel
(95% recovery yield). Rf ¼ 0 (SiO2, CH2Cl2/isopropanol 9:1,
UV); IR (CH2Cl2 liquid film on NaCl) nmax (cmꢂ1): 3326,
2940, 2870, 1718, 1606, 1521, 1458, 1347, 1260, 1134,
4.1.2.2. [1-(4-Ethyl-piperazine-1-carbonyl)-4-(N0-methyl-gua-
nidino)-butyl]-carbamic acid tert-butyl ester (Boc-Arg(NO2)-
A4). This product was obtained from 5.0 g (15.7 mmol,
1 equiv) of N-Boc-N-nitro-arginine, 3.18 g (31.3 mmol,
2 equiv) of NMM, 2.14 g (15.7 mmol, 1 equiv) of isobutyl
chloroformate, 1.78 g (15.7 mmol, 1 equiv) of ethyl piperazine
and again 1.60 g (15.7 mmol, 1 equiv) of NMM in 80 mL of
THF. The solid residue obtained after workup was purified
by two column chromatographies (CH3CN/isopropanol 9:1,
CH3CN/isopropanol 5:5) to obtain 2.61 g of an orange gel
1
1
1105, 854, 739; H NMR (300 MHz, CDCl3) d (ppm): 2.47
(yield 40%). H NMR (300 MHz, CDCl3) d (ppm): 1.11 (t,
(m, 4H), 2.59 (t, 2H, J ¼ 5.7 Hz), 2.89 (m, 4H, J ¼ 4.8 Hz),
3.40 (q, 2H, J ¼ 5.3 Hz), 3.57 (t, 2H, J ¼ 5.7 Hz), 3.61 (m,
6H, J ¼ 5.3 Hz), 5.20 (s, 2H), 5.62 (t, 1H, J ¼ 5.0 Hz), 7.52
(d, 2H, J ¼ 8.8 Hz), 8.22 (d, 2H, J ¼ 8.8 Hz); HRMS-ESI,
m/z: 397.2072 (calcd. for C18H29N4O6 ¼ 397.2087).
3H, J ¼ 7.1 Hz), 1.42 (s, 9H), 1.64 (m, 7H), 1.74 (m, 8H),
2.48 (m, 6H), 3.27 (m, 1H), 3.48 (m, 2H), 3.58 (m, 1H),
3.66 (m, 2H), 4.56 (m, 1H), 5.84 (d, 1H, 7.5 Hz), 7.81 (br,
2H), 8.89 (br, 1H); 13C NMR (125 MHz, CDCl3) d (ppm):
11.58, 24.27, 28.28, 31.20, 40.18, 41.88, 45.22, 48.54,
52.00, 52.46, 80.37, 156.54, 159.30, 169.74; IR (CH2Cl2 liq-
uid film on NaCl) nmax (cmꢂ1): 3400, 2976, 2938, 2826,
1992 (C]O st), 1634 (C]N), 1531 (N]O as), 1455, 1255,
1165.
4.1.2. General procedure for the preparation of 5-(3-nitro-
guanidino)-2(S )-tert-butoxycarbonylamino-1-(azacyclo-
hexan-1-yl)pentan-1-one (Boc-Arg(NO2)-Ai; Scheme 1,
step i).
N-a-Boc-N-a-nitro-L-arginine (1.00 g, 3.13 mmol, 1 equiv)
was dissolved in dry THF (10 mL) and NMM (0.64 g,
6.26 mmol, 2 equiv). Isobutyl chloroformate (0.43 g,
3.13 mmol, 1 equiv) was then added dropwise at ꢂ20/
ꢂ25 ꢀC. After 30 min azacyclohexane (3.13 mmol, 1 equiv)
in dry THF (2 mL) was added dropwise at ꢂ20 ꢀC and stirred
for additional 20 min. The solution was allowed to warm up to
room temperature and stirred for 1 h. The solvent was then
evaporated and the solid residue partitioned between EtOAc
and brine. The organic layer was dried over MgSO4 and con-
centration under vacuum gave crude product that was either
submitted directly to deprotection, or purified by column chro-
matography on SiO2.
4.1.2.3. [(S )-1-(4-Acetyl-piperazine-1-carbonyl)-4-(N0-nitro-
guanidino)-butyl]-carbamic acid tert-butyl ester (Boc-
Arg(NO2)-A5). This product was obtained from 5.0 g
(15.7 mmol, 1 equiv) of N-Boc-N-nitro-arginine, 3.18 g of
NMM (31.3 mmol, 2 equiv), 2.14 g (15.7 mmol, 1 equiv) of
isobutyl chloroformate, 2.0 g (15.7 mmol, 1 equiv) of acetyl
piperazine and again 1.60 g (15.7 mmol, 1 equiv) of NMM
in 80 mL of THF. The solid residue was purified by column
chromatography to obtain 3.26 g of a white solid (46% yield).
1H NMR (300 MHz, CDCl3) d (ppm): 1.41 (s, 9H), 1.64 (m,
2H), 1.73 (m, 2H), 2.10 (s, 3H), 3.48 (m, 2H), 3.10e3.80
(m, 8H), 4.59 (m, 1H), 5.80 (m, 1H), 7.73 (br, 2H), 8.82 (br,
1H); 13C NMR (125 MHz, CDCl3) d (ppm): 21.15, 21.21,
24.27, 28.20, 30.74, 40.31, 40.82, 41.14, 45.61, 45.92,
41.77, 41.94, 45.00, 45.26, 48.88, 80.27, 156.20, 159.30,
169.20, 169.24, 170.26, 170.41; IR (CH2Cl2 liquid film on
Compounds Boc-Arg(NO2)-Ai with i being 1, 3, 6 and 9
have been described elsewhere [41]. Rf, MS/EA for all follow-
ing compounds are reported in Table 3.

48
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
NaCl) nmax (cmꢂ1): 3420, 2976, 2932, 2870, 1705, 1622,
1521, 1436, 1366, 1253, 1166.
dissolution in NaOH 1 N, and extraction of the aqueous solution
with EtOAc (about 90% product recovery).
Compounds H2N-Arg(NO2)-Ai with i being 1, 3, 6 and 9
have been described elsewhere [41]. Rf, MS/EA for all follow-
ing compounds are reported in Table 3.
4.1.2.4. (12-{4-[(S )-2-tert-Butoxycarbonylamino-5-(N0-nitro-
guanidino)-pentanoyl]-piperazin-1-yl}-12-oxo-dodecyl)-carba-
mic acid 4-nitro-benzyl ester (Boc-Arg(NO2)-A7). This product
was prepared from N-Boc-N-nitro-arginine (1.71 g, 5.34 mmol,
1 equiv), NMM (1.62 g, 16 mmol, 3 equiv), isobutyl chlorofor-
mate (0.73 g, 5.34 mmol, 1 equiv) and (12-oxo-12-piperazin-1-
yl-dodecyl)-carbamic acid 4-nitro-benzyl ester (3.08 g,
5.34 mmol, 1 equiv) to give a brown solid (3.67 g) containing
the desired product (83% by NMR, 75% raw yield). The raw
product could be submitted to the next reaction step without
further purifications. Pure product was obtained by column
chromatography (74% isolated yield). 1H NMR (300 MHz,
CDCl3) d (ppm): 1.26 (br, 12H), 1.43 (s, 9H), 1.49 (m, 2H),
1.64 (m, 4H), 1.75 (m, 2H), 2.33 (t, 2H, J ¼ 7.2 Hz), 3.18
(q, 2H, J ¼ 6.6 Hz), 3.26 (m, 1H), 3.48 (m, 6H), 3.70 (m, 2H),
4.60 (m, 1H), 4.85 (m, 1H), 5.18 (s, 2H), 5.85 (dd, 1H,
J ¼ 8.2 Hz), 7.50 (d, 2H, J ¼ 8.7 Hz), 7.65 (br, 2H), 8.20
(d, 2H, J ¼ 8.7 Hz), 8.80 (br, 1H).
4.1.3.1. N-((S )-4-Amino-5-morpholine-4-yl-5-oxo-pentyl)-N0-
nitro-guanidine (H2N-Arg(NO2)-A2). This product was
obtained from 0.50 g of Boc-Arg(NO2)-A2 (1.31 mmol) and
10 mL of TFA/CH2Cl2 solution. 0.515 g of product as trifluor-
oacetate salt was isolated (quantitative yield). 1H NMR
(300 MHz, CDCl3) d (ppm): 1.75 (m, 2H), 1.94 (m, 2H),
3.36 (m, 2H), 3.63 (m, 4H), 3.75 (m, 2H), 3.81 (m, 2H),
4.58 (t, 1H); IR (CH2Cl2 liquid film on NaCl) nmax (cmꢂ1):
1684, 1447, 1263, 1103, 1037, 850, 721.
4.1.3.2. N-[(S )-4-Amino-5-(4-ethyl-piperazin-1-yl)-5-oxo-pen-
tyl]-N0-nitro-guanidine (H2N-Arg(NO2)-A4). This product
was obtained from 4.70 g of Boc-Arg(NO2)-A4 (11 mmol)
and 20 mL of solution of TFA/CH2Cl2 solution. 2.92 g of
the product as free amine was isolated (80% yield). 1H
NMR (300 MHz, CDCl3) d (ppm): 1.09 (t, 3H, J ¼ 7.1 Hz),
1.43 (s, 9H), 1.65 (m, 7H), 1.76 (m, 8H), 2.43 (m, 6H),
3.28e3.73 (m, 6H), 4.56 (m, 1H), 5.85 (d, 1H, J ¼ 7.5 Hz),
7.80 (br, 2H), 8.76 (br, 1H); 13C NMR (125 MHz, CDCl3)
d (ppm): 10.4, 27.7, 31.20, 41.5, 42.1, 44.4, 52.2, 52.9, 54.2,
162.1, 169.8; IR (CH2Cl2 liquid film on NaCl) nmax (cmꢂ1):
1710e1630, 1439, 1294, 1205, 1131.
4.1.2.5. (12-{4-[(S )-2-tert-Butoxycarbonylamino-5-(N0-nitro-
guanidino)-pentanoyl]-piperazin-1-yl}-12-oxo-dodecyl)-carba-
mic acid 4-nitro-benzyl ester (Boc-Arg(NO2)-A8). This
product was obtained from 1.84 g (5.78 mmol, 1 equiv) of
N-Boc-N-nitro-arginine, 1.17 g (11.6 mmol, 2 equiv) of NMM,
0.79 g (5.8 mmol, 1 equiv) of isobutyl chloroformate, 2.29 g
(5.8 mmol, 1 equiv) of product A8 and again 0.58 g
(5.8 mmol, 1 equiv) of NMM in 20 mL of THF. The solid res-
idue was purified by two column chromatographies (EtOAc/
isopropanol 8:2, and then CH3CN/isopropanol 6:4) in order
to obtain 2.32 g of pure product (58% yield). 1H NMR
(300 MHz, CDCl3) d (ppm): 1.43 (s, 9H), 1.62 (m, 2H), 1.73
(m, 2H), 2.53 (m, 4H), 2.64 (m, 2H), 3.26 (m, 1H), 3.39 (m,
2H), 3.44 (m, 2H), 3.56 (m, 2H), 3.61 (m, 9H), 4.55 (m,
1H), 5.19 (s, 2H), 5.48 (m, 1H), 5.82 (m, 1H), 7.51 (d, 2H,
JHH ¼ 8.6 Hz), 7.79 (br, 2H), 8.20 (d, 2H, JHH ¼ 8.6 Hz),
8.86 (br, 1H); 13C NMR (125 MHz, CDCl3) d (ppm): 24.2,
28.3, 31.3, 40.1, 40.9, 41.8, 45.1, 48.5, 52.8, 53.2, 57.3, 65.0,
68.5, 69.8, 70.1, 70.2, 80.4, 123.6, 128.0, 144.0, 147.4,
155.9, 156.6, 159.3, 169.7; IR (CH2Cl2 liquid film on NaCl)
nmax (cmꢂ1) 3328, 2930, 2870, 1707, 1628, 1518, 1451,
1341, 1252, 1158, 1100, 723.
4.1.3.3.
N-[5-(4-Acetyl-piperazin-1-yl)-4-(S )-amino-5-oxo-
pentyl]-N0-nitro-guanidine (H2N-Arg(NO2)-A5). This product
was obtained from 5.09 g of raw Boc-Arg(NO2)-A5
(9.12 mmol) and 50 mL of solution of TFA/CH2Cl2. The prod-
uct was isolated as trifluoroacetate white solid (4.1 g, quantita-
1
tive yield). H NMR (300 MHz, CDCl3) d (ppm): 1.69 (m,
2H), 1.93 (m, 2H), 2.14 (s, 3H), 3.33 (m, 2H), 3.50e3.71 (m,
8H), 4.58 (t, 1H, J ¼ 4.5 Hz); 13C NMR (125 MHz, CDCl3)
d (ppm): 20.8, 27.5, 40.4, 41.4, 41.8, 42.4, 42.7, 45.1, 45.3,
45.8, 46.2, 50.8, 159.2, 168.1, 173.1; IR (CH2Cl2 liquid film
on NaCl) nmax (cmꢂ1): 3400e2900, 1691, 1634, 1518, 1439,
1368, 1267, 1202, 1132, 1000, 835, 800, 722.
4.1.3.4.
(12-{4-[2-(S )-Amino-5-(N0-nitro-guanidino)-penta-
noyl]-piperazin-1-yl}-12-oxo-dodecyl)-carbamic acid 4-nitro-
benzyl ester (H2N-Arg(NO2)-A7). This product was obtained
from raw Boc-Arg(NO2)-A7 (3.67 g) dissolved in TFA/DCM
1:1 (50 mL). The product was isolated as trifluoroacetate
salt (3.538 g, quantitative yield). 1H NMR (500 MHz,
CD3OD) d (ppm): 1.30 (m, 12H), 1.50 (2H, m), 1.61 (m,
2H, J ¼ 7.3 Hz), 1.72 (m, 2H), 1.88 (t, 2H, J ¼ 7.2), 2.41
(m, 2H), 3.11 (q, 2H, J ¼ 6.6 Hz), 3.31 (m, 2H), 3.64 (m,
8H), 4.49 (m, 2H), 5.18 (s, 2H), 7.57 (d, 2H, J ¼ 8.7 Hz),
8.20 (d, 2H, J ¼ 8.7 Hz); 13C NMR (125 MHz, CDCl3)
d (ppm): 26.41, 28.79, 27.78, 30.37, 30.43, 30.50, 30.61,
30.62, 30.84, 34.08, 41.18, 41.86, 42.21, 42.69, 43.18,
43.59, 46.19, 46.54, 46.61, 51.44, 65.87, 124.56, 129.03,
4.1.3. General procedure for the preparation of 2(S )-
amino-5-nitro-guanidino-1-(azacyclohexan-1-yl)-pentan-1-
one (H2N-Arg(NO2)-Ai; Scheme 1, step ii).
Product Boc-Arg(NO2)-Ai (1.25 mmol) was dissolved in
a 1:1 mixture of TFA/CH2Cl2 (10 mL) and stirred at room tem-
perature for 3 h. The solution was concentrated at reduced pres-
sure and the obtained orange gel was suspended and triturated in
Et2Ountilprecipitationofthetrifluoroacetatesaltasacrystalline
solid occurred. The precipitate was washed with Et2O and used
directly in the next reaction (quantitative yield). For spectro-
scopic analysis the product was converted to the free amine by

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
49
146.39, 148.86, 158.42, 158.42, 168.71, 168.68, 174.47,
177.54.
4.1.4.3.
(12-{4-[5-(N0-Nitro-guanidino)-2-(S )-(3-trifluoro-
methyl-benzenesulfonylamino)-pentanoyl]-piperazin-1-yl}-12-
oxo-dodecyl)-carbamic acid 4-nitro-benzyl ester (S1-
Arg(NO2)-A7). This product was obtained from 0.154 g
(0.21 mmol, 1 equiv) of the precursor NH2-Arg(NO2)-A7,
0.063 g of Et3N (0.64 mmol, 3 equiv) and 0.060 g of 3-trifluor-
omethyl-benzenesulfonyl chloride (0.24 mmol, 1.2 equiv).
After workup the residue was purified by column chromatogra-
phy to obtain the desired product (0.106 g, yield 67%). IR
(CH2Cl2 liquid film on NaCl) nmax (cmꢂ1): 1707, 1627, 1522,
4.1.3.5. {2-[2-(2-{4-[2(S )-Amino-5-(N0-nitro-guanidino)-pen-
tanoyl]-piperazin-1-yl}-ethoxy)-ethoxy]-ethyl}-carbamic acid
4-nitro-benzyl ester (H2N-Arg(NO2)-A8). This product was
obtained from 3.38 g of Boc-Arg(NO2)-A8 (4.08 mmol) and
50 mL of TFA/CH2Cl2. The product was isolated as trifluoroa-
cetate salt (4.805 g, quantitative yield). The product was then
neutralized for the next reaction step by dissolution in NaOH
1 N and extraction with CH2Cl2 off the aqueous phase satu-
1
1427, 1346, 1326, 1163, 1130; H NMR (300 MHz, CDCl3)
1
rated with NaCl. H NMR (300 MHz, CDCl3) d (ppm): 1.70
d (ppm): 1.26 (m, 14H), 1.51 (m, 4H), 1.59 (m, 4H), 2.29
(m, 2H), 3.10e3.70 (m, 12H), 4.18 (m, 1H), 4.90 (m, 1H),
5.19 (s, 2H), 7.51 (d, 2H, J ¼ 8.1 Hz), 7.68 (t, 1H, J ¼ 8.7 Hz),
7.82 (d, 1H, J ¼ 8.7 Hz), 8.06 (m, 2H), 8.21 (d, 2H,
J ¼ 8.1 Hz); 13C NMR (125 MHz, CD3OD) d (ppm): 25.4,
26.9, 29.5, 29.6, 29.7, 29.8, 30.2, 33.4, 40.5, 41.4, 42.2, 42.3,
42.4, 45.3, 45.4, 46.3, 52.8, 65.2, 123.2 (JCF ¼ 273 Hz), 123.8,
124.3 (JCF ¼ 4.3 Hz), 128.2, 129.6, 130.2 (JCF ¼ 4.3 Hz),
131.0, 140.7, 144.4, 147.5, 156.0, 159.3, 172.7, 177.7.
(m, 2H), 1.88 (m, 2H), 3.33 (m, 8H), 3.42 (m, 2H), 3.60 (m,
2H), 3.68 (t, 4H), 3.83 (m, 4H), 4.57 (m, 1H), 5.20 (s, 2H),
7.55 (d, 2H, J ¼ 8.7 Hz), 8.20 (d, 2H, J ¼ 8.7 Hz); 13C NMR
(125 MHz, CDCl3) d (ppm): 39.5, 40.4, 40.8, 42.5, 48.9,
50.7, 51.6, 56.6, 63.7, 65.8, 69.6, 69.7, 69.9, 124.0, 127.9,
144.8, 147.3, 158.3, 159.1, 168.2; IR (CH2Cl2 liquid film on
NaCl) nmax (cmꢂ1): 3328, 2936, 1676, 1608, 1522, 1450,
1349, 1271, 1203, 1132, 836, 800, 740, 722.
4.1.4. General procedure for the preparation of arylsulfonic
acid [4-guanidino-1(S )-(azacyclohexan-1-ylcarbonyl)
butyl]amide (Sj-Arg(NO2)-Ai; Scheme 1, step iii).
4.1.4.4. {2-[2-(2-{4-[(S )-5-Guanidino-2-(3-trifluoromethyl-ben-
zenesulfonylamino)-pentanoyl]-piperazin-1-yl}-ethoxy)-ethoxy]-
ethyl}-carbamic acid 4-nitrobenzyl ester (S1-Arg(NO2)-A8).
This product was obtained from 0.22 g of NH2-Arg(NO2)-A8
(0.37 mmol, 1 equiv), 0.149 g of Et3N (1.48 mmol, 4 equiv)
and 0.108 g of 3-trifluoromethyl-benzenesulfonyl chloride
(0.44 mmol, 1.2 equiv). After workup, the residue was purified
by flash chromatography (gradient of EtOAc in CH3CN from
1:9 to 2:8) to obtain 0.1772 g of desired product (yield 60%).
1H NMR (500 MHz, CDCl3, 44 ꢀC) d (ppm): 1.60 (m, 2H),
1.67 (m, 2H), 2.12 (m, 1H), 2.22 (m, 1H), 2.37 (m, 1H), 2.44
(m, 1H), 3.17 (m, 2H), 3.30 (m, 2H), 3.40 (m, 4H), 3.52 (m,
2H), 3.60 (m, 8H), 4.10 (t, 2H), 5.21 (s, 2H), 5.38 (br, 1H),
6.24 (br, 1H), 7.14 (br, 2H), 7.50 (d, 2H, J ¼ 8.7 Hz), 7.65 (t,
1H), 7.82 (d, 1H), 8.02 (d, 1H), 8.05 (s, 1H), 8.20 (d, 2H,
J ¼ 8.7 Hz); 13C NMR (125 MHz, CDCl3, 40 ꢀC) d (ppm):
24.28, 30.03, 41.05, 40.23, 42.13, 45.10, 52.28, 52.77, 53.02,
57.26, 65.14, 68.80, 69.78, 70.29, 123.65, 124.20, 128.03,
129.48, 129.98, 131.72, 140.53, 144.04, 147.66, 155.98,
159.49, 168.19; IR (CH2Cl2 liquid film on NaCl) nmax
(cmꢂ1): 3328, 2936, 1716, 1634, 1608, 1522, 1430, 1347,
1326, 1262, 1165, 1132, 1100.
Product H2N-Arg(NO2)-Ai (0.54 mmol, 1 equiv) was dis-
solved in CH2Cl2 (5 mL) and Et3N (0.169 g, 1.67 mmol,
3.1 equiv). Then arylsulfonyl chloride (0.64 mmol, 1.2 equiv)
in CH2Cl2 (1 mL) was added dropwise with stirring at 0e
5 ꢀC. The solution was stirred for additional 2 h at room tem-
perature, then the mixture was washed with brine, dried over
MgSO4, evaporated under reduced pressure and purified by
column chromatography on SiO2 or SFC (supercritical fluid
chromatography, Mettler-Toledo facilities).
Compounds S1-Arg(NO2)-Ai with i being 1, 3, 6 and 9
have been described elsewhere [41]. Rf, MS/EA for all follow-
ing compounds are reported in Table 3.
4.1.4.1.
N-[4-(S )-(N0-Nitro-guanidino)-1-(morpholine-4-
carbonyl)-butyl]-3-trifluoromethyl-benzenesulfonamide (S1-Arg
(NO2)-A2). This product was obtained from 0.60 mmol of
NH2-Arg(NO2)-A2 and 0.60 mmol of 3-trifluoromethyl-ben-
zenesulfonyl chloride. After workup 0.256 g of raw product
was obtained and submitted to next reaction step without fur-
1
ther purification. H NMR (300 MHz, CDCl3) d (ppm): 1.64
(m, 2H), 1.84 (m, 2H), 3.30 (m, 8H), 3.54 (m, 2H), 4.14 (m,
1H), 6.65 (d, 1H, J ¼ 8.6 Hz), 7.55 (br, 2H), 7.68 (t, 1H,
J ¼ 7.7 Hz), 7.84 (d, 1H, J ¼ 7.7 Hz), 8.05 (d, 1H), 8.07 (s,
1H), 8.65 (br, 1H); IR (CH2Cl2 liquid film on NaCl) nmax
(cmꢂ1): 3233, 2925, 2859, 1634, 1538, 1429, 1327, 1268,
1165, 1133, 1113, 1071, 1034.
4.1.5. General procedure for the preparation of arylsulfonic
acid [4-guanidino-1(S )-(azacyclohexan-1-ylcarbonyl)butyl]
amide (AiSj; Scheme 1, step iv).
Product Sj-Arg(NO2)-Ai (1.01 mmol, 1 equiv) was dis-
solved in a 3:1 mixture of EtOH/AcOH (10 mL). The flask
was purged with nitrogen and the catalyst Pd/C 10%
(0.091 g, 0.086 mmol, 0.085 equiv) was added. The reaction
mixture was then stirred at 50 ꢀC for 12 h under hydrogen at-
mosphere. The catalyst was filtered off and the solution was
concentrated under reduced pressure. The solid residue was
crystallized from a solution of H2O/MeOH 1:1 and washed
4.1.4.2. N-[1-(4-Ethyl-piperazine-1-carbonyl)-4-(N0-nitro-gua-
nidino)-butyl]-3-trifluoromethyl-benzenesulfonamide (S1-Arg
(NO2)-A4). This product was obtained from 0.60 mmol of
NH2-Arg(NO2)-A4 and 0.76 mmol of 3-trifluoromethyl-ben-
zenesulfonyl chloride. After workup only traces of product
were isolated (yield <10%).

50
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
with Et2O. After drying under vacuum, the product was iso-
lated as acetate salt (about 90% yield).
Compounds AiSj with i being 1, 3, 6 and 9 have been de-
scribed elsewhere [41]. Rf, MS/EA for all following com-
pounds are reported in Table 3.
4.1.6. General procedure for the preparation of the sulfon-
amide library by parallel synthesis (see Tables 1 and 2)
3 mmol of each amine (4 equiv) and 9 mmol of NEt3
(12 equiv) were dissolved in 25 mL of CH2Cl2. 3 mmol
(4 equiv) of 3-trifluoromethyl-benzenesulfonyl chloride (S1)
and 3-methylbenzenesulfonyl chloride (S2) were each dis-
solved in 10 mL of CH2Cl2. 1-Naphthalenesulfonyl chloride
(S3) was dissolved in 8 mL of CH2Cl2 and 0.01 mL of
DMF. Quinolinesulfonyl chloride (S4) was dissolved in
20 mL of pyridine (not soluble in other solvents). The Mini-
blockÔ synthesizer was plugged in the MinimapperÔ solvent
handling workstation, connecting the refrigerating circulator
to the metallic jacket of the synthesizer. 5 mL of each amine
solution was transferred in the four positions of a row of the
synthesizer. Then 2 mL of S1, S2 and S3 solutions were trans-
ferred dropwise in the first three columns, and 5 mL of S4 in
the last column. The MiniblockÔ synthesizer was then un-
plugged from the MinimapperÔ and shaken for 2 h, shutting
down the refrigerating system after 1 h. The reaction mixtures
were collected in round bottomed tubes by transfer through the
filtration manifold and submitted to liquideliquid extraction
on AllexisÔ workstation. The organic layer was washed
with brine, and then filtered. The formation of the sulfon-
amides was confirmed by TLC, MS, and NMR (when re-
quired). Typical sulfonamides’ analysis profile is detailed for
series A5. Rf, MS/EA for all following compounds are re-
ported in Table 3.
4.1.5.1. N-[1-(S )-(4-Acetyl-piperazine-1-carbonyl)-4-guani-
dino-butyl]-3-trifluoromethyl-benzenesulfonamide
(A2S1).
This product was obtained from 0.058 g of product S1-
Arg(NO2)-A2 (0.11 mmol). 0.0481 g was isolated for a final
1
yield of 81%. H NMR (500 MHz, CD3OD) d (ppm): 1.62
(m, 2H), 1.72 (m, 2H), 2.09 (s, 3/2H), 2.10 (s, 3/2H), 3.18
(m, 2H), 3.10e3.70 (m, 8H), 4.32 (m, 1H), 7.77 (t, 1H,
J ¼ 8.1 Hz), 7.93 (d, 1H, J ¼ 8.1 Hz), 8.10 (d, 1H,
J ¼ 8.1 Hz), 8.12 (s, 1H); 13C NMR (125 MHz, CD3OD)
d (ppm): 21.06, 21.12, 26.0, 31.0, 41.8, 42.0, 42.6, 46.6,
45.9, 42.7, 43.1, 46.2, 47.1, 53.9, 125.1, 130.3, 131.3, 132.0,
158.7; IR (CH2Cl2 liquid film on NaCl) nmax (cmꢂ1): 3600e
3000, 1637, 1431, 1328, 1166, 1134.
4.1.5.2.
N-{1-[4-(12-Amino-dodecanoyl)-piperazine-1-car-
bonyl]-4-guanidino-butyl}-3-trifluoromethyl-benzenesulfonamide
(A7S1). This product was obtained from 0.269 g of the
sulfonamide precursor S1-Arg(NO2)-A7 (0.31 mmol, 1 equiv).
The product was isolated as acetate salt (0.161 g, 68% yield).
IR (KBr), nmax (cmꢂ1): 3417, 3339, 2927, 2855, 1628, 1467,
1432, 1326, 1165, 1130; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.35 (m, 14H), 1.57 (m, 2H), 1.61 (m, 2H), 1.66
(m, 2H), 1.70 (m, 2H), 2.37 (m, 2H), 2.90 (m, 2H), 3.18 (m,
2H), 3.10e3.70 (m, 8H), 4.32 (m, 1H), 7.77 (t, 1H,
J ¼ 8.7 Hz), 7.93 (d, 1H, J ¼ 8.7 Hz), 8.11 (d, 1H), 8.12 (s,
1H); 13C NMR (125 MHz, CD3OD) d (ppm): 25.94, 26.38,
28.66, 27.48, 30.23, 30.40, 30.49, 30.53, 30.59, 30.94,
33.92, 33.98, 40.74, 41.70, 42.69, 42.05, 43.16, 46.01,
46.41, 46.58, 53.76, 124.90, 125.10, 130.40, 131.42, 132.09,
132.25, 143.37, 158.77, 170.80, 170.92, 174.34, 174.47.
4.1.6.1.
N-[5-(4-Acetyl-piperazin-1-yl)-4-(S )-amino-5-oxo-
pentyl]-N0-nitro-guanidine (S1-Arg(NO2)-A5). This product
was obtained from 0.60 mmol of NH2-Arg(NO2)-A5 and
0.76 mmol of 3-trifluoromethyl-benzenesulfonyl chloride. Af-
ter workup 0.126 g of raw product was obtained and submitted
to SFC for purification. 0.058 g of pure product was isolated
1
for an overall yield of 18%. H NMR (300 MHz, CD3OD)
d (ppm): 1.59 (m, 4H), 2.02 (s, 3/2H), 2.00 (s, 3/2H), 2.74e
3.54 (m, 10H), 4.34 (m, 1H), 4.56 (m, 1H), 7.68 (t, 1H,
J ¼ 7.5 Hz), 7.83 (d, 1H, J ¼ 7.5 Hz), 8.01 (d, 1H), 8.04 (s,
1H).
4.1.5.3. N-[1-(4-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-pipera-
zine-1-carbonyl)-4-guanidino-butyl]-4-trifluoromethyl-benze-
nesulfonamide (A8S1). This product was obtained from S1-
Arg(NO2)-A8 (0.483 g, 0.56 mmol, 1 equiv), Pd/C 10%
(0.09 g, 0.085 mmol, 0.015 equiv) and a mixture of EtOH/
AcOH 1:1 (10 mL). 0.315 g of product was isolated for a
4.1.6.2. N-[(S)-1-(4-Acetyl-piperazine-1-carbonyl)-4-(N0-nitro-
guanidino)-butyl]-3-methyl-benzenesulfonamide (S2-Arg(NO2)-
A5). This product was obtained from 0.60 mmol of
NH2-Arg(NO2)-A5 and 0.76 mmol of 3-toluenesulfonyl chlo-
ride. After workup 0.191 g of raw product was obtained and
submitted to SFC for purification. 0.097 g of pure product
was isolated for an overall yield of 33%. H NMR (300 MHz,
CD3OD) d (ppm): 1.59 (m, 4H), 2.01 (s, 3H), 2.32 (s, 3H),
2.74e3.54 (m, 10H), 4.15 (m, 1H), 7.34 (m, 1H), 7.58 (m, 1H).
1
final yield of 75%. H NMR (500 MHz, CD3OD) d (ppm):
1
1.55e1.75 (m, 4H), 2.09e2.50 (m, 4H), 2.54 (m, 2H,
J ¼ 5.4 Hz), 3.08 (m, 2H), 3.18 (m, 2H), 3.21 (m, 1H),
3.34 (m, 1H), 3.43 (m, 2H, J ¼ 5.1 Hz), 3.61 (m, 2H,
J ¼ 5.4 Hz), 3.63 (m, 2H), 3.66 (m, 2H), 3.68 (m, 2H,
J ¼ 5.1 Hz), 4.30 (m, 2H, J ¼ 4.2e9.4 Hz), 7.77 (t, 1H),
7.94 (d, 1H), 8.09 (d, 1H), 8.10 (s, 1H); 13C NMR
(125 MHz, CDCl3) d (ppm): 25.93, 31.02, 40.72, 41.69,
42.74, 46.19, 53.63, 53.88, 54.60, 58.41, 68.48, 69.39,
71.35, 124.81, 125.11, 130.39, 131.46, 132.08, 132.34,
143.32, 158.78, 170.40; IR (CH2Cl2 liquid film on NaCl)
nmax (cmꢂ1): 1651, 1555, 1407, 1328, 1167.
4.1.6.3. Naphthalene-1-sulfonic acid [(S )1-(4-acetyl-pipera-
zine-1-carbonyl)-4-(N0-methyl-guanidino)-butyl]-amide (S3-
Arg(NO2)-A5). This product was obtained from 0.60 mmol
of NH2-Arg(NO2)-A5 and 0.76 mmol of naphthalene-1-sulfo-
nyl chloride. After workup 0.210 g of raw product was ob-
tained and submitted to SFC for purification. 0.0920 g of
1
pure product was isolated for an overall yield of 30%. H

C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
51
NMR (300 MHz, CD3OD) d (ppm): 1.42 (m, 4H), 1.98 (s, 3H),
2.95e3.40 (m, 10H), 4.12 (m, 1H), 7.12e7.61 (m, 3H), 7.91
(d, 1H, J ¼ 7.5 Hz), 8.07 (d, 1H, J ¼ 8.7 Hz), 8.17 (d, 1H,
J ¼ 7.5 Hz), 8.62 (d, 1H, J ¼ 8.1 Hz).
4.1.7.2. N-[1-(S )-(4-Acetyl-piperazine-1-carbonyl)-4-guani-
dino-butyl]-3-methylbenzenesulfonamide (A5S2). This product
was obtained from 0.097 g of S2-Arg(NO2)-A5. 0.0847 g of
product was isolated for a yield of 85%. 1H NMR
(300 MHz, CDCl3) d (ppm): 1.56 (m, 4H), 1.99 (s, 3/2H),
2.00 (s, 3/2H), 2.32 (s, 3H), 3.06e3.53 (m, 8H), 4.10 (m,
1H), 7.33 (m, 2H), 7.56 (m, 2H).
4.1.6.4. Quinoline-8-sulfonic acid [1-(S )-(4-acetyl-piperazine-
1-carbonyl)-4-(N0-nitro-guanidino)-butyl]-amide (S4-Arg(NO2)-
A5). This product was obtained from 0.60 mmol of NH2-Arg
(NO2)-A5 and 0.76 mmol of naphthalene-1-sulfonyl chloride.
After workup 0.286 g of raw product was obtained and sub-
mitted to SFC for purification. 0.0872 g of pure product was
isolated for an overall yield of 28%. 1H NMR (300 MHz,
CD3OD) d (ppm): 1.56 (m, 4H), 1.99 (s, 3/2H), 2.02 (s, 3/2H),
2.84e3.38 (m, 10H), 4.20 (m, 1H), 7.61e7.70, 8.17, 8.33e8.40,
9.01 (m, 6H).
4.1.7.3. Naphthalene-1-sulfonic acid [1-(S )-(4-acetyl-pipera-
zine-1-carbonyl)-4-guanidino-butyl]-amide (A5S3). This prod-
uct was obtained from 0.092 g of S3-Arg(NO2)-A5 (0.20 mmol).
0.0319 gofproduct wasisolatedforafinalyieldof34%. 1H NMR
(300 MHz, CD3OD) d (ppm): 1.43 (m, 4H), 1.94 (s, 3/2H), 1.95
(s, 3/2H), 2.70e3.40 (m, 10H), 4.10 (m, 1H), 7.46e7.61 (m,
3H), 7.89 (d, 1H, J ¼ 7.7 Hz), 8.05 (d, 1H, J ¼ 7.7 Hz), 8.14 (d,
1H, J ¼ 7.7 Hz), 8.60 (d, 1H, J ¼ 7.7 Hz).
4.1.7. General procedure for the final deprotection step by
parallel synthesis
4.1.7.4. 1,2,3,4-Tetrahydro-quinoline-8-sulfonic acid [1-(S )-
(4-acetyl-piperazine-1-carbonyl)-4-guanidino-butyl]-amide
(A5S4). This product was obtained from 0.0872 g
(0.21 mmol) of S4-Arg(NO2)-A5. 0.0839 g of product was iso-
Basic requirements for combined heating, H2 fluxing and
vigorous stirring for catalyst suspension were not suitable
for MiniblockÔ synthesizer. We decided to perform hydroge-
nation in round bottomed cylindrical vials providing individ-
ual magnetic stirring. We fixed them on a solid support
made with a perforated polystyrene block (25 ꢄ 25 cm2,
5 mm width) providing 16 positions. The support was used
to allow the simultaneous immersion of the reaction vials in
a single oil bath for heating. The vials containing the sulfon-
amides (0.45e0.75 mmol), 0.02 g of Pd/C catalyst, in 5 mL
of a 3:1 mixture of EtOH/AcOH were sealed with a septum,
purged with nitrogen and filled with H2. Two cow receivers
for distillation were used to connect two H2 filled balloons
to eight tubes, reducing the number of required balloons to en-
sure H2 atmosphere saturation in the reaction vials. The reac-
tion mixtures were stirred at 50 ꢀC for 12 h under hydrogen
atmosphere. The catalyst was filtered off, and the filtrates, col-
lected in round bottomed flasks, were concentrated under re-
duced pressure. The solid residues were crystallized from
a solution of H2O/MeOH 1:1 and washed with Et2O. The sol-
vent was evaporated and the product was isolated as acetate
salt (about 90% yield).
1
lated for a final yield of 70%. H NMR (300 MHz, CD3OD)
d (ppm): 1.50 (m, 4H), 1.76 (m, 2H), 1.97 (s, 3H), 2.65 (m,
2H), 3.03 (m, 2H), 3.10e3.45 (m, 10H), 4.10 (d, 1H,
J ¼ 5.7 Hz), 6.40 (m, 1H, J ¼ 7.7 Hz), 6.96 (d, 1H,
J ¼ 7.7 Hz), 7.32 (d, 1H, J ¼ 7.7 Hz).
4.2. Biological assays
4.2.1. Enzyme kinetics
Substrate H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitro-
anilide dihydrochloride (S2238) was obtained from Chromo-
genix. Human thrombin was obtained from Hypen BioMed.
Enzyme kinetics studies were performed on a Cary 210
spectrophotometer.
The inhibition activities were measured by adapting the
protocol developed by Lottenberg et al. [47]: 100 mL of the so-
lution of substrate S2238 (0.1 mM in water) and 100 mL of
a water solution of the tested compound (50e200 mM final
concentration) were diluted in 2 mL of TriseHepes Buffer
(Tris 0.01 M, Hepes 0.01 M, NaCl 0.5 M, PEG 6000 0.1%
m/v, pH 7.8). Finally 10 mL of water solution of thrombin
(10 NIH/mL, 75.2 nM) was added to start the reaction. The ap-
pearance of the substrate hydrolysis product ( p-nitroaniline)
was measured at 405 nm as a function of time. From the slope
(m) of the linear curve used to fit the plots of V/Vi versus in-
hibitor concentration (ratio of hydrolysis in the absence and
in the presence of inhibitors) we calculated the inhibition con-
stants from the equation Ki ¼ Km/m(1/([S] þ Km), where Km is
the Michaelis constant of the system as reported by Lottenberg
(1.5 mM [47]) and [S] is the substrate concentration. The ob-
tained inhibition constants are shown in Table 2. Two series
of independent measurements are reported.
Typical deprotected sulfonamides’ analysis profile is de-
tailed for series A5. Rf, MS/EA for all following compounds
are reported in Table 3.
4.1.7.1. N-[1-(S )-(4-Acetyl-piperazine-1-carbonyl)-4-guani-
dino-butyl]-3-trifluoromethyl-benzenesulfonamide
(A5S1).
This product was obtained from 0.058 g of S1-Arg(NO2)-A5
(0.11 mmol). 0.0481 g of product was isolated for a reaction
1
yield of 81%. H NMR (500 MHz, CD3OD) d (ppm): 1.62
(m, 2H), 1.72 (m, 2H), 2.09 (s, 3/2H), 2.10 (s, 3/2H), 3.18
(m, 2H), 3.10e3.70 (m, 8H), 4.32 (m, 1H), 7.77 (t, 1H,
J ¼ 8.1 Hz), 7.93 (d, 1H, J ¼ 8.1 Hz), 8.10 (d, 1H,
J ¼ 8.1 Hz), 8.12 (s, 1H); 13C NMR (125 MHz, CDCl3)
d (ppm): 21.1, 21.1, 26.0, 31.0, 41.8, 42.0, 42.6, 46.6, 45.9,
42.7, 43.1, 46.2, 47.1, 53.9, 125.1, 130.3, 131.3, 132.0,
158.7; IR (CH2Cl2 liquid film on NaCl) nmax (cmꢂ1): 3600e
3000, 1637, 1431, 1328, 1166, 1134.
4.2.2. Surface grafting
Native PET track-etched membranes from Cyclopore-Lou-
vain la neuve (thickness 12 mm, density 1.39 g/cm2, pore size

52
C. Salvagnini et al. / European Journal of Medicinal Chemistry 42 (2007) 37e53
0.49 mm, 1.45 ꢄ 10ꢂ6 pores/cm2) were cut in disks of 13 mm
of diameter. The polymer samples (10 disks) were stirred at
60 ꢀC for 1 h into a mixture of acetone (50 mL), pyridine
(1.06 mL) and p-toluenesulfonyl chloride (2.5 g). The samples
were washed successively with acetone (50 mL, 5 min) and
water (50 mL, 5 min). The resulted activated samples (hy-
droxyl functions of chain-ends transformed into tosylates)
were individually immersed in 1 mL of a 10ꢂ3 M solution of
inhibitor in a phosphate buffer (PBS, pH 8)/CH3CN mixture
(1:1) and incubated 2 h at 20 ꢀC with shaking. The samples
were washed with PBS/CH3CN (1 mL, 2 ꢄ 10 min), water
(1 mL, 2 ꢄ 5 min), 5 ꢄ 10ꢂ3 M HCl (1 mL, 2 ꢄ 5 min) and
water (1 mL, 2 ꢄ 10 min). The resulting inhibitor grafted
membranes were finally dried over filter paper and analysed
by XPS within 24 h. The blank samples were prepared in
the same way but without addition of p-toluenesulfonyl chlo-
ride in the activation step.
polymer coated with heparin (Leo, 5000 I.E./U.I./mL, 100 mL)
was used as anti-clotting reference sample.
Acknowledgments
This work has been supported by the FNRS (Fonds Na-
tional de la Recherche Scientifique, Belgium) and the Commu-
´
naute Franc¸aise de Belgique (Action de Recherche Concertee
no. 99/04-240). The authors thank the ‘‘Unite de chimie des
´
´
interfaces, UCL’’ (Pr. P. Rouxhet and M. Genet) for access
to the XPS facilities and Dr. R. Touillaux for NMR spectra in-
terpretation. Mettler-Toledo GMbH Autochem (Switzerland)
is acknowledged for parallel synthesis and SFC facilities.
We thank Shimadzu Benelux (Australielaan 14, NL-5232
BB’s Hertogenbosch, Netherlands) for providing financial sup-
port for the acquisition of the FTIR-8400S. J.M.-B. is senior
research associate of the FNRS.
The surface molar fractions were measured by XPS analysis:
PET grafted with A6S1: C 1s ¼ 70.34%, F 1s ¼ 1.23% (F/
C ¼ 0.0175); PET blank: C 1s ¼ 68.99%, F 1s ¼ 0.83% (F/
C ¼ 0.0120); corrected F/C ꢄ 100 ¼ 0.55. The concentration of
F tagged molecule on the surface was calculated from the cor-
rected F/C ratio. Considering the polymer repeated unit of PET
-CO-C6H4-CO2-CH2CH2-O- (C10H8O4) and the derivatized (by
grafting of compound A6S1) chain unit -CO-C6H4-CO2-
CH2CH2-A6S1 (C39H57F3N7O6S) we calculated the percentage
of derivatized units as follows. For a mixture 98.1% (C10H8O4)
and 1.9% (C39H51F3N7O6S): F/C ꢄ 100 ¼ [(1.9 ꢄ 3)/(98.1 ꢄ
10 þ 1.9ꢄ39)] ꢄ 100 ¼ 5.7/1055.1 ꢄ 100 ¼ 0.54 (experimental
value 0.55). We previously calculated on the basis of PET crystal-
lographic data and simple geometrical considerations [10] an av-
erage of 1.72 ꢄ 1015 PET monomer units per cm2 of surface
covering 10 atomic layers (the depth analysed by XPS) or about
2850 pmol/cm2. Thus, 1.9% of derivatized surface monomer
units corresponds to about 54 pmol/cm2 of fixed compound
A6S1.
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