Fluorinated benzyloxyphenyl piperidine-4-carboxamides with dual function against thrombosis: Inhibitors of factor xa and platelet aggregation

Article abstract of DOI:10.1021/jm801141f

A series of benzyloxy anilides of nipecotic (5, 6) and isonipecotic (7, 8) acids were synthesized and assayed in vitro as inhibitors of ADP-induced platelet aggregation and the blood coagulation enzymes factor Xa (FXa) and thrombin (Flla). An exploration of effects of the amidine group attached at the piperidine nitrogen, position and substitution (F, phenyl) of the benzyloxy group, and addition of fluorine/s on the second (distal) phenyl ring, led us to single out some promising isonipecotamide derivatives 7. Addition of meta-F and para-CF₃ on the distal phenyl ring resulted in a 6-to-18-fold enhancement of the FXa potency and in 2-to- 4-fold increase of the antiplatelet potency, the last depending to a large extent upon lipophilicity. Two congeners of N-{[3-(1,1′-biphenyl-4-yl)methoxy]phenyljpiperidine-4-carboxamide (7m and 7p) proved to be potent FXa-selective inhibitors (K_i = 130 and 57 nM, respectively) and antiplatelet agents and were identified as leads for developing new dual function antithrombotic drugs.

Full text of DOI:10.1021/jm801141f

1018
J. Med. Chem. 2009, 52, 1018–1028
Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides with Dual Function against
Thrombosis: Inhibitors of Factor Xa and Platelet Aggregation
Modesto de Candia,† Francesco Liantonio,† Andrea Carotti,†,§ Raimondo De Cristofaro,‡ and Cosimo Altomare*,†
Dipartimento Farmaco-chimico, UniVersity of Bari, Via Orabona 4, I-70125, Bari, Italy, Hemostasis Research Centre, Institute of Internal
Medicine and Geriatrics, Catholic UniVersity School of Medicine, Largo F. Vito 1, I-00168 Rome, Italy
ReceiVed September 12, 2008
A series of benzyloxy anilides of nipecotic (5, 6) and isonipecotic (7, 8) acids were synthesized and assayed
in vitro as inhibitors of ADP-induced platelet aggregation and the blood coagulation enzymes factor Xa
(FXa) and thrombin (FIIa). An exploration of effects of the amidine group attached at the piperidine nitrogen,
position and substitution (F, phenyl) of the benzyloxy group, and addition of fluorine/s on the second (distal)
phenyl ring, led us to single out some promising isonipecotamide derivatives 7. Addition of meta-F and
para-CF3 on the distal phenyl ring resulted in a 6-to-18-fold enhancement of the FXa potency and in 2-to-
4-fold increase of the antiplatelet potency, the last depending to a large extent upon lipophilicity. Two
congeners of N-{[3-(1,1′-biphenyl-4-yl)methoxy]phenyl}piperidine-4-carboxamide (7m and 7p) proved to
be potent FXa-selective inhibitors (Ki ) 130 and 57 nM, respectively) and antiplatelet agents and were
identified as leads for developing new dual function antithrombotic drugs.
Introduction
Anticoagulant therapy, used to treat a variety of conditions
including cure and prevention of venous thromboembolism
(VTE) and long-term prevention of ischemic stroke in patients
with atrial fibrillation, involves heparins and the vitamin K
antagonist warfarin.
Thrombotic events are leading causes of cardiovascular-
associated death in the developed world.1,2 Prophylaxis and
treatment of arterial thrombosis in patients with cardiovascular
diseases (e.g., atherosclerotic vascular disease, acute coronary
syndrome (ACSa) including myocardial infarctions and unstable
angina) are achieved using antiplatelet drugs,3 whereas venous
thrombosis (e.g., deep vein thrombosis (DVT) and pulmonary
embolism (PE)) is treated with anticoagulant drugs, which inhibit
various proteases in the blood coagulation cascade.4-6
The main side effect of most of the available antithrombotic
drugs is bleeding, whereas therapy of insufficient intensity
(aspirin, clopidogrel) can entail a risk of recurrent thrombosis.
A number of drug-class-specific adverse reactions include, for
instance, aspirin-related gastrointestinal ulcers and bleeding,
thrombotic thrombocytopenic purpura that may be associated
to thienopyridines,14 heparin-induced thrombocytopenia (re-
duced with low-molecular weight heparins),15 warfarin-induced
skin necrosis.16 Moreover, warfarin has a narrow therapeutic
window, and its activity is affected by diet and genetic makeup,
so requiring continuous monitoring for accurate dosing.17 For
all these reasons, the search for new antiplatelet and antico-
agulant drugs, with better therapeutic windows and easier-to-
use than the existing ones, still represents a challenging goal.5,18
Platelets are primary components of hemostasis,7 but their
activation is involved in the pathogenesis of thromboembolic
diseases8 and complications following percutaneous coronary
intervention.9 They can be activated by a number of agonists
such as adenosine 5′-diphosphate (ADP), thromboxane A2
(TxA2), thrombin, platelet activating factor (PAF), epinephrine
(EPN), and collagen.10 Antiplatelet drugs include the most
commonly used aspirin, the thienopyridine derivative clopi-
dogrel, and its more powerful analogue prasugrel, which are
antagonists of the cell-surface ADP receptor P2Y12,11 the R2ꢀ3-
integrin antagonists (also known as glycoprotein IIb/IIIa-GpIIa/
IIIb-receptor antagonists), such as the specific antibody abcix-
imab, the oligopeptide eptifibatide and nonpeptide inhibitor
tirofiban,12 and the recently discovered antagonists of the
thrombin receptor PAR-1 (protease-activated receptor-1) such
as SCH 530348 undergoing phase-III clinical trials.13
Among the novel antithrombotics in development, several
orally administered direct inhibitors of thrombin (FIIa)19 and
factor Xa (FXa)20 offer the promise of better activities and lesser
side effects. Two notable examples are the thrombin inhibitor
dabigatran etexilate,21 which is as effective as enoxaparin at
reducing the risk of VTE,22 and the FXa-selective inhibitor
rivaroxaban,23 which has shown a favorable safety/efficacy
balance for preventing VTE after major orthopedic surgery.24
The coagulation enzyme FXa has been recognized as a promis-
ing target for the development of new antithrombotic drugs.
FXa inhibitors seemed to have a lower risk of bleeding than
heparin and warfarin.25 Indeed, they do not block thrombin
directly but decrease the amplified generation of thrombin,
allowing the existing levels of thrombin to ensure primary
hemostasis.26
* To whom correspondence should be addressed. Phone: +39-080-
5442781. Fax: +39-080-5442230. E-mail: altomare@farmchim.uniba.it.
†
Dipartimento Farmaco-chimico, University of Bari.
Hemostasis Research Centre, Catholic University School of Medicine.
Present address: Dipartimento di Chimica e Tecnologia del Farmaco,
‡
§
University of Perugia, Via del Liceo 1, I-06123 Perugia, Italy.
a Abbreviations: ACS, acute coronary syndrome; ADP, adenosine 5′-
diphosphate; aPTT, activated partial tromboplastin time; R-CT, R-chymot-
rypsin; DAG, 1,2-diacylglycerol; DVT, deep vein thrombosis; EPN,
epinephrine; FIIa, thrombin; FXa, factor Xa; GpIIb/IIIa, glycoprotein IIb/
IIIa; IP3, inositol 1,4,5-triphosphate; PAF, platelet activating factor; PAR-
1, protease-activated receptor-1; PE, pulmonary embolism; PLs, phospho-
lipids; PRP, platelet-rich plasma; PPP, platelet-poor plasma; TxA2,
thromboxane A2; VTE, venous thromboembolism.
The combined use of antiplatelet and anticoagulant agents
showed additional benefits over anticoagulants alone in patients
with prosthetic hearth valves,27 as well as in patients with atrial
fibrillation.28 Furthermore, old and recent literature showed that
VTE and arterial disease share common risk factors.29 Although
10.1021/jm801141f CCC: $40.75
2009 American Chemical Society
Published on Web 02/04/2009

Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1019
Chart 1. Progression and Design of Nipecotamide-Based
Composite Antithrombotics
antiplatelet activity by a parabolic relationship between inhibi-
tion data (IC50) and log P-related parameters.33
In this work, we synthesized other anilide derivatives of
nipecotic and isonipecotic acid, replacing in particular the
n-hexyloxy groups in the para position with R-substituted
benzyloxy ones, our investigation pursuing a 2-fold goal: (i) to
improve the antiplatelet activity of this series and (ii) to further
modify the structure of the most promising antiplatelet candidate,
used as a scaffold, for building up compounds, which addition-
ally can attain FXa inhibition. Thus, we initially replaced the
chiral nipecotic with the achiral isonipecotic acid, then shifted
the substitution position of the benzyl group on the -HN-
C6H4-O- linker from the para to meta position, and finally
attempted to improve binding of the P1 and P4 groups into the
“arginine recognition pocket” S1 and “aryl binding site” S3/S4
of FXa, respectively.
Our early binding models, based on docking calculation (QXP
software34) between the designed molecules and the 3D structure
of human FXa (PDB code: 1FJS),35 suggested that meta-R-
substituted (e.g., R ) phenyl, 7d) benzyloxy anilides of
isonipecotic acid have potential for binding into the FXa
catalytic cavity. Figure 1 shows the QXP-calculated docking
pose of the representative isonipecotanilide inhibitor N-{[3-(1,1′-
biphenyl-4-yl)methoxy]phenyl}piperidine-4-carboxamide (7d)
into the binding pocket of human FXa, highlighting the
importance of interactions (salt bridge, H-bonds) between the
protonated piperidine (P1) and Asp189 in S1, whereas the P4
phenyl group is embedded into the S3/S4 site, which is a box-
shaped aromatic/hydrophobic cavity lined by residues Trp215,
Tyr99, and Phe174.
We systematically explored the effects on the antiplatelet
activity and FXa/thrombin inhibition of attaching the basic
amidine group at P1, and taking into account the effect of
aromatic fluorine/s in increasing the potency of FXa36 and
thrombin37 inhibitors, we introduced various fluorinated 1,1′-
biphenyl as P3/P4 moiety. Herein, we report synthesis, in vitro
biological activity, structure-activity relationships (SARs), and
molecular modeling, highlighting our progress toward the above
goal.
numerous investigations suggest the existence of an association
between VTE and atherosclerosis, the nature of this relation
has not been clarified. It seems, however, that VTE, promoting
inflammatory processes, could exert a causative role in the
pathogenesis of atherothrombosis. Indeed, there are many
examples of conditions accounting for both arterial and venous
thromboembolic disorders, such as antiphospholipid antibodies,
hyperhomocysteinemia, factor V Leiden or prothrombin gene
mutation, high levels of factor VIII:C, myeloproliferative
disorders, and HIV infection.
Even though the appropriateness of a combined anticoagulant
and antiplatelet therapy for patients with multiple disorders is
still discussed,30 compounds combining in the same molecule
anticoagulant and platelet antiaggregatory activities are believed
to be promising drugs. Compared to a combination of antiplatelet
and anticoagulant drugs, a single composite (dual function) agent
should be advantageous, thanks to an expected less complex
pharmacokinetics, a likely lower incidence of side effects, and
less demanding clinical studies.31
Chemistry
The nipecotamide- (5, 6) and isonipecotamide-based deriva-
tives (7, 8) investigated in this study were synthesized as shown
in Schemes 1 and 2. According to Scheme 1, the preparation
of 1-(benzyloxy)-3-nitrobenzene (3a), its para isomer, and
related congeners (3b-e, structural details in Table 1) was
achieved upon reaction of 3(or 4)-nitrophenol with suitable
benzyl bromide in dry DMF in the presence of K2CO3 (3a-c),
or alternatively (3b-e) through a Mitsunobu reaction between
3(or 4)-nitrophenol and 1,1′-biphenyl-4-ylmethanol, using triph-
enylphosphine and diisopropyl azodicarboxylate (DIAD).38
Reduction of the nitro group in 3a-e with hydrazine hydrate
and Raney nickel in refluxing MeOH39 afforded the correspond-
ing anilines 4a-e, which were coupled in the subsequent step
with the N-Boc-protected nipecotic and isonipecotic acids,33
previously activated via acyl chloride (fluorinated derivatives
b and c) or mixed anhydride formation (a, d, and e) using benzyl
chloroformate, with satisfactory yields (45-85%). The Boc
group was then removed with HCl gas in CHCl3 solutions of
the Boc-protected intermediates, affording the target anilide
derivatives 5a-e and 7a-e as hydrochloride salts. Reaction of
compounds 5b,d and 7b,d with N,N′-bis-Boc-S-methylthi-
opseudourea and HgCl2 in dry DMF, and subsequent Boc
removal with HCl gas, yielded the corresponding guanidine
derivatives 6 and 8, respectively.
In our ongoing research addressed toward new antithrombotic
agents endowed with dual antithrombotic function, we used as
a suitable scaffold for design piperidine-3(or 4)-carboxylic acid
phenyl amide. In previous studies by Gollamudi et al.,32
derivatives of piperidine-3-carboxamide, such as the bis-
nipecotamide derivatives 1 (Chart 1), proved to be inhibitors
of platelet aggregation in vitro and in vivo. From a mechanistic
point of view, type 1 compounds showed ability to penetrate
the platelet membranes and to interact with anionic phospho-
lipids (PLs), mainly phosphatidylinositides, decreasing their
turnover, release of second messengers (i.e., inositol 1,4,5-
triphosphate IP3 and 1,2-diacylglycerol DAG), intracellular
calcium levels, and consequently inhibiting platelet activation
and aggregation. A few years ago, we also have reported a large
series of nipecotamides, among which N-[4-(hexyloxy)phe-
nyl]piperidine-3-carboxamide (2b) proved to be in vitro inhibitor
of human platelet aggregation induced by ADP, arachidonic
acid, and EPN, with a potency comparable to that of 1 (Chart
1), and demonstrated the impact of lipophilicity on their

1020 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
de Candia et al.
Figure 1. QXP highest scored docking pose of the designed isonipecotanilide inhibitor 7d in stick (blue: nitrogen; red: oxygen; light blue: carbon
atoms) into the binding pocket of human FXa (PDB: 1FJS). The protein surface is shown together with the main residues of the subsites S1
(magenta) and S3/S4 (cyan). The catalytic triad (red-colored residues) and Q192 (green) are also shown. The figure was drawn by PyMOL.36
Unfortunately, following the pathway illustrated in Scheme
1, the isonipecotamide derivatives bearing fluorinated 1,1′-
biphenyl as P4 moieties (7f-p) were obtained in poor yields.
They were then synthesized with moderate-to-good yields
through an alternative pathway (Scheme 2), where the Boc-
protected N-[3-(benzyloxy)phenyl]piperidine-4-carboxamide
7a was first debenzylated by hydrogenolysis and the hydrox-
yphenyl intermediate 9 was alkylated with the suitable fluori-
nated 4-(bromomethyl)-1,1′-biphenyl. All the F-substituted 4′-
bromomethylbiphenyl derivatives used for preparing compounds
7f-p were already known,40 and were synthesized in this study
according to a reported Pd-catalyzed Suzuki cross-coupling
reaction,41 followed by bromination of the 4′-Me group with
N-bromosuccinimmide (NBS).
response relation. The nipecotanilide derivative 2b has been
reassayed in this study as reference standard, and in Table 1,
two other previously reported compounds (2a and 2c)33 have
been included for comparison.
As a matter of fact, all the compounds tested against both
agonists attained 50% inhibition activity of the EPN-induced
PRP aggregation at concentrations 1.3-2.5 times lower than
those required for inhibiting the ADP-induced aggregation; the
EPN and ADP pIC50 values resulted linearly correlated with a
close-to-one slope value and high correlation coefficient (r2 )
0.954). It is known that the first low deflection of the aggregation
wave in the EPN-induced platelet aggregation accounts for the
formation of small aggregates, whereas their fusion into larger
ones contributes to the second phase, which largely depends
upon TxA2 production and ADP release. Taking into account
previous findings and the results of this study, such high
correlation between EPN and ADP IC50 values suggests that
most likely they exert inhibitory effects on EPN-induced
aggregation, mainly interfering with the ADP-related signal
transduction pathway. The overall amount of ADP released
during EPN-induced aggregation is expected to be quite smaller
than that used in our ADP assay, and this may explain the
magnitude of EPN IC50 values, which were found lower than
the values correspondingly determined in the ADP-induced
aggregation test.
Results and Discussion
The newly synthesized compounds were assayed for their
antiplatelet activity on the in vitro aggregation of human platelet-
rich plasma (PRP) induced by 10 µM ADP, using the Born’s
turbidimetric method.42 Ten compounds were also tested for
their activities against 10 µM EPN-induced PRP aggregation.
The aggregation tracing relative to the controls with both
agonists revealed a typical biphasic aggregation wave, which
is more pronounced with EPN. At higher concentrations of
nipecotamides (5) and isonipecotamides (7), both phases were
inhibited. The platelet aggregation inhibition data, expressed
as the IC50 (µM) values, represent the means ( SEM of at least
three individual determinations, each performed in duplicate.
Only compound 6d, showing much less than 50% inhibitory
effects at 1 mM concentration, was not examined for dose-
The lipophilicity of the investigated compounds was assessed
by a reversed-phase (RP)-HPLC method,33b,43 and the log values
of polycratic capacity factors (log k′w), that is capacity factors
extrapolated at 100% aqueous mobile phase (pH 5.0), reported
in Table 1.

Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1021
Scheme 1a
a Reagents and conditions: (a) X ) Br, K2CO3, dry DMF, room temperature, 12 h, 85%; (b) X ) OH, PPh3, DIAD, dry THF, room temperature, 48 h,
62-66%; (c) H4N2 ·H2O, Raney-Ni, MeOH, reflux, 2-4 h, 58-98%; (d) R ) H or C6H5, BnOCOCl, TEA, dry THF, room temperature, 12 h; (e) R ) F,
SOCl2, dry THF, TEA, room temperature, 12 h; (f) CHCl3, HCl gas, 0 °C, 45-85%; (g) N,N′-bis-Boc-S-methylthiopseudourea, HgCl2, dry DMF, 0 °C to
room temperature, 24 h, 63-75%.
Scheme 2a
tives (7b and 7d) displaying better antiplatelet properties than
the para positional isomers (7c and 7e).
Interestingly, a fluorine substituent at the para position of the
benzyl group favorably affected the potency of the examined
piperidine carboxamides, such an effect being particularly strong
with 3-(4-fluorobenzyl)oxy isonipecotanilide 7b, which resulted
the most promising antiplatelet agent in Table 1 (IC50 values of
68 and 36 µM against ADP- and EPN-induced PRP aggrega-
tions, respectively). In contrast, a sharp decrease of antiplatelet
potency was observed for the amidine-bearing (N1-C(NH)NH2)
derivatives 6 and 8; their potency was 3-10 times lower than
that of the corresponding more lipophilic N1-H derivatives 5
and 7.
In agreement with previous findings,32,33b most of the N1-H
nipecotamides (2 and 5) and isonipecotamides (7) lie on a
parabolic curve correlating pIC50 with the lipophilicity parameter
log k′w, and indeed the following parabolic equation, explaining
more than 80% of the variance in the activity data, was obtained
a Reagents and conditions: (a) H2, 10% Pd/C, EtOH, room temperature,
12 h, 98%. (b) (i) NaH, dry DMF, room temperature, overnight; (ii) CHCl3,
HCl gas, 0 °C, 48-62%.
by regression analysis of the N1-H data set:
From the SAR viewpoint, data in Table 1 show that, albeit
less lipophilic, benzyloxy anilides are almost equipotent with
the n-hexyloxy congeners. With one exception (7a), the isonipe-
cotoyl derivatives are equipotent or slightly more potent than
the isomeric nipecotoyl congeners, the meta-substituted deriva-
ꢀ
(
)
pIC50 ) 1.03 (0.16 log k w
-
ꢀ
2
(
)
(
)
0.15 (0.03 log k
+ 2.29 (0.22
(
)
w
n ) 13 r2 ) 0.8435 s ) 0.1226 F ) 26.95 (1)

1022 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
de Candia et al.
Table 1. Platelet Aggregation Inhibitory Activities, Inhibition Constants for Factor Xa (FXa) and Thrombin (FIIa) and Lipophilicity Parameters of
Benzyloxyphenyl Piperidine Carboxamidesa
IC50,c µM
Ki,d µM
b
compd
3 or 4 position
R
log k′w
ADP
EPN
n.d.g
43 ( 9
n.d.
FXa
FIIa
2ae
2be
2ce
5a
5b
5c
5d
5e
6b
6d
7a
7b
7c
4
4
3
3
3
4
3
4
3
3
3
3
4
3
4
3
3
0.79
3.53
3.75
2.44
2.67
2.41
4.50
4.30
2.82
4.57
2.47
2.61
2.35
4.45
4.18
2.76
4.67
983 ( 20***
85 ( 12
113 ( 17
128 ( 19
103 ( 16
116 ( 6
153 ( 12*
125 ( 15
274 ( 26*
>1000
229 ( 30**
68 ( 13
121 ( 11
111 ( 8*
135 ( 20
768 ( 40***
400 ( 20**
n.d.
f
f
f
260
f
2.50
f
57.0
2.00
f
n.d.
f
f
f
f
f
f
f
160
31.3
f
f
f
f
f
H
n.d.
F
F
70 ( 8
90 ( 11*
87 ( 13
n.d.
n.d.
n.d.
C6H5
C6H5
F
C6H5
H
n.d.
F
F
36 ( 3
93 ( 10*
54 ( 6
n.d.
n.d.
244 ( 8**
f
f
7d
7e
8b
8d
C6H5
C6H5
F
1.00
126
33.8
5.20
20.8
40.4
C6H5
a General structures in Scheme 1. b Log of RP-HPLC polycratic capacity factor, i.e., capacity factor extrapolated at 100% aqueous mobile phase (variable
fraction of MeOH in 40 mM ammonium acetate solution, pH ) 5.0); stationary phase: Phenomenex Gemini C18 column (150 mm × 4.6 mm i.d., 5 µm
particles). c Compound concentration which inhibits ADP- and EPN-induced platelet aggregation by 50% in a PRP assay. Data are means ( SEM of at least
three different determinations, each performed in duplicate; *p < 0.05, **p < 0.01, ***p < 0.001 significantly different from compound 2b. d In vitro
inhibition constants from amydolitic assay. e Previously reported33 N-(4-methoxyphenyl)- (2a), N-(4-hexyloxyphenyl)- (2b), and N-(3-hexyloxyphenyl)piperidine-
3-carboxamides (2c); compd 2b has been tested in this study as a reference standard. f Less than 30% inhibition at the maximum concentration tested (250
µM). g n.d. ) not determined.
where n represents the number of data points, r2 the squared
correlation coefficient, s the standard deviation of the regression
equation, F the statistical significance of fit; 95% confidence
intervals of the regression coefficients are given in parentheses.
The above relationship between antiplatelet potency data and
lipophilicity parameters of piperidine-3 (or 4-)-carboxamides
is consistent with the postulated mechanism of action32,33 in
which (i) penetration through the lipid bilayer of the platelet
membrane should be a critical step and (ii) interaction with
anionic PLs (in particular with phosphoinositides, which are
major components of the platelet membrane) triggers a number
of processes leading to the inhibition of platelet activation and
aggregation. In both steps, kinetics of adsorption to and
translocation through platelet membranes, as well as micelle
formation and/or limited solubility of more lipophilic analogues,
could be responsible for the parabolic lipophilicity-activity
relationship.
The four guanidine derivatives 6b,d and 8b,d do not fit the
parabolic eq 1. An argument that can shed light on the different
behavior shown by amine or guanidine derivatives is offered
by a recent study of Kooijman et al.,44 who investigated specific
interactions between lysine (primary amine) and arginine
(guanidine) side chains with the phosphate headgroup of
phosphatidic acid (PA), a minor but important PL playing a
central role in several key cellular processes through specific
interactions with proteins. Lys and Arg residues increase the
charge of PA from -1 to -2, predominantly by forming
hydrogen bonds (HBs), thereby stabilizing the protein-lipid
interactions. Lys residues, which are likely stronger HB donors
than Arg residues, as on the other hand supported by search of
Cambridge Structural database and molecular dynamics simula-
tions, proved to be more effective in docking the phosphomo-
noester headgroup of PA. Phosphomonoesters occur not only
in PA, but also in other anionic PLs, such as phosphatidyl
inositol mono-, bi-, and triphosphate, bearing -OPO3H- moi-
ety(ies) at the position 3, 4, and/or 5 of the inositol fragment.
Binding of our N1-H and N1-C(NH)NH2 (iso)nipecotamide
derivatives, both protonated at physiological pH, to the phos-
phomonoester moiety(ies) of the anionic PLs should be reason-
ably regulated by similar principles. A stronger interaction of
the secondary amino group in the (iso)nipecotamide derivatives
2, 5, and 7 with the phosphate headgroup in the anionic PLs of
the platelet membrane could likely explain their higher activity
compared with that of the guanidine derivatives 6 and 8.
The compounds in Table 1 were then evaluated in vitro for
inhibition of FXa and thrombin and their potencies expressed
as inhibition constants (Ki, µM). Only four compounds, namely
5d, 6d, 7d, and 8d, all bearing 1,1′-biphenyl as P3-P4 moiety,
showed FXa inhibition constants in the low micromolar range
(Ki values ranging from 1.00 to 5.20 µM). Interestingly, the
meta-substituted derivatives (5d and 7d) proved to be hundred-
fold more potent than the para-substituted ones (5e and 7e).
Although guanidine derivatives 6d and 8d were almost equi-
potent with 5d and 7d against FXa, the selectivity versus
thrombin was only 15-fold and 8-fold, respectively. The achiral
isonipecotoyl derivative 7d is noteworthy for FXa inhibition
potency and selectivity and was chosen as lead for further
optimization.
In an attempt to improve binding of the P4 moiety of 7d into
the “aryl binding site” of FXa, we replaced hydrogen/s of the
distal phenyl with fluorine/s. Actually, fluorine substitution has
become increasingly a successful strategy for lead optimization
in several cases.45 The substitution of hydrogen with fluorine
generally results in minimal steric effects, while a number of
properties contributing to the ability of a molecule to engage
in intermolecular interactions, such as dipole moment, electro-
static distribution, aromatic-aromatic interactions, hydrogen
bonding, and lipophilicity, can be significantly modulated.46
Evidence has been reported in literature proving implication of
aromatic F in the enzyme-ligand binding affinity and selectivity
of thrombin38,47 and factor Xa inhibitors.37
Eleven fluorinated 3-(biphenyl-4-ylmethoxy)phenyl piperi-
dine-4-carboxamides (7f-p), including monofluoro (7f-h),
difluoro (7j-m), trifluoro (7n-o), and one 4-trifluoromethyl
(7p) congeners, were tested in vitro for inhibition of FXa,
thrombin (FIIa), and R-chymotrypsin (R-CT), and some of them,
including the most potent FXa inhibitors, for inhibition of
platelet aggregation (Table 2).

Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1023
Table 2. Platelet Aggregation Inhibitory Activities, Inhibition Constants for FXa, FIIa, and R-Chymotrypsin (R-CT), and Lipophilicity Parameters of
Fluorinated 3-(Biphenyl-4-ylmethoxy)phenyl Piperidine-4-carboxamidesa
IC50, µMc
Ki, µMd
b
compd
X
log k′w
ADP
EPN
FXa
FIIa
R-CT
7f
4′-F
3′-F
2′-F
4.74
5.07
4.87
5.06
5.05
4.98
4.72
4.93
4.61
5.21
5.93
35 ( 15**
34.7 ( 5**
62.7 ( 9
98.8 ( 14
n.d.
n.d.
n.d.
27.8 ( 4***
n.d.
19 ( 5*
14.4 ( 3***
n.d.f
n.d.
n.d.
n.d.
n.d.
17 ( 3***
n.d.
3.87
e
e
e
54.0
e
e
e
e
e
31.0
e
e
60.0
e
e
60.3
7g
7h
7j
7k
7i
0.177
0.767
0.512
0.548
1.27
1.45
0.130
9.47
2′,4′-F2
2′,3′-F2
2′,5′-F2
2′,6′-F2
3′,5′-F2
2′,4′,6′-F3
3′,4′,5′-F3
4′-CF3
7l
7m
7n
7o
7p
26.7
e
e
e
e
e
76.7 ( 15
65.7 ( 13
n.d.
n.d.
0.666
0.057
a General structure in Scheme 2. b Log of RP-HPLC polycratic capacity factor, i.e., capacity factor extrapolated at 100% aqueous mobile phase (variable
fraction of MeOH in 40 mM ammonium acetate solution, pH ) 5.0); stationary phase: Phenomenex Gemini C18 column (150 mm × 4.6 mm i.d., 5 µm
particles). c Compound concentration which inhibits ADP- and EPN-induced platelet aggregation by 50% in a PRP assay. Data are means ( SEM of at least
three different determinations, each performed in duplicate; *p< 0.05, **p < 0.01, ***p < 0.001 significantly different from compound 2b. d In vitro
inhibition constants from amydolitic assay. e Less than 30% inhibition at the maximum concentration tested (250 µM). f n.d. ) Not determined.
Among the monofluorinated derivatives, when compared with
the unsubstituted parent compound 7d, only the 3′-F congener
(7g) resulted in 6-fold (∆∆G ≈ 1 kcal ·mol-1) greater potency
although showing limited selectivity against R-CT, whereas the
2′-F isomer (7h) did not show any appreciable increase of FXa
inhibitory potency and the 4′-F one (7f) proved to be about
4-fold less potent. Our results showed that di- and trifluorinated
derivatives, with the exception of the 3′,5′-difluorinated congener
with about 8-fold increase of potency (7m), have a potency
similar to or lower than that of nonfluorinated 7d. Double
fluorination at the ortho positions (7l), and even more addition
of three fluorines in the ortho and para positions (7n), proved
to be deleterious, the last showing a drop of FXa potency and
selectivity against thrombin. As opposed to the para-monoflu-
orinated derivative 7f, the 4′-CF3 congener 7p was much more
potent (∆∆G ≈ 2 kcal ·mol-1) than the parent analogue 7d.
Compound 7p, with a Ki value of 57 nM, is to date our most
potent FXa inhibitor, displaying more than 4400-fold selectivity
against both FIIa and R-CT.
The binding mode into the binding site of FXa of the most
active inhibitors 7g, 7m, and 7p, compared with that of the
parent compound 7d, was investigated by docking calculations.
The X-ray structure of human FXa (PDB code 1FJS; 1.92 Å
resolution),35 in complex with the inhibitor ZK-807834 (CI-
1031), was retrieved from the Brookhaven Protein Data Bank
and used in the calculations with QXP software.34 The docking
poses corresponding to the highest scored solutions of 7m (very
similar to those of 7d and 7g) and 7p are shown in Figure 2. In
the lowest-energy enzyme-ligand complexes, the conformations
of all the investigated compounds docked to the FXa are similar
with respect to the bend between the protonated piperidine (P1)
and the fluorinated 1,1′-biphenyl (P3-P4) moieties.
All the binding models show two main common interactions:
(i) salt bridge, strengthen by HBs, between the piperidinium
group, in “chair” conformation, and the carboxylate of Asp189
at the bottom of the enzyme S1 pocket (average distance 3.3
Å), and (ii) HB between the amide carbonyl of the ligand and
the catalytic residue Ser195 of FXa (O · · ·O average distance
and O-H· · ·O angle about 2.8 Å and 160°, respectively). The
models show a possible aromatic ring-stacking between the
distal phenyl group (P4) and the S4 pocket, that is a narrow
hydrophobic channel lined by aromatic side chains of Tyr99,
Phe174, and Trp215. In particular, for 7d, 7g, and 7m, which
adopt closely superimposed conformations, the distal phenyl
ring, deeply inside the S4 aryl binding site, is sandwiched
between the aromatic rings of Tyr99 and Phe174, likely through
π-π interactions, and makes CH-π interactions with the indole
ring of Trp215. In contrast, the distal 4′-CF3-phenyl of 7p is
shifted ahead and rotated, achieving face-to-face aromatic
interaction with Trp215.
We believe that the positioning of the F atoms in 7g, 7m,
and 7p, with their propensity to engage polar interactions with
the backbone CO (and NH) groups inside the S4 pocket (i.e.,
the so-called “fluorophilic” interactions), may be a determinant
for the increase of their inhibition potency. In particular, for
the most potent inhibitor 7p, the computational model identifies
two middle-range contacts between C(sp3)-F and CO moieties
of Thr98 (d: 2.9 Å, θ: 40°) and Glu97 (d: 3.8 Å, θ: 25°), whose
geometries (e.g., θ values less than the magic angle, which is
approximately 54.7°) would be suitable for allowing attractive
C(sp3)-F· · ·CdO dipole-dipole interactions.
The prolongation of the activated partial tromboplastin time
(aPTT) in human plasma was also measured for the most potent
inhibitors 7g, 7m, and 7p in order to determine their in vitro
anticoagulant activity (defined as the inhibitor concentration
required to increase aPTT by 2-fold). The aPTT identifies the
time interval required for clot formation in response to a
nonphysiological stimulus, such as kaolin, which leads to
primary activation of the intrinsic or contact pathway. In our
assay conditions, the aPTT was doubled at concentrations only
in the high micromolar range (140 µM for 7p, 190 µM for 7m,
and 205 µM for 7g; 7a did not show any effect on the aPTT up
to 1 mM concentration).
Finally, a number of fluorinated isonipecotamide derivatives
7 in Table 2 were assayed for the ability to inhibit in vitro the
ADP (and EPN in a fewer cases)-induced human platelet
aggregation. All the tested compounds inhibited platelet ag-
gregation with potency higher than that of the unsubstituted
analogue 7d, their IC50 values ranging from 28 to ca. 100 µM.
Such an increase in antiplatelet potency was not expected based
only on the small increase of lipophilicity and the parabolic eq
1. We recalculated the second-order curve regression with log
k′w for the whole series of the N1-H piperidine-(3 and
4)-carboxamides (i.e., combining the antiaggregatory activity
data of compounds 2, 5, and 7 from Table 1 and fluorinated
congeners 7 from Table 2) and incorporated into the regression
model favorable and detrimental effects, using the following
indicator (or category) variables: IF accounting for fluorination
(it assumes the value 1 for fluorinated and 0 for nonfluorinated
compounds), and Io accounting for detrimental F-substitution

1024 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
de Candia et al.
Figure 2. Overlay of compounds 7m and 7p docked into the binding site of factor Xa (PDB code: 1FJS). The QXP highest scored docking poses
are shown. The figure was drawn by PyMOL.36 The protein is shown in ribbon, and the ligands and potentially interacting residues in stick (blue:
nitrogen; red: oxygen; green: carbon atoms in 7m; orange: carbon atoms in 7p).
in the ortho positions (-1 for ortho-substituted congeners, 0
for the others). A stepwise multiple regression analysis yielded
the following equation:
>4400-fold selectivity for FXa versus thrombin and chymot-
rypsin, is noteworthy.
Conclusions
ꢀ
ꢀ
2
)
(
)
(
)
pIC50 ) 0.64 (0.13 log k w - 0.07 (0.02 log k
+
(
w
Introducing suitable modifications into the structure of
lipophilic nipecotic acid anilides,33 previously identified as in
vitro inhibitors of blood platelet aggregation, we succeeded in
the purpose of empowering these molecules with anticoagulant
activity, achieved by the selective inhibition of factor Xa of
the blood coagulation cascade. The combination of FXa
inhibition and platelet antiaggregatory activities into one
molecule, which per se should be advantageous (as for phar-
macokinetics, drug interactions and toxicity, less demanding
clinical studies, etc.), can enhance its potential as antithrombotic
agent in patients with prosthetic heart valves and atrial
fibrillation.
(
)
(
)
0.34 (0.06 I - I + 2.54 (0.21
(
)
F
o
n ) 20 r2 ) 0.8458 s ) 0.1406 F ) 29.26 (2)
The substitution of H with F can improve the interaction of
these derivatives (predominantly in the piperidinium form at
the physiological pH) with platelet membranes, more than
expected from the lipophilicity parameter alone. Indeed, studies
of transport kinetics of lipophilic ions across plasma membranes
proved the translocation of the F-substituted congeners to be
slower than that of the parent hydrogenated ones by at least 1
order of magnitude.48 Such a kind of “extralipophilic” effect
due to the F-substitution should add significantly to the
antiplatelet activity of the 4-carboxypiperidinium derivatives 7.
Our study led to the development of some fluorinated
benzyloxyphenyl piperidine-4-carboxamides (7), able of acting
in vitro both as FXa inhibitors, with potency in the nanomolar
range, and inhibitors of ADP-induced platelet aggregation, with
potency in the micromolar range.
Taking all the above findings into account, three isonipeco-
tamide-containing compounds, namely the meta-fluorinated
derivatives 7g (Ki (FXa) ) 0.177 µM, IC50 (ADP) ) 34.7 µM)
and 7m (Ki (FXa) ) 0.130 µM, IC50 (ADP) ) 27.8 µM), and
the para-trifluoromethyl congener 7p (Ki (FXa) ) 0.057 µM,
IC50 (ADP) ) 65.7 µM) as well, which proved to be good FXa
and moderate platelet aggregation inhibitors, can be chosen as
leads for further optimization of novel dual-function antithrom-
botic agents. Among them, the 4′-CF3 congener 7p, displaying
A major outcome of this study was the identification of two
fluorinated derivatives of N-[3-(biphenyl-4-yl)-phenyl]piperi-
dine-4-carboxamide, 7m (3′,5′-F2 congener) and 7p (4′-CF3
congener), which proved to be potent and selective inhibitors
of FXa, additionally endowed with a moderate-to-good anti-
platelet activity. These new dual-function agents, albeit still
requiring optimization of potency and selectivity, can be

Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1025
3-[(1,1′-Biphenyl-4-yl)methoxy]aniline (4d). A mixture of 3d
(1.0 g, 3.28 mmol), hydrazine hydrate (1.1 mL, 22.9 mmol), and
activated Raney nickel suspension (1 mL) in 100 mL of methanol
was heated at reflux for 4 h until starting material disappeared on
TLC. The cooled mixture was filtered through a celite pad and the
filtrate concentrated under reduced pressure to afford an oil residue,
which was purified by chromatography on silica gel, eluting with
petroleum ether/ethyl acetate (90:10, v/v), to afford the crude title
compound as a pale-brown solid (0.56 g, 62% yield). The raw
material of 4d was used in the next coupling reaction without further
considered promising leads in the search for new efficacious
and safe antithrombotic drugs.
Experimental Section
Chemistry. Melting points were determined by using the
capillary method on a Stuart Scientific SMP3 electrothermal
apparatus and are uncorrected. Elemental analyses (C, H, N) were
performed on an Euro EA3000 analyzer (Eurovector, Milan, Italy)
using the Analytical Laboratory Service of the Dipartimento
Farmaco-chimico of the University of Bari, and the results agreed
to within (0.40% of the theoretical values. Mass spectra were
recorded on Agilent GC-MS 689-973. IR spectra were recorded
using KBr disks on a Perkin-Elmer Spectrum One FT-IR spec-
trophotometer (Perkin-Elmer Ltd., Buckinghamshire, UK), and the
most significant absorption bands are listed. 1H NMR spectra were
recorded at 300 MHz on a Varian Mercury 300 instrument.
Chemical shifts are expressed in δ and the coupling constants J in
hertz. The following abbreviations are used: s, singlet; d, doublet;
t, triplet; m, multiplet. Signal due to NH and OH protons were
located by deuterium exchange with D2O.
Chromatographic separations were performed on silica gel 60
for column chromatography (Merck 70-230 mesh, or alternatively
15-40 mesh for flash chromatography). Unless otherwise stated,
starting materials, and all chemicals and solvents as well, were
purchased from Sigma-Aldrich. Several compounds were synthe-
sized according to known procedures with slight modifications; their
melting points and spectral data were in full agreement with those
reported in literature, and no effort was made at this stage to
optimize the yields.
General Procedure for Synthesis of 3- or 4-[(4-R-benzyl)-
oxy]anilines (4). The synthesis of 3-benzyloxyaniline (4a) is
reported as an example. Dry potassium carbonate (1.2 g, 8.63 mmol)
was added to a solution of 3-nitrophenol (1.0 g, 7.19 mmol) in 7
mL of dry DMF. After 30 min, a solution of benzyl bromide (0.94
mL, 7.91 mmol) in 3 mL of dry DMF was added dropwise, and
the suspension was stirred at room temperature overnight. The
reaction was quenched by addition of 100 mL of water, and the
resulting mixture was extracted with ethyl acetate (3 × 20 mL).
The combined organic extracts were then washed with brine, dried
(Na2SO4), and concentrated under reduced pressure. The resultant
yellow solid was crystallized from ethanol to afford 1.4 g (85%
yield) of 1-(benzyloxy)-3-nitrobenzene (3a).
A mixture of 3a (1.4 g, 6.10 mmol), hydrazine hydrate (1.5 mL,
30.3 mmol), and activated Raney nickel suspension (1 mL) in 100
mL of methanol was heated at reflux for 2 h, until starting material
disappeared on TLC. The cooled mixture was filtered through a
pad of celite and the filtrate concentrated under reduced pressure
to afford the target compound 4a in almost quantitative yield.
Physical data, IR and 1H NMR spectra of 1-(benzyloxy)-3-
nitrobenzene (3a), 1-[(4-fluorobenzyl)oxy]-3-nitrobenzene (3b),
1-[(4-fluorobenzyl)oxy]-4-nitrobenzene (3c), and corresponding
amino derivatives (4a-c) were in good agreement with the reported
ones.40,49-51
1
purification; mp 115-118 °C, from petroleum ether/AcOEt. H
NMR (CDCl3) δ 7.67-7.59 (m, 5H), 7.52-7.42 (m, 4H), 7.35 (d,
1H, J ) 6.9 Hz), 6.89 (d, 1H, J ) 6.6 Hz), 6.69-6.85 (m, 2H),
5.13 (s, 2H), 4.00 (s, 2H). IR (cm-1) 3462, 3368, 1624, 1596, 1190,
1010, 831, 752.
4-[(1,1′-Biphenyl-4-yl)methoxy]aniline (4e). Compound 4e was
synthesized by reduction of 3e according to the procedure described
for 4d.
General Procedures for Synthesis of R-Substituted Benzy-
loxyphenyl Piperidine-4 (or 3-)-Carboxamides. Method A
(5b-c, 7b-c, 7f). The synthesis of N-{3-[(4-fluorobenzyl)oxy]-
phenyl}piperidine-3-carboxamide hydrochloride (5b) is reported as
an example.
Thionyl chloride (1.4 mL, 19.3 mmol) was added to a cooled
solution of N-Boc-nipecotic acid (1.1 g, 4.83 mmol) in 20 mL of
dry THF. The reaction mixture was stirred at room temperature
for 3 h, and the solvent was removed by evaporation under reduced
pressure to give the acyl chloride product as a pale-yellow oil, which
was dried in nitrogen atmosphere. The crude oil dissolved in 10
mL of dry THF was added dropwise to a solution of 4-(benzy-
loxy)aniline (0.70 g, 3.22 mmol) and triethylamine (1.35 mL, 9.66
mmol) in 15 mL of dry THF, and reaction mixture was stirred
overnight at room temperature. The solid precipitate was filtered
off and the filtrate concentrated under reduced pressure. The oil
residue was dissolved in 50 mL of ethyl acetate and washed with
1 N HCl (3 × 20 mL), 5% (w/v) NaHCO3 (3 × 20 mL) and finally
with brine (3 × 20 mL). The organic phase was dried (Na2SO4)
and evaporated in vacuo, yielding an oil residue, which was purified
by silica gel column chromatography, eluting with petroleum ether/
ethyl acetate (65:35, v/v) to afford the target N-Boc-protected anilide
as a solid product. A solution of 1.0 mmol of the N-Boc-protected
anilide in 30 mL of chloroform was cooled to 0-5 °C, saturated
with HCl gas. Then the solvent was evaporated under reduced
pressure, and the crude solid was purified by crystallization from
ethanol/ethyl acetate to afford the hydrochloride salt of N-{3-[(4-
fluorobenzyl)oxy]phenyl}piperidine-3-carboxamide (5b) as a white
microcrystalline solid (85% yield); mp 219-220 °C. 1H NMR
(DMSO-d6) δ 10.27 (s, 1H), 8.60 (s, 2H), 7.46 (t, 1H, J ) 6 Hz),
7.38 (s,1H), 7.10-7.23 (m, 5H), 6.69 (d, 1H, J ) 8 Hz), 5.02 (s,
2H), 3.29-3.32 (m, 2H), 3.13-3.17 (m, 2H), 2.46-2.49 (m, 1H),
1.48-1.77 (m, 4H). IR (cm-1) 3541, 3322, 1663, 1225, 1154, 1039,
857, 825, 770. Anal. (C19H22N2O2FCl) C, H, N.
Method B (5a, 5d-e, 7a, 7d-e). . The synthesis of N-{[3-(1,1′-
biphenyl-4-yl)methoxy]phenyl}piperidine-4-carboxamide hydro-
chloride (7d) is reported as an example.
(1,1′-Biphenyl-4-yl)methyl 3-nitrophenyl ether (3d). Diiso-
propyl azodicarboxylate (DIAD, 1.42 mL, 7.19 mmol) was added
dropwise at 0 °C to a stirred solution of 3-nitrophenol (1.0 g, 7.19
mmol), triphenylphosphine (1.9 g, 7.19 mmol), and (1,1′-biphenyl-
4-yl)methanol (1.3 g, 7.19 mmol) in 20 mL of dry THF, and the
mixture was stirred for about 48 h at room temperature. The solvent
was removed by evaporation under reduced pressure, and the oil
residue was purified by chromatography on silica gel, eluting with
petroleum ether/ethyl acetate (90:10, v/v), to afford 1.44 g (66%
yield) of the crude product as a pale-yellow solid; mp 126-129
°C, from petroleum ether/AcOEt. 1H NMR (CDCl3) δ 7.96 (d, 1H,
J ) 8.0 Hz), 7.5-7.68 (m, 8H), 7.45 (t, 1H, J ) 7.7 Hz), 7.37 (d,
1H, J ) 7.1 Hz), 7.15 (d, 1H, J ) 8.2 Hz), 7.05 (t, 1H, J ) 8.0
Hz), 5.28 (s, 2H). IR (cm-1) 1532, 1518, 1353, 1247, 1028, 861,
814, 763.
A solution of benzyl chloroformate (0.63 mL, 4.36 mmol) in 5
mL of dry THF was added dropwise to a cooled solution of N-Boc-
nipecotic acid (1.0 g, 4.36 mmol) and triethylamine (1.22 mL, 8.72
mmol) in 15 mL of dry THF. After 30 min of stirring at room
temperature, a solution of 3-[(1,1′-biphenyl-4-yl)methoxy]aniline
(1.2 g, 4.36 mmol) in 10 mL of dry THF was added, and the
reaction mixture was stirred at room temperature overnight. The
solid precipitate was filtered off and the filtrate concentrated under
reduced pressure. The oil residue was dissolved in 50 mL of ethyl
acetate and washed with 1 N HCl (3 × 20 mL), 5% w/v NaHCO3
(3 × 20 mL), and finally with brine (3 × 20 mL). The organic
phase was dried (Na2SO4) and evaporated in vacuo, yielding an oil
residue, which was purified by silica gel column chromatography
(mobile phase: petroleum ether/ethyl acetate, 80:20, v/v) to afford
the target N-Boc-protected anilide as a solid product. The N-Boc
carboxamide was deprotected with HCl gas, following a procedure
(1,1′-Biphenyl-4-yl)methyl 4-nitrophenyl ether (3e). Com-
pound 3e was synthesized from 4-nitrophenol according to the
procedure described for 3d.

1026 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
de Candia et al.
similar to that described above for 5b, and the resultant residue
was purified by crystallization from ethanol/ethyl acetate to give
N-{[3-(1,1′-biphenyl-4-yl)methoxy]phenyl}piperidine-4-carboxam-
ide hydrochloride salt (7d) as pale-brown crystals in 60% yield;
mL of dry DMF was slowly added dropwise, and the reaction
mixture was stirred for 6-8 h at room temperature. The reaction
was quenched by adding 5 mL of 5% (w/v) KHSO4 and, after 5
min, diluted with 50 mL of distilled water. The aqueous phase was
then extracted with ethyl acetate (3 × 15 mL), and the combined
organic extracts were washed twice with brine, dried (Na2SO4),
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography, eluting with petroleum ether/
ethyl acetate (90:10, v/v), and the chromatographically pure N-Boc
protected derivative was treated with HCl gas to afford 0.50 g of
N-{3-[(3′-fluoro-1,1′-biphenyl-4-yl)methoxy]phenyl}piperidine-4-
carboxamide hydrochloride salt (7g), which was purified by
crystallization: pale-brown crystals, 60% yield; mp 248-249 °C,
from EtOH/EtOAc.
1
mp 189-192 °C. H NMR (DMSO-d6) δ 10.12 (s, 1H), 8.78 (s,
2H), 7.67-7.63 (m, 4H), 7.51-7.32 (m, 6H), 7.21-7.12 (m, 2H),
6.70 (d, 1H, J ) 9 Hz), 5.09 (s, 2H), 3.32-3.27 (m, 1H), 2.88 (t,
2H, J ) 9 Hz), 2.58-2.65 (m, 1H), 1.71-1.95 (m, 5H). IR (cm-1
)
3466, 1663, 1284, 1224, 1156, 791, 753. Anal. (C23H27N2O2Cl·
H2O) C, H, N.
General Procedure for Guanylation of Compounds 5 (6b,
6d) and 7 (8b, 8d). The synthesis of 1-[amino(imino)methyl]-N-
{3-[(4-fluorobenzyl)oxy]phenyl}piperidine-4-carboxamide hydro-
chloride (8b) is reported as an example.
1H NMR (CDCl3) δ 10.11 (s, 1H), 8.87 (s,
N,N′-bis-Boc-S-methylthiopseudourea (0.62 g, 2.14 mmol), tri-
ethylamine (0.75 mL, 5.34 mmol), and mercury(II) chloride (0.58
g, 2.14 mmol) were added in a sealed tube containing a cooled
solution of 7b (650 mg, 1.78 mmol) in 6 mL of dry DMF, and the
reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. Ethyl acetate (15 mL) was then added to the reaction
mixture, and stirring was continued for 15 min. The precipitate
was filtered off and washed 3 times with 15 mL of ethyl acetate.
The combined organic phases were washed with brine (3 × 20
mL), dried (Na2SO4), and concentrated under reduced pressure to
afford a residue, which was purified by silica gel column chroma-
tography, eluting with petroleum ether/ethyl acetate (90:10, v/v).
The Boc group was removed (HCl gas) to afford 0.46 g (63% yield)
of the product HCl salt 8b as white microcrystalline solid; mp
2H), 7.71 (d, 2H, J ) 8 Hz), 7.53-7.48 (m, 5H), 7.42 (d, 1H, J )
2 Hz), 7.21-7.11 (m, 3H), 6.71 (d, 1H, J ) 7 Hz), 5.10 (s, 2H),
3.30 (d, 2H, J ) 14 Hz), 2.88 (t, 2H, J ) 14 Hz), 2.66-2.58 (m,
1H), 1.94-1.88 (m, 2H), 1.85-1.75 (m, 2H). IR (cm-1) 3284, 1794,
1695, 1220, 1162, 858, 792. Anal. (C25H26N2O2FCl) C, H, N.
In Vitro Inhibition of Platelet Aggregation. Human blood was
obtained from healthy volunteers (25-45 years of age), who had
not ingested any platelet inhibitory drug for at least 1 week prior
to donation. All subjects provided informed consent. Blood and
blood products were handled in plastic ware, whereas siliconized
glass cuvettes and stir bars were used in the aggregation assay.
Human platelet-rich plasma (PRP) was obtained from the
supernatant after centrifugation of venous blood (9 mL), mixed with
0.129 mol/L sodium citrate (1:9 to blood) to prevent it from clotting,
at 800 rpm for 15 min at 21 °C. Platelet-poor plasma (PPP) was
obtained after centrifugation of the venous blood at 3200 rpm for
10 min. Platelet counts were adjusted to the constant value of
230000 µL-1 by using PPP.
Aggregation was measured by the turbidimetric method of Born,
using a four-channel aggregometer (PACKS-4, Helena Laboratories
S.p.A., Beaumont, TX).43 The transmittance of PPP was taken as
100% aggregation. PRP (250 µL) was preincubated with the test
compounds (5 µL solutions; final concentrations in the PRP
solutions ranging from 10 µM to 1 mM) or with controls (to
eliminate the effect of the solvent on the aggregation and release
reaction of platelets, the final concentration of DMSO was fixed at
0.5%, v/v) at 37 °C for 5 min, during which the suspension was
stirred at 800 rpm. Then, 50 µL of plasma isotonic solution (0.9%,
w/v, NaCl) containing adenosine 5′-diphosphate (ADP) or epi-
nephrine (EPN) was added to the stirred sample (10 µM final
concentration), and the change in transmittance at 640 nm was
recorded for 5 min. The control cuvette containing the vehicle-
treated PRP followed the same sequence of events. The above
concentrations of ADP and EPN proved to elicit a full response in
all our PRP aggregation assays.
1
239-241 °C, from EtOH. H NMR (DMSO-d6) δ 10.37 (s, 1H),
7.54 (s, 4H), 7.49-7.37 (m, 3H), 7.22-7.11 (m, 4H), 6.66 (d, 1H,
J ) 7 Hz), 5.02 (s, 2H), 3.88 (d, 2H, J ) 14 Hz), 3.05 (d, 1H, J
) 14 Hz), 3.01 (d, 1H, J ) 14 Hz), 2.70-2.55 (m, 1H), 1.90-1.75
(m, 2H) 1.58- 1.45 (m, 2H). IR (cm-1) 3333 1654, 1224 1154 1039,
825, 775. Anal. (C20H24N4O2FCl·H2O) C, H, N.
tert-Butyl 4-[N-(3-Hydroxyphenyl)carbamoyl]piperidine-1-
carboxylate(9).Asuspensionoftert-butyl4-{[3-(benzyloxy)phenyl]-
carbamoyl}piperidine-1-carboxylate 7a (2.1 g, 5.24 mmol) and
0.10 g of 10% palladium on carbon charcoal in 50 mL of ethanol
was stirred at room temperature in hydrogen atmosphere until
starting material disappeared on TLC. The reaction mixture was
filtered through a pad of celite, and the solvent was removed by
evaporation under reduced pressure to give 9 as a brown solid in
almost quantitative yield. Compound 9 was used in the subsequent
reactions without further purification. 1H NMR (DMSO-d6) δ 9.75
(s, 1H), 9.30 (s, 1H), 7.15 (m, 1H), 7.02 (t, 1H, J ) 9 Hz), 6.93 (d,
1H, J ) 9 Hz), 6.40 (d, 1H, J ) 9 Hz), 3.97 (d, 2H, J ) 14 Hz),
2.80-2.73 (m, 2H), 2.55-2.45 (m, 1H), 1.80-1.65 (m, 2H),
1.52-1.48 (m, 2H), 1.38 (s, 9H). IR (cm-1) 3240, 1663, 1278, 1222,
1160, 781.
Synthesis of Fluorinated 4′-(Bromomethyl)-1,1′-biphenyl
Congeners. The preparation of 4′-(bromomethyl)-3-fluoro-1,1′-
biphenyl is reported as representative example. N-bromosuccinim-
mide (1.5 g, 8.70 mmol) was added to a solution of 3-fluoro-4′-
methyl-1,1′-biphenyl (1.6 g, 8.70 mmol), which in turn was prepared
according to a reported method52 in 30 mL of CCl4. The reaction
mixture was refluxed for 20 h and then allowed to cool to room
temperature. The insoluble succinimmide was filtered off and the
solvent was evaporated under reduced pressure to yield 2.1 g (90%
yield) of the target compound as a crude oil, which was used without
All the aggregation experiments were completed within 3 h from
thebloodcollection,whereaseachseriesofdataforconcentration-response
curves in duplicate was collected within 1 h. Inhibition of
aggregation was expressed as a percent difference of the maximum
aggregatory response, and IC50 values (i.e., the concentration
effecting 50% inhibition of aggregation), obtained by nonlinear
(sigmoid) regression of aggregation inhibition (% A.I.) on log
concentration of test compounds (r2 > 0.80), were reported as means
( SEM of at least three different determinations.
Inhibition Assays of Factor Xa and Related Serine Proteases.
The target compounds were assessed for their inhibitory activity
toward factor Xa (fXa), thrombin (fIIa), and R- chymotrypsin (R-
CT) by kinetic analysis using chromogenic substrates monitored
at 405 nm. All buffer salts were obtained from Sigma-Aldrich Co.
(Milan, Italy). Enzymes and substrates were obtained as follows:
for fXa kinetic studies, 2 nM human factor Xa and 0.04 mM S-2765
(Z-D-Arg-Gly-Arg-p-nitroanilide) from Chromogenix AB-Instru-
mentation Laboratories (Milan, Italy); for fIIa kinetic studies, 0.41
U/mL bovine thrombin from Sigma-Aldrich (Milan, Italy) and 0.050
mM S-2238 (D-Phe-Pip-Arg-p-nitroanilide) from Chromogenix AB-
Instrumentation Laboratories (Milan, Italy); for R-CT kinetic studies,
1
further purification. H NMR (CDCl3) δ 7.71 (d, 2H, J ) 8 Hz),
7.53-7.48 (m, 4H), 7.21-7.11 (m, 2H). IR (cm-1) 2950, 1485,
820, 765.
General Procedure for Synthesis of Compounds 7f-p. The
synthesis of N-{3-[(3′-fluoro-1,1′-biphenyl-4-yl)methoxy]phenyl}-
piperidine-4-carboxamide·HCl (7g) is reported as a representative
example.
A solution of tert-butyl 4-[N-(3-hydroxyphenyl)carbamoyl]pip-
eridine-1-carboxylate 9 (0.60 g, 1.89 mmol) in 5 mL of dry DMF
was added to a suspension of NaH (0.68 g, 2.84 mmol) in 3 mL of
dry DMF at room temperature. After 15 min, a solution of
4′-(bromomethyl)-3-fluoro-1,1′-biphenyl (0.50 g, 1.89 mmol) in 5

Fluorinated Benzyloxyphenyl Piperidine-4-carboxamides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1027
0.4 µg/mL bovine R-chymotrypsin and 0.185 mM N-succinyl-Ala-
Ala-Pro-Phe-p-nitroanilide from Sigma-Aldrich (Milan, Italy).
In kinetic studies with fXa or fIIa, 500 µL of the enzyme solution
in a 10 mM Tris buffer (pH 8), containing 0.15 M NaCl and 0.1%
of PEG 6000, was mixed with 10 µL of solution of the test
compound, its final concentration ranging from 10 nM to 0.25 mM;
DMSO, derived from inhibitory compound stock solution, did not
exceed a final concentration of 2% v/v (10 µL of DMSO in control
assays). The enzymes were incubated with the test compound or
its solvent (DMSO) for 30 min at 25 °C, and the reactions were
initiated by the addition of 500 µL of the appropriate substrate
solution in buffer. Increase in absorbance due to proteolytic cleavage
of the substrate was monitored at 25 °C and 405 nm for 5 min.
In kinetic studies with R-CT, 845 µL of 50 mM Tris buffer (pH
7.5), containing 50 mM of CaCl2, were added to 50 µL of enzyme
solution in buffer and 5 µL of solution of the test compound (final
concentration ranging from 10 nM to 0.25 mM) or its solvent
(DMSO). After 10 min of incubation at 25 °C, 100 µL of the
substrate solution in buffer was added and the absorbance increase
at 405 nm was monitored at 25 °C for 5 min.
Initial velocities were determined by analysis of the initial linear
portion of the kinetic curve, and values were plotted against log
inhibitor concentration. IC50 values were obtained by nonlinear
(sigmoid) regression calculation on dose-response plots in at least
three different determinations, and used in the Cheng-Prusoff
equation for calculating inhibition constants (Ki).53
In Vitro Coagulation Assays. Commercially available pooled
lyophilized human plasma (100 µL), aPTT reagent (100 µL) and
0.025 mol/L CaCl2 (100 µL) (Futura System s.r.l., Formello, Rome,
Italy) were used to determine the concentration of the test compound
required for doubling the activated partial thromboplastin time
(aPTT). Increasing concentration of the inhibitor or solvent were
added to plasma and incubated for 3 min at 37 °C. Clotting times
(seconds) were determined in a coagulometer (Behnk Elektronik
GmbH, Norderstedt, Germany) and compared with those from the
appropriate human control plasma. Each measurement was per-
formed in triplicate, and concentrations of the test compound which
caused a 2-fold prolongation of aPTT (2 × aPTT) were calculated
from concentration-response curves.
Determination of Lipophilicity Parameters by RP-HPLC.
Polycratic capacity factors (log k′w) were determined according to
a previously reported RP-HPLC method.33b,44 Compounds, dis-
solved in MeOH (1 mg/mL), were injected onto a Gemini C18
column (150 mm × 4.6 mm i.d., 5 µm particles) from Phenomenex
Italia Srl (Castel Maggiore, Bologna, Italy) and retention data
measured at regular increments of the volume fraction of methanol
in 40 mM ammonium acetate aqueous solution (pH 5.0). All the
measurements were made at temperature of 25 ( 1 °C, flow-rate
of 1.0 mL/min and at 254 nm wavelength, on a Waters HPLC 1525
multisolvent delivery system, equipped with a Waters 2487 variable
wavelength UV detector (Waters Assoc., Milford, MA). Capacity
factors (k′) of each compound at different mobile phase composi-
tions (0.05 increments of MeOH volume fraction, ranging between
0.80 and 0.40) were calculated as: k′ ) (tR - t0)/t0, where tR is the
retention time of the solute and t0 is the column dead time, measured
as the elution time of MeOH. In all the cases, the log k′ values
increased linearly with decreasing MeOH volume fraction. Linear
regression analysis (r2 > 0.97) was performed on at least five data
points and the linear relationship extrapolated to 100% aqueous
mobile phase to yield log k′w values.
free protein was kept rigid, except for the side chains of the residues
lying within a distance of 4 Å from ZK-807834 in the original
PDB complex (1FJS) retrieved by the Brookhaven Protein Data
Bank (http://www.rcsb.org/pdb/), which were settled free to relax
during the minimization of the binding complexes found by the
calculation routine. Finally, oxygen atom of Ser195 was marked
for indicating the binding cavity and the inhibitor added and settled
as completely free to move. To allow a good sampling of the
conformational space, 1000 runs of Monte Carlo search, followed
by conjugate gradient minimization, were performed, and the
highest-scored docking hypotheses were taken into account.
Acknowledgment. We acknowledge the financial support
by the Italian Ministry for Education Universities and Research
(MIUR, Rome, Italy; PRIN 2005, grant no. 2005064838-002).
Supporting Information Available: Physicochemical data, IR
and NMR spectra, and elemental analyses (C, H, N) of the
intermediates and target compounds (3e, 4e, 5a, 5c-e, 6b, 6d,
7a-c, 7e-p, 8d). This material is available free of charge via the
Internet at http://pubs.acs.org.
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