Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo

Article abstract of DOI:10.1021/jm900553j

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3- dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl) piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900553j

5826 J. Med. Chem. 2009, 52, 5826–5836
DOI: 10.1021/jm900553j
Discovery of Novel Protease Activated Receptors 1 Antagonists with Potent Antithrombotic
Activity in Vivo
Michel Perez,*,§ Marie Lamothe,§ Catherine Maraval,§ Etienne Mirabel,§ Chantal Loubat,§ Bruno Planty,§ Clemens Horn,§
Julien Michaux,§ Sebastien Marrot,§ Robert Letienne,# Christophe Pignier,# Arnaud Bocquet,# Florence Nadal-Wollbold,#
Didier Cussac,‡ Luc de Vries,‡ and Bruno Le Grand#
§Medicinal Chemistry 4 Division and #Cardiovascular 2 Division and ‡Department of Cellular and Molecular Biology,
Pierre Fabre Research Center, 17 Avenue Jean Moulin, 81106 Castres Cedex, France
Received April 30, 2009
Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled
receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin
activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets
via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one
subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these
receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-
dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone
39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human
platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after
iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often
observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new
class of antithrombotic agents.
Introduction
particular intramolecular activation mechanism, termed “teth-
ered ligand mechanism”, makes this target particularly difficult
to address. Indeed, the aim of this project was not to inhibit
thrombin-mediated receptor cleavage but to compete with the
intramolecular activation of the receptor by the tethered ligand,
an entropically favored interaction compared to an external
antagonist ligand. Inhibiting thrombin-mediated platelet acti-
vation without affecting thrombin’s role in the coagulation
cascade could lead to a new promising class of antiplatelet
drugs with a limited impact on bleeding.8 Since all known
antithrombotic therapies suffer from drawbacks mainly asso-
ciated with hemorrhagic side effects, the lack of bleeding with
PAR1 antagonists could constitute an essential advantage.
The proof of concept for this new antiaggregant approach
and its low impact on bleeding has been recently established in
a clinical trial with SCH-530348 (Figure 1), which is currently
in phase 3 for the treatment of acute coronary syndrome.9
Today, this is the only antithrombotic treatment by a PAR1
antagonist to have been demonstrated in humans.
Antiaggregant drugs such as ADP antagonists, thrombox-
ane A2 inhibitors, or GPIIb/IIIaa antagonists constitute very
important classes of antithrombotic therapies.1,2 These drugs
are essential for the treatment of life-threatening pathologies
such as stroke, acute coronary syndrome, or myocardial
infarction. Platelets are activated by a variety of agonists such
as thrombin, ADP, thromboxane A2, collagen, serotonin, or
epinephrine. Among these, thrombin is probably the most
potent activator of platelet aggregation. Thrombin is also a
well-known serine protease involved in the coagulation cascade
that converts soluble fibrinogen to fibrin. The aggregation of
platelets by thrombin is mediated via proteolytic activation of
specific cell surface receptors known as protease activated
receptors (PARs) or thrombin receptors.3-5 Four PARs have
been identified so far: PAR1, PAR2, PAR3, and PAR4. PAR1,
PAR3, and PAR4 are activated by thrombin, while PAR2 is
activated by trypsin. PAR1 is the major thrombin-activated
receptor on human, monkey, and guinea pig platelets.6
Other compounds have been published as potent PAR1
antagonists without reaching the clinic. Among them ER-
121958-06 (Figure 1) has been described as a potent antiag-
gregant compound on human platelets (IC50 = 21 nM).8e In a
search for novel antagonists of PAR1, we have performed a
screening of our proprietary library and found an interesting
hit that, after a first optimization step, gave lead compound 4
derived from cinnamoylpiperazine (Scheme 1).
PARs are activated by a unique mechanism in which a
proteolytic enzyme such as thrombin (or trypsin) binds to the
receptor and then cleaves its extracellular domain between
Arg41 and Ser42.7 The newly unmasked amino terminus
binds intramolecularly to the proximally located transmem-
brane loop of the GPCR, eliciting intracellular signaling. This
*To whom correspondence should be addressed. Phone: (þ33)
563714296. Fax: (þ33) 563714299. E-mail: michel.perez@pierre-fabre.
com.
Chemistry
a Abbreviations: PARs, protease activated receptors; GPCRs,
G-protein-coupled receptors; iv, intravenous; po, per os; ADP, adeno-
sine diphosphate; GPIIb/IIIa, glycoprotein IIb/IIIa; FLIPR, fluoro-
metric imaging plate reader.
Lead compound 4 was prepared according to route A des-
cribed in Scheme 1. Cinnamic acid 1a was first transformed into
an acid chloride, which was reacted with tert-butylpiperazine
r
pubs.acs.org/jmc
Published on Web 09/16/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5827
Scheme 1. Synthesis of Cinnamoyl Derivatives 4-11a
a Conditions: (a) SOCl2, reflux; (b) Boc-piperazine, CH2Cl2, Et3N, room temp; (c) TFA, toluene, room temp; (d) benzyl bromide, CH2Cl2, Et3N,
room temp; (e) cinnamic acids 1a-e, CH2Cl2, PS-carbodiimide, HOBT, room temp.
Table 1. SAR for the Substitution of the Aromatic Rings
% antagonism at
10 μM ( SEMa
compd
R1
R2
4
2-NO2
2,6-diF
2-Br
4-F
88.6 ( 3.8
86.2 ( 4.3
93.1 ( 0.9
85.4 ( 1.9
65.3 ( 4.6
73.1 ( 3.6
72.4 ( 5.3
0.7 ( 6.8
5
3-Me
4-F
4-F
6
Figure 1. Structures of SCH-530348 and ER-121958-06.
7
2-CN
8
2,6-diF
2,6-diF
2-NO2
3-Cl
3,4-diMe
3,4-diF
3-Me
4-F
1-carboxylate in the presence of Et3N and then deprotected
with TFA to give piperazine 2a. This intermediate provided
compound 4 after treatment with 4-fluorobenzyl bromide in
the presence of Et3N.
9
10
11
12
2-NH2
4-F
7.4 ( 4.3
Optimization of compound 4 relied, in the first instance, on
a screening assay based on the inhibition of fluorescent
calcium release induced by the PAR1 selective agonist peptide
(SFLLR-NH2) in CHO cells (FLIPR).10 This peptide, also
termed thrombin receptor activating peptide (TRAP), has
been shown to be sufficient to mimic human receptor activa-
tion by thrombin.11-15 Table 1 summarizes the results ob-
tained by modification of the substitution of the two aromatic
rings of compound 4. Compounds 5-11 were prepared
according to the synthetic routes depicted in Scheme 1. Com-
pound 12 was prepared by reduction of compound 4 with tin
chloride. The ortho substitution on the left-hand phenyl gives
the best results as exemplified by the comparison of com-
pound 11 (3-Cl, 0.7%) with compound 6 (2-Br, 93.1%) or
compound 39 (2-Cl, 92.8%) depicted in Table 4. The nature of
the substituent in this position is particularly important, as
exemplified by the comparison of compounds 4 (2-NO2,
88.6%) and 12 (2-NH2, 7.4%). Other analogues have been
prepared with substitutions in meta or para positions that
confirmed that the ortho position afforded the most active
products (data not shown).
a Inhibition of calcium release induced by 1 μM SFLLR-NH2.
3-(2-nitrophenyl)propionic acid and (2-nitrophenyl)propy-
noic acid. Diene analogue 17 was synthesized according to
Scheme 2 from commercially available (E)-3-(2-nitrophenyl)-
propenal.
A number of analogues were obtained by substitution
or replacement of the piperazine ring. 2,5-Dimethylpiperazine
(18), piperazine-2-one (19), homopiperazine (20, 22), and 4-
aminopiperidine (21) analogues were prepared using synthetic
approaches similar to the one depicted in Scheme 1. Introduc-
tion of a piperidine ring was performed according to Scheme 3.
Piperidine-4-carboxylic acid 23 was protected by a BOC
group and then condensed with N,O-dimethylhydroxylamine
after activation of the acid by EDCI and HOOBT. Boc
deprotection of Weinreb amide 24 with TFA followed by
reductive amination with 4-F-benzaldehyde afforded inter-
mediate 25. Diethyl methylphosphonate was first deproto-
nated with n-BuLiat-50°Cand then treated withamide 25to
give intermediate 26. Horner-Wadsworth-Emmons reac-
tion between this phoshonate and 2-Cl-benzaldehyde gave
compound 27. The evaluation in the PAR1 screening assay of
these compounds is summarized in Table 2.
We then focused our attention on the modification of
the unsaturated linker of compound 4. Alkane and al-
kyne analogues 13 and 14, respectively (Table 2), were pre-
pared following a synthetic scheme similar to that used for
compounds 4-11 but starting with commercially available
We also studied the replacement of the 4-F-benzyl part of
compound 4by a substituted phenyl (28, 29) or a heterocycle (30).

5828 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Table 2. SAR of Analogues of Compound 4a
Perez et al.
Table 3. SAR on the Piperazine Substitutiona
a The asterisk (/) indicates inhibition of calcium release induced by
1 μM SFLLR-NH2.
on the inhibition of calcium release model (FLIPR assay) in
order to classify the potency of these compounds as PAR1
antagonists. Since these compounds behave as competitive
antagonists, pA2 values were calculated for each of them.
Second, antiaggregant activity was evaluated using an
SFLLR-induced human platelet aggregation model.6 Follow-
ing this, an arteriovenous extracorporal shunt model in
anesthetized rats was performed to address the antithrombo-
tic potential of these compounds in vivo.16 In this model, a
shunt is realized between the left carotid artery and the right
jugular vein. A silk thread, placed in the central portion of the
shunt, was used as thrombogenic substrate. A thermal mi-
croprobe was placed onto the central part of the shunt to
determine the time for occlusion to occur (indicated by a
dramatic decrease in temperature). This assay was performed
by either intravenous or oral administration in order to obtain
the first evaluation of the bioavailability of the molecules. An
antithrombotic agent is able to increase the occlusion time in
such model.
Among the compounds listed in Table 4, several are potent
antagonists of PAR1 with pA2 or pKb of >7 (i.e., 34, 35, 37).
However, there is no clear relationship between this activity
and the inhibition of human platelet aggregation. Thus,
compound 39 has a weaker antagonistic activity compared
to compound 34 (pA2 of 6.42 vs 7.16, respectively) but a very
comparable inhibitory activity on platelet aggregation (pKb of
5.52 vs 5.50, respectively). The reference compound ER-
121958-06 displays a moderate pKb of 6.85 (noncompetitive
displacement curve) but a very potent antiaggregant activity.
Surprisingly, compound 38 was completely inactive in the
aggregation model and very weakly active in the shunt model
despite a good antagonistic activity (pA2 of 6.67). Since there is no
a The asterisk (/) indicates inhibition of calcium release induced by
1 μM SFLLR-NH2.
The synthesis of these compounds was accomplished follow-
ing the same experimental routes as described in Scheme 1
except that we used an aryl- or heteroarylpiperazine instead
of the Boc piperazine. In addition, we also synthesized
amides, sulfonamides, or ureas in this position via the reac-
tion of an acyl chloride, sulfonyl chloride, or isocyanate with
intermediates 2, as exemplified by compounds 31-33. The
evaluation in the PAR1 screening assay of these compounds
is summarized in Table 3.
The results displayed in Table 3 show that replacement of the
4-F-benzyl group of compound 4 by either a 3-Cl-phenyl or a
cycloheptylcarbonyl leads to potent compounds (28 and 31).
On the other hand, close analogues such as 29, 30, or 32 are
completely inactive. Urea analogue 33 gives very moderate
activity.
Finally, we combined the different modifications to further
optimize the activity. The most interesting compounds, ob-
tained by synthetic routes similar to the one described in
Schemes 1 and 2, are summarized in Table 4.
Results and Discussion
In order to more precisely determine the activity of these
compounds, additional pharmacological evaluations were
conducted. First, a concentration-response was performed

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5829
Table 4. SAR of Optimized Analogues of Compound 4
% antagonism
at 10 μM ( SEMa pA2a [limit values]
hum plat (pKb)b
[limit values]
AV shunt
AV shunt
compd
R1
n
R2
(iv, %) ( SEMc (po, %) ( SEMd
ER-121958-06
6.85e [ND]
7.16 [ND]
8.18 [8.09-8.49] 27 ( 13
5.50 [5.34-6.11] 44 ( 8
5.65 [5.27-5.71] 3 ( 5
5.30 [5.22-5.49] 58 ( 17
5.17 [5.07-5.27] 19 ( 6
43 ( 11
7 ( 4
ND
34
35
36
37
38
39
40
2-NO2
2-NO2
2-CN
2-CN
2,6-diF
2-Cl
2
2
2
1
1
1
1
c-pentyl
phenyl
91.7 ( 2.8
100.0 ( 0.2
98.7 ( 0.5
92.6 ( 5.3
98.9 ( 0.6
92.8 ( 0.9
7.23 [ND]
6.49 [6.40-6.55]
7.06e [ND]
2-pyridine
CO-cHeptyl
CO-c-heptyl
4-F-benzyl
54 ( 14
ND
6.67 [6.28-6.88]
6.67 [6.34-7.14]
6.31 [6.28-6.36]
0 [ND]
14 ( 3
0 ( 4
59 ( 11
ND
5.52 [5.32-5.97] 28 ( 6
5.31 [4.80-5.82] 29 ( 4
2-NO2
CH2-c-hexyl 91.5 ( 3.0
a Inhibition of calcium release induced by 1 μM SFLLR-NH2: % at 10 μM or pA2. b Inhibition of SFLLR-induced human platelet aggregation.
c % increase of the occlusion time in an arteriovenous shunt in rat at 1.25 mg/kg iv. d % increase of the occlusion time in an arteriovenous shunt in
rat at 40 mg/kg po. e pKb value (noncompetitive profile).
Scheme 2. Synthesis of Diene Compound 17a
a Conditions: (a) ethyl 2-(diethoxyphosphoryl)acetate, NaH, THF, room temp, 92%; (b) KOH, EtOH, 70 °C, 98%; (c) 4-F-benzylpiperazine,
HOOBT, EDCI, DIEA, CH2Cl2, room temp, 77%.
Scheme 3. Synthesis of Piperidine Analogue 27a
a Conditions: (a) (BOC)2O, K2CO3, H2O-THF, 99%; (b) EDCI, HOOBT, DMF, DIEA, MeONHMe HCl, 88%; (c) TFA, toluene, room temp,
3
74%; (d) 4-F-benzaldehyde, NaBH(OAc)3, AcOH, 1,2-DCE, room temp, 77% ; (e) (EtO)2P(O)Me, n-BuLi, THF, -50 °C; (f) þamide 25, THF; (g) 2-Cl-
benzaldehyde, K2CO3, CH3CN, room temp, 68% (three steps).
basic nitrogen in this compound (as well as in compound 37),
formation of a salt was not possible. Solubility issues are
probably the main reason for the poor activity observed in
most assays. Similarly and despite the fact that compound 35
was tested as a hydrochloride salt, its poor water solubility
(<1 mg/mL) could also explain the weak activity observed in
the shunt model. In this model, after intravenous administra-
tion, uneven results were obtained. Compounds 34 and 36
displayed the most potent activities in these conditions with
44% and 58% increases in the occlusion time, respectively.
The activity of compound 34 after iv administration was not
confirmed after oral dosing (44% iv vs 7% po), probably
because of a limited bioavailability. Inversely, compound 39
gave a much better activity after oral administration at 40 mg/kg
(59%) compared to 1.25 mg/kg iv (28%), while compound 36
gave very homogeneous results (58% iv vs 54% po). Inter-
estingly, the reference compound ER-121958-06 is slightly less
potent in the in vivo shunt model compared to compound 36
(both routes of administration) and to a lesser extent to
compound 39, despite very potent antiaggregant activity
(pKb = 8.18). Overall, these results demonstrate a potent anti-
thrombotic effect of molecules 36 and 39 in this model.
The selectivity of these compounds versus PAR2and PAR4
was addressed. For that purpose, we used a serum response
element (SRE) dependent luciferase activity model18 in Cos-7
cells expressing PAR2, PAR4, or PAR1. We evaluated the
ability of the compounds 36 and 39 to antagonize the increase
of SRE-luciferase activity mediated by each selective agonist
peptides. Thus, for PAR2, the activation by SLIGRL was
weakly antagonized with pKb values ((SEM, n = 3) of 4.39 (
0.12 and 4.45 ( 0.07 for compounds 36 and 39, respectively.
For PAR4, the activation by AYPGKF was antagonized with
pKb values ((SEM, n = 3) of 4.93 ( 0.01 and 4.91 ( 0.11, res-
pectively. For a clearcomparisonpurpose, wealsodetermined
PAR1 antagonistic activity of these two compounds in the
same SRE-luciferase model using SFLLR as selective agonist.

5830 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Perez et al.
Table 5. ADME Profile of the Two Lead Compounds 36 and 39
added tert-butyl piperazine-1-carboxylate (4.18 g, 22.43 mmol).
After being stirred overnight at room temperature, the reac-
tion mixture was diluted with 1 N aqueous NaOH, extracted
with CH2Cl2 (ꢀ2), dried with MgSO4, and concentrated
under reduced pressure. Purification of the crude material by
flash column chromatography (SiO2, CH2Cl2/MeOH/NH4OH,
98/1.5/0.5) gave (E)-tert-butyl 4-(3-(2-nitrophenyl)acryloyl)-
piperazine-1-carboxylate (6.94 g, 86%): 1H NMR (DMSO-d6)
δ 1.42 (s, 9H), 3.37 (broad s, 4H), 3.56 (broad s, 2H), 3.73 (broad
s, 2H), 7.50 (d, J = 15 Hz, 1H), 7.62 (d, J = 15 Hz, 1H), 7.70 (t,
J = 8 Hz, 1H), 8.19 (m, 2H), 8.63 (s, 1H); HPLC (Symmetry)
36
39
water solubilitya (mg/mL)
plasma T1/2b (min)
Fa (%)
2.5
1.7
167 ( 16
42
0.36
130 ( 30
43
2.4
CLa ((L/h)/kg)
a For the hydrochloride salt. b Dosed iv (1 mg/kg) and po (5 mg/kg) in
male rats (mean value over three rats).
Compounds 36 and 39 displayed antagonistic activity with
pKb values ((SEM, n = 3) of 5.60 ( 0.03 and 5.55 ( 0.04, res-
pectively. Thus, in this model, these compounds displayed a
selectivity of about 15-fold vs PAR2 and 5-fold vs PAR4. It is
noteworthy that none of these molecules displayed any ago-
nistic activity in this model.
To address the impact on bleeding time, which is often a
limitation of antithrombotic agents, these compounds were
evaluated in a well-established tail cut rat model17 in which
they were devoid of significant effect (data not shown).
Compounds 36 (F 16618) and 39 (F 16357), being the most
potent compounds after oral administration in the rat, were
further evaluated in an ADME protocol (Table 5). Both
compounds have reasonable water solubility and a good
bioavailability and clearance, while the plasma half-lives in
the rat are moderate.
t
R = 4.86 min, 97.8%; MS (ESI) m/z = 362 [MHþ].
To a solution of this intermediate (6.17 g, 17.07 mmol) in
toluene (50 mL) was added TFA (47 mL, 614 mmol). After being
stirred 2 h at room temperature, the reaction mixture was
concentrated under reduced pressure. The reaction mixture
was diluted with 1 N aqueous NaOH, extracted with CH2Cl2
(ꢀ2), dried with MgSO4, and concentrated under reduced
pressure to give the desired compound 2a (3.9 g, 93%). This
intermediate was used without further purification for the next
step: 1H NMR (DMSO-d6) δ 3.69 (broad s, 4H), 3.50 (broad s,
2H), 3.65 (broad s, 2H), 7.48 (d, J = 15 Hz, 1H), 7.59 (d, J = 15
Hz, 1H), 7.69 (t, J = 8 Hz, 1H), 8.19 (m, 2H), 8.61 (s, 1H); MS
(ESIþ) m/z = 262 [MHþ].
(E)-1-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-(2-nitrophenyl)pro-
penone (4). To a solution of intermediate 2a (441 mg, 1.79 mmol)
and triethylamine (375 μL, 2.68 mmol) in CH2Cl2 (10 mL) was
added 4-F-benzyl bromide (268 μL, 2.15 mmol). After being
stirred 5 h at room temperature, the reaction mixture was diluted
with 1 N aqueous NaOH, extracted with CH2Cl2 (ꢀ2), dried
with MgSO4, and concentrated under reduced pressure. Purifi-
cation of the crude material by flash column chromatography
(SiO2, CH2Cl2/MeOH, 100/0 to 97/3) gave the desired product
(428 mg, 65%). Treatment of this product by aqueous HCl
Conclusions
We have identified new PAR1 antagonists that inhibit
SFLLR-induced human platelet aggregation and are active
by both iv and oral routes in a rat thrombosis model without
any significant impact on bleeding time. Among them, com-
pounds 36 and 39 are the most promising in terms of
antithrombotic activity and ADME profile. Furthermore,
these compounds are particularly easy to synthesize, which
could constitute an interesting advantage compared with
other known PAR1 antagonists such as SCH-530348 or
ER-121958-06. Further pharmacological characterization of
these compounds is currently ongoing.
1
afforded the hydrochloride salt as a yellow powder: H NMR
(DMSO-d6) δ 3.01 (broad m, 2H), 3.20 (broad t, J = 12 Hz, 1H),
3.36 (m, 2H), 3.62 (broad t, J = 12 Hz, 1H), 4.35 (broad s, 2H),
4.52 (broad t, J = 16 Hz, 2H), 7.27-7.34 (m, 3H), 7.66 (t, J = 7
Hz, 3H), 7.76-7.82 (m, 2H), 8.03 (dd, J=7, 15 Hz, 1H), 11.32
(broad s, 1H); HPLC (Xterra MS) tR=3.52 min, 98.5%; MS
(ESIþ) m/z=370 [MHþ]. Anal. Calcd for C20H20F1N3O3 HCl:
3
C, 59.19; H, 5.22; N, 10.35. Found: C, 58.80; H, 5.22; N, 10.15.
Representative Procedure B for the Synthesis of Cinnamoyl
Derivatives. 1-(4-Fluorobenzyl)piperazine (3f). To a solution of
tert-butyl piperazine-1-carboxylate (2.0 g, 10.74 mmol) and
triethylamine (2.25 mL, 11.81 mmol) in CH2Cl2 (40 mL) was
added 4-F-benzyl bromide (1.5 mL, 16.11 mmol). After being
stirred overnight at room temperature, the reaction mixture was
diluted with water, extracted with CH2Cl2 (ꢀ2), dried with
MgSO4, and concentrated under reduced pressure. Purification
of the crude material by flash column chromatography (SiO2,
PE/AcOEt, 80/20) gave the desired product (2.1 g, 60%). To
a solution of this intermediate (2.1 g, 7.13 mmol) in toluene
(40 mL) was added TFA (16 mL, 214 mmol). After being stirred
for 1.2 h at room temperature, the reaction mixture was
concentrated under reduced pressure. The reaction mixture
was diluted with 1 N aqueous NaOH, extracted with CH2Cl2
(ꢀ2), dried with MgSO4, and concentrated under reduced
pressure to give the desired compound 3f as a yellow syrup
(1.39 g, 99%): 1H NMR (DMSO-d6) δ 2.26 (broad s, 4H),
2.67 (broad s, 4H), 3.39 (s, 2H), 7.12 (t, J = 8 Hz, 2H), 7.31
(t, J = 8 Hz, 2H)
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and were not corrected. 1H NMR
spectra were recorded on a Bruker Avance 400 spectrometer
operating at 400 MHz for 1H and 100 MHz for 13C. Chemical
shifts are reported in δ value (ppm) relative to an internal
standard of tetramethylsilane. Microanalyses were obtained
on a Fison EA 1108/CHN analyzer. Mass spectra (TSQ 7000
Finnigan, Thermoelectron Corporation) were determined by
electron spray ionization (ESI). Only 100% relative intensity
peaks are given. HPLC analysis were performed on a Waters
instrument with a photodiode array (PDA) detector (UV detec-
tion with chromatogram extracted at 220 nm). The columns
used were either a C18 Symmetry, 50 mm ꢀ 4.6 mm, 5 μm, or a
C18 XTerra MS, 50 mm ꢀ 4.6 mm, 5 μm (both from Waters),
eluting at 3 mL/min with a 6 min gradient from 0 to 100%
CH3CN (þ0.05% TFA) followed by 1 min at 100% CH3CN.
The purity of final compounds was determined by either ele-
mental analysis or analytical HPLC. All compounds described
hereafter have a minimum purity of 95%.
(E)-1-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-(2-bromophenyl)-
propenone (6). To a solution of 2-bromocinnamic acid (140 mg,
0.62 mmol) and intermediate 3f (101 mg, 0.52 mmol) in CH2Cl2
(4 mL) was added PS-carbodiimide (780 mg, 1.6 mmol/g,
1.24 mmol) and HOBT (104 mg, 0.78 mmol).19 After being
stirred overnight at room temperature, the reaction mixture was
diluted with 1 N aqueous NaOH, extracted with CH2Cl2 (ꢀ2),
dried with MgSO4, and concentrated under reduced pressure.
Representative Procedure A for the Synthesis of Cinnamoyl
Derivatives. (E)-3-(2-Nitrophenyl)-1-(piperazin-1-yl)prop-2-en-
1-one (2a). (E)-2-Nitrocinnamic acid (5.2 g, 26.92 mmol) was
treated with thionyl chloride (50 mL) under reflux for 6 h. The
mixture was then concentrated under reduced pressure to give
the crude acid chloride. To a solution of this intermediate and
triethylamine (4.7 mL, 33.6 mmol) in CH2Cl2 (60 mL) was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5831
Purification of the crude material by flash column chromatog-
raphy (SiO2, CH2Cl2/MeOH, 100/0 to 95/5) gave the desired
product (129 mg, 57%). Treatment of this product by aqueous
HCl afforded the hydrochloride salt as a white powder:
1H NMR (DMSO-d6) δ 2.90-3.10 (m, 2H), 3.20 (broad t, J=
11 Hz, 1H), 3.34 (broad d, J=11 Hz, 2H), 3.62 (broad t, J=11 Hz,
1H), 4.34 (broad s, 2H), 4.52 (broad t, J=11 Hz, 2H), 7.25-
7.40 (m, 4H), 7.45 (t, J = 8 Hz, 1H), 7.64-7.72 (m, 3H), 7.80
(d, J=15 Hz, 1H), 7.99 (dd, J = 1 Hz, 8 Hz, 1H), 11.40 (s, 1H);
HPLC (Xterra MS) tR = 3.86 min, 99%; MS (ESIþ) m/z =
403 [MHþ].
pressure. Purification of the crude material by flash column
chromatography (SiO2, CH2Cl2/MeOH/Et3N, 95/5/0.1) gave
the desired product (370 mg, 48%) isolated as an HCl salt as a
yellow powder: 1H NMR (DMSO-d6) of the base: δ 2.36 (broad
s, 4H), 3.49 (s, 2H), 3.56 (broad s, 2H), 3.65 (broad s, 2H), 5.75
(s, 2H), 6.52 (t, J = 7 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 6.94 (d,
J = 15 Hz, 1H), 7.02 (t, J = 7 Hz, 1H), 7.15 (t, J = 8 Hz, 2H),
7.35 (m, 2H), 7.47 (d, J = 7 Hz, 1H), 7.67 (d, J = 15 Hz, 1H);
HPLC (Xterra MS) tR = 3.22 min, 96%; MS (ESIþ) m/z = 340
[MHþ].
1-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-(2-nitrophenyl)propan-
1-one (13). To a solution of 3-(2-nitrophenyl)propionic acid (11
mg, 0.056 mmol) and intermediate 3f (12 mg, 0.061 mmol) in
CH2Cl2 (2 mL) was added EDCI (12 mg, 0.061 mmol), HOOBT
(10 mg, 0.061 mmol), and DIEA (19 μL, 0.11 mmol). After being
stirred overnight at room temperature, the reaction mixture was
diluted with 1 N aqueous NaOH, extracted with CH2Cl2 (ꢀ2),
dried with MgSO4, and concentrated under reduced pressure.
Purification of the crude material by flash column chromatog-
raphy (SiO2, CH2Cl2/MeOH, 100/0 to 85/15) gave the desired
product (20 mg, 95%). Treatment of this product by aqueous
HCl afforded the hydrochloride salt as a white powder: 1H
NMR (DMSO-d6) δ 2.69-277 (m, 2H), 2.87-2.96 (m, 2H), 3.03
(t, J = 8 Hz, 3H), 3.27 (broad t, J = 11 Hz, 2H), 3.45 (t, J = 13
Hz, 1H), 4.03 (broad d, J = 14 Hz, 1H), 4.15 (broad s, 2H), 4.43
(broad d, J = 14 Hz, 1H), 7.31 (t, J = 8 Hz, 2H), 7.47 (dt, J = 1,
8 Hz, 1H), 7.55 (d, J = 8 Hz, 1H), 7.62-7.67 (m, 3H), 7.92 (dd,
J = 1, 8 Hz, 1H); HPLC (Xterra MS) tR = 3.56 min, 99%; MS
(ESIþ) m/z = 372 [MHþ].
(E)-3-(2,6-Difluorophenyl)-1-[4-(3-methylbenzyl)piperazin-
1-yl]propenone (5). According to representative procedure A
described above, compound 5 was isolated as an orange powder:
1H NMR (DMSO-d6) δ 2.30 (s, 3H), 2.36 (broad s, 4H), 3.46 (s,
2H), 3.59 (broad s, 4H), 7.00-7.30 (m, 7H), 7.40-7.55 (m, 2H);
HPLC (Symmetry) tR = 3.75 min, 98.7%; MS (ESIþ) m/z =
357 [MHþ].
2-{(E)-3-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-oxopropenyl}-
benzonitrile (7). According to representative procedure A de-
scribed above, compound 7 was isolated as an HCl salt as an
orange powder: 1H NMR (DMSO-d6) δ 2.90-3.15 (m, 2H), 3.22
(broad t, J=11 Hz, 1H), 3.35 (broad d, J=11 Hz, 2H), 3.64
(broad t, J = 11 Hz, 1H), 4.34 (broad s, 2H), 4.53 (broad t, J =
11 Hz, 2H), 7.32 (t, J = 8 Hz, 2H), 7.53 (d, J = 15 Hz, 1H), 7.60
(t, J = 8 Hz, 1H), 7.66 (broad s, 2H), 7.74 (d, J = 15 Hz, 1H),
7.79 (t, J = 8 Hz, 1H), 7.92 (d, J = 7 Hz, 1H), 8.17 (d, J = 7 Hz,
1H), 11.40 (s, 1H); HPLC (Xterra MS) tR = 3.45 min, 98%; MS
(ESIþ) m/z = 350 [MHþ].
(E)-3-(2,6-Difluorophenyl)-1-[4-(3,4-dimethylbenzyl)pipera-
zin-1-yl]propenone (8). According to representative procedure A
described above, compound 8 was isolated as an HCl salt as an
orange powder: 1H NMR (DMSO-d6) of the base δ 2.19 (s, 3H),
2.20 (s, 3H), 2.36 (broad s, 4H), 3.41 (s, 2H), 3.57 (broad s, 4H),
7.00 (d, J = 8 Hz, 1H), 7.05-7.1 (m, 2H), 7.15-7.25 (m, 3H),
7.40-7.55 (m, 2H); HPLC (Symmetry) tR = 3.85 min, 98%; MS
(ESIþ) m/z = 371 [MHþ].
1-(4-(4-Fluorobenzyl)piperazin-1-yl)-3-(2-nitrophenyl)prop-
2-yn-1-one (14). 1-Iodo-2-nitrobenzene (10 g, 40 mmol) was
dissolved in 120 mL of THF under argon atmosphere. Methyl
propiolate (14.3 mL, 160 mmol) was added along with copper
iodide (300 mg, 1.6 mmol), K2CO3 (11 g, 80 mmol), and
Pd(PPh3)4 (920 mg, 0.8 mmol). The mixture was heated at 65 °C
for 1.5 h and was concentrated under reduced pressure. The
residue was taken up into EtOAc, washed with water, dried over
MgSO4, filtered, and concentrated under reduced pressure.
Purification of the crude material by flash column chromato-
graphy (SiO2, CH2Cl2/PE, 40/60 to 50/50) gave methyl 3-(2-
(E)-1-[4-(3,4-Difluorobenzyl)piperazin-1-yl]-3-(2,6-difluoro-
phenyl)propenone (9). According to representative procedure A
described above, compound 9 was isolated as an HCl salt as a
yellow powder: 1H NMR (DMSO-d6) of the base δ 2.39 (broad
s, 4H), 3.50 (s, 2H), 3.59 (broad s, 4H), 7.15-7.25 (m, 4H),
7.35-7.55 (m, 4H); HPLC (Symmetry) tR = 3.64 min, 99%; MS
(ESIþ) m/z = 379 [MHþ].
1
nitrophenyl)propiolate (1.53 g, 19%): H NMR (DMSO-d6) δ
3.82 (s, 3H), 7.80-7.90 (m, 2H), 7.97 (dd, J = 2, 8 Hz, 1H), 8.26
(dd, J = 2 Hz, 8 Hz, 1H). This compound (1.53 g, 7.4 mmol) was
dissolved in 22 mL of THF and was treated at room temperature
with 11.2 mL of an aqueous solution of LiOH (1 M) until
completion of the reaction (about 3 h). The solution was then
concentrated, acidified with HCl (1 N), and extracted twice with
EtOAc. The organic phases were combined, dried over MgSO4,
filtered, and concentrated under reduced pressure to give 3-(2-
nitrophenyl)propiolic acid (1.12 g, 78%): 1H NMR (DMSO-d6)
δ 7.80-7.90 (m, 2H), 7.93 (dd, J = 2, 8 Hz, 1H), 8.23 (dd, J = 2,
8 Hz, 1H). This compound (1.12 g, 5.8 mmol) was dissolved in
45 mL of CH2Cl2 under nitrogen atmosphere. EDCI (1.12 g, 5.8
mmol), HOOBT (950 mg, 5.8 mmol), DIEA (1.8 mL, 10.6
mmol), and 1-Boc-piperazine (990 mg, 5.3 mmol) were added,
and the mixture was stirred at room temperature for 20 h. The
solution was concentrated under reduced pressure, then diluted
with EtOAc and washed with NaOH (1 M). The organic phase
was dried over Na2SO4, filtered, and concentrated under re-
duced pressure. Purification of the crude material by flash
column chromatography (SiO2, EtOAc/CH2Cl2, 5/95 to 10/90)
gave 1.37 g (71%) of tert-butyl 4-(3-(2-nitrophenyl)propio-
(E)-1-[4-(3-Methylbenzyl)piperazin-1-yl]-3-(2-nitrophenyl)-
propenone (10). According to representative procedure A de-
scribed above, compound 10 was isolated as an HCl salt as a
brown powder: 1H NMR (DMSO-d6) of the base δ 2.30 (s, 3H),
2.39 (broad s, 4H), 3.47 (s, 2H), 3.57 (broad s, 2H), 3.70 (broad s,
2H), 7.05-7.15 (m, 3H), 7.21 (t, J=7 Hz, 1H), 7.26 (d, J=15 Hz,
1H), 7.63 (t, J = 8 Hz, 1H), 7.71 (d, J = 15 Hz, 1H), 7.77 (t, J=
7 Hz, 1H), 8.00-8.05 (m, 2H); HPLC (Symmetry) tR=3.65 min,
99%; MS (ESIþ) m/z = 366 [MHþ].
(E)-1-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-(3-chlorophenyl)-
propenone (11). According to representative procedure B de-
scribed above, compound 11 was isolated as an HCl salt as
1
a white powder: H NMR (DMSO-d6) δ 2.90-3.15 (m, 2H),
3.20 (broad t, J = 11 Hz, 1H), 3.33 (broad d, J = 11 Hz, 2H),
3.62 (broad t, J = 11 Hz, 1H), 4.34 (broad s, 2H), 4.53 (broad d,
J = 11 Hz, 2H), 7.32 (t, J = 9 Hz, 2H), 7.36 (d, J = 15 Hz, 1H),
7.45 (broad s, 2H), 7.51 (d, J = 15 Hz, 1H), 7.66 (broad s, 3H),
7.92 (s, 1H), 11.53 (s, 1H); HPLC (Xterra MS) tR = 3.88 min,
99%; MS (ESIþ) m/z = 359 [MHþ].
1
loyl)piperazine-1-carboxylate: H NMR (DMSO-d6) δ 1.42 (s,
(E)-1-[4-(4-Fluorobenzyl)piperazin-1-yl]-3-(2-aminophenyl)-
propenone (12). To a solution of compound 4 (834 mg,
2.26 mmol) in ethanol (25 mL) was added tin chloride dihydrate
(2.55 g, 11.3 mmol). After being stirred for 7 h under reflux, the
reaction mixture was concentrated under reduced pressure,
diluted with aqueous NaHCO3, extracted with ethyl acetate
(ꢀ2), dried with Na2SO4, and concentrated under reduced
9H), 3.37 (broad s, 2H), 3.44 (broad s, 2H), 3.54 (broad s, 2H),
3.79 (broad s, 2H), 7.79 (t, J = 8 Hz, 1H), 7.86 (t, J = 8 Hz, 1H),
7.96 (d, J = 8 Hz, 1H), 8.24 (d, J = 8 Hz, 1H). This compound
(1.25 g, 3.4 mmol) was dissolved in 13 mL of a saturated solution
of HCl in EtOAc. The mixture was stirred at room temperature
for 14 h. The white precipitate formed was recovered, and the
residue was concentrated and treated again with 7 mL of a

5832 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Perez et al.
saturated solution of HCl in EtOAc. The white precipitate
formed was recovered, and the residue was concentrated and
treated again with 14 mL of a saturated solution of HCl in
EtOAc. The three white solids were combined to give 682 mg
(66%) of 3-(2-nitrophenyl)-1-(piperazin-1-yl)prop-2-yn-1-one
hydrochloride: 1H NMR (DMSO-d6) δ 3.14 (t, J = 5 Hz, 2H),
3.23 (t, J = 5 Hz, 2H), 3.79 (t, J = 5 Hz, 2H), 4.03 (t, J = 5 Hz,
2H), 7.80 (t, J = 8 Hz, 1H), 7.87 (t, J = 8 Hz, 1H), 7.97 (d, J=
8 Hz, 1H), 8.26 (d, J = 8 Hz, 1H), 9.40 (broad s, 2H). A fraction
of this compound (60 mg, 0.20 mmol) was dissolved in 3 mL of
CH2Cl2 under nitrogen atmosphere. Triethylamine (51 μL, 0.36
mmol) and 4-fluorobenzyl bromide (30 μL, 0.24 mmol) were
added, and the mixture was stirred at room temperature for 24 h.
More CH2Cl2 was added, and the mixture was washed once with
NaOH (2 N), dried over Na2SO4, filtered, and concentrated
under reduced pressure. Purification of the crude material by
flash column chromatography (SiO2, NH4OH/MeOH/CH2Cl2,
0.5/4.5/95), followed by treatment with an excess of HCl in
EtOAc gave 32 mg (36%) of the hydrogen chloride salt of the
title compound as an off-white powder: 1H NMR (DMSO-d6) δ
2.9-3.6 (m, 5H), 3.74 (broad t, J = 11 Hz, 1H), 4.30-4.55 (m,
4H), 7.32 (t, J = 9 Hz, 2H), 7.66 (broad s, 2H), 7.81 (td, J=
1 Hz, 8 Hz, 1H), 7.87 (td, J=1 Hz, 8 Hz, 1H), 7.97 (dd, J=
1 Hz, 8 Hz, 1H), 8.26 (dd, J=1 Hz, 8 Hz, 1H), 11.40 (broad s,
1H); HPLC (XTerra MS) tR = 3.60 min, 95%; MS (ESIþ) m/z
= 368 [MHþ].
(2E,4E)-5-(2-Nitrophenyl)penta-2,4-dienoic Acid (16).20 NaH
(270 mg, 6.77 mmol) was put in suspension in 20 mL of anhy-
drous THF under nitrogen atmosphere. Ethyl 2-(diethoxy-
phosphoryl)acetate (1.23 mL, 6.21 mmol) dissolved in 5 mL of
THF was added dropwise at room temperature over 10 min. (E)-
3-(2-Nitrophenyl)acrylaldehyde (5.2 g, 26.92 mmol) dissolved in
20 mL of THF was then added, and the mixture was stirred until
completion (about 3 h). Then the reaction mixture was sequen-
tially quenched with a few drops of water, diluted with more
water, extracted twice with EtOAc, dried over MgSO4, filtered,
and concentrated under reduced pressure. Purification of the
crude material by flash column chromatography (SiO2, CH2Cl2/
PE, 70/30) gave (2E,4E)-ethyl 5-(2-nitrophenyl)penta-2,4-
dienoate (1.29 g, 92%) as a yellow powder: 1H NMR (DMSO-
d6) δ 1.24 (t, J = 7 Hz, 3H), 4.16 (q, J = 7 Hz, 2H), 6.20 (d, J =
15 Hz, 1H), 7.17 (dd, J = 11, 15 Hz, 1H), 7.35 (d, J = 15 Hz,
1H), 7.46 (dd, J = 11, 15 Hz, 1H), 7.59 (t, J = 8 Hz, 1H), 7.75 (t,
J = 8 Hz, 1H), 7.89 (d, J = 8 Hz, 1H), 7.99 (d, J = 8 Hz, 1H).
This compound (1.29 g, 5.2 mmol) was dissolved in 20 mL of
ethanol, and a solution of aqueous KOH (1 N, 6 mL) was added.
The mixture was heated at 70 °C until completion (about 1 h).
The mixture was cooled to room temperature, then quenched
with HCl (1 N) and extracted 3 times with CH2Cl2/MeOH
(90/10). The organic phases were combined, dried over Na2SO4,
filtered, and concentrated under reduced pressure to give 1.12 g
of the title compound (98%) as a yellow powder: 1H NMR
(DMSO-d6) δ 6.12 (d, J = 15 Hz, 1H), 7.16 (dd, J = 11, 15 Hz,
1H), 7.30 (d, J = 15 Hz, 1H), 7.38 (dd, J = 11, 15 Hz, 1H), 7.58
(t, J = 8 Hz, 1H), 7.75 (t, J = 8 Hz, 1H), 7.90 (d, J = 8 Hz, 1H),
7.99 (d, J = 8 Hz, 1H), 12.42 (s, 1H); HPLC (XTerra MS) tR=
4.05 min, 99.8%.
(DMSO-d6) δ 3.08 (broad s, 2H), 3.16 (broad s, 1H), 3.38 (m,
2H), 3.59 (broad s, 1H), 4.32-4.51 (m, 4H), 6.86 (d, J = 15 Hz,
1H), 7.11 (dd, J = 11, 15 Hz, 1H), 7.25-7.34 (m, 3H), 7.58 (t,
J = 7 Hz, 1H), 7.64-7.67 (m, 2H), 7.76 (t, J = 7 Hz, 1H), 7.85
(d, J = 8 Hz, 1H), 7.99 (d, J = 8 Hz, 1H), 11.29 (broads, 1H);
HPLC (XTerra MS) tR = 3.75 min, 98%; MS (ESIþ) m/z = 396
[MHþ].
(E)-3-(2,6-Difluorophenyl)-1-(4-(4-fluorobenzyl)-2,5-dimethyl-
piperazin-1-yl)prop-2-en-1-one (18). 2,5-Dimethylpiperazine
(1.5 g, 2.62 mmol) was dissolved in 17 mL of CH2Cl2 under
nitrogen atmosphere. Triethylamine (551 μL, 2.62 mmol) was
added, and the mixture was cooled to 0 °C. 4-Fluorobenzyl
bromide (360 μL, 2.62 mmol), diluted into 3 mL of CH2Cl2, was
added dropwise over a 30 min period. Then the cold bath was
removed, and the mixture was stirred at room temperature for
1 h. More CH2Cl2 was then added, and the mixture was washed
once successively with NaOH (1 M), water, and brine. The
aqueous phases were combined and extracted twice with
CH2Cl2. The organic phases were combined, dried over Na2SO4,
filtered, and concentrated under reduced pressure. Purification
of the crude material by flash column chromatography (SiO2,
NH4OH/MeOH/CH2Cl2, 0.5/6.5/93) gave 437 mg (75%) of
1-(4-fluorobenzyl)-2,5-dimethylpiperazine as a yellow powder:
1H NMR (DMSO-d6) δ 0.81 (d, J = 6 Hz, 3H), 1.02 (d, J = 6
Hz, 3H), 1.52 (t, J = 11 Hz, 1H), 1.91 (broad s, 1H), 2.05-2.15
(m, 1H), 2.36 (t, J = 11 Hz, 1H), 2.45-2.60 (m, 2H), 2.75 (dd,
J = 3, 12 Hz, 1H), 3.00 (d, J = 14 Hz, 1H), 3.98 (d, J = 14 Hz,
1H), 7.12 (t, J=9 Hz, 2H), (dd, J=6, 8 Hz), 7.30; MS (ESIþ) m/z
= 223 [MHþ]. This compound (50 mg, 0.22 mmol) was dis-
solved in 2 mL of CH2Cl2 under nitrogen atmosphere. EDCI (47
mg, 0.24 mmol), HOOBT (40 mg, 0.24 mmol), DIEA (79 μL,
0.45 mmol), and (E)-3-(2,6-difluorophenyl)acrylic acid (50 mg,
0.27 mmol) were added, and the mixture was stirred for 20 h. The
solution was diluted with CH2Cl2 and washed with NaOH (1
M). The organic phase was dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification of the crude
material by flash column chromatography (SiO2, MeOH/
CH2Cl2, 0/100 to 5/95) followed by treatment with an excess
of HCl in EtOAc gave 82 mg (85%) of the hydrogen chloride salt
of the title compound as a white powder: HPLC (XTerra MS) tR
= 3.85 min, 95%; MS (ESIþ) m/z = 389 [MHþ].
(E)-1-(4-Fluorobenzyl)-4-(3-(2-nitrophenyl)acryloyl)piperazin-
2-one (19). (E)-3-(2-Nitrophenyl)acrylic acid (1.27 g, 6.59 mmol)
was dissolved in 45 mL of CH2Cl2 under nitrogen atmosphere.
EDCI (1.26 g, 6.59 mmol), HOOBT (1.07 g, 6.59 mmol), DIEA
(2 mL, 11.9 mmol), and piperazin-2-one (600 mg, 5.99 mmol)
were added, and the mixture was stirred for 20 h. The solution
was diluted with CH2Cl2 and washed with NaOH (1 M). The
organic phase was dried over Na2SO4, filtered, and concentrated
under reduced pressure. Purification of the crude material by
flash column chromatography (SiO2, NH4OH/MeOH/CH2Cl2,
0.5/2.5/97 to 0.5/4.5/95) gave 1.35 g (75%) of (E)-4-(3-(2-
1
nitrophenyl)acryloyl)piperazin-2-one: H NMR (DMSO-d6) δ
3.22 (broad s, 1H), 3.30 (broad s, 1H), 3.72 (broad s, 1H), 3.90
(broad s, 1H), 4.07 (s, 1H), 4.33 (s, 1H), 7.24-7.32 (m, 1H), 7.65
(t, J=8 Hz, 1H), 7.75-7.80 (m, 2H), 8.0-8.2 (m, 3H). This com-
pound (140 mg, 0.50 mmol) was dissolved in 15 mL of THF, and
grounded KOH (29 mg, 0.51 mmol) was added along with 3
drops of water. 4-Fluorobenzyl bromide (53 μL, 0.42 mmol) was
then added. The mixture was stirred at room temperature for
24 h, heated under reflux conditions for 4 h, and then concentrated
under reduced pressure. Purification of the crude material by
flash column chromatography (SiO2, NH4OH/MeOH/CH2Cl2,
0.5/2.5/97 to 0.5/4.5/95) gave 31 mg (15%) of the title compound
as an off-white powder: 1H NMR (DMSO-d6) δ 3.35 (broad s,
2H), 3.79 (broad s, 1H), 3.96 (broad s, 1H), 4.23 (s, 1H), 4.50 (s,
1H), 4.56 (s, 2H), 7.17 (t, J = 8 Hz, 2H), 7.26 (d, J = 15 Hz, 1H),
7.32 (dd, J = 6, 8 Hz, 2H), 7.64 (t, J = 8 Hz, 1H), 7.75-7.84 (m,
2H), 8.00-8.15 (m, 2H); HPLC (XTerra MS) tR = 4.36 min,
95%; MS (ESIþ) m/z = 384 [MHþ].
(2E,4E)-1-(4-(4-Fluorobenzyl)piperazin-1-yl)-5-(2-nitrophenyl)-
penta-2,4-dien-1-one (17). Compound 16 (70 mg, 0.31 mmol)
was dissolved in 3 mL of CH2Cl2 under nitrogen atmosphere.
EDCI (67 mg, 0.35 mmol), HOOBT (57 mg, 0.35 mmol), DIEA
(84 μL, 0.48 mmol), and 1-(4-fluorobenzyl)piperazine (68 mg,
0.35 mmol) were added, and the mixture was stirred for 20 h. The
solution was diluted with CH2Cl2 and washed with NaOH
(1 M). The organic phase was dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification of the crude
material by flash column chromatography (SiO2, NH4OH/
MeOH/CH2Cl2, 0/0/100 to 1/10/90), followed by treatment with
an excess of HCl in EtOAc gave 106 mg (77%) of the hydrogen
chloride salt of the title compound as a yellow powder: 1H NMR

Article
(E)-1-(4-(4-Fluorobenzyl)-1,4-diazepan-1-yl)-3-(2-nitrophe-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5833
over MgSO4, filtered, and concentrated under reduced pressure.
Purification of the crude material by flash column chromatog-
raphy (SiO2, NH4OH/MeOH/CH2Cl2, 1/9/90) gave 676 mg
(60%) of the title compound as an off-white powder:
1H NMR (DMSO-d6) δ 1.57 (broad q, J = 10 Hz, 2H), 2.19
(broad d, J = 10 Hz, 2H), 2.72 (broad t, J = 12 Hz, 1H), 3.13
(broad t, J = 12 Hz, 1H), 3.33 (broad s, 1H), 4.19 (broad s, 2H),
4.40 (broad d, J = 14 Hz, 1H), 4.55 (broad d, J = 14 Hz, 1H),
7.29 (t, J = 9 Hz, 2H), 7.34 (d, J = 15 Hz, 1H), 7.60-7.69 (m,
3H), 7.73 (d, J = 15 Hz, 1H), 7.78 (t, J = 8 Hz, 1H), 8.05 (td, J=
1 Hz, 7 Hz, 2H), 9.40 (broad s, 2H); HPLC (XTerra MS) tR=
3.60 min, 98%; MS (ESIþ) m/z = 384 [MHþ].
nyl)prop-2-en-1-one (20). tert-Butyl 1,4-diazepane-1-carboxy-
late (275 mg, 1.37 mmol) was dissolved in 7 mL of CH2Cl2
under nitrogen atmosphere. Triethylamine (288 μL, 1.94 mmol)
was added followed by 4-fluorobenzyl bromide (286 mL, 1.51
mmol). The mixture was stirred at room temperature for 18 h.
More CH2Cl2 was added, and the mixture was washed once
successively with NaOH (1 M), water, and brine. The organic
phase was dried over Na2SO4, filtered, and concentrated under
reduced pressure. Purification of the crude material by flash
column chromatography (SiO2, CH2Cl2) gave 324 mg (77%) of
tert-butyl 4-(4-fluorobenzyl)-1,4-diazepane-1-carboxylate as a
colorless oil: 1H NMR (DMSO-d6) δ 1.40 (s, 9H), 1.69 (broad s,
2H), 2.45-2.60 (m, 4H), 3.35 (broad s, 4H), 3.58 (s, 2H), 7.13 (t,
J = 8 Hz, 2H), 7.33 (dd, J = 6, 8 Hz, 2H); MS (ESIþ) m/z = 309
[MHþ]. This compound (324 mg, 1.05 mmol) was dissolved in
8.5 mL of toluene under nitrogen atmosphere. TFA (2.4 mL,
32 mml) was added, and the reaction was stirred until comple-
tion (about 1 h). It was then concentrated under reduced
pressure, diluted into CH2Cl2, washed once successively with
NaOH (1 M), water, and brine, then dried over Na2SO4, filtered,
and concentrated under reduced pressure. Purification of the
crude material by flash column chromatography (SiO2,
NH4OH/MeOH/CH2Cl2, 1/9/90) gave 192 mg (88%) of 1-(4-
fluorobenzyl)-1,4-diazepane as a colorless oil: 1H NMR
(DMSO-d6) δ 1.64 (pentet, J = 6 Hz, 2H), 2.53 (broad s, 2H),
2.58 (t, J = 6 Hz, 2H), 2.71 (t, J = 5 Hz, 2H), 2.79 (t, J = 8 Hz,
2H), 3.1-3.5 (m, 1H, NH), 3.58 (s, 2H), 7.12 (t, J = 8 Hz, 2H),
7.34 (dd, J = 6, 8 Hz, 2H); MS (ESIþ) m/z = 209 [MHþ]. This
compound (50 mg, 0.24 mmol) was dissolved in 2 mL of CH2Cl2
under nitrogen atmosphere. EDCI (51 mg, 0.26 mmol), HOOBT
(43 mg, 0.26 mmol), DIEA (84 μL, 0.48 mmol), and (E)-3-(2-
nitrophenyl)acrylic acid (56 mg, 0.29 mmol) were added, and the
mixture was stirred for 20 h. The solution was diluted with
CH2Cl2 and washed with NaOH (1 M). The organic phase was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. Purification of the crude material by flash column
chromatography (SiO2, MeOH/CH2Cl2, 0/100 to 10/90) fol-
lowed by treatment with an excess of HCl in EtOAc gave 81 mg
(80%) of the hydrogen chloride salt of the title compound as a
white powder: 1H NMR (DMSO-d6) δ 2.12 (broad s, 1H), 2.37
(broad s, 1H), 3.0-3.2 (m, 2H), 3.3-3.6 (m, 3H), 3.7-3.9 (m,
2H), 4.15-4.4 (m, 3H), 7.22 (m, 1H), 7.31 (t, J = 9 Hz, 2H),
7.60-7.75 (m, 3H), 7.75-7.81 (m, 2H), 8.04 (td, J = 1 Hz, 8 Hz,
2H), 10.90 (s, 1H); HPLC (XTerra MS) tR = 3.64 min, 99%; MS
(ESIþ) m/z = 384 [MHþ].
tert-Butyl 4-(Methoxy(methyl)carbamoyl)piperidine-1-car-
boxylate (24). Synthesis of the title compound has already been
reported.21 Boc protection of piperidine-4-carboxylic acid was
performed using the described protocol, but a different metho-
dology was used for the amidation step. Therefore, 1-(tert-
butoxycarbonyl)piperidine-4-carboxylic acid (8.63 g, 37.6
mmol) was dissolved in 250 mL of DMF under nitrogen atmo-
sphere. EDCI (7.93 g, 41.3 mmol), HOOBT (6.75 g, 41.3 mmol),
DIEA (19.7 mL, 113 mmol), and MeONHMe HCl (4.1 g, 41.3
3
mmol) were added, and the mixture was stirred for 24 h at 50 °C.
The solution was cooled to room temperature, then hydrolyzed
with NaOH (1 M) and extracted twice with CH2Cl2. The organic
phase was washed once with HCl (1 N), dried over MgSO4,
filtered, and concentrated under reduced pressure. Purification
of the crude material by flash column chromatography (SiO2,
EtOAc/PE, 30/70 to 60/40) gave 9.05 g (88%) of the title
1
compound as a yellow oil: H NMR (DMSO-d6) δ 1.3-1.45
(m, 11H), 1.63 (broad d, J = 12 Hz, 2H), 2.6-2.95 (m, 3H), 3.09
(s, 3H), 3.68 (s, 3H), 3.94 (broad d, J = 12 Hz, 2H).
1-(4-Fluorobenzyl)-N-methoxy-N-methylpiperidine-4-carboxa-
mide (25). Compound 24 (2.95 g, 10.8 mmol) was dissolved in
36 mL of toluene under nitrogen atmosphere and cooled at 0 °C
prior to the addition of 18 mL of TFA. The cooling bath was
removed, and the mixture was stirred for 1 h at room tempera-
ture. The solvent was removed under reduced pressure, and the
residue was dissolved in CH2Cl2, then quenched by the addition
of NaOH (1 M). The aqueous phase was extracted 6 times with
CH2Cl2. The organic phase was dried over MgSO4, filtered, and
concentrated under reduced pressure to give 1.38 g (74%) of
N-methoxy-N-methylpiperidine-4-carboxamide as a pale-yellow-
ish oil: 1H NMR (DMSO-d6) δ 1.42 (ddd, J = 4, 12, 24 Hz, 2H),
1.55 (broad d, J = 11 Hz, 2H), 2.4-2.6 (m, 2H), 2.94 (broad d,
J = 12 Hz, 2H), 2.9-3.4 (m, 1H, NH), 3.08 (s, 3H), 3.66 (s, 3H);
MS (ESIþ) m/z = 173 [MHþ]. A fraction of this compound (750
mg, 4.3 mmol) was dissolved in 20 mL of 1,2-dichloroethane
under nitrogen atmosphere. 4-Fluorobenzaldehyde (595 mg, 4.8
mmol) was added, followed by 1.4 mL of acetic acid. The
mixture was stirred for half an hour at room temperature prior
to the addition of NaBH(OAc)3 (1.02 g, 4.8 mmol). After 24 h of
reaction, the starting material was not fully consumed; there-
fore, an aliquot of 4-fluorobenzaldehyde (216 mg) and NaBH-
(OAc)3 (370 mg) was added. Twenty-four hours later, the
reaction was quenched with 30 mL of water, basified with
NaOH (1 M), and extracted 3 times with CH2Cl2. The organic
phase was dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification of the crude material by flash
column chromatography (SiO2, MeOH/CH2Cl2, 0/100 to 5/95)
(E)-1-(4-(4-Fluorobenzylamino)piperidin-1-yl)-3-(2-nitrophe-
nyl)prop-2-en-1-one (21). (E)-3-(2-Nitrophenyl)acrylic acid (2 g,
10.3 mmol) was dissolved in 75 mL of CH2Cl2 under nitro-
gen atmosphere. EDCI (1.98 g, 10.3 mmol), HOOBT (1.68 g,
10.3 mmol), DIEA (4.9 mL, 28 mmol), and the TFA salt of
piperidin-4-one (2.0 g, 9.38 mmol) were added, and the mixture
was stirred for 20 h. The solution was diluted with CH2Cl2 and
washed with NaOH (1 M) and brine. The organic phase was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. Purification of the crude material by flash column
chromatography (SiO2, acetone/CH2Cl2, 5/95 to 10/90) gave
2.22 g (86%) of (E)-1-(3-(2-nitrophenyl)acryloyl)piperidin-4-
one: 1H NMR (DMSO-d6) δ 2.4-2.5 (m, 4H), 3.85 (t, J =
6 Hz, 2H), 4.00 (t, J = 6 Hz, 2H), 7.17 (d, J = 15 Hz, 1H), 7.64 (t,
J = 8 Hz, 1H), 7.75-7.80 (m, 2H), 8.06 (t, J = 8 Hz, 2H); HPLC
(XTerra MS) tR = 3.51 min, 98%; MS (ESIþ) m/z = 275
[MHþ]. A fraction of this compound (804 mg, 2.93 mmol) was
dissolved in 13 mL of 1,2-dichloroethane under nitrogen atmo-
sphere. 4-Fluorobenzylamine (337 μL, 2.93 mmol) was added,
followed by 918 μL of acetic acid. The mixture was stirred for
half an hour at room temperature prior to the addition of
NaBH(OAc)3 (684 mg, 3.2 mmol). After 18 h the reaction was
quenched with 30 mL of water, basified with saturated NaH-
CO3, and extracted with CH2Cl2. The organic phase was dried
1
gave 940 mg (77%) of the title compound as a yellow oil: H
NMR (DMSO-d6) δ 1.5-1.75 (m, 4H), 1.95 (td, J = 2, 11 Hz,
2H), 2.61 (broad t, 1H), 2.80 (d, J = 11 Hz, 2H), 3.08 (s, 3H),
3.43 (s, 2H), 3.66 (s, 3H), 7.13 (t, J=8 Hz, 2H), 7.31 (dd, J=
8, 6 Hz, 2H); HPLC (XTerra MS) tR =2.97 min, 100%; MS
(ESIþ) m/z = 281 [MHþ].
(E)-3-(2-Chlorophenyl)-1-(1-(4-fluorobenzyl)piperidin-4-yl)-
prop-2-en-1-one (27). Diethyl methylphosphonate (1.1 mL,
7.8 mmol) was dissolved in 20 mL of anhydrous THF under
nitrogen atmosphere and cooled at -50 °C prior to the dropwise
addition n-buthyllithium (1.6 M in hexane, 5.0 mL, 8 mmol).

5834 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Perez et al.
The resulting white suspension was stirred for 1 h between
-40 and -50 °C. In a separated flask, compound 25 (880 mg,
3.1 mmol) was dissolved in 11 mL of anhydrous THF under
nitrogen atmosphere. This clear solution was slowly added into
the above cold suspension while maintaining the temperature
between -45 and -43 °C. The resulting reaction mixture was
stirred for 1 h at -45 °C, then quenched with a few drops of
water prior to the removal of the cooling bath. It was then
diluted with 20 mL of HCl (1 N) and washed 3 times with
EtOAc. The aqueous phase was brought to pH 10 with con-
centrated NaOH and was extracted 3 times with CH2Cl2. The
organic phases were combined, dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue (26) was
taken up into 25 mL of CH3CN under nitrogen atmosphere.
2-Chlorobenzaldehyde (700 μL, 6.2 mmol) and potassium car-
bonate (850 mg, 6.2 mmol) were added, and the mixture was
stirred at room temperature until completion (about 24 h). The
reaction mixture was filtered and concentrated under reduced
pressure. Purification of the crude material by flash column
chromatography (SiO2, NH4OH/MeOH/CH2Cl2, 0/0/100 to
0.5/4.5/95) gave 611 mg (68%) of the title compound as a
colorless oil: 1H NMR (DMSO-d6) δ 1.52 (ddd, J = 3, 12,
24 Hz, 2H), 1.83 (broad d, J=12 Hz, 2H), 2.02 (td, J=2, 11 Hz,
2H), 2.68 (tt, J = 4, 11 Hz, 1H), 2.82 (broad d, J=12 Hz, 2H),
3.46 (s, 2H), 7.05-7.25 (m, 3H), 7.33 (dd, J = 6, 8 Hz, 2H),
7.1-7.46 (m, 2H), 7.55 (dd, J = 2, 8 Hz, 1H), 7.82 (d, J = 16 Hz,
1H), 7.98 (dd, J = 2, 8 Hz, 1H); HPLC (XTerra MS) tR=4.10
min, 100%; MS (ESIþ) m/z=358 [MHþ].
7.45 (t, J = 8 Hz, 1H), 7.51 (td, J = 2 Hz, 8 Hz, 1H), 7.64 (td,
J = 1 Hz, 8 Hz, 1H), 7.70 (d, J = 15 Hz, 1H), 7.74-7.82 (m,
3H), 7.99 (d, J = 7 Hz, 1H), 8.02 (dd, J = 1 Hz, 8 Hz, 1H);
HPLC (XTerra MS) tR = 4.78 min, 95%; MS (ESIþ) m/z =
420 [MHþ].
(E)-4-(3-(2-Nitrophenyl)acryloyl)piperazine-1-carboxylic Acid
Cyclohexylamide (33). According to representative procedure A
described above, compound 33 was isolated in 66% yield as a
white powder: 1H NMR (DMSO-d6) δ 1.00-1.30 (m, 5H),
1.50-1.80 (m, 5H), 3.33 (broad s, 4H), 3.41 (broad s, 1H), 3.54
(broad s, 2H), 3.67 (broad s, 2H), 6.28 (d, J = 8 Hz, 1H), 7.29 (d,
J = 15 Hz, 1H), 7.64 (td, J = 1 Hz, 9 Hz, 1H), 7.72-7.80 (m, 2H),
8.02 (d, J = 8 Hz, 2H); HPLC (XTerra MS) tR = 4.38 min, 99%;
MS (ESIþ) m/z = 381 [MHþ].
(2E,4E)-1-(4-Cyclopentylpiperazin-1-yl)-5-(2-nitrophenyl)penta-
2,4-dien-1-one (34). According to compound 17 synthesis described
above, compound 34 was isolated in 87% yield as an HCl salt and a
yellow powder: 1H NMR (DMSO-d6) δ 1.56 (broad s, 2H), 1.73
(broad s, 4H), 2.05 (broad s, 2H), 2.85-3.25 (m, 3H), 3.4-3.75 (m,
4H), 4.28 (broad s, 1H), 4.53 (broad s, 1H), 6.90 (d, J = 15 Hz,
1H), 7.13 (dd, J = 11 Hz, 15 Hz, 1H), 7.27 (d, J = 15 Hz, 1H), 7.34
(dd, J=11 Hz, 15 Hz, 1H), 7.58 (t, J=8 Hz, 1H), 7.75 (t, J =
8 Hz, 1H), 7.86 (d, J = 8 Hz, 1H), 7.99 (d, J = 8 Hz, 1H), 10.87
(broad s, 1H, NHþ); HPLC (XTerra MS) tR=3.62 min, 99%;
MS (ESIþ) m/z = 356 [MHþ]. Anal. Calcd for C20H25N3O3 HCl:
3
C, 61.30; H, 6.69; N, 10.72. Found: C, 60.91; H, 6.72; N, 10.94.
(2E,4E)-5-(2-Nitrophenyl)-1-(4-phenylpiperazin-1-yl)penta-
2,4-dien-1-one (35). According to compound 17 synthesis de-
scribed above, compound 35 was isolated in 70% yield as an
(E)-1-[4-(3-Chlorophenyl)piperazin-1-yl]-3-(2-nitrophenyl)pro-
penone (28). According to compound 13 synthesis described
above, compound 28 was isolated in 75% yield as an HCl salt as
1
HCl salt and a yellow powder: H NMR (DMSO-d6) δ 3.26
(broad s, 4H), 3.82 (broad s, 4H), 5.4-6.4 (broad m, 1H, NHþ),
6.9-7.0 (m, 2H), 7.05-7.4 (m, 7H), 7.58 (t, J = 8 Hz, 1H), 7.75
(t, J = 8 Hz, 1H), 7.87 (d, J = 8 Hz, 1H), 7.99 (d, J = 8 Hz, 1H);
HPLC (XTerra MS) tR = 4.60 min, 97.8%; MS (ESIþ) m/z =
1
a yellow powder: H NMR (DMSO-d6) δ 3.24 (broad s, 4H),
3.72 (broad s, 2H), 3.87 (broad s, 2H), 6.82 (d, J = 8 Hz, 1H),
6.95 (d, J = 8 Hz, 1H), 7.00 (s, 1H), 7.24 (t, J = 8 Hz, 1H), 7.35
(d, J = 15 Hz, 1H), 7.64 (t, J = 8 Hz, 1H), 7.74-7.87 (m, 2H),
8.05 (t, J = 8 Hz, 1H); HPLC (XTerra MS) tR = 5.18 min, 98%;
364 [MHþ]. Anal. Calcd for C21H21N3O3 HCl: C, 63.08; H,
3
5.55; N, 10.51. Found: C, 63.17; H, 5.61; N, 10.81.
MS (ESI) m/z = 372 [MHþ]. Anal. Calcd for C19H18N3O3Cl1
HCl: C, 55.89; H, 4.69; N, 10.29. Found: C, 55.77; H, 4.77;
N, 10.03.
2-((1E,3E)-5-Oxo-5-(4-(pyridin-2-yl)piperazin-1-yl)penta-1,3-
dienyl)benzonitrile (36). Bromobenzonitrile (910 mg, 5 mmol)
was dissolved in 10 mL of dimethylacetamide under nitrogen
atmosphere. DIEA (1 mL), 3,3-diethoxyprop-1-ene (2.3 mL,
15 mmol), and Herrmann’s catalyst (90 mg, 0.1 mmol) were
added, and the mixture was heated at 90 °C for 18 h. The mixture
was then cooled to room temperature, diluted with ether,
washed with HCl (1 N), dried with Na2SO4, filtered, and
concentrated under reduced pressure. Flash column chroma-
tography of the residue (SiO2, CH2Cl2) gave 314 mg of (E)-2-(3-
oxoprop-1-enyl)benzonitrile partially purified. 1H NMR
(DMSO-d6) δ 7.07 (dd, J=7.5, 15 Hz, 1H), 7.67 (t, J=8 Hz,
1H), 7.81 (t, J = 8 Hz, 1H), 7.87 (d, J = 15 Hz, 1H), 7.98 (d, J=8
Hz, 1H), 8.13 (d, J=8 Hz, 1H), 9.82 (d, J=8 Hz, 1H). This al-
dehyde was carried out in the following step without further
purification. This compound (314 mg) was dissolved in 10 mL of
toluene under nitrogen atmosphere, and (carbethoxymethyl-
ene)triphenylphosphorane (740 mg, 2.1 mmol) was added. The
mixture was stirred under reflux conditions for 4 h, then con-
centrated under reduced pressure. Purification of the crude
material by flash column chromatography (SiO2, EtOAc/PE,
10/90) gave 383 mg (33%, two steps) of a mixture of (2E,4E)-
and (2E,4Z)-ethyl 5-(2-cyanophenyl)penta-2,4-dienoates (80/20).
This mixture was dissolved in 4 mL of CH3CN and treated
with a catalytic amount of iodine (4 mg) for 3 h at room
temperature. The mixture was then concentrated under reduced
pressure, diluted with CH2Cl2, washed with an aqueous solution
of Na2SO3 (1%), dried over MgSO4, filtered, and concentrated
under reduced pressure to give 333 mg (87%) of (2E,4E)-ethyl
3
(E)-3-(2-Nitrophenyl)-1-[4-(4-cyanophenyl)piperazin-1-yl]pro-
penone (29). According to compound 13 synthesis described
above, compound 29 was isolated in 95% yield as a red-brown
powder: 1H NMR (DMSO-d6) δ 3.43 (broad s, 4H), 3.72 (broad
s, 2H), 3.88 (broad s, 2H), 7.05 (d, J = 8 Hz, 1H), 7.34 (d, J = 15
Hz, 1H), 7.60-7.66 (m, 3H), 8.05 (t, J = 8 Hz, 2H); HPLC
(Symmetry) tR = 4.87 min, 98%; MS (ESIþ) m/z = 363 [MHþ].
(E)-3-(2-Nitrophenyl)-1-(4-pyridin-4-ylmethylpiperazin-1-yl)-
propenone (30). According to representative procedure A de-
scribed above, compound 30 was isolated in 77% yield as an
orange powder: 1H NMR (DMSO-d6) δ 2.30-2.45 (m, 4H), 3.56
(s, 2H), 3.58 (broad s, 2H), 3.71 (broad s, 2H), 7.28 (d, J=15 Hz,
1H), 7.38 (dd, J = 4 Hz, 8 Hz, 1H), 7.62 (td, J=1 Hz, 8 Hz,
1H), 7.70-7.79 (m, 3H), 8.00-8.05 (m, 2H), 8.45-8.55 (m,
2H); HPLC (Symmetry) tR=2.94 min, 95%; MS (ESIþ) m/z=
353 [MHþ].
(E)-1-(4-Cycloheptanecarbonylpiperazin-1-yl)-3-(2-nitrophe-
nyl)propenone (31). According to representative procedure A
described above, compound 31 was isolated in 92% yield as a
white powder: 1H NMR (DMSO-d6) δ 1.44-1.56 (m, 8H),
1.67-1.72 (m, 4H), 2.74-2.77 (m, 1H), 3.53-3.69 (broad m,
6H), 3.69-3.74 (broad m, 2H), 7.29 (d, J = 15 Hz, 1H), 7.64 (t,
J = 8 Hz, 1H), 7.75 (d, J = 15 Hz, 1H), 7.79 (d, J = 8 Hz, 1H),
8.04 (d, J = 8 Hz, 1H); HPLC (XTerra MS) tR = 4.77 min, 98%;
MS (ESIþ) m/z = 386 [MHþ]. Anal. Calcd for C21H27N3O4: C,
65.44; H, 7.06; N, 10.90. Found: C, 65.49; H, 7.21; N, 10.84.
(E)-1-[4-(2-Fluorobenzenesulfonyl)piperazin-1-yl]-3-(2-nitro-
phenyl)propenone (32). According to representative procedure A
described above, compound 32 was isolated in 50% yield as an
1
5-(2-cyanophenyl)penta-2,4-dienoate: H NMR (DMSO-d6) δ
1.25 (t, J = 7 Hz, 3H), 4.16 (q, J = 7 Hz, 2H), 6.21 (d, J = 15 Hz,
1H), 7.25-7.40 (m, 2H), 7.45-7.56 (m, 2H), 7.74 (t, J=8 Hz,
1H), 7.87 (d, J=8 Hz, 1H), 7.96 (d, J=8 Hz, 1H). This com-
pound (2.0 g, 8.8 mmol) was dissolved in 50 mL of ethanol, and a
1
off-white powder: H NMR (DMSO-d6) δ 3.10 (broad s, 4H),
3.67 (broad s, 2H), 3.81 (broad s, 2H), 7.24 (d, J = 15 Hz, 1H),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5835
solution of aqueous KOH (1 N, 13.2 mL) was added. The
mixture was heated under refluxing conditions until completion
(about 1.5 h). The mixture was cooled to room temperature,
then quenched with HCl (1 N), filtered, and concentrated under
reduced pressure to give 1.63 g of (2E,4E) 5-(2-cyanophe-
nyl)penta-2,4-dienoic acid (93%): 1H NMR (DMSO-d6) δ
6.14 (d, J=15 Hz, 1H), 7.22 (d, J=15 Hz, 1H), 7.30-7.48 (m,
2H), 7.52 (t, J=8 Hz, 1H), 7.73 (t, J=8 Hz, 1H), 7.86 (d, J=
8 Hz, 1H), 7.96 (d, J = 8 Hz, 1H); HPLC (XTerra MS) tR = 4.00
min, 95%; MS (ESI-) m/z = 198 [M - H]. This compound was
used according to compound 17 synthesis described above. The
HCl salt of compound 36 was obtained in 78% yield as a yellow
powder: 1H NMR (DMSO-d6) δ 3.81 (broad s, 8H), 3.60-4.30
(broad m, 1H), 6.9-7.0 (m, 2H), 7.10-7.45 (m, 4H), 7.51 (t, J=8
Hz, 1H), 7.75 (t, J = 8 Hz, 1H), 7.87 (d, J = 8 Hz, 1H), 7.91 (d,
J = 8 Hz, 1H), 7.97 (t, J=5 Hz, 1H), 8.07 (d, J = 5 Hz, 1H);
HPLC (XTerra MS) tR=3.45 min, 99%; MS (ESI) m/z = 345
[MHþ]. Anal. Calcd for C21H20N4O1 HCl 1.5H2O: C, 61.84;
Supporting Information Available: Experimental protocols
for the biological assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
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3
3
H, 5.93; N, 13.74. Found: C, 61.65; H, 5.60; N, 13.45.
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white powder: 1H NMR (DMSO-d6) δ 1.44-1.53 (m, 8H),
1.67-1.73 (m, 4H), 2.75 (broad s, 1H), 3.53-3.70 (broad m,
6H), 3.70-3.75 (broad m, 2H), 7.53 (d, J = 15 Hz, 1H), 7.58 (t,
J = 8 Hz, 1H), 7.73 (d, J = 15 Hz, 1H), 7.78 (d, J = 8 Hz, 1H),
7.90 (d, J = 8 Hz, 1H), 8.18 (broad s, 1H); HPLC (XTerra MS)
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for C22H27N3O2 0.6H2O: C, 70.22; H, 7.55; N, 11.17. Found: C,
3
70.28; H, 7.57; N, 11.07.
(E)-1-(4-Cycloheptanecarbonylpiperazin-1-yl)-3-(2,6-difluoro-
phenyl)propenone (38). According to representative procedure A
described above, compound 38 was isolated in 27% yield as a
white powder: 1H NMR (DMSO-d6) δ 1.44-1.55 (m, 8H),
1.67-1.72 (m, 4H), 2.67 (broad s, 1H), 3.51-3.61 (broad m,
8H), 7.20-7.27 (m, 3H), 7.48-7.53 (m, 2H); HPLC (XTerra
MS) tR = 4.86 min, 98%; MS (ESIþ) m/z = 377 [MHþ]. Anal.
Calcd for C21H26N2F2O2: C, 67.00; H, 6.96; N, 7.44. Found: C,
66.82; H, 7.13; N, 7.44.
(E)-3-(2-Chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]-
propenone (39). According to representative procedure A de-
scribed above, compound 39 was isolated in 69% yield as an
HCl salt as a white powder: 1H NMR (DMSO-d6) δ 3.01-3.17
(m, 3H), 3.34-3.38 (m, 2H), 3.59 (broad t, 1H), 4.34 (broad s,
1H), 4.53 (broad t, 1H), 7.31-7.35 (m, 2H), 7.41-7.44 (m, 1H),
7.53-7.55 (m, 1H), 7.64 (broad s, 2H), 7.84 (d, J = 15 Hz, 1H),
7.99-8.01 (m, 1H), 11.1 (s, 1H); HPLC (XTerra MS) tR = 3.73
min, 99%; MS (ESIþ) m/z = 359 [MHþ]. Anal. Calcd for
C20H20N2Cl1F1O1 0.9HCl 0.2H2O: C, 60.78; H, 5.43; N, 7.09.
(10) Andrade-Gordon, P.; Maryanoff, B.; Derian, C. K.; Zhang, H.-C.;
Addo, M. F.; et al. Design, synthesis, and biological characteriza-
tion of a peptide-mimetic antagonist for a tethered-ligand receptor.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (22), 12257–12262.
(11) Scarborough, R.; Naughton, M.; Teng, W.; Hung, D.; Rose, J.; Vu,
T. K.; Wheaton, V.; Turck, C.; Coughlin, S. Tethered ligand
agonist peptides: structural requirements for thrombin receptor
activation reveal mechanism of proteolytic unmasking of agonist
function. J. Biol. Chem. 1992, 267, 13146–13149.
3
3
Found: C, 60.36; H, 5.44; N, 7.02.
(E)-1-(4-Cyclohexylmethylpiperazin-1-yl)-3-(2-nitrophenyl)-
propenone (40). According to representative procedure A de-
scribed above, compound 40 was isolated in 73% yield as an
HCl salt as a yellow powder: 1H NMR (DMSO-d6) δ 0.92-1.00
(m, 2H), 1.07-1.30 (m, 3H), 1.61-1.70 (m, 3H), 1.81 (broad s,
3H), 2.09-3.03 (m, 3H), 3.27-3.30 (m, 1H), 3.53 (broad s, 2H),
3.70 (broad t, 1H), 4.48 (t, J = 14 Hz, 2H), 7.31 (d, J = 15 Hz,
1H), 7.66 (t, J = 8 Hz, 1H), 7.78 (m, 2H), 8.03 (d, J = 8 Hz, 1H),
(12) Vassallo, R.; Kieber-Emmons, T.; Cichowski, K.; Brass, L. Struc-
ture-function relationships in the activation of platelets thrombin
receptors by receptor-derived peptides. J. Biol. Chem. 1992, 267,
6081–6085.
(13) Chao, B.; Kalkunte, S.; Maraganore, J.; Stone, S. Essential groups
in synthetic agonist peptides for activation of the platelet thrombin
receptor. Biochemistry 1992, 31, 6175–6178.
(14) Hui, K.; Jakubowski, J.; Wyss, V.; Angleton, E. Minimal sequence
requirement of thrombin receptor agonist peptide. Biochem. Bio-
phys. Res. Commun. 1992, 184, 790–796.
8.05 (d, J = 8 Hz, 1H), 10.18 (s, 1H). HPLC (XTerra MS) tR
=
3.65 min, 99%; MS (ESIþ) m/z = 358 [MHþ]. Anal. Calcd for
(15) Natarajan, S.; Riexinger, D.; Cambardella, M.; Seiler, S. “Tethered
ligand” derived pentapeptide agonists of thrombin receptor: a
study of side chain requirements for platelet aggregation. Int. J.
Pept. Protein Res. 1995, 45, 145–151.
C20H27N3O3 HCl 0.6H2O: C, 60.68; H, 7.17; N, 10.43. Found:
C, 60.30; H, 7.19; N, 10.36.
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3
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(16) Letiennet, R.; Leparq-Panissie, A.; Bocquet, A.; Calmettes, Y.;
ꢀ
Culie, C.; Le Grand, B. PAR1 antagonist mediated antithrombotic
Acknowledgment. The authors thank the Chemistry De-
partment of J.-P. Ribet and more specifically P. Zalavari,
M. Pelissou, R. Pena for analytical support and J.-L. Maurel,
S. Brunel, andJ. Beziatfor synthetic support. The authors also
thank the ADMET Department of C. Filaquier for PK
evaluation of compounds 36 and 39.
activity in extracorporeal arterio-venous shunt in the rat. Thromb.
Res., in press.
(17) Wollny, T.; Jacoviello, L.; Buczko, W.; De Gaetano, G.; Donati,
M. B. Prolongation of bleeding by acute hemolysis in rats: a role for
nitric oxide. Am. J. Physiol. 1997, 242, H2875–H2884.
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receptor-1 by a serum responsive element-dependent reporter gene
assay: major role of calmodulin. Biochem. Pharmacol. 2006, 71,
1449–1458.
2-methyl and 3-diene groups in the Adda residue and the effect of
macrocyclic conformational restraint. J. Chem Soc., Perkin Trans.
1 2001, 1696–1708.
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purification-free, preparation of amides using polymer-supported
reagents. Tetrahedron Lett. 2001, 42, 6703–6705.
(21) Klein, S. I.; Molino, B. F.; Czekaj, M.; Gardner, C. J.; Chu, V.;
Brown, K.; Sabatino, R. D.; Bostwick, J. S.; Kasiewski, C.;
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tein phophatase inhibitors derived from nodularin: role of the