Discovery and structure-activity relationship of 3-methoxy-N-(3-(1-methyl-1 H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide (APD791): A highly selective 5-hydroxytryptamine2A receptor inverse agonist for the treatment of arterial thrombo

Article abstract of DOI:10.1021/jm100044a

Serotonin, which is stored in platelets and is released during thrombosis, activates platelets via the 5-HT_2A receptor. 5-HT_2A receptor inverse agonists thus represent a potential new class of antithrombotic agents. Our medicinal pro

Full text of DOI:10.1021/jm100044a

4412 J. Med. Chem. 2010, 53, 4412–4421
DOI: 10.1021/jm100044a
Discovery and Structure-Activity Relationship of 3-Methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-
morpholinoethoxy)phenyl)benzamide (APD791): A Highly Selective 5-Hydroxytryptamine2A Receptor
Inverse Agonist for the Treatment of Arterial Thrombosis
Yifeng Xiong,* Bradley R. Teegarden, Jin-Sun Karoline Choi, Sonja Strah-Pleynet, Marc Decaire, Honnappa Jayakumar,
Peter I. Dosa, Martin D. Casper, Lan Pham, Konrad Feichtinger, Brett Ullman, John Adams, Diane Yuskin, John Frazer,
Michael Morgan, Abu Sadeque, Weichao Chen, Robert R. Webb, Daniel T. Connolly, Graeme Semple, and
Hussien Al-Shamma
Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, California 92121
Received January 13, 2010
Serotonin, which is stored in platelets and is released during thrombosis, activates platelets via the
5-HT2A receptor. 5-HT2A receptor inverse agonists thus represent a potential new class of antithrom-
botic agents. Our medicinal program began with known compounds that displayed binding affinity for
the recombinant 5-HT2A receptor, but which had poor activity when tested in human plasma platelet
inhibition assays. We herein describe a series of phenyl pyrazole inverse agonists optimized for
selectivity, aqueous solubility, antiplatelet activity, low hERG activity, and good pharmacokinetic
properties, resulting in the discovery of 10k (APD791). 10k inhibited serotonin-amplified human
platelet aggregation with an IC50 = 8.7 nM and had negligible binding affinity for the closely related
5-HT2B and 5-HT2C receptors. 10k was orally bioavailable in rats, dogs, and monkeys and had an
acceptable safety profile. As a result, 10k was selected further evaluation and advanced into clinical
development as a potential treatment for arterial thrombosis.
Introduction
The 5-HT2A receptor is one of 15 different serotonin re-
ceptor subtypes.2 In the cardiovascular system, modulation of
5-HT2A receptors on vascular smooth muscle cells and plate-
lets is thought to play an important role in the regulation of
cardiovascular function.3,4 Platelets are activated by a variety
of agonists such as ADP, thrombin, thromboxane, serotonin,
epinephrine, and collagen. Upon platelet activation at the site
of blood vessel injury, a number of factors including serotonin
(5-HT) are released. Although by itself serotonin is a weak
activator of platelet aggregation, in vitro it can amplify aggre-
gation induced by other agonists as mentioned above.5,6 There-
fore, serotonin released from activated platelets may induce
further platelet aggregation and enhance thrombosis.
The 5-HT2A receptor antagonist ketanserin (Figure 1) was
shown in clinical studies to reduce early restenosis7 and dec-
rease myocardial ischemia during coronary balloon angio-
plasty.8 However, in another study, ketanserin did not signi-
ficantly improve clinical outcomes, and the rate of adverse
events was higher than that observed in the control group.9
Some of the adverse events reported in the latter study could
be specific to ketanserin and resulted from its lack of 5-HT2A
receptor selectivity. Other 5-HT2A antagonists with improved
selectivity profiles have shown promise in clinical studies. For
example, sarpogrelate (Figure 1) was shown to inhibit rest-
enosis following coronary stenting.10
Arterial thrombosis is the formation of a blood clot or
thrombus inside an artery or arteriole that restricts or blocks
the flow of blood and, depending upon location, can result in
acute coronary syndrome or stroke. The formation of a thro-
mbus is usually initiated by blood vessel injury, which triggers
platelet aggregation and adhesion of platelets to the vessel
wall. Treatments aimed at inhibiting platelet aggregation have
demonstrated clear clinical benefits in the setting of acute
coronary syndrome and stroke. Current antiplatelet therapies
include aspirin, which irreversibly inhibits cyclooxygenase
(COXa) and results in reduced thromboxane production,
clopidogrel and prasugrel, which inhibit platelet adenosine
diphosphate (ADP) P2Y12 receptors, and platelet glycopro-
tein IIb/IIIa receptor antagonists. Another class of antiplatelet
drugs, protease-activated thrombin receptor (PAR-1) antago-
nists, are also being evaluated in the clinic for the treatment of
acute coronary syndrome.1a The most advanced candidate in
this class, N-[(1R,3aR,4aR,6R,8aR,9S,9aS)-9-{2-[5-(3-fluoro-
phenyl)pyridin-2-yl]vinyl}-1-methyl-3-oxoperhydro-naphtho-
[2,3-c]furan-6-yl]-carbamic acid ethyl ester (SCH-530348),
is currently in phase 3 trials for the prevention of arterial
thrombosis.1b,c
*To whom correspondence should be addressed. Phone: þ1 858-453-
7200. Fax: þ1 858-453-7210. E-mail: yxiong@arenapharm.com.
a Abbreviations: COX, cyclooxygenase; ADP, adenosine dipho-
sphate; SAR, structure-activity relationship; hERG, human ether-a-
go-go-related gene; CNS, central nervous system; 5-HT, serotonin;
AUC, area under the plasma concentration time curve, iv, intravenous;
IP, inositol phosphate.
Because the 5-HT2A receptor is expressedboth inperipheral
tissues and in the central nervous system (CNS), compounds
withlimited CNSpartitioningwould be preferredtomaximize
cardiovascular and blood platelet pharmacological activity
while minimizing CNS effects. In addition, because 5-HT2A
r
pubs.acs.org/jmc
Published on Web 05/10/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4413
Figure 1. Serotonin and known 5-HT2A receptor antagonists.
Figure 2. Phenyl-pyrazole urea based 5-HT2A receptor inverse agonists.
Scheme 1. Synthesis of Phenyl-Pyrazole Amide Derivativesa
a Reagents and conditions: (a) n-BuLi, THF at -78 °C, then ZnCl2; (b) 2-iodo-4-nitroanisole, Pd(PPh3)4; (c) AlCl3, 1,2-dichloroethane; (d) 1,2-
dibromoethane, K2CO3, DMSO, (e) HOCH2CH2NR1R2, Ph3P, DIAD, THF; (f) HNR1R2, K2CO3, DMF; (g) zinc dust, NH4Cl, THF; (h) R-PhCO2H,
HATU, triethylamine, DMF or R-Ph-COCl, triethylamine, THF; (i) NBS or NCS, MeOH or DMF.
receptor inverse agonists are thought to reduce thrombus
formation via inhibition of serotonin-mediated amplification
of platelet aggregation without inhibiting agonist driven agg-
regation per se, it is possible that this class of inhibitors will
have an improved bleeding risk side effect profile compared to
what has been observed with other classes of antithrombotic
drugs.
We have previously identified a series of phenyl-pyrazole
urea-based 5-HT2A inverse agonists (e.g., compound 1,
Figure 2).11 This structure-activity relationship (SAR) effort
led to the discovery of 2 (Nelotanserin, Figure 2), which was
advanced into clinical trials for the treatment of insomnia.12
Although such ureas are potent 5-HT2A inverse agonists, they
were found to possess limited aqueous solubility, a property
thatwethought wouldbeimportant for goodantiplatelet acti-
vity. We have since reported on modifications to this urea
series in which we incorporated amino moieties into the scaf-
fold. The resulting series of phenyl-pyrazole ureas as exem-
plified by 313-15 were significantly more soluble in aqueous
solution. Moreover these compounds also showed improved
inhibition of platelet aggregation.14 Unfortunately, many of
these solubilized ureas had suboptimal selectivity profiles and
poor pharmacokinetic properties. To further explore the scope
of the soluble phenyl-pyrazole scaffold, with a particular goal

4414 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Xiong et al.
of improving the pharmacokinetic and pharmaceutical char-
acteristics, the SAR was expanded from ureas to sulfonami-
des, carbamates, and amides. Herein, we describe SAR efforts
that led to the identification of 10k (APD791), a highly selec-
tive 5-HT2A inverse agonist with high receptor binding affi-
nity, potent activity on human platelets, a clean safety profile,
and outstanding pharmaceutical properties.
Table 1. Binding Affinities of Compounds 10a-u at Human 5-HT2A
and 5-HT2C Receptors, Activity of Inhibition of Serotonin Mediated
Amplification of ADP-Stimulated Human Platelet Aggregation and
Inhibition of hERG Channel
Chemistry
The syntheses of the 1-N-alkyl-5-phenyl-pyrazole scaffold
4, its subsequent modification to introduce a basic amine
moiety to provide intermediates 8, and formation of final
amides 10 are outlined in Scheme 1. Starting with commer-
cially available N-methyl pyrazole, lithiation with n-BuLi
followed by a Negishi coupling with 2-iodo-4-nitroanisolefur-
nished intermediate 4.13 Demethylation with aluminum tri-
chloride afforded phenol 5. Reaction with 1,2-dibromoethane
gave the bromo-tethered compound 6. Alkylation of 6 with a
series of amines introduced basic functionalities into the mole-
cules providing 8. Alternatively, 8 could also be prepared via
Mitsunobu reaction from phenol 5 and the appropriate amino-
ethanol derivatives. Reduction of the nitro group with zinc
dustafforded anilines9, whichwerethencoupledwithacids or
acid chlorides to furnish the desired amides 10.13 Halogena-
tion of the pyrazole ring, where appropriate, could be effected
at different stages. Bromo-tethered analogue 6 was treated
with either N-chlorosuccinimide (NCS) or N-bromosuccinimide
(NBS) to form chloro or bromo pyrazoles 7a and 7b, respec-
tively. Amination, reduction, and amidation of 7 led to the
final products 10. Alternatively, halogens could be introduced
after the amine moiety was incorporated from 8, and thus
halogenation, subsequent reduction, and amidation of 8 pro-
vided an alternative approach to final products 10.
Results and Discussion
In vitro competitive binding assays with radiolabeled 2,5-
dimethoxy-4-iodoamphetamine (125I-DOI) were used to deter-
mine Ki values for all compounds against recombinant h5-HT2A
and h5-HT2C receptors (Table 1). Compounds were also
tested for inhibition of serotonin-induced amplification of
ADP-stimulated platelet aggregation in human platelet rich
plasma, andin selected cases, for humanether-a-go-go-related
gene (hERG) inhibition in a patch clamp assay (Table 1). As
previously observed in the phenyl-pyrazole urea series, the
phenyl-pyrazole amides showed excellent affinity for the 5-HT2A
receptor. In addition, and in contrast to the urea series,14 the
amides generally showed a very high degree of selectivity for
the 5-HT2A receptor over the 5-HT2C receptor. In all the ex-
amples tested, the selectivity over the 5-HT2B receptor was
uniformly high and thus affinity for this receptor was not ex-
amined on a routine basis.
A wide range of monosubstitutions on the phenyl ring was
evaluated, all well tolerated regardless of the electron with-
drawing or donating groups. The meta-substituted analogues
were found to be higher affinity ligands for the 5-HT2A re-
ceptor than the para-substituted analogues. For example, the
meta-F analogue 10e and meta-CF3 analogue 10i had slightly
higher affinity than the corresponding para-F analogue 10f
and para-CF3 analogue 10h. Furthermore, the meta-substi-
tuted analogues were more selective for the 5-HT2A receptor
than the para-substituted analogues. For instance, 10b (meta-
CF3) had lower affinity at the 5-HT2C receptor and slightly
higher affinity at the 5-HT2A receptor than 10a (para-CF3),
a Ki values are the mean of at least three experiments performed in
triplicate, determined from 10 concentrations, all Ki values were calcu-
lated from IC50 values using the method of Cheng and Prusoff 22 with
standard deviation <0.4 log units. b hERG patch clamp assays were
performed at Aviva Biosciences, San Diego, CA (http://www.avivabio.
com). c Measured at 1 μM concentration. d Not determined. e IC50=4.1μM.
f IC50 =0.9 μM. g IC50 =11 μM. h IC50 =4.1 μM
making 10b much more selective at the 5-HT2A receptor
(1900-fold) than 10a (118-fold). Therefore, during lead opti-
mization at other positions in the molecules (pyrazole halo-
genations and incorporation of amino functionalities), we
focused more heavily on the meta- than para-substituted
analogues. Among the different meta-substituents, lipophilic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4415
CF3 groups significantly enhanced the 5-HT2A receptor affi-
nity. For example, 10i (meta-CF3) had 6- to 7-fold higher
affinity than 10k (meta-OMe) or 10e (meta-F) analogues.
The effect of pyrazole halogenation on receptor affinity was
also examined. In general, bromination or chlorination of the
pyrazole ring led to increased affinity for the 5-HT2A receptor
when compareddirectly tothe nonhalogenated pyrazoles. For
example, chlorination of 10l provided 10m, which showed a
more than 50-fold increase in the 5-HT2A receptor affinity. In
another example, addition of a chlorine to 10e resulted in 10g,
which showed a 8-fold improvement in 5-HT2A receptor
affinity.
A wide range of amino substituents was also evaluated. As
shown in Table 1, almost all amino groups provided com-
pounds with good binding affinity at the 5-HT2A receptor
with the exception of the oxo-piperazinyl group (10l), which
was 20- to 480-fold weaker compared to other amino groups
with the same phenyl ring and pyrazole ring substitutions. As
discussed earlier, binding affinity with this amino group was
improved by introducing a chlorine atom onto the pyrazole
(10m). Whereas all amino groups showed good (e.g., 118-fold,
10a) to excellent (e.g., 2040-fold, 10k) selectivity for the
5-HT2A receptor vs the 5-HT2C receptor, a morpholino group
appeared to be most beneficial, showing very little affinity for
the 5-HT2C receptor even with halogen substitutions on the
pyrazole.
On the basis of their structural similarity to our previous
series of 5-HT2A receptor ligands (including 2), which were all
functional inverse agonists,12 we expected the compounds in
these current series to have the same functional response in
vitro. In this current investigation, we confirmed this expecta-
tion for selected compounds using inhibition of total inositol
phosphate (IP) accumulation as a functional assay to deter-
mine the inverse agonist potency.19 For example, 10b had an
IC50 of 23 nM in the IP assay, and 10k inhibited IP accumula-
tion with an IC50 of 5.2 nM.
After this initial examination of their receptor binding
affinities and selectivity profile, compounds were then eval-
uatedina second functional(antagonist) assayfor theirability
to inhibit serotonin-mediated amplification of ADP-stimu-
lated human platelet aggregation. As shown in Table 1, IC50
values for inhibition of platelet aggregation ranged from 2 to
189 nM. In general, platelet activity did not correlate well with
binding affinity, but compounds were usually somewhat less
potent in the platelet inhibition assay than in the binding
assay. The degree of difference between the Ki value in binding
and the IC50 value in the platelet assay varied from 1- to 2-fold
(10a and 10k) to 30-fold (10n) and depended on compound
structure. Interestingly, removal of serum proteins from
platelet rich plasma resulted in a left shift in the IC50 for inhi-
bition of platelet aggregation, suggesting that protein binding
may play a significant role in the observed differences between
platelet aggregation activity and receptor affinity. Hence, the
platelet assayservedasa useful guidetoreceptoractivityinthe
presence of serum proteins, and only compounds with a modest
shift between the values for the two assays were considered for
further evaluation.
activities, as exemplified by 10p and 10o. Although, in another
example, pyrazole chlorination was more successful, 10g
(chloro-pyrazole) being superior to 10e in binding affinity
as well as platelet activity. We reasoned that as compounds
became more lipophilic after introduction of a halogen, they
would likely be more protein bound, which in turn would
result in a significantly greater shift between Ki in the binding
assay and IC50 values in the platelet assay. In contrast, we
could discern no clear trend on how the substitutions on the
phenyl ring affected the platelet activity.
After introducing basic amines into the scaffold, both
aqueous solubility and platelet activity were improved, but
one of the liabilities we observed was the introduction of a
significant interaction with the hERG channel. As a result,
compounds of interest were screened in a patch clamp elec-
trophysiology assay at a single concentration of 3 or 1 μM
(Table 1).
Several previously well-explored approaches were em-
ployed toattenuatehERG activity.16,17 The first was toreduce
the pKa of the basic nitrogen, which proved to be quite eff-
ective in reducing hERG activity while still maintaining the
receptor affinity and platelet activity. As discussed earlier,
both morpholino- and pyrrolidino-containing compounds
trended toward improved potency in the platelet aggregation
assay. However, due to the high pKa of the pyrrolidine nitro-
gen, many of the pyrrolidino-containing compounds had very
high activity in the hERG channel assay and were therefore
dropped from further evaluation. In contrast, compounds
with a morpholino-substituent had somewhat reduced acti-
vity at the hERG channel. For example, 10b containing a
pyrrolidine (pKa 9.56)18 showed 80% inhibition at 1 μM while
the analogous 10i, which incorporated a morpholino-substi-
tuent (pKa 6.38), showed only 50% inhibition at the same
concentration. The pKa of the basic nitrogen could also be
reduced by introducing electron withdrawing groups adjacent
to the amine moiety, again resultinginreducedhERG channel
blockade. Compound 10c, which contained a gem-difluoro-
pyrrolidine (pKa 4.91), had a further attenuation of hERG
activity to 50% inhibition at 3 μM. Increasing the polarity of
molecules (lowering clogP) is another well explored method of
decreasing hERG activity. Compounds 10q, 10r, and 10s all
containedpolar groups attached tothe aminemoietyandeach
had significantly lower hERG inhibition, typically less than
30% at 3 μM. In addition, 10q (meta-OMe on phenyl ring,
24% inhibition at 3 μM) had lower hERG activity than 10n
(meta-CF3 on phenyl ring, 75% inhibition at 3 μM), showing
that making changes to other areas of the molecule to reduce
clogP (in this case from 3.16 to 2.22) was also able to assist in
decreasing hERG activity. This strategy of optimization of
receptor affinity and selectivity and the subsequent manage-
ment of platelet activity and hERG activity led us to the initial
identification of our lead compound. 10k was a selective, high
affinity 5-HT2A receptor inverse agonist with excellent activity
in the platelet aggregation assay. In addition, 10k showed only
moderate hERG inhibition of 32% at 3 μM in the screening
assay, and this translated into an IC50 of 11 μM when tested in
the same patch clamp assay in full concentration-response
mode.
In general, compounds containing pyrrolidino and mor-
pholino moieties had smaller shifts compared to compounds
containing other basic amines. For example, 10j and 10k had
same receptor affinity as 10m and 10n, but 10j and 10k, which
had a morpholino moiety, are much more potent than 10m or
10n in the platelet assay. Halogen substitutions of the pyrazole
usually increased binding affinities but not necessarily platelet
Another challenge encountered after introducing the basic
amine functionality was the poor microsomal stability of
compounds in rodents, which resulted in poor in vivo rodent
pharmacokinetic parameters. Pharmacokinetic profiles of
selected compounds in rat are shown in Table 2. In general,
as expected with poor microsomal stability, most compounds

4416 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Xiong et al.
Table 2. Rat Pharmacokinetic Parameters of Selected Compounds after 10 mg/kg Oral Dosea
compd
Cmax (ng/mL)
AUClast (h ng/mL)
3
T1/2 (h)
IV CLtotal (L/h/kg)
Vss (L/kg)
F (%)b
10d
10i
56
202
453
171
288
1.5
1.8
2.8
14
11.7
5.1
22
27
43
9.6
1.1
10a
3831
5.4
a Values represent the mean of n = 3 animals. b % F calculated relative to a 2 mg/kg iv dosing.
Table 3. Pharmacokinetic Profile of 10k in Preclinical Species after 10 mg/kg Oral Dosea
species
Cmax (ng/mL)
AUClast (h ng/mL)
3
T1/2 (h)
IV CLtotal (L/h/kg)
Vss (L/kg)
F (%)b
rat
dog
58
2280
1462
43
3544
1922
1.0
0.9
2.1
22
29
10
45
16
1.2
0.9
1.3
0.6
monkey
a Values represent the mean of n = 3 animals. b % F calculated relative to a 2 mg/kg i.v. dosing.
Figure 3. Compound 10k and its active metabolites 10t and 10u.
tested exhibited very high plasma clearance, low exposure,
and poor oral bioavailability. Compound 10d was a typical
example (Table 2). We observed that a lipophilic CF3 group
sometimes improved these pharmacokinetic parameters. For
example, 10i, witha CF3 groupon the phenylring, hadslightly
lower clearance than 10g and improved Cmax and AUC
compared to 10d. However, the hERG activity of 10i pre-
vented this compound from moving forward. The combina-
tion of a CF3 group on the phenyl ring and a pyrrolidino
group (10a) resulted in much lower clearance, very good ex-
posure, and oral bioavailability. However, due to the high
hERG activity and the significant 5-HT2C receptor affinity,
10a was not suitable for further development despite its potent
platelet activity and good pharmacokinetic profile.
The pharmacokinetic profile of 10k in preclinical species
was initially somewhat unpromising (Table 3). Compound
10k had modest pharmacokinetic characteristics in rats, but
in monkeys and dogs, 10k had quite acceptable pharmacoki-
netic properties. Plasma clearance was low, oral bioavailabi-
lity was moderate to good, and overall exposure was high. In
addition, two major active metabolites of 10k were identified
(Figure 3). 10t and 10u were generated upon incubation of 10k
with human, dog and rat liver microsomes and were also
observed in vivo across all species tested. These two metabo-
lites were essentially equipotent with 10k in receptor affinity,
inhibition of platelet aggregation, and target selectivity
(Table 1). Remarkably, after lengthy struggles to control
hERG inhibition within the series, the metabolites of 10k also
turned out to be only weak inhibitors of the hERG channel.
Thus, there were no known off-target effects of these major
metabolites that would preclude the further development of
10k, and indeed, it was envisioned that their generation in vivo
could extend the duration of biological activity for 10k.
Neither metabolite was suitable as a development candidate
in its own right, however, as neither showed good exposure
after oral administration, which we ascribed to lack of intest-
inal absorption.
The in vivo activity of 10k was tested in beagle dogs using
inhibition of ex vivo serotonin induced platelet aggregation
and inhibition of arterial thrombosis in the Folts model, a
classic canine model of recurrent coronary thrombosis, con-
sidered to mimic many of the key pathophysiologic features
of unstable angina, as previously described.19,20 Levels of
serotonin-mediated amplification of collagen-stimulated pla-
telet aggregation were determined in whole blood following a
single oral dose of 10 mg/kg of 10k. Complete inhibition of
serotonin- mediated aggregation was observed 1 h after dos-
ing of 10k and was maintained at the 6, 12, and 24 h time
points with 86%, 57%, and 58% inhibition, respectively.
Similar results were obtained using the ex vivo assay following
10 days of repeat dosing of 10k at a 1 mg/kg dose.19 In the
Folts model, 10k attenuated recurrent thrombosis in anesthe-
tized dogs either pretreated with 10k or treated with 10k after
the onset of recurrent thrombosis, at a dose of 0.07 mg/kg iv
bolus followed by continuous iv fusion of 1.16 μg/kg/min.
Flow-time area averaged 58-59% following administration
of 10k vs 21-28% in saline controls.20 Overall, this profile,
including the potential for an extended duration of action via
the generation of active metabolites, was deemed sufficiently
attractive for us to select 10k for further development.
Summary
10k is a novel 5-HT2A inverse agonist with high receptor
binding affinity (Ki=4.9 nM) and functional inverse agonism
of inositol phosphate accumulation (IC50 = 5.2 nM).19 10k
had excellent selectivity for the 5-HT2A receptor versus
5-HT2C (Ki >10000 nM) and 5-HT2B (Ki >10000 nM) rec-
eptors in competition binding assays (>2000-fold selectivity
on both receptors). 10k had limited CNS partitioning, with a
brain to plasma ratio of 0.05 (based on Cmax at 30 mg/kg dose)
in rat. Further profiling against a panel of 70 human GPCRs,
ion channels, and transporters (Cerep) showed that 10k had
no appreciable activities on any other receptors or transpor-
ters tested. Patch clamp analysis suggested that 10k had

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4417
limited hERG channel inhibition potential, with an IC50 of 11
μM. On the basis of CYP450 inhibition assays using human
liver microsomes, 10k appeared to have little potential for
drug-drug interactions. 10k exhibited potent inhibition of
serotonin mediated amplification of ADP-stimulated human
and dog platelet aggregation (IC50 = 8.7 and 23.1 nM, res-
pectively). Oral administration of 10k to beagle dogs resulted
in acute (1 h) and subchronic (10 day) inhibition of serotonin
mediated amplification of collagen-stimulated platelet aggre-
gation in whole blood ex vivo. In the Folts model, 10k impro-
ved coronary patency even after the onset of recurrent throm-
bosis. Taken together, these data suggest that 10k, a high
affinity, selective, orally bioavailable 5-HT2A receptor inverse
agonist, represents a promisingcompound for development as
an antiplatelet agent.
combined organic phases were dried over MgSO4, filtered, and
partly concentrated until a solid started to precipitate. Hexane
(approximately 3 volumes) was added and the resultant precipitate
collected by filtration and washed with a cold 3:1 mixture of
hexane/ethyl acetate to afford 4 (8.6 g, 59%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ: 3.74 (s, 3H), 3.96 (s, 3H), 6.31 (d,
J=1.6 Hz, 1H), 7.08 (d, J=9.2 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H),
8.19 (d, J = 2.8 Hz, 1H), 8.34 (dd, J = 2.8, 9.2 Hz, 1H). LCMS
m/z= 234.1 [M þ H]þ.
2-(2-Methyl-2H-pyrazol-3-yl)-4-nitro-phenol (5). A mixture
of 4 (3 g, 12.9 mmol) in 1,2-dichloroethane (50 mL) was cooled
to 0 °C with stirring. Aluminum trichloride (6.9 g, 51.6 mmol)
was added. The resulting mixture was heated at 80 °C for 3.5 h.
The crude mixture was diluted with ethyl acetate and the solid
removed by filtration. The resultant filtrate was washed with a
10% solution of potassium tartrate and the solvent removed by
evaporation. The residue was purified by HPLC to give 5 as a
yellow solid (1.08 g, 38%). 1H NMR (400 MHz, DMSO-d6) δ:
3.74 (s, 3H), 6.38 (d, J=1.6 Hz, 1H), 7.16 (d, J =9.2 Hz, 1H),
7.48 (d, J=2.0 Hz, 1H), 8.05 (d, J=2.8 Hz, 1H), 8.24 (dd, J=
2.8, 9.2 Hz, 1H), 12.0 (s, 1H). LCMS m/z=220.1 [M þ H]þ.
5-[2-(2-Bromo-ethoxy)-5-nitro-phenyl]-1-methyl-1H-pyrazole
(6). To a suspension of 5 (1.042 g, 4.76 mmol) in THF (50 mL) at
rt was added triphenylphosphine (2.138 g, 8.15 mmol) and
2-bromoethanol (0.580 mL, 8.21 mmol). Diisopropyl azodicar-
boxylate (DIAD) (1.58 mL, 7.81 mmol) was added dropwise to
the stirring mixture. On completion of the addition, the reaction
mixture was stirred at rt for a further 2 h and then concentrated
to yield an oil which was purified by SiO2 column chromatogra-
phy (eluent: 40% ethyl acetate/60% hexanes) to afford 6 (1.40 g,
90%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ: 3.73 (s,
3H), 3.82-3.77 (m, 2H), 4.59-4.54 (m, 2H), 6.43 (d, J=1.8 Hz,
1H), 7.42 (d, J=9.2 Hz, 1H), 7.51 (d, J=1.8 Hz, 1H), 8.12 (d,
J = 2.9 Hz, 1H), 8.36 (dd, J = 9.2, 2.9 Hz, 1H). LCMS m/z =
326.1/328.1 [M þ H]þ.
Experimental Section
Chemistry. All reagents were commercially available and used
without further purification unless stated otherwise. Microwave
irradiations were performed using either a Smith Synthesizer or
an Emrys Optimizer (Biotage). Column chromatography was
carried out on prepacked silica gel columns using KP-Sil
supplied by Biotage and on silica gel columns using Kieselgel
60, 0.063-0.200 mm (Merck). Proton nuclear magnetic reso-
nance (1H NMR) spectra were recorded on a Bruker Avance-
400 equipped with a Quad Nucleus probe (QNP) or a Broad-
band Inverse (BBI) probe and z-gradient. Chemical shifts (δ) are
given in parts per million (ppm), with the residual solvent signal
used as a reference. Coupling constants (J) are reported in Hz.
NMR abbreviations are used as follows: s=singlet, d=doublet,
t=triplet, q=quartet, m=multiplet, dd=doublet of doublets,
dt=doublet of triplets, b=broad. Melting points were recorded
on differential scanning calorimetry (DSC).
Analytical HPLC/MS was conducted on a PE Sciex API
150EX mass spectrometer with an electrospray source, using a
Shimadzu Inc. LC-10A VP UV detector monitoring at 214 nm,
Analyst 1.2 software, and either (a) a Gilson 215 autosampler
and an Alltech Prevail C18 column (5 μ, 250 mm ꢀ4.6 mm),
using a gradient of 5% v/v CH3CN (containing 1% v/v TFA) in
H2O (containing 1% v/v TFA) (t=0.0 min) gradient to 95% v/v
CH3CN in H2O (t = 6.0 min), 3.5 mL/min, or (b) a PE 200
autosampler and a Supelco Discovery C18 column (5 μ, 50 mm ꢀ
2.1 mm), using a gradient of 5% v/v CH3CN (containing 1% v/v
TFA) in H2O (containing 1% v/v TFA) (t=0.0 min) gradient to
95% v/v CH3CN in H2O (t = 5.0 min), 0.75 mL/min. Prepara-
tive HPLC was conducted on a Varian Prostar reverse phase
HPLC using either (a) a Phenomenex Luna C18 column (10 μ,
250 mm ꢀ 21.2 mm), 5% (v/v) CH3CN (containing 0.1% v/v
TFA) in H2O (containing 0.1% v/v TFA) gradient to 95%
CH3CN, 20 mL/min, λ = 220 nm, or (b) a Phenomenex Luna
C18 column (10 μ, 250 mm ꢀ 50 mm), 5% (v/v) CH3CN
(containing 0.1% v/v TFA) in H2O (containing 0.1% v/v TFA)
gradient to 95% CH3CN, 50 mL/min, λ = 220 nm. The purity
of all tested compounds was g95% (peak area % at 214 nm)
using the analytical methods described above unless stated
otherwise.
5-(2-Methoxy-5-nitro-phenyl)-1-methyl-1H-pyrazole (4). A solu-
tion of N-methylpyrazole (5.12 g, 62.4 mmol) in THF (100 mL)
was cooled to -78 °C and 2.5 M n-butyllithium in hexane
(27.5 mL, 68.8 mmol) was added. After stirring for 30 min at the
same temperature, 0.5 M zinc chloride in THF (107 mL, 53.5 mmol)
and 1 M zinc chloride in diethyl ether (15.3 mL, 15.2 mmol) were
added. The mixture was allowed to warm up to room temperature
and 2-iodo-4-nitroanisole (17.4 g, 62.4 mmol) followed by tetrakis-
(triphenylphospine)palladium (3.6 g, 3.1 mmol) were added. The
reaction mixture was heated to 60 °C overnight. Once the reaction
was complete, the solvents were removed under reduced pressure
and the residue partitioned between ethyl acetate and brine. The
5-[2-(2-Bromo-ethoxy)-5-nitro-phenyl]-4-chloro-1-methyl-1H-
pyrazole (7a). A mixture of 6 (470 mg, 1.44 mmol) and N-chloro-
succinimide (192 mg, 1.44 mmol) in DMF (2 mL) was heated
under microwave irradiation at 100 °C for 20 min. The mixture
was quenched with water, extracted with ethyl acetate, and the
combined extracts dried in vacuo to give 7a as a yellow solid
(462 mg, 89%) which was used in subsequent reactions without
purification. 1H NMR (400 MHz, CDCl3) δ: 3.55-3.61 (m, 2H),
3.78 (s, 3H), 4.44 (t, J = 5.6 Hz, 2H), 7.13 (d, J = 9.1 Hz, 1H),
7.57 (s, 1H), 8.23 (s, 1H), 8.40 (dd, J=2.8, 9.1 Hz, 1H). LCMS
m/z =360.1/362.1 [M þ H]þ.
4-Bromo-5-[2-(2-bromo-ethoxy)-5-nitro-phenyl]-1-methyl-1H-
pyrazole (7b). A mixture of 6 (200 mg, 0.61 mmol) and N-bromo-
succinimide (109 mg, 0.61 mmol) in DMF (2 mL) was heated
under microwave irradiation at 100 °C for 10 min. The mixture
was quenched with water, extracted with ethyl acetate, and the
combined extracts dried in vacuo to give 7b as a yellow solid
(200 mg, 81%), which was used in subsequent reactions without
purification. 1H NMR (400 MHz, CDCl3) δ: 3.53-3.64 (m, 2H),
3.78 (s, 3H), 4.38-4.49 (m, 2H), 7.11 (d, J=9.1 Hz,1H), 7.57 (s,
1H), 8.23 (s, 1H), 8.39 (dd, J = 2.8, 9.1 Hz, 1H). LCMS m/z =
404.1/406.1/407.9 [M þ H]þ.
General Procedure A, Preparation of Intermediates 8 via
Alkylation: 1-Methyl-5-{5-nitro-2-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-
1H-pyrazole (8a). A mixture of 6 (390 mg, 1.20 mmol), pyrro-
lidine (104 mg, 1.46 mmol), and potassium carbonate (218 mg,
1.58 mmol) in DMF (4 mL) was heated under microwave
irradiation at 100 °C for 15 min. The resulting mixture was
purified by HPLC to give the TFA salt of 8a as a yellow solid
(400 mg, 77%). 1H NMR (400 MHz, CD3CN) δ: 1.80-1.88 (m,
2H), 1.94-2.00 (m, 2H), 2.79-2.89 (m, 2H), 3.37-3.45 (m, 2H),
3.48-3.53 (m, 2H), 3.72 (s, 3H), 4.43-4.46 (m, 2H), 6.40 (d, J=
2.0 Hz, 1H), 7.25 (d, J=9.2 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H),
8.20 (d, J=2.9 Hz, 1H), 8.38 (dd, J=2.9, 9.2 Hz, 1H). LCMS m/
z =317.2 [M þ H]þ.

4418 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Xiong et al.
5-{2-[2-(3,3-Difluoropyrrolidin-1-yl)ethoxy]-5-nitrophenyl}-1-
methyl-1H-pyrazole (8c). 8c was prepared in a manner similar to
that described for 8a, using 6 (50 mg, 0.15 mmol) and 3,3-
difluoropyrrolidine hydrochloride (26 mg, 0.18 mmol) as start-
ing materials, to give the TFA salt of 8c as a yellow solid (16 mg,
1-{4-[2-(4-Amino-2-(1-methyl-1H-pyrazol-5-yl)phenoxy)ethyl]-
piperazin-1-yl}ethanone (9n). 9n was prepared from 8n (373 mg,
0.67 mmol) in a manner similar to that described for 9a, pro-
viding 9n as a white solid (180 mg, 78%). LCMS m/z = 344.1
[M þ H]þ. The product was used in the next step without further
purification.
1
22%). H NMR (400 MHz, CD3CN) δ: 2.35-2.45 (m, 2H),
3.31-3.43 (m, 4H), 3.49-3.56 (m, 2H), 3.69 (s, 3H), 4.46-4.49
(m, 2H), 6.34-6.36 (m, 1H), 7.22 (d, J=9.3 Hz, 1H), 7.49-7.50
(m, 1H), 8.18 (d, J=3.0 Hz, 1H), 8.38 (dd, J=2.9, 9.0 Hz, 1H).
LCMS m/z =353.3 [M þ H]þ.
General Procedure C, Preparation of Amides 10 via HATU
Coupling: N-[3-(2-Methyl-2H-pyrazol-3-yl)-4-(2-pyrrolidin-1-yl-
ethoxy)-phenyl]-4-trifluoromethyl-benzamide (10a). A mixture of
9a (57 mg, 0.2 mmol), 4-trifluoromethyl-benzoic acid (42 mg,
0.22 mmol), N,N,N0,N0-tetramethyl-O-(7-azabenzotriazol-1-yl)-
uronium hexafluorophosphate (HATU, 91 mg, 0.24 mmol) and
triethylamine (0.1 mL) in DMF (2 mL) was heated under
microwave irradiation at 100 °C for 15 min. After removal of
excess solvent, the crude product was purified by HPLC to give
4-{2-[2-(2-Methyl-2H-pyrazol-3-yl)-4-nitro-phenoxy]-ethyl}-
morpholine (8d). A mixture of triphenylphosphine (7.5 g,
28.6 mmol) and diisopropyl azodicarboxylate (5.66 mL, 28.6 mmol)
in THF (50 mL) was stirred at 0 °C for 10 min, then 5 (3.5 g,
15.9 mmol) and 2-morpholin-4-yl-ethanol (3.66 mL, 28.6 mmol)
were added at 0 °C. The reaction mixture was allowed to warm to
rt and was stirred for another hour. The solvent was removed in
vacuo, and the crude mixture was purified by HPLC to pro-
1
the TFA salt of 10a as a white solid (65 mg, 71%). H NMR
(DMSO-d6, 400 MHz) δ: 1.73-1.76 (m, 2H), 1.87-1.92 (m, 2H),
2.85-2.90 (m, 2H), 3.29-3.32 (m, 2H), 3.53-3.57 (m, 2H), 3.69
(s, 3H), 4.32 (t, J=4.8 Hz, 2H), 6.32 (d, J=2.0 Hz, 1H), 7.26 (d,
J = 8.6 Hz, 1H), 7.50 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 2.5 Hz,
1H), 7.88-7.94 (m, 3H), 8.15 (d, J =8.8 Hz, 2H), 9.81 (s, 1H),
10.5 (s, 1H). LCMS m/z=459.4 [M þ H]þ.
1
vide 8g as a yellow solid (9.44 g, 89%). H NMR (400 MHz,
DMSO-d6) δ: 2.92-3.58 (m, 6H), 3.69 (s, 3H), 3.75-3.85 (m, 2H),
4.54-4.56 (m, 2H), 4.77-4.85 (m, 2H), 6.39 (d, J = 1.8 Hz, 1H),
7.45 (d, J = 9.2 Hz, 1H), 7.52 (d, J = 1.8 Hz, 1H), 8.16 (d,
J=2.9 Hz, 1H), 8.43 (dd, J=9.2, 2.9 Hz, 1H), 8.88 (s, 1H). LCMS
m/z= 333.4 [M þ H]þ.
[3-(2-Methyl-2H-pyrazol-3-yl)-4-(2-pyrrolidin-1-yl-ethoxy)-
phenyl]-3-trifluoromethyl-benzamide (10b). 10b was prepared
in a manner similar to that described for 10a, using 9a (57 mg,
0.2 mmol) and 3-trifluoromethyl-benzoic acid (57 mg, 0.3 mmol)
4-{2-[2-(1-Methyl-1H-pyrazol-5-yl)-4-nitrophenoxy]ethyl}-
piperazin-2-one (8l). 8l was prepared in a manner similar to that
described for 8a, using 6 (500 mg, 1.53 mmol) and piperazin-2-
one (153 mg, 1.53 mmol) as starting materials. The TFA salt of
8l was isolated as a yellow solid (353 mg, 67%). 1H NMR (400
MHz, DMSO-d6) δ: 2.07 (s, 2H), 3.11-3.23 (m, 4H), 3.42-3.52
(m, 2H), 3.60-3.66 (m, 1H), 3.69 (s, 3H), 4.51-4.56 (m, 2H),
6.40 (d, J = 1.9 Hz, 1H), 7.45 (d, J = 9.3 Hz, 1H), 7.52 (d, J =
1.9 Hz, 1H), 8.16 (d, J=2.9 Hz, 1H), 8.43 (dd, J=9.2, 2.9 Hz,
1H). LCMS m/z=346.1 [M þ H]þ.
1
to give the TFA salt of 10b as a white solid (42 mg, 46%). H
NMR (CD3CN, 400 MHz) δ: 1.25-1.40 (m, 2H), 1.81-1.88 (m,
2H), 2.79-2.82 (bs, 2H), 3.36-3.46 (m, 4H), 3.71 (s, 3H), 4.32 (t,
J = 4.8 Hz, 2H), 6.29 (d, J = 1.9 Hz, 1H), 7.13 (d, J = 8.9 Hz,
1H), 7.46 (d, J=1.9 Hz, 1H), 7.64 (d, J=2.8 Hz, 1H), 7.72 (t, J=
7.8 Hz, 1H), 7.80 (dd, J=2.9, 8.9 Hz, 1H), 7.89 (d, J=7.9 Hz,
1H), 8.17 (d, J=7.9 Hz, 1H), 8.23 (s, 1H), 8.95 (s, 1H). LCMS
m/z =459.4 [M þ H]þ.
1-{4-[2-(2-(1-Methyl-1H-pyrazol-5-yl)-4-nitrophenoxy)ethyl]-
piperazin-1-yl}ethanone (8n). 8n was prepared in a manner
similar to that described for 8a, using 6 (325 mg, 1.0 mmol)
and 1-(piperazin-1-yl)ethanone (153 mg, 1.2 mmol) as starting
materials, to give the TFA salt of 8n as a yellow solid (423 mg,
100%). 1H NMR (400 MHz, DMSO-d6) δ: 2.02 (s, 3H), 2.74-
3.40 (m, 6H), 3.50-3.60 (m, 2H), 3.68 (s, 3H), 3.75-4.35 (m,
2H), 4.50-4.60 (m, 2H), 6.40 (d, J = 1.9 Hz, 1H), 7.45 (d, J =
9.3 Hz, 1H), 7.54 (d, J=1.9 Hz, 1H), 8.16 (d, J=2.9 Hz, 1H),
8.43 (dd, J= 9.2, 2.9 Hz, 1H). LCMS m/z= 374.3 [M þ H]þ.
General Procedure B, Reduction of Nitro Group to Intermedi-
ates 9: 3-(1-Methyl-1H-pyrazol-5-yl)-4-[2-(pyrrolidin-1-yl)ethoxy]-
aniline (9a). To a solution of 8a (2.52 g, 7.98 mmol) in saturated
ammonium chloride (4 mL), water (4 mL), and THF (10 mL)
was added zinc dust (2.81 g, 43 mmol) at 0 °C. The mixture was
stirred at rt for 1 h and then filtered through a pad of celite to
remove the solids. The filtrate was concentrated in vacuo to give
9a as a white solid in essentially quantitative yield (2.24 g).
LCMS m/z=287.2 [M þ H]þ. The product was used in the next
step without purification.
4-[2-(3,3-Difluoropyrrolidin-1-yl)ethoxy]-3-(1-methyl-1H-pyrazol-
5-yl)aniline (9c). 9c was prepared from 8c (258 mg, 0.73 mmol) in
a manner similar to that described for 9a, providing 9c as a white
solid in quantitative yield (230 mg). LCMS m/z = 323.2 [M þ
H]þ. The product was used in the next step without further
purification.
3-(1-Methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)aniline
(9d). 9d was prepared from 8d (1.91 g, 5.75 mmol) in a manner
similar to that described for 9a, providing 9d as a white solid in
quantitative yield (1.87 g). LCMS m/z = 303.4 [M þ H]þ. The
product was used in the next step without further purification.
4-{2-[4-Amino-2-(1-methyl-1H-pyrazol-5-yl)phenoxy]ethyl}-
piperazin-2-one (9l). 9l was prepared from 8l (353 mg, 1.02 mmol)
in a manner similar to that described for 9a, providing 9l as a white
solid (245 mg, 76%). LCMS m/z=315.8 [M þ H]þ. The product
was used in the next step without further purification.
General Procedure D, Preparation of Amides 10 via Acid
Chloride Coupling: N-{4-[2-(3,3-Difluoropyrrolidin-1-yl)ethoxy]-
3-(1-methyl-1H-pyrazol-5-yl)phenyl}-3-(trifluoromethyl)-benz-
amide (10c). A mixture of 9c (64 mg, 0.2 mmol), 3-trifluoro-
methylbenzoyl chloride (62 mg, 0.3 mmol), and triethylamine
(0.1 mL) in THF (2 mL) was stirred at room temperature for
20 min. The solvent was evaporated and the crude product was
purified by HPLC to give the TFA salt of 10c as a white solid
(33 mg, 33%). 1H NMR (acetone-d6, 400 MHz) δ: 2.43-2.52 (m,
2H), 3.38-3.46 (m, 4H), 3.56-3.61 (m, 2H), 3.75 (s, 3H), 4.45 (t,
J = 4.5 Hz, 2H), 6.29 (s, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.44 (s,
1H), 7.79-7.81 (m, 2H), 7.91-7.95 (m, 2H), 8.30 (d, J=8.3 Hz,
2H), 9.82 (s, 1H). LCMS m/z =495.3 [M þ H]þ.
2,4-Difluoro-N-[3-(2-Methyl-2H-pyrazol-3-yl)-4-(2-morpholin-
4-yl-ethoxy)-phenyl]-benzamide. (10d). 10d was prepared in a man-
ner similar to that described for 10c, using 9d (200 mg, 0.66 mmol)
and 2,4-difluorobenzoyl chloride (140 mg, 0.79 mmol) to give the
TFA salt of 10d as a white solid (99 mg, 34%); mp (HCl salt,
1
recrystallized from iPrOH) 140-142 °C. H NMR (acetone-d6,
400 MHz) δ: 2.92-3.06 (m, 2H), 3.13-3.17 (m, 2H), 3.50-3.58
(m, 2H), 3.68 (s, 3H), 3.82-3.85 (m, 4H), 4.36 (t, 2H), 6.29 (s, 1H),
7.24 (d, J = 9.1 Hz, 2H), 7.44 (dt, J = 10.1, 2.8 Hz, 1H), 7.49 (s,
1H), 7.67(s, 1H), 7.72-7.79 (m, 1H), 7.81 (dd, J=9.1, 2.8 Hz, 1H),
9.43 (s, 1H). LCMS m/z=443.5 [M þ H]þ.
3-Fluoro-N-[3-(2-methyl-2H-pyrazol-3-yl)-4-(2-morpholin-4-yl-
ethoxy)-phenyl]-benzamide (10e). 10e was prepared in a manner
similar to that described for 10c, using 9d (120 mg, 0.40 mmol)
and 3-fluorobenzoyl chloride (76 mg, 0.47 mmol) to give the
TFA salt of 10e as a white solid (102 mg, 61%). 1H NMR
(acetone-d6, 400 MHz) δ: 2.99-3.25 (m, 2H), 3.25-3.49 (m,
2H), 3.69 (t, J = 4.5 Hz, 2H), 3.75 (s, 3H), 3.77-3.96 (m, 4H),
4.59 (t, J=4.8 Hz, 2H), 6.30 (t, J=1.5 Hz, 1H), 7.23 (d, J=9.1Hz,
1H), 7.36 (dt, J = 2.5, 8.6 Hz, 2H), 7.49 (t, J = 1.8 Hz, 1H),
7.54-7.61 (m, 1H), 7.74 (dt, J=2.0,9.8Hz,1H), 7.82(t,J=2.4Hz,
1H), 7.84 (d, J=7.8 Hz, 1H), 7.94 (dt, J=2.4, 9.1 Hz, 1H), 9.64
(s, 1H). LCMS m/z =425.4 [M þ H]þ.

Article
4-Fluoro-N-[3-(2-methyl-2H-pyrazol-3-yl)-4-(2-morpholin-4-yl-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4419
(52 mg, 41%). 1H NMR (CD3CN, 400 MHz) δ: 3.16-3.21 (m,
2H), 3.30-3.36 (m, 2H), 3.48 (t, J = 4.8 Hz, 2H), 3.65 (s, 2H),
3.73 (s, 3H), 4.40 (t, J = 4.4 Hz, 2H), 6.33 (d, J = 1.8 Hz, 1H),
6.65 (s, 1H), 7.14 (d, J=9.0 Hz, 1H), 7.51 (d, J =1.7 Hz, 1H),
7.65 (d, J=2.6 Hz, 1H), 7.72 (t, J=7.8 Hz, 1H), 7.81 (dd, J=
2.6, 8,8 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.8 Hz,
1H), 8.23 (s, 1H), 8.93 (s, 1H). LCMS m/z =488.2 [M þ H]þ.
N-{3-(4-Chloro-2-methyl-2H-pyrazol-3-yl)-4-[2-(3-oxo-piper-
azin-1-yl)-ethoxy]-phenyl}-3-trifluoromethyl-benzamide (10m). 10m
was prepared in a manner similar to that described for 10c, using
4-{2-[4-amino-2-(4-chloro-1-methyl-1H-pyrazol-5-yl)phenoxy]-
ethyl}piperazin-2-one (88 mg, 0.25 mmol) and 3-trifluoromethyl
benzoyl chloride (52 mg, 0.25 mmol) to give the TFA salt of 10m as
a white solid (70 mg, 53%). 1H NMR (DMSO-d6, 400 MHz) δ:
3.20-3.24 (m, 4H), 3.62-3.78 (m, 8H), 4.31-4.38 (m, 2H), 7.31 (d,
J=9.1 Hz, 1H), 7.65 (s, 1H), 7.71 (d, J=2.8 Hz, 1H), 7.77-7.81
(m, 1H), 7.93-7.99 (m, 2H), 8.25-8.29 (m, 2H), 10.5 (s, 1H).
LCMS m/z=522.3/524.2 [M þ H]þ.
ethoxy)-phenyl]-benzamide (10f). 10f was prepared in a manner
similar to that described for 10a, using 9d (60 mg, 0.20 mmol)
and 4-fluorobenzoic acid (36 mg, 0.26 mmol) to give the TFA
salt of 10f as a white solid (22 mg, 26%). 1H NMR (DMSO-d6,
400 MHz) δ: 3.02 (m, 2H), 3.14-3.17 (m, 2H), 3.50-3.59 (m,
4H), 3.69 (s, 3H), 3.82-3.85 (m, 2H), 4.36 (t, J = 4.8 Hz, 2H),
6.29 (s, 1H), 7.24 (d, J = 9.1 Hz, 1H), 7.37 (t, J = 8.8 Hz, 2H),
7.49 (s, 1H), 7.72 (s, 1H), 7.88 (dd, J=2.8, 9.1 Hz, 1H), 8.03 (dd,
J = 5.6, 8.8 Hz, 2H), 9.86 (s, 1H), 10.3 (s, 1H). LCMS m/z =
425.4 [M þ H]þ.
N-[3-(4-Chloro-2-methyl-2H-pyrazol-3-yl)-4-(2-morpholin-4-
yl-ethoxy)-phenyl]-3-fluoro-benzamide (10g). 10g was prepared
in a manner similar to that described for 10c and was obtained as
the TFA salt as an off-white solid (44%). 1H NMR (DMSO-d6,
400 MHz) δ 3.10 (m, 4H), 3.59 (m, 4H), 3.67 (s, 3H), 3.87 (m,
2H), 4.34 (m, 1H), 7.31 (d, J=9.1 Hz, 1H), 7.46 (dt, J=2.5, 9.0
Hz, 1H), 7.59 (m, 1H), 7.67 (s, 1H), 7.73 (d, J=2.6 Hz, 1H), 7.77
(m, 1H), 7.82 (m, 1H), 7.96 (dd, J=2.6, 9.0 Hz, 1H), 10.3 (s, 1H).
LCMS m/z =459/461 [M þ H]þ.
N-{4-[2-(4-Acetylpiperazin-1-yl)ethoxy]-3-(1-methyl-1H-pyr-
azol-5-yl)phenyl}-3-(trifluoromethyl)benzamide (10n). 10n was
prepared in a manner similar to that described for 10c, using
9n (60 mg, 0.18 mmol) and 3-trifluoromethylbenzoyl chloride
(36 mg, 0.18 mmol) to give the TFA salt of 10n as a white solid
N-[3-(2-Methyl-2H-pyrazol-3-yl)-4-(2-morpholin-4-yl-ethoxy)-
phenyl]-4-trifluoromethyl-benzamide (10h). 10h was prepared
in a manner similar to that described for 10a, using 9d (60 mg,
0.20 mmol) and 4-trifluoromethylbenzoic acid (49 mg, 0.26 mmol)
1
(36 mg, 40%, 92% purity by LCMS). H NMR (CD3CN, 400
1
to give the TFA salt of 10h as a white solid (27 mg, 28%). H
MHz) δ: 1.94 (s, 3H), 3.32 (t, J = 4.8 Hz, 2H), 3.50-3.85 (m,
8H), 3.62 (s, 3H), 4.29 (t, J=4.8 Hz, 2H), 6.23 (s, 1H), 7.03 (d,
J = 9.1 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.55-7.82 (m, 4H),
8.08-8.10 (m, 1H), 8.15 (s, 1H), 8.86 (s, 1H). LCMS m/z=516.4
[M þ H]þ.
NMR (DMSO-d6, 400 MHz) δ: 3.00-3.03 (m, 2H), 3.16-3.19
(m, 2H), 3.52-3.60 (m, 4H), 3.70 (s, 3H), 3.84-3.87 (m, 2H),
4.39 (t, J = 5.0 Hz, 2H), 6.31 (s, 1H), 7.28 (d, J = 9.0 Hz, 1H),
7.51 (s, 1H), 7.75 (s, 1H), 7.94 (d, J=8.3 Hz, 2H), 8.16 (d, J=8.8
Hz, 2H), 8.89 (s, 1H), 9.88 (s, 1H), 10.5 (s, 1H). LCMS m/z =
475.4 [M þ H]þ.
N-{4-[2-(4-Acetylpiperazin-1-yl)ethoxy]-3-(1-methyl-1H-pyr-
azol-5-yl)phenyl}-3-fluorobenzamide (10o). 10o was prepared in
a manner similar to that described for 10c, using 9n (100 mg,
0.29 mmol) and 3-fluorobenzoyl chloride (46 mg, 0.29 mmol) to
give the TFA salt of 10o as a white solid (38 mg, 28%); mp (free
base, recrystallized from iPrOH/heptane=70/30) 139-140 °C.
1H NMR (CD3CN, 400 MHz) δ: 2.09 (s, 3H), 3.32-3.38 (m,
6H), 3.48 (t, J = 4.8 Hz, 4H), 3.79 (s, 3H), 4.43 (t, J = 4.8 Hz,
2H), 6.39 (d, J=2.0 Hz, 1H), 7.18 (d, J=9.1 Hz, 1H), 7.40 (dt,
J = 7.4, 8.6 Hz, 1H), 7.57-7.63 (m, 2H), 7.70-7.74 (m, 2H),
7.80-7.82 (m, 1H), 7.85 (dd, J=2.8, 9.1 Hz, 1H), 8.93 (s, 1H).
LCMS m/z =466.4 [M þ H]þ.
N-[3-(2-Methyl-2H-pyrazol-3-yl)-4-(2-morpholin-4-yl-ethoxy)-
phenyl]-3-trifluoromethyl-benzamide (10i). 10i was prepared in
a manner similar to that described for 10c, using 9d (50 mg,
0.16 mmol) and 3-trifluoromethylbenzoyl chloride and was obt-
ained as a white solid (72 mg, 92%). 1H NMR (DMSO-d6,
400 MHz) δ: 2.34 (t, J = 4.0 Hz, 4H), 2.61 (t, J = 8.0 Hz, 2H),
3.51 (t, J=4.0 Hz, 4H), 3.71 (s, 3H), 4.12 (t, J=8.0 Hz, 2H), 6.26
(d, J=1.8 Hz, 1H), 7.21 (d, J=9.0 Hz, 1H), 7.45 (d, J=1.8 Hz,
1H), 7.69 (d, J=2.6 Hz, 1H), 7.81-7.77 (m, 1H), 7.85-7.82 (m,
1H), 7.97 (d, J=7.8 Hz, 1H), 8.26 (d, J= 7.9 Hz, 1H), 8.29 (s,
1H), 10.4 (bs, 1H). LCMS m/z= 475 [M þ H]þ.
N-{4-[2-(4-Acetylpiperazin-1-yl)ethoxy]-3-(4-chloro-1-methyl-
1H-pyrazol-5-yl)phenyl}-3-fluorobenzamide (10p). 10p was pre-
pared in a manner similar to that described for 10c, using
1-{4-[2-(4-amino-2-(4-chloro-1-methyl-1H-pyrazol-5-yl)phenoxy)-
ethyl]piperazin-1-yl}ethanone (86 mg, 0.23 mmol) and 3-fluor-
obenzoyl chloride (43 mg, 0.27 mmol) to give the TFA salt of
3,4-Difluoro-N-[3-(2-methyl-2H-pyrazol-3-yl)-4-(2-morpholin-
4-yl-ethoxy)-phenyl]-benzamide (10j). 10j was prepared in a
manner similar to that described for 10c, using 9d (120 mg,
0.40 mmol) and 3,4-difluorobenzoyl chloride (84 mg, 0.48 mmol)
to give the TFA salt of 10j as a white solid (110 mg, 63%). 1H
NMR (acetone-d6, 400 MHz) δ: 3.11 (bs, 2H), 3.37 (s, 2H), 3.68
(t, J=4.8 Hz, 2H), 3.75 (s, 3H), 3.86 (bs, 4H), 4.59 (t, J=4.8 Hz,
2H), 6.30 (s, 1H), 7.23 (d, J=8.8 Hz, 1H), 7.48 (m, 2H), 7.80 (s,
1H), 7.87-7.92 (m, 2H), 7.93-7.99 (m, 1H), 9.67 (s, 1H). LCMS
m/z =443.3 [M þ H]þ.
1
10p as an off-white solid (33 mg, 29%). H NMR (CD3CN,
400 MHz) δ: 1.97 (s, 3H), 2.24-2.38 (m, 4H), 2.60-2.69 (m,
2H), 3.17 (d, J = 4.8 Hz, 2H), 3.31-3.36 (m, 2H), 3.67 (s, 3H),
4.07-4.13 (m, 2H), 7.25 (d, J=9.0 Hz, 1H), 7.44 (t, J=8.5 Hz,
1H), 7.56-7.62 (m, 1H), 7.63 (s, 1H), 7.71 (d, J =2.5 Hz, 1H),
7.77 (d, J=9.8 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.90 (dd, J=
9.0, 2.5 Hz, 1H), 10.3 (s, 1H). LCMS m/z=500.7 [M þ H]þ.
N-{4-[2-(4-Acetylpiperazin-1-yl)ethoxy]-3-(1-methyl-1H-pyr-
azol-5-yl)phenyl}-3-methoxybenzamide (10q). Step 1: Preparation
of 4-(2-Bromo-ethoxy)-3-(2-methyl-2H-pyrazol-3-yl)-phenylamine.
To a solution of N-[4-(2-bromo-ethoxy)-3-(2-methyl-2H-pyrazol-
3-yl)-phenyl]-acetamide (3 g, 8.9 mmol) in methanol (6 mL) at 0 °C
was added sulfuric acid (2.2 mL, 42 mmol). The reaction mixture
was then heated to reflux for 1.5 h. The mixture was cooled,
quenched with water, and cautiously neutralized with sodium
hydroxide solution until the pH was around 7. The mixture was
extracted twice with ethyl acetate, and the combined organic layers
concentrated in vacuo to give the title compound as an off-white
solid (2.6 g, 99%). LCMS m/z = 296.1/298.1 [M þ H]þ. The
intermediate was used in the next step without further purification.
Step 2: Preparation of N-[4-(2-Bromo-ethoxy)-3-(2-methyl-
2H-pyrazol-3-yl)-phenyl]-3-methoxy-benzamide. A mixture of
the intermediate obtained from step 1 (1.01 g, 3.42 mmol),
3-Methoxy-N-[3-(2-methyl-2H-pyrazol-3-yl)-4-(2-morpholin-
4-yl-ethoxy)-phenyl]-benzamide (10k). 10k was prepared in a
manner similar to that described for 10c, using 9d (120 mg,
0.40 mmol) and 3-methoxybenzoyl chloride (81 mg, 0.48 mmol)
to give the TFA salt of 10k as a white solid (88 mg, 51%); mp
1
(HCl salt, recrystallized from iPrOH) 214-216 °C. H NMR
(acetone-d6, 400 MHz) δ: 2.99-3.21 (m, 2H), 3.22-3.45 (m,
2H), 3.66 (t, J=4.8 Hz, 2H), 3.75 (s, 3H), 3.85 (s, 3H), 3.79-3.89
(m, 4H), 4.58 (t, J=4.8 Hz, 2H), 6.29 (d, J=2.0 Hz, 1H), 7.13
(dd, J = 2.5, 8.3 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.42 (t, J =
7.8 Hz, 1H), 7.47 (d, J = 1.7 Hz, 1H), 7.52 (t, J = 1.7 Hz, 1H),
7.56 (d, J=7.0 Hz, 1H), 7.80-7.83 (m, 1H), 7.91-7.96 (m, 1H),
9.54 (s, 1H). LCMS m/z = 437.5 [M þ H]þ.
N-{3-(2-Methyl-2H-pyrazol-3-yl)-4-[2-(3-oxo-piperazin-1-yl)-
ethoxy]-phenyl}-3-trifluoromethyl-benzamide (10l). 10l was pre-
pared in a manner similar to that described for 10c, using 9l
(79 mg, 0.25 mmol) and 3-trifluoromethyl benzoyl chloride
(63 mg, 0.30 mmol) to give the TFA salt of 10l as a white solid

4420 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Xiong et al.
3-methoxy-benzoyl chloride (700 mg, 4.10 mmol), and triethyl-
amine (1.0 mL) in THF (5 mL) was stirred at room temperature
for 10 min. Excess solvent was evaporated and the crude
product redissolved in acetonitrile. H2O was added slowly to
eventually provide an approximately 3/2 CH3CN/H2O v/v
mixture, and the resulting precipitate was collected by filtra-
tion and dried to give the title compound as an off-white solid
(957 mg, 65%). LCMS m/z=430.3/432.3 [M þ H]þ. This was
used in the next step without further purification.
human 5-HT2A and 5-HT2C receptors were developed using
crude plasma membranes prepared from HEK293 cells stably
expressing these receptors.21 The 5-HT2 agonist [125I]DOI (0.5nM)
was used as radioligand and nonspecific radioligand binding
was determined in the presence of 10 μM cold DOI. Competition
experiments consisted of addition of 95 μL of assay buffer (20 mM
HEPES, pH 7.4, 10 mM MgCl2), 50 μL of membranes (5-25 μg
protein), 50 μL of [125I]DOI (0.5 nM final assay concentration),
and 5 μL of test compound diluted in assay buffer (final con-
centrations ranging from 1 pM to 10 μM) to 96-well Perkin-
Elmer GF/C microtiter plates, and incubations were performed
for one hour at room temperature. Each radioligand competi-
tion study consisted of testing eight different concentrations in
which triplicate determinations were performed for each test
compound concentration. Assay incubations were terminated
by rapid filtration of microtiter plates under vacuum pressure
using a Brandell cell harvester, followed by washing filter plates
several times with ice-cold wash buffer (50 mM Tris-HCl, pH
7.4). Plates were then dried at 45 °C in an oven for a minimum of
two hours, 25 μL of BetaScint scintillation cocktail was added to
each well, and microtiter plates were counted in a Packard
TopCount scintillation counter.
Human Platelet Aggregation Assay. Platelet aggregation was
measured turbidometrically at 37 °C. Platelet rich plasma was
prepared from anticoagulated whole blood obtained from male
and female donors. Platelet concentrations in the platelet rich
plasma were adjusted to 250000 platelets/μL using platelet poor
plasma, then 500 μL of platelet rich plasma was preincubated at
37 °C and stirred at 1200 rpm with inhibitors for 1 min before
induction of aggregation by the simultaneous addition of 5-HT
(final concentration 1 μM) and ADP (final concentration 1 μM).
The maximal amplitude of aggregation response within 3 min
was measured in triplicate using the Chronolog model 490
aggregometer. Percent inhibition of aggregation was calculated
from the maximum decrease in optical density of the controls
and of the samples containing inhibitor.
Step 3: Preparation of N-{4-[2-(4-Acetylpiperazin-1-yl)ethoxy]-
3-(1-methyl-1H-pyrazol-5-yl)phenyl}-3-methoxybenzamide (10q).
A mixture of the intermediate obtained from step 2 (90 mg,
0.21 mmol), 1-piperazin-1-yl-ethanone (32 mg, 0.25 mmol), and
K2CO3 (116 mg, 0.84 mmol) in DMF (2 mL) was heated under
microwave irradiation at 120 °C for 20 min. The crude reaction
mixture was purified by HPLC to give the TFA salt of 10q as a
1
white solid (97 mg, 97%). H NMR (acetone-d6, 400 MHz) δ:
2.07 (s, 3H), 3.30 (bs, 4H), 3.71 (t, J=4.8 Hz, 2H), 3.75 (s, 3H),
3.79 (bs, 2H), 3.87 (s, 3H), 4.60 (t, J=4.8 Hz, 2H), 4.99 (bs, 2H),
6.32 (s, 1H), 7.13 (d, J=8.0 Hz, 1H), 7.23 (d, J= 9.0 Hz, 1H),
7.42 (t, J=8.0 Hz, 1H), 7.53 (m, 2H), 7.56 (d, J=7.8 Hz, 1H),
7.82 (s, 1H), 7.94 (dd, J=8.8, 2.5 Hz, 1H), 9.56 (s, 1H). LCMS
m/z =478.3 [M þ H]þ.
4-{2-[4-(3-Methoxy-benzoylamino)-2-(2-methyl-2H-pyrazol-
3-yl)-phenoxy]-ethyl}-piperazine-1-carboxylic Acid Amide (10r).
10r was prepared in a manner similar to that described for 10q,
using the intermediate from step 2 of 10q (90 mg, 0.21 mmol) and
piperazine-1-carboxamide (32 mg, 0.25 mmol) to give the TFA
1
salt of 10r as a white solid (52 mg, 52%). H NMR (CD3CN,
400 MHz) δ: 2.70-3.25 (m, 6H), 3.30-3.37 (m, 1H), 3.40 (t, J=
4.8 Hz, 2H), 3.71 (s, 3H), 3.86 (s, 3H), 4.36 (t, J =4.8 Hz, 2H),
5.13 (bs, 2H), 6.30 (s, 1H), 7.11 (d, J=9.0 Hz, 1H), 7.14 (d, J=
8.0 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.45 (s, 1H), 7.49 (d, J =
7.8 Hz, 1H), 7.51 (s, 1H), 7.63 (s, 1H), 7.80 (d, J=9.0 Hz, 1H),
8.75 (s, 1H). LCMS m/z = 479.4 [M þ H]þ.
3-Methoxy-N-{3-(1-methyl-1H-pyrazol-5-yl)-4-[2-(4-(methyl-
sulfonyl)piperazin-1-yl)ethoxy]phenyl}benzamide (10s). 10s was
prepared in a manner similar to that described for 10q, using
the intermediate from step 2 of 10q (90 mg, 0.21 mmol) and
1-(methylsulfonyl)-piperazine (41 mg, 0.25 mmol) to give the
TFA salt of 10s as a white solid (65 mg, 61%). 1H NMR
(CD3CN, 400 MHz) δ: 2.85 (s, 3H), 3.05-3.67 (m, 8H), 3.44
(t, J=4.7 Hz, 2H), 3.72 (s, 3H), 3.86 (s, 3H), 4.37 (t, J=4.7 Hz,
2H), 6.32 (d, J=1.8 Hz, 1H), 7.10-7.16 (m, 2H), 7.41-7.55 (m,
4H), 7.65 (d, J=2.4 Hz, 1H), 7.82 (dd, J=8.8, 2.5 Hz, 1H), 8.76
(s, 1H). LCMS m/z =514.5 [M þ H]þ.
Rat Pharmacokinetics. Male Sprague-Dawley rats were
dosed orally or intravenously at 10 and 2 mg/kg, respectively,
in 80% PEG400 and 20% phosphate buffeted saline. Animals
were fasted overnight prior to oral dose administration. Whole
blood samples were collected from the jugular vein over a 24 h
period postdose. Plasma was prepared from sodium heparin
treated whole blood and separated by centrifugation. Plasma
samples were assayed using a selective HPLC/MS/MS method.
The HPLC/MS/MS was operated in multiple reaction monitor-
ing (MRM) mode under optimized conditions for detection of
selected compounds and the internal standard using positive
ions formed by electrospray ionization. Quantitation was de-
termined using a weighted regression analysis of peak area ratios
of analyte and internal standard. The method provided a lower
limit of quantitation of 1 ng/mL and an upper limit of quantita-
tion of 2000 ng/mL. Serial sampling (at 0.25, 0.5, 1, 2, 4, 6, 8, 10,
12,15, 18 and 21 h post dosing) was used to define the plasma
concentration vs time profile.
N-{4-[2-(2-Hydroxyethylamino)ethoxy]-3-(1-methyl-1H-pyr-
azol-5-yl)phenyl}-3-methoxybenzamide (10t). 10t was prepared
in a manner similar to that described for 10q, using the inter-
mediate from step 2 of 10q (200 mg, 0.46 mmol) and ethanol-
amine (43 mg, 0.70 mmol) to give the TFA salt of 10t as a white
solid (150 mg, 78%). 1H NMR (DMSO-d6, 400 MHz) δ: 2.82-
2.90 (m, 2H), 3.30-3.36 (m, 2H), 3.50-3.55 (m, 2H), 3.69 (s,
3H), 3.83 (s, 3H), 4.24-4.28 (m, 2H), 6.32 (d, J = 1.8 Hz, 1H),
7.14-7.18 (m, 1H), 7.21 (d, J=9.1 Hz, 1H), 7.42-7.55 (m, 4H),
7.61 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 9.0, 2.5 Hz, 1H), 7.89-
7.95 (br s, 2H), 10.2 (s, 1H). LCMS m/z = 411.4 [M þ H]þ.
N-[4-(2-Aminoethoxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl]-
3-methoxybenzamide (10u). 10u was prepared in a manner
similar to that described for 10q, using the intermediate from
step 2 of 10q (500 mg, 1.16 mmol) and ammonia in methanol
(11.6 mmol) to give the TFA salt of 10u as a white solid (226 mg,
Acknowledgment. We thank Juan Ramirez, Chen Liaw,
William J. Thomsen, and Joel Gatlin for additional technical
support.
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