The discovery of 4-{l-[({2,5-dimethyl-4-[4-(trifluoromethyl)benzyl]- 3thienyl}carbonyl)amino]cyclopropyl}benzoic acid (MK-2894), a potent and selective prostaglandin e2 subtype 4 receptor antagonist

Article abstract of DOI:10.1021/jm901771h

The discovery of highly potent and selective second generation EP4 antagonist MK-2894 (34d) is discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.

Full text of DOI:10.1021/jm901771h

J. Med. Chem. 2010, 53, 2227–2238 2227
DOI: 10.1021/jm901771h
The Discovery of 4-{1-[({2,5-Dimethyl-4-[4-(trifluoromethyl)benzyl]-3-
thienyl}carbonyl)amino]cyclopropyl}benzoic Acid (MK-2894), A Potent and Selective
Prostaglandin E2 Subtype 4 Receptor Antagonist†
Marc Blouin,*,‡ Yongxin Han,*,‡ Jason Burch,‡ Julie Farand,‡ Christophe Mellon,‡ Mireille Gaudreault,§ Mark Wrona,§
§
Jean-Franc-ois Levesque, Danielle Denis, Marie-Claude Mathieu, Rino Stocco, Erika Vigneault, Alex Therien,
ꢀ
Patsy Clark,^ Steve Rowland,^ Daigen Xu,^ Gary O’Neill, Yves Ducharme,‡ and Rick Friesen‡
‡Department of Medicinal Chemistry, §Department of DMPK, Department of In Vitro Sciences, and ^Department of In Vivo Sciences,
Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada Ltd., 16771 Trans-Canada Highway, Kirkland, QC, Canada H9H 3L1
Received November 30, 2009
The discovery of highly potent and selective second generation EP4 antagonist MK-2894 (34d) is
discussed. This compound exhibits favorable pharmacokinetic profile in a number of preclinical species
and potent anti-inflammatory activity in several animal models of pain/inflammation. It also shows
favorable GI tolerability profile in rats when compared to traditional NSAID indomethacin.
Introduction
PGE2 exerts its biological effects through four subtype EP
receptors, EP1-4. In a mouse model of experimental arthritis
(CAIA), the EP4-/- mice showed a remarkable resistance to
both the incidences and symptom scores (paw swelling/
redness, ankylosis) of arthritis compared to the wild type
controls while the EP1-3-/- mice showed no effect, suggesting
that the effect of PGE2 in chronic inflammation was mediated
predominantly by the EP4 receptor.5 It is worth mentioning
that EP4 may not be the only mediator of chronic inflamma-
tion, as both IP signaling and combined EP2/EP4 signaling
werereportedtomediate joint inflammationin amousemodel
of CIA.6 Lin et al. demonstrated that EP4, not EP1-3, con-
tributed to inflammatory pain hypersensitivity in rats.7 Using
highly selective EP1, EP3, and EP4 antagonists, we demon-
strated that EP4, not EP1 or EP3, is the primary receptor
involved in joint inflammation and pain in rodent models of
rheumatoid and osteoarthritis,8 further supporting EP4 an-
tagonism as a valid strategy for treating inflammatory pain. It
is plausible that a selective EP4 antagonist may ameliorate
symptoms of chronic inflammation without the potential CV
side effects observed with NSAIDs and COX-2 inhibitors
because it does not interfere with the biosynthesis of any of the
prostanoids, including prostacyclin and thromboxanes.
In addition to its role in inflammation, the EP4 receptor has
also been implicated in destabilizing atherosclerotic plaques in
human,9 in developing tumor metastasis,10 in migraine head-
ache,11 and in Alzheimer’s disease.12 Therefore, EP4 antagon-
ism represents a potential promising new therapeutic approach
for treating inflammatory pain, atherosclerosis, cancer, mi-
graine, and Alzheimer’s disease and has been the subject of
recent research interests.
Prostanoids (prostaglandins and thromboxanes) are im-
portant lipid hormones formed from arachidonic acid meta-
bolism. PGE2,a in particular, is the principal proinflammatory
prostanoid and is implicated in the pathogenesis of a number
of diseases such as pain, fever, arthritis and cancer. Inhibition
of PGE2 production by NSAIDs and COX-2 inhibitors
(coxibs) relieves arthritis symptoms and thus is the basis of
widespread uses of these drugs as analgesics.1 Unfortunately,
the therapeutic utilities of these drugs are limited by their
potential to cause either GI toxicity (by NSAIDs)2 or CV side
effects (by both NSAIDs and coxibs).3 The CV adverse events
associated with these drugs are not clearly understood,
although it is speculated that inhibition of prostacyclin bio-
synthesis may cause the prothrombotic and hypertensive
effects.4 Therefore, there is a vast unmet medical need to
discover safer alternatives for treating chronic ailments such
as arthritis.
†Previously disclosed as MF482 at the ACS National Meeting,
Washington, DC, August 16-20, 2009.
*To whom correspondence should be addressed. For M.B.: phone,
(514) 428-3240; fax, (514) 428-4900; E-mail, marc_blouin@merck.com.
For Y.H.: phone, (514) 428-3301; fax, (514) 428-4900; E-mail, yongxin_han@
merck.com.
a Abbreviations: AIA, adjuvent-induced arthritis; CAIA, collagen
antibody-induced arthritis; cAMP, cyclic adenosine monophosphate;
CIA, collagen-induced arthritis; CFA, complete Freund’s adjuvant;
COX, cyclooxygenase; CV, cardiovascular; CYP, cytochrome P450;
DIEA, N,N-diisopropylethylamine; DP1-2, prostaglandin D2 receptor
subtypes 1 and 2; EBNA, Epstein-Barr nuclear antigen; EDTA,
ethylenediaminetetraacetic acid; EP1-4, prostaglandin E2 receptor sub-
types 1, 2, 3, and 4; FP, prostaglandin F2a receptor; GI, gastrointestinal;
GSH, reduced glutathione; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N0,
N0-tetramethyluronium hexafluorophosphate; HEK, human embryonic
kidney; HS, human serum; HWB, human whole blood; IP, prostaglan-
din I2 receptor; IP-10, interferon-inducible protein 10; NADPH, re-
duced nicotinamide adenine dinucleotide phosphate; NBS, N-
bromosuccinimide; NCS, N-chlorosuccinimide; NSAID, nonsteroidal
anti-inflammatory drug; PGE2, prostaglandin E2; SD, Sprague-Daw-
ley; TFA, trifluoroacetic acid; TNF, tumor necrosis factor; TP, throm-
boxane A2 receptor.
Previously, we disclosed the discovery of our first genera-
tion EP4 antagonists MF498 (1)8 and MF310 (2)13 (Figure 1).
While 2 is an extremely potent compound in the rat AIA
model with an ED50 of 0.003 mg/kg/day, it suffers from CYP
3A mediated hydrolysis of the acylsulfonamide moiety, giving
rise to the corresponding sulfonamide M1. This metabolite
r
2010 American Chemical Society
Published on Web 02/17/2010
pubs.acs.org/jmc

2228 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Blouin et al.
Figure 1. Chemical structure of EP4 antagonists 1, 2, 34d, and 37, metabolite M1, and COX-2 inhibitor 41.
has the potential to cause drug-drug interaction due to the
fact it is a moderate time-dependent CYP 3A4 inhibitor and a
CYP 3A4 inducer and has the potential to accumulate.14 SAR
effort to prevent the acylsulfonamide hydrolysis was unsuc-
cessful, so we turned our attention to nonacylsulfonamide
analogues which resulted in the discovery of a highly selective
and potent second generation EP4 antagonist, 34d (MK-2894)
(Figure 1). We herein describe the SAR leading to 34d, its
in vitro profile and pharmacokinetic properties, its in vivo
efficacy in models of pain and inflammation, and its GI
tolerability profile in rats.
15 and 16 via monolithiation using n-butyllithium followed
by quenching the anionic intermediates with appropriately
substituted benzaldehydes. The thiophenecarboxylic acids
19 and 20 were obtained using similar procedures described
for the transformation of alcohol 7 to acid 9 in Scheme 1. To
prepare the 2,5-dichlorothiophene-containing analogues 27,
the desired carboxylic acid 21 was obtained by the treatment
of the 2,5-unsubstituted precursor 19 with NCS in hot acetic
acid.17 Compounds 26-28 were obtained from acids 19-21
in a similar manner as described in Scheme 1 using enantio-
merically pure (S)-R-methylbenzylamine 22.16
Chemistry. The synthetic routes developed for the synth-
esis of the thiophene-based EP4 antagonists are outlined in
Schemes 1-4.
The cyclopropylbenzamide derivatives 34 were prepared
by first submitting 1,4-dicyanobenzene (29) to the
Szymoniak variation18 of the Kulinkovich reaction in
which one of the cyano groups was modified to afford
the cyclopropylamine derivative 30 (Scheme 3). The other
cyano group was then hydrolyzed in boiling 6 N hydro-
chloric acid, and the resulting carboxylic acid 31 was
esterified to give the methyl ester 32. The latter was reacted
with the 2,5-dimethylthiophenecarboxylic acids 20 under
the same conditions described above to provide the EP4
antagonists 34 after hydrolysis of the methyl esters 33.
The synthesis of the tetrazole derivative 36 was conveni-
ently accomplished by coupling of carboxylic acid 20d with
cyclopropylamine 30, followed by treating the resultant
benzonitrile 35 with azidotributyltin in refluxing toluene
(Scheme 4).19
Compound 12 was prepared according to Scheme 1.
Commercially available 3-thiophenemethanol (3) was bro-
minated at the 2-position using NBS15 to afford alcohol 4
that was subsequently treated with NCS15 to provide the 2,5-
dihalogenated thiophene derivative 5. Oxidation under the
Swern conditions gave aldehyde 6 that was reacted with
3-chlorophenylmagnesium bromide to yield alcohol 7.
Deoxygenation of 7 by treatment with TFA/triethylsilane
yielded bromide 8. Metal-bromine exchange and subse-
quent trapping of the resultant organometallic species with
carbon dioxide gave the 2-thiophenecarboxylic acid 9. Cou-
pling of acid 9 with the racemic R-methylbenzylamine 10
using HATU as the coupling reagent,16 followed by hydro-
lysis of the obtained methyl ester 11 under standard condi-
tions furnished 2-thiophenecarboxamide 12.
Results and Discussion
The synthesis of the compounds bearing carboxamide and
benzyl groups at the 3,4-positions of the thiophene core is
illustrated in Scheme 2. 3,4-Dibromothiophene derivatives
13 and 14 were converted to the secondary benzylic alcohols
SAR. EP4 antagonists bearing a benzoic acid moiety were
reported recently.20 For example, CJ-042,794 (37), illu-
strated in Figure 1, was reported to be a potent and selective

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2229
Scheme 1. Synthesis of 2,3-Substituteda Thiophene 12b
a The 2,3-substitution refers to the carboxamide and benzyl groups respectively. b Reagents and conditions: (a) NBS, THF, H2O, 0 ꢀC to rt; (b) NCS,
THF, H2O, rt; (c) DMSO, (COCl)2, Et3N, CH2Cl2, -78 ꢀC to rt; (d) 3-chlorophenylmagnesium bromide, THF, Et2O, -78 ꢀC; (e) TFA, Et3SiH, CH2Cl2,
rt; ( f ) n-BuLi, CO2, THF, -78 to 0 ꢀC; (g) HATU, DIEA, DMF, 0 ꢀC; (h) 1 M aq LiOH, THF, MeOH, rt.
EP4 antagonist with good pharmacokinetic profile and was
shown to be efficacious in two rat models of pain and
inflammation.20c
and selectivity. Interestingly, compound 28a also behaved as
a weak partial agonist. We further observed that the agonist/
antagonist balance could be influenced dramatically by the
substitution pattern of the benzyl group. Shifting the sub-
stituent from the meta-position to the para-position, as seen
in compounds 28c and 28d (Table 1, entries 5 and 6), gave rise
to full antagonists with further improved potency and selec-
tivity. Interestingly, we observed that the affinity and selec-
tivity profile of enantiomers 27a (Table 1, entry 2) and 39
(Figure 3 and Table 1, entry 10) were essentially identical,
suggesting that the chiral center was not critical for either
affinity or selectivity. Removing the methyl group in 27a
gave compound 40 (Figure 3 and Table 1, entry 11), which
maintained the EP4 affinity but lost selectivity against the
EP2 receptor. Substituting the chiral R-methylbenzamide
moiety in 28d with an achiral cyclopropylbenzamide furnis-
hed the title compound 34d with further improved affinity
and selectivity. 34d is a high affinity full antagonist against
the EP4 receptor with a Ki of 0.56 nM and an IC50 of 2.5 nM.
It is not significantly shifted in the presence of 10% HS in
either the binding assay or the functional assay (Table 2). It
also exhibits exquisite selectivity against all other prostanoid
receptors (>7000-fold) (Table 2) and does not display sig-
nificant activity (IC50 < 10 μM) against a panel of >170
enzymes and receptors in a MDS Pharma screen. As shown
in Table 3, 34d exhibits favorable pharmacokinetic profile in
preclinical species with moderate bioavailability (F =
18-32%), good elimination half-lives (T1/2 = 4.5-19 h) and
slow to moderate clearance rate (CL = 3.2-23 mL/min/kg).
We also prepared the corresponding tetrazole analogue 36
(Scheme 4 and Table 1, entry 9) for comparison because
tetrazole is a classical carboxylic acid bioisostere. While 36
Our efforts focused on compounds bearing suitable het-
erocyclic templates in place of the central phenyl core in 37.
By screening a number of monocyclic or bicyclic hetero-
cycles, we discovered that thiophene is an excellent replace-
ment. Interestingly, we observed that the regiochemistry of
the carboxamide and benzyl substituents, in compounds
such as 12 (Scheme 1 or Figure 2) and 26a (Scheme 2 or
Table 1, entry 1), had a profound effect on the agonist/
antagonist properties of these compounds. For instance,
while the 2,3-substituted analogue 12 (Figure 2) behaved as
a full agonist in the EP4 functional assay, the 3,4-substituted
analogue 26a (Table 1, entry 1) was a partial agonist
(10% maximum agonist activity vs PGE2) when the 2- and
5-positions are not substituted. This compound, however,
was not selective against the EP4 receptor. Switching over
the carboxamide and benzyl substituent of 12, such as in
the fluorinated analogue 38 (Figure 2), produced a 4-fold
decrease in EP4 binding affinity with an IC50 of 12 nM versus
3.0 nM for 12. Substituting the 2- and 5-positions of 26a with
chlorine atoms gave compound 27a (Table 1, entry 2) with
significantly improved potency and selectivity. It also be-
haved as a full antagonist in the EP4 functional assay. This
compound, unfortunately, suffered from extensive covalent
protein binding when incubated with human or rat liver
microsomes in vitro in the presence of cofactor NADPH, as
indicated by extensive formation of a GSH adduct. Replac-
ing the chlorine atoms with methyl groups as in com-
pound 28a (Table 1, entry 3) completely circumvented the
covalent protein labeling issue while maintaining potency

2230 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Blouin et al.
Scheme 2. Synthesis of 3,4-Substituteda Thiophene-containing EP4 Antagonists 26-28b
a The 3,4-substitution refers to the carboxamide and benzyl groups respectively. b Reagents and conditions: (a) n-BuLi, YC6H4CHO, Et2O, -78 to
0 ꢀC; (b) TFA, Et3SiH, CH2Cl2, 0 ꢀC; (c) n-BuLi, CO2, Et2O/THF, -78 to 0 ꢀC; (d) NCS, AcOH, 110 ꢀC; (e) HATU, DIEA, DMF, 0 ꢀC; (f ) 1 M aq
LiOH, THF, MeOH, rt or 50 ꢀC.
Scheme 3. Synthesis of Cyclopropylbenzamide EP4 Antagonists 34a
a Reagents and conditions: (a) Ti(OiPr)4, EtMgBr, BF3 Et2O, CH2Cl2, rt; (b) 6 N HCl, reflux; (c) cat. HCl, MeOH, reflux; (d) HATU, DIEA, DMF,
3
0 ꢀC; (e) 1 M aq LiOH, THF, MeOH, 50 ꢀC.
maintained comparable affinity, selectivity (except for EP1,
Ki = 500 nM), and functional potency in vitro, it exhibited
poor oral bioavailability (F = 1%) in rat, and as a result, was
not pursued further.
Biology. The radio ligand binding assays to determine
affinity and selectivity of compounds were performed ac-
cording to reported literature procedures in membranes pre-
pared from HEK 293(EBNA) clonal cell lines overexpressing

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2231
Scheme 4. Synthesis of Tetrazole Derivative 36a
a Reagents and conditions: (a) HATU, DIEA, DMF, 0 ꢀC; (b) Bu3SnN3, toluene, reflux.
exhibited comparable maximum efficacy but were much less
potent with ED50s of 0.7 and 0.5 mg/kg/day, respectively. In
addition, the potency and efficacy of EP4 antagonist 34d is
clearly superior to that reported for 37 (Figure 1) in the same
model (ED80 = 11.5 mg/kg).20c
As illustrated in Figure 5, 34d exhibited excellent GI
tolerability in the rat model of GI mucosal permeability.
Dosing 34d at 3, 10, and 30 mg/kg/day for 5 days did not
cause significant 51Cr leakage, while dosing indomethacin at
5 mg/kg/day for three days resulted in significant increase in
urinary 51Cr excretion due to increased GI mucosal perme-
ability. This result corroborates well with results obtained
with 18 and 37,20b further demonstrating that a highly
selective EP4 antagonist may have superior GI tolerability
profile compared to traditional NSAIDs.
Figure 2. Structure of 2,3-Substituted thiophene-containing EP4
ligands 12 and 38.
the human prostanoid receptors EP1-4, DP1-2, TP, FP, and
IP and their respective 3H-prostaglandins or thrombox-
ane,21 and was further described in detail in our recent
publication.8 The EP4 functional assay (PGE2-induced
cAMP accumulation in the same cell line) to determine EP4
agonist/antagonist activity was performed according to the
procedure reported by Clark et al.8 A partial agonist gen-
erally is unable to completely inhibit the PGE2 effect, and can
partially potentiate cAMP accumulation in the absence of
PGE2. The EP4 binding and functional assays were per-
formed in the absence and/or presence of 10% HS in order
to assess the extent of serum protein binding of compounds.
The HWB assay (blockade of inhibition of TNFR-induced
IP-10 release by EP4 agonist L-902688 in HWB) was per-
formed similarly to the procedure reported by Murase
et al.,20d except that a specific EP4 agonist, L-902688, was
used instead of PGE2.22
In Vivo Efficacy and Tolerability. The AIA model and GI
tolerability model in male SD rats were described in ref 8.
The acute carrageenan-induced mechanical hyperalgesia
model in SD rats was performed according to a published
literature procedure.23 In this model, 34d displayed a dose-
dependent inhibition of pain response when measured at 3 h
post subplantar injection of carrageenan and 1 h post oral
dose of compound (Figure 4a). The ED50 was established as
0.36 mg/kg with a corresponding EC50 of 250 nM. The
maximum inhibition reached was 60-70% [vs 70% for
COX-2 inhibitor MF-tricyclic (41)24 (Figure 1) in the same
experiment] when compared to the vehicle control.
Conclusion
We have described the discovery and preclinical character-
ization of the highly potent and selective EP4 antagonist 34d.
In rodent models of pain and inflammation, 34d exhibited
comparable maximum efficacy to coxibs and NSAIDs but
was significantly more potent. In the rat GI tolerability model
measuring mucosal permeability, 34d exhibited no obvious
effect to the GI mucosal integrity at doses up to 30 mg/kg for
5 days while indomethacin caused significant GI mucosal
damage in 3 days. Overall, 34d displays the desired potency,
selectivity, pharmacokinetic, and GI tolerability profile for
further development and represents a potential safer alter-
native for treating pain and inflammation than traditional
NSAIDs and coxibs.
Experimental Section
Chemistry. All commercial chemicals were used without
further treatment. Anhydrous solvents were purchased from
Sigma-Aldrich (Milwaukee, WI), Acros Organics (Fisher Scien-
tific, Nepeau, ON, Canada), or American Chemicals Ltd.
(Montreal, QC, Canada). All air-sensitive reactions were carried
out under an atmosphere of dry nitrogen. TLC analyses were
performed on Merck 60 F254 silica gel glass-backed plates, with
spots detected visually under ultraviolet irradiation (254 nm).
Purification of the crude reaction mixtures was performed as
indicated using chromatography on silica gel, either by standard
flash method (glass column, Merck Kieselgel 60, 230-400 mesh)
or by using a CombiFlash apparatus (Teledyne Isco, Lincoln,
NE) equipped with columns of appropriate size (Teledyne Isco,
Lincoln, NE, or Silicycle, Quebec City, QC, Canada). All tested
compounds possess a purity g95% (except 38, 91%) as deter-
mined by reversed-phase HPLC and/or combustion analysis.
Proton NMR spectra were obtained using a Bruker Avance 400
spectrometer, at 400 MHz, and were recorded in the indicated
solvent. Chemical shifts (δ) were recorded in parts per million
(ppm) and reported relative to the solvent peak. High-resolution
In the rat AIA model, 34d exhibited potent activity in
inhibiting chronic paw swelling, in both the primary paw and
the secondary paw, in a dose-dependent manner, with an
ED50 of 0.02 mg/kg/day and an EC50 at 24 h after the last
dose of ∼1 nM, reflecting its potent in vitro activity
(Figure 4b, only data from the secondary paw is shown).
Complete inhibition of the secondary paw swelling was
achieved at an ED100 of 0.1 mg/kg/day with a plasma
concentration of 4 nM at 24 h after the final dose. In
comparison, potent COX-2 inhibitors such as rofecoxib25 and
41 (Figure 1),24 in the same model but different experiments,

2232 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Blouin et al.
Table 1. In Vitro Profile of 3,4-Substituteda Thiophene-Containing EP4 Antagonistsb
binding affinity
(Ki, nM)c
EP4 functional
potency (IC50, nM)e
entry
compd
X
Y
R1
CH3
R2
A
EP4
EP2
othersd
1
26a
27a
28a
28b
28c
28d
34b
34d
36
H
Cl
m-Cl
m-Cl
H
H
H
H
H
H
p-COOH
p-COOH
p-COOH
p-COOH
p-COOH
p-COOH
p-COOH
p-COOH
5-tetrazolyl
p-COOH
p-COOH
6.4 ( 2.6
1.3 ( 0.6
3.1 ( 0.4
2.5 ( 0.2
2.9 ( 0.7
16 ( 2
746 ( 231
467 ( 228
800 ( 187
202 ( 40
>7282
>456
12 ( 5
3.6 ( 2.0
8.6 ( 1.0
4.7 ( 3.5
5.4
2
CH3
CH3
3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
m-Cl
m-CF3
p-Cl
>2226
>3461
>2117
>5474
>4992
>3900
>501
4
CH3
CH3
5
6
p-CF3
m-CF3
p-CF3
p-CF3
m-Cl
CH3
1.4 ( 0.4
1194 ( 429
>29142
4.5 ( 1.2
nd f
7
-CH2-
-CH2-
-CH2-
H
-CH2-
-CH2-
-CH2-
CH3
0.75 ( 0.20
0.56 ( 0.10
0.63 ( 0.11
0.87 ( 0.20
0.72 ( 0.29
8
5900 ( 3300
>29034
2.5 ( 0.7
2.3
1.8
9
10
11
39
40
384 ( 108
43 ( 12
>224
>628
Cl
m-Cl
H
H
1.6
a The 3,4-substitution refers to the carboxamide and benzyl groups, respectively. b Values are means from at least three experiments, except for EP4
functional potency for compounds 28c, 36, 39, and 40 (n = 1). c For details on the binding assay for all PG receptors, see ref 8. d Other PG receptors refers
to EP1, EP3, DP1, DP2, IP, TP, FP. e The EP4 functional assay measured the inhibition of PGE2-induced cAMP accumulation in HEK 293 cells; for
details, see ref 8. f nd: not done.
Table 2. In Vitro Profile of 34da
binding affinityb
receptor
EP4
Ki, nM
0.56 ( 0.10
0.97 ( 0.23
>5600
EP4 þ 10% HS
EP1
EP2
EP3
DP1
DP2
IP
5900 ( 3300
>7900
>3900
Figure 3. (R)-Enantiomer (39) and unsubstituted analogue (40) of
(S)-R-methylbenzamide derivative 27a.
>21000
>22000
>6800
mass spectra (HRMS) were performed in the ESI mode using an
Agilent Technologies MSD TOF mass spectrometer coupled to
an Agilent Technologies 1200 series LC. Reversed-phase HPLC
purity determinations were performed at three different wave-
lengths (λmax, 220 and 254 nm) on an Agilent Technologies 1100
series system equipped with a Zorbax RxC18 column (150 mm ꢀ
4.6 mm, 5 μ) using an acetonitrile/water gradient containing
0.1% formic acid. Elemental analyses were performed by Pre-
valere Life Sciences, Inc. (Whitesboro, NY). LC/MS/MS ex-
periments for the pharmacokinetic studies were conducted using
a Perkin-Elmer Sciex API 2000 instrument equipped with a
Phenomenex Luna C18 column (50 mm ꢀ 2 mm, 5 μ) using an
acetonitrile/water gradient containing 0.1% formic acid.
Pharmacokinetic Studies. All aspects of housing, care and use
of the animals were in accordance with the guidelines of the
Canadian Council on Animal Care and the “Guide to the Care
and Use of Experimental Animals.” SD rats, C57BL/6 mice,
beagle dogs, and cynomolgus monkeys were fasted overnight
and prior to dosing given either by oral gavage or intravenously.
At various time points, blood samples were collected, and this
blood was centrifuged to provide plasma samples. The plasma
was stored at -10 ꢀC until time of analysis. At this time, aliquots
of plasma samples were treated with an equal amount of
acetonitrile, containing 0.1% formic acid, to precipitate pro-
teins. The samples thus obtained were vortexed and centrifuged.
TP
FP
>24000
0.70 ( 0.16
>29000
7200
rEP4
rEP1
rEP2
rEP3
3300
EP4 functional potencyc
cell line
IC50, nM
HEK 293
HEK 293 þ 10% HS
2.5 ( 0.7
2.9 ( 1.1
11 ( 9
HWB
a Values are means from at least three experiments, except for rEP2
and rEP3 (n = 1); HS, human serum; r, rat. b For details on the binding
assay for all PG receptors, see ref 8. c The EP4 functional potency was
evaluated in HEK 293 cells, where the inhibition of PGE2-induced cAMP
accumulation is measured (for details, see ref 8) or in human whole blood
(HWB) where the blockade of inhibition of TNFR-induced IP-10 release by
an EP4 agonist was measured (for details, see ref 24).
The resulting supernatants were transferred into sample vials
and subsequently analyzed by LC/MS/MS in the presence of an
internal standard. Reference standard samples for each test
compounds were prepared as described above, using plasma
derived from the blood of untreated animals and subsequently

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2233
spiked with known concentrations of test compounds in an
appropriate range.
Receptor Binding, Functional and HWB Assays. The receptor
binding assays and the EP4 funtional assay were conducted
according to the procedure described in ref 8. The EP4 HWB
assay was perfomed as described in ref 22.
was added oxalyl chloride (1.90 mL, 26.8 mmol) dropwise, and
the mixture was stirred for 30 min at room temperature. 5
(4.50 g, 19.8 mmol) in CH2Cl2 (25 mL) was added, and the
resulting solution was stirred for 30 min. Triethylamine (6.40
mL, 45.5 mmol) was then added in one portion, and the mixture
was stirred at -78 ꢀC for 30 min and allowed to warm slowly
in air. After concentration in vacuo, the residue was suspended
in Et2O and then filtered. The filtrate was concentrated in vacuo
to give 6. The crude product was used in the next step directly,
In Vivo Efficacy and Tolerability Models. The rat AIA model
was performed under the conditions published in ref 8. The rat
carrageenan-induced mechanical hyperalgesia model was con-
ducted according to the procedure described in ref 23. The GI
tolerability model in rat was described previously in ref 8 and
performed accordingly.
2-Bromo-3-hydroxymethylthiophene (4). To a solution of
commercially available 3-thiophenemethanol (3) (8.20 g, 71.8
mmol) in THF (150 mL) at 0 ꢀC was added water (10 mL)
followed by solid NBS (12.8 g, 71.8 mmol), and the solution was
stirred at room temperature for 1 h. Most of the solvent was
evaporated in vacuo, and the residue was redissolved in EtOAc
and washed with water (3ꢀ) and brine. The organic layer was
dried (MgSO4), filtered, and concentrated to give 4 as a yellow-
ish oil. The crude product was used in the next step directly,
without further purification. 1H NMR (400 MHz, acetone-d6) δ
7.50 (d, 1H), 7.12 (d, 1H), 4.55 (s, 2H).
1
without further purification. H NMR (400 MHz, CDCl3) δ
9.81 (s, 1H), 7.20 (s, 1H).
(2-Bromo-5-chloro-3-thienyl)(3-chlorophenyl)methanol (7). To
a solution of 6 (2.50 g, 11.1 mmol) in a mixture of THF (20 mL)
and Et2O (20 mL) at -78 ꢀC was added 3-chlorophenylmagne-
sium bromide (0.5 M in THF, 26.6 mL, 13.3 mmol) within 2 min.
The mixture was stirred at the same temperature for 5 min,
quenched with saturated NH4Cl, and extracted with EtOAc.
The organic layer was washed with brine, dried (MgSO4), and
filtered. The filtrate was concentrated, and the residue was
purified by flash chromatography (EtOAc/hexane 10:90 to
20:80) to give 7 (2.51 g, 67%). 1H NMR (400 MHz, acetone-d6)
δ 7.52 (s, 1H), 7.39 (d, J = 5.1 Hz, 2H), 7.32 (m, 1H), 7.00 (s, 1H),
5.89 (d, J = 4.1 Hz, 1H), 5.36 (d, J = 4.1 Hz, 1H).
(2-Bromo-5-chloro-3-thienyl)methanol (5). To a solution of 4
(13.0 g, 67.3 mmol) in a mixture of THF (100 mL) and water (10
mL) was added NCS (9.88 g, 74.0 mmol), and the resulting
mixture was stirred at room temperature for 5 h and concentrated
in vacuo. The residue was redissolved in EtOAc and washed with
water (3ꢀ) and brine. The organic layer was dried (MgSO4),
filtered, and concentrated to give 5. The crude product was used
2-Bromo-5-chloro-3-(3-chlorobenzyl)thiophene (8). To a solu-
tion of 7 (2.50 g, 7.40 mmol) in CH2Cl2 (30 mL) at room
temperature was added TFA (5.68 mL, 74.0 mmol) (red solution
formed) followed by triethylsilane (5.91 mL, 37.0 mmol) (red
solution turned to yellow). The mixture was stirred at room
1
in the next step directly, without further purification. H NMR
(400 MHz, acetone-d6) δ 7.03 (s, 1H), 4.52 (d, 2H), 4.45 (t, 1H).
2-Bromo-5-chlorothiophene-3-carbaldehyde (6). To a solution
of DMSO (2.10 mL, 29.7 mmol) in CH2Cl2 (50 mL) at -78 ꢀC
Table 3. Pharmacokinetic Parameters of EP4 antagonist 34da
dose po, iv
F
Cmax T1/2
CL
Vdss
species
(mg/kg) (%) ( μM) (h) (mL/min/kg) (L/kg)
mouseb
ratc
20, 5
20, 5
5, 1
21 1.4 15
23
9.2
7.6
2.6
0.91
7.9
29 4.5
32 3.3
4.5
8.8
dogc
3.2
0.91
cynomolgus monkeyc
5, 1
18 0.05 19
Figure 5. GI tolerability of compound 34d in rats vs NSAID
indomethacin based on urinary 51Cr-excretion after a single oral
dose of 51Cr-EDTA on day 4 for 34d treated animals and on day
2 for indomethacin treated animals (n = 5/group for compound
treatment and 4/group for vehicle; **p < 0.05 vs vehicle, 1 way
ANOVA/Dunnett’s test). Indomethacin: 5 mg/kg once daily in
0.5% methocel for 3 days; 34d: 3, 10, and 30 mg/kg in 0.5%
methocel once daily for 5 days.
a Oral (po) dose administered in 0.5% methocel. Intravenous (iv) dose
administered in 60% PEG 200. F denotes bioavailability, Cmax denotes
the maximum concentration reached and is determined from the po
data, T1/2 is the half-life in plasma and is determined from the iv data, CL
is plasma clearance, Vdss is the volume of distribution. b The correspond-
ing sodium salt was used in this experiment. c The free acid was used in
this experiment.
Figure 4. In vivo activity of compound 34d. (a) Dose-dependent inhibition of hyperalgesic response in the rat carrageenan-induced
hyperalgesia model (red) in comparison to COX-2 inhibitor 41 at 10 mg/kg (green) from the same experiment (n = 10-20/group, *p <
0.01 vs vehicle by 1 way ANOVA/Dunnett’s test, at single oral doses of g0.1 mg/kg at 3 h after carrageenan challenge and 1 h after oral dosing
of compounds). (b) Inhibition of secondary paw swelling in female Lewis rats with AIA (red) in comparison with COX-2 inhibitor 41 (green)
from a separate experiment (n = 7/group for compound and vehicle, *p < 0.02 vs vehicle by 1 way ANOVA/Dunnett’s test at doses g0.01 mg/
kg; compounds were dosed in 0.5% methocel once daily for 9 days starting from day 9 after injection of CFA in the primary paw on day 0).

2234 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Blouin et al.
temperature for 30 min and concentrated. Residual TFA was
coevaporated with toluene and the residue dried under high
vacuum. The crude was purified by flash chromatography
(hexane) to give 8 as a colorless oil (1.57 g, 66%). H NMR
(400 MHz, acetone-d6) δ 7.39-7.21 (m, 4H), 6.93 (s, 1H), 3.95
(s, 2H).
and brine, dried (Na2SO4), filtered, and concentrated. The crude
product was purified by flash chromatography (toluene/hexane
5:95) to afford 17a as a colorless oil (110 mg, 81%). H NMR
(400 MHz, acetone-d6) δ 7.57 (d, J = 3.4 Hz, 1H), 7.37-7.20 (m,
5H), 4.01 (s, 2H).
1
1
4-(3-Chlorobenzyl)thiophene-3-carboxylic Acid (19a). To a
solution of n-BuLi (2.5 M in hexane, 8.65 mL, 21.6 mmol) in
Et2O (40 mL) at -78 ꢀC was added a solution of 17a (6.22 g, 21.6
mmol) in THF (10 mL) dropwise, and the resulting yellow
solution was stirred for 30 min at the same temperature. An
excess of CO2 gas was bubbled into the reaction mixture, and the
latter was stirred at -78 ꢀC for 30 min, allowed to warm to 0 ꢀC,
and quenched with 1 N NaOH. The aqueous layer was washed
with Et2O (2ꢀ) and acidified (pH 2) with 6 N HCl. The off-white
precipitate was extracted with CHCl3 (5ꢀ), and the combined
organic layers were washed successively with water and brine,
dried (Na2SO4), filtered, and concentrated give 19a as an off-
5-Chloro-3-(3-chlorobenzyl)thiophene-2-carboxylic Acid (9).
To a solution of 8 (1.56 g, 4.84 mmol) in THF (15 mL) at
-78 ꢀC was added n-BuLi (2.5 M in hexane, 2.13 mL, 5.32
mmol) dropwise, and the mixture was stirred for 5 min at the
same temperature. An excess of CO2 gas was bubbled into the
reaction mixture, and the latter was allowed to warm to 0 ꢀC,
quenched with 1 N HCl, and extracted with EtOAc. The organic
layer was washed successively with saturated NH4Cl and brine,
dried (MgSO4), and filtered. The filtrate was concentrated and
the residue was recrystallized from Et2O/hexane to give 9 as a
1
white solid (1.26 g, 91%). H NMR (400 MHz, acetone-d6) δ
1
white solid (4.75 g, 87%). H NMR (400 MHz, acetone-d6) δ
7.39-7.23 (m, 4H), 7.04 (s, 1H), 4.40 (s, 2H).
11.05 (br s, 1H), 8.32 (d, J = 3.4 Hz, 1H), 7.33-7.27 (m, 2H),
7.23-7.18 (m, 3H), 4.31 (s, 2H).
Methyl 4-[1-({[5-Chloro-3-(3-chlorobenzyl)-2-thienyl]carbonyl}-
amino)ethyl]benzoate (11). To a solution of 9 (200 mg, 0.696 mmol)
and (()-1-[4-(methoxycarbonyl)phenyl]ethanaminium chloride
(10) [prepared from (()-1-(4-bromophenyl)ethylamine according
to ref 16] (180 mg, 0.835 mmol) in DMF (3 mL) at 0 ꢀC were added
successively HATU (317 mg, 0.835 mmol) and DIEA (304 μL,
1.74 mmol) dropwise. The mixture was stirred at 0 ꢀC for 15 min
and diluted with a mixture of water, EtOAc, and Et2O. The
organic layer was washed with water and brine, dried (MgSO4),
filtered, and concentrated. The crude was purified by chromatog-
raphy using the CombiFlash system (EtOAc/hexane 10:90 to
Methyl 4-[(1S)-1-({[4-(3-Chlorobenzyl)-3-thienyl]carbonyl}-
amino)ethyl]benzoate (23a). To a solution of 19a (380 mg, 1.50
mmol) and (1S)-1-[4-(methoxycarbonyl)phenyl]ethanaminium
chloride (22)16 (294 mg, 1.36 mmol) in DMF (1.5 mL) at 0 ꢀC
were added successively HATU (518 mg, 1.36 mmol) and DIEA
(548 μL, 3.14 mmol) dropwise. The mixture was stirred at 0 ꢀC
for 1 h, poured into 1:1 saturated NaHCO3/water (15 mL), and
extracted with EtOAc (3ꢀ). The combined organic layers were
washed with water and brine, dried (Na2SO4), filtered, and
concentrated. The crude product was purified by flash chroma-
tography (EtOAc/hexane 40:60 to 95:5) to give 23a as a white
solid (520 mg, 92%). 1H NMR (400 MHz, acetone-d6) δ
7.99-7.90 (m, 4H), 7.51 (d, J = 8.0 Hz, 2H), 7.25-7.12 (m,
1
40:60, 15 min) to give 11 as a white solid (300 mg, 96%). H
NMR (400 MHz, acetone-d6) δ 7.99 (d, J = 8.3 Hz, 2H), 7.82 (br
d, J = 7.6 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.34-7.20 (m, 4H),
6.98(s,1H),5.30(m,1H),4.30(d,JAB = 14.6 Hz, 1H), 4.26 (d, JAB =
5H), 5.26 (m, 1H), 4.25 (d, JAB = 15.0 Hz, 1H), 4.21 (d, JAB
15.0 Hz, 1H), 3.89 (s, 3H), 1.52 (d, J = 7.1 Hz, 3H).
=
14.7 Hz, 1H), 3.89 (s, 3H), 1.57 (d, J = 7.1 Hz, 3H).
4-[1-({[5-Chloro-3-(3-chlorobenzyl)-2-thienyl]carbonyl}amino)-
ethyl]benzoic Acid (12). To a solution of 11 (81.0 mg, 0.181 mmol)
in a mixture of THF (1.5 mL) and MeOH (1.5 mL) was added a
solution of LiOH (aq 1 M, 900 μL, 0.900 mmol). The resulting
mixture was stirred at room temperature for 18 h and concen-
trated. The residue was diluted with 1 N HCl and extracted with
EtOAc. The organic layer was dried (Na2SO4), filtered, and
concentrated to give 12 as a white solid (78 mg, 99%). 1H
NMR (400 MHz, acetone-d6) δ 11.20 (br s, 1H), 8.02 (d, J =
8.2 Hz, 2H), 7.79 (br d, J = 7.7 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H),
7.34 (s, 1H), 7.31-7.20 (m, 3H), 6.98 (s, 1H), 5.34-5.26 (m, 1H),
4.31 (d, JAB = 14.7 Hz, 1H), 4.26 (d, JAB = 14.7 Hz, 1H), 1.57 (d,
J = 7.1 Hz, 3H). HRMS calcd for C21H16Cl2NO3S [(M - H)-],
432.0233; found, 432.0229.
(4-Bromo-3-thienyl)(3-chlorophenyl)methanol (15a). Com-
mercially available 3,4-dibromothiophene (13) (15.5 g, 64.1
mmol) was added dropwise to a solution of n-BuLi (2.5 M in
hexane, 25.6 mL, 64.1 mmol) in Et2O (50 mL) at -78 ꢀC. After
1.5 h, 3-chlorobenzaldehyde (7.29 mL, 64.1 mmol) was added
dropwise to the beige suspension. The resulting solution was
stirred at -78 ꢀC for 1 h and allowed to warm to 0 ꢀC. After 1 h,
the reaction was quenched by the addition of 25% aqueous
NH4OAc. The aqueous layer was extracted with EtOAc (2ꢀ),
and the combined organics were washed with water and brine,
dried (Na2SO4), filtered, and concentrated to afford the 15a as
a pale-yellow oil (19.5 g, 100%). The crude product was used
directly without further purification. 1H NMR (400 MHz,
acetone-d6) δ 7.54 (m, 2H), 7.47 (s, 1H), 7.39-7.28 (m, 3H),
5.89 (d, J = 4.6 Hz, 1H), 5.12 (d, J = 4.5 Hz, 1H).
4-[(1S)-1-({[4-(3-Chlorobenzyl)-3-thienyl]carbonyl}amino)ethyl]-
benzoic Acid (26a). To a solution of 23a (100 mg, 0.242 mmol) in a
mixture of THF (2.5 mL) and MeOH (1.25 mL) was added a
solution of LiOH (aq 0.5 M, 1.45 mL, 0.725 mmol). The resulting
mixture was stirred at room temperature for 60 h, acidified with 6 N
HCl (1 mL), diluted with Et2O (30 mL), and washed with water and
brine. CH2Cl2 (50 mL) was added to the organic layer to dissolve
the suspended portion of product. The aqueous layer was further
extracted with CH2Cl2, and the combined organic fractions were
dried (Na2SO4), filtered, and concentrated to provide pure 26a as a
white solid (87 mg, 90%). 1H NMR (400 MHz, methanol-d4) δ8.64
(d, J = 7.8 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 3.2 Hz,
1H), 7.34 (d, J = 8.2 Hz, 2H), 7.16-7.10 (m, 3H), 7.07 (s, 1H),
7.02-6.98 (m, 1H), 5.10 (m, 1H), 4.16 (d, JAB = 15.3 Hz, 1H), 4.10
(d, JAB = 15.3 Hz, 1H), 1.45 (d, J = 7.1 Hz, 3H). HRMS calcd for
C21H17ClNO3S [(M - H)-], 398.0623; found, 398.0616. Anal.
(C21H18ClNO3S) H, N. C: calcd, 63.07; found, 62.36.
2,5-Dichloro-4-(3-chlorobenzyl)thiophene-3-carboxylic Acid
(21a). NCS (4.46 g, 33.4 mmol) was added to 19a (4.02 g, 15.9
mmol) in AcOH (40 mL), and the resulting mixture was heated
at 110 ꢀC for 2 h and allowed to cool to room temperature. The
solvent was coevaporated with toluene (3ꢀ), and the residue was
partitioned between CHCl3 (250 mL) and water (100 mL). The
organic layer was washed with water (3ꢀ) and brine, dried
(Na2SO4), filtered, and concentrated. The crude product was
purified by flash chromatography (EtOAc/hexane 20:80 to
30:70 containing 0.5% AcOH) to afford 21a as a beige solid
(3.60 g, 70%) after residual AcOH was coevaporated with
1
3-Bromo-4-(3-chlorobenzyl)thiophene (17a). To 15a (144 mg,
0.474 mmol) in CH2Cl2 (1 mL) at 0 ꢀC were successively added
triethylsilane (303 μL, 1.90 mmol) quickly and TFA (364 μL,
4.74 mmol) dropwise. After 30 min, the reaction mixture was
concentrated to dryness and the residue was dissolved in CHCl3
and washed with 5% NaHCO3. The aqueous layer was extracted
with CHCl3, and the combined organics were washed with water
toluene. H NMR (400 MHz, acetone-d6) δ 11.91 (br s, 1H),
7.34-7.27 (m, 1H), 7.26-7.18 (m, 2H), 7.19-7.11 (m, 1H), 4.27
(s, 2H).
Methyl 4-[(1S)-1-({[2,5-Dichloro-4-(3-chlorobenzyl)-3-thie-
nyl]carbonyl}amino)ethyl]benzoate (24a). The acid 21a (1.90 g,
5.91 mmol), (1S)-1-[4-(methoxycarbonyl)phenyl]ethanaminium
chloride (22)16 (1.27 g, 5.91 mmol), HATU (2.25 g, 5.91 mmol),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2235
and DIEA (2.35 mL, 13.6 mmol) were reacted in DMF (28 mL)
under the conditions described above for the preparation of 23a.
The crude product was purified by chromatography using the
CombiFlash system (EtOAc/toluene 2:98 to 5:95, 20 min) to
afford 24a as a white solid (2.14 g, 75%). 1H NMR (400 MHz,
acetone-d6) δ 8.11 (br d, J = 7.8 Hz, 1H), 7.95 (d, J = 8.0 Hz,
2H), 7.50 (d, J = 8.0 Hz, 2H), 7.28-7.18 (m, 3H), 7.12-7.07 (m,
(MeOH/CH2Cl2 0:100 to 2:98) to provide 25a as a white solid
(130 mg, 85%). 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.0
Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.11-7.17 (m, 2H), 7.03 (s,
1H), 6.93 (d, J = 6.6 Hz, 1H), 5.69 (br d, J = 7.9 Hz, 1H), 5.18
(m, 1H), 3.94 (s, 2H), 3.92 (s, 3H), 2.44 (s, 3H), 2.31 (s, 3H),
1.35 (d, J = 7.0 Hz, 3H).
4-[(1S)-1-({[4-(3-Chlorobenzyl)-2,5-dimethyl-3-thienyl]carbonyl}-
amino)ethyl]benzoic Acid (28a). The ester 25a (130 mg, 0.294 mmol)
in THF (3 mL) and MeOH (1.5 mL) was treated with LiOH (aq 0.5
M, 1.76 mL, 0.882 mmol) for 17 h, under the conditions described
above for the preparation of 27a, but at room temperature. Pure 28a
was obtained as a white solid without purification (115 mg, 91%).
1H NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H), 8.68 (d, J = 8.1
Hz, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H),
7.23-7.17 (m, 2H), 7.09 (s, 1H), 7.04-6.99 (m, 1H), 5.09 (m, 1H),
3.90 (d, JAB = 15.3 Hz, 1H), 3.83 (d, JAB = 15.3 Hz, 1H), 2.35 (s,
3H), 2.29 (s, 3H), 1.34 (d, J = 7.0 Hz, 3H). HRMS calcd for
C23H21ClNO3S [(M - H)-], 426.0936; found, 426.0930.
(4-Bromo-2,5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]-
methanol (16b). A solution of 3,4-dibromo-2,5-dimethylthio-
phene26 (14) (3.00 g, 11.1 mmol) in a mixture of Et2O (40 mL)
and THF (20 mL) was treated with n-BuLi (2.5 M in hexane,
4.44 mL, 11.1 mmol) and 3-(trifluoromethyl)benzaldehyde (1.48
mL, 11.1 mmol) under the conditions described above for the
preparation of 15a. The pale-brown oil obtained (16b) was used
without further purification (3.93 g, 97%). 1H NMR (400 MHz,
acetone-d6) δ 7.83 (s, 1H), 7.63-7.53 (m, 3H), 6.17 (d, J = 4.4
Hz, 1H), 5.18 (d, J = 4.2 Hz, 1H), 2.34 (s, 6H).
3-Bromo-2,5-dimethyl-4-[3-(trifluoromethyl)benzyl]thiophene
(18b). Triethylsilane (5.63 mL, 35.3 mmol) and TFA (6.77 mL,
88.2 mmol) were successively added to a solution of 16b (3.22 g,
8.82 mmol) in CH2Cl2 (20 mL) under the conditions described
above for the preparation of 18a. The crude product was
purified by chromatography using the CombiFlash system
(EtOAc/hexane 0:100 to 2:98, 26 min) to afford 18b as a colorless
liquid (2.53 g, 82%). 1H NMR (400 MHz, acetone-d6) δ
7.57-7.48 (m, 3H), 7.44 (m, 1H), 4.08 (s, 2H), 2.42 (s, 3H),
2.36 (s, 3H).
1H), 5.23 (m, 1H), 4.07 (d, JAB = 15.3 Hz, 1H), 4.00 (d, JAB
15.3 Hz, 1H), 3.89 (s, 3H), 1.48 (d, J = 7.1 Hz, 3H).
=
4-[(1S)-1-({[2,5-Dichloro-4-(3-chlorobenzyl)-3-thienyl]carbonyl}-
amino)ethyl]benzoic Acid (27a). To a solution of 24a (2.11 g, 4.37
mmol) in a mixture of THF (90 mL) and MeOH (45 mL) was added
a solution of LiOH (aq 1 M, 13.1 mL, 13.1 mmol). The resulting
mixture was heated at 50 ꢀC for 4 h, cooled to room temperature,
and acidified with 1 N HCl (14.0 mL). Water was added until
precipitation, and the solid was extracted with EtOAc (3ꢀ). The
combined organics were washed with water and brine, dried
(Na2SO4), filtered, and concentrated. The crude product was stirred
in Et2O, collected by filtration, rinsed with the same solvent, and
dried to provide pure 27a as a white solid (1.84 g, 90%). 1H NMR
(400 MHz, acetone-d6) δ 11.17 (br s, 1 H), 8.11 (br d, J = 7.8 Hz,
1H), 7.99 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.29-7.18
(m, 3H), 7.13-7.07 (m, 1H), 5.24 (m, 1H), 4.07 (d, JAB = 15.3 Hz,
1H), 4.01 (d, JAB = 15.3 Hz, 1H), 1.48 (d, J = 7.0 Hz, 3H). HRMS
calcd for C21H15Cl3NO3S [(M - H)-], 465.9844; found, 465.9836.
Anal. (C21H16Cl3NO3S) C, H, N.
(4-Bromo-2,5-dimethyl-3-thienyl)(3-chlorophenyl)methanol (16a).
A solution of 3,4-dibromo-2,5-dimethylthiophene26 (14) (3.00 g,
11.1 mmol) in THF (50 mL) was treated with n-BuLi (2.5 M in
hexane, 4.44 mL, 11.1 mmol) and 3-chlorobenzaldehyde (1.26 mL,
11.1 mmol) under the conditions described above for the prepara-
tion of 15a. The crude product was purified by chromatography
using the CombiFlash system (EtOAc/hexane 2.98 to 12:88, 20 min)
to afford 16a as a yellow oil (2.92 g, 79%). 1H NMR (400 MHz,
acetone-d6) δ 7.47 (s, 1H), 7.39-7.25 (m, 3H), 6.08 (d, J = 4.4 Hz,
1H), 5.07 (d, J = 4.3 Hz, 1H), 2.35 (s, 6H).
3-Bromo-4-(3-chlorobenzyl)-2,5-dimethylthiophene (18a). Tri-
ethylsilane (5.55 mL, 34.7 mmol) and TFA (6.68 mL, 86.8
mmol) were successively added to a solution of 16a (2.88 g,
8.68 mmol) in CH2Cl2 (20 mL) at 0 ꢀC. The mixture was stirred
at the same temperature for 30 min and concentrated to dryness.
The residue was dissolved in CH2Cl2 and washed with 5%
aqueous NaHCO3, water, and brine, dried (Na2SO4), filtered,
and concentrated. The crude product was purified by chroma-
tography using the CombiFlash system (EtOAc/hexane 2:98 to
5:95, 20 min) to afford 18a as a colorless oil (2.42 g, 88%). 1H
NMR (400 MHz, acetone-d6) δ 7.31 (t, J = 7.8 Hz, 1H), 7.23 (d,
J = 8.2 Hz, 1H), 7.17 (s, 1H), 7.13 (d, J = 7.77 Hz, 1H), 3.98 (s,
2H), 2.40 (s, 3H), 2.35 (s, 3H).
2,5-Dimethyl-4-[3-(trifluoromethyl)benzyl]thiophene-3-car-
boxylic Acid (20b). A solution of 18b (2.50 g, 7.16 mmol) in THF
(30 mL) was treated with n-BuLi (2.5 M in hexane, 2.86 mL, 7.16
mmol) and CO2 gas under the conditions described above for the
preparation of 20a. The crude product was purified by flash
chromatography (EtOAc/hexane 20:80 to 25:75 containing
0.25% AcOH) to afford 20b as a beige solid (1.20 g, 53%) after
residual AcOH was coevaporated with toluene. 1H NMR (400
MHz, acetone-d6) δ 11.03 (br s, 1H), 7.51-7.43 (m, 3H), 7.39 (m,
1H), 4.32 (s, 2H), 2.64 (s, 3H), 2.36 (s, 3H).
Methyl 4-{(1S)-1-[({2,5-Dimethyl-4-[3-(trifluoromethyl)-
benzyl]-3-thienyl}carbonyl)amino]ethyl}benzoate (25b). The acid
20b (200 mg, 0.636 mmol), (1S)-1-[4-(methoxycarbonyl)phenyl]-
ethanaminium chloride (22)16 (137 mg, 0.636 mmol), HATU
(242 mg, 0.636 mmol), and DIEA (253 μL, 1.46 mmol) were
reacted in DMF (3.5 mL) under the conditions described above
for the preparation of 23a. The crude product was purified by
chromatography using the CombiFlash system (EtOAc/toluene
2:98 to 10:90, 20 min) to provide 25b as an off-white solid (209 mg,
4-(3-Chlorobenzyl)-2,5-dimethylthiophene-3-carboxylic Acid
(20a). A solution of 18a (2.39 g, 7.57 mmol) in THF (30 mL)
was treated with n-BuLi (2.5 M in hexane, 3.03 mL, 7.57 mmol)
and CO2 gas under the conditions described above for the
preparation of 19a. The reaction was quenched with 25%
aqueous NH4OAc and extracted with EtOAc. The combined
organics were washed with water and brine, dried (Na2SO4),
filtered, and concentrated. The crude product was purified by
flash chromatography (EtOAc/hexane 25:75 containing 0.25%
AcOH) to afford 20a as a beige solid (1.23 g, 58%) after residual
1
69%). H NMR (400 MHz, acetone-d6) δ 7.92 (d, J = 8.0 Hz,
2H), 7.70 (br d, J = 8.0 Hz, 1H), 7.49-7.34 (m, 6H), 5.22 (m, 1H),
4.07 (d, JAB = 15.6 Hz, 1H), 4.02 (d, JAB=15.6 Hz, 1H), 3.88
(s, 3H), 2.41 (s, 3H), 2.32 (s, 3H), 1.45 (d, J=7.1 Hz, 3H).
4-{(1S)-1-[({2,5-Dimethyl-4-[3-(trifluoromethyl)benzyl]-3-
thienyl}carbonyl)amino]ethyl}benzoic Acid (28b). The ester 25b
(202 mg, 0.425 mmol) in THF (8 mL) and MeOH (4 mL) was
treated with LiOH (aq 1 M, 1.27 mL, 1.27 mmol) for 4 h under
the conditions described above for the preparation of 27a. The
crude solid was triturated in 1:4 CHCl3/hexane, collected by
filtration, rinsed with hexane, and dried to afford pure 28b as a
white solid (162 mg, 82%). 1H NMR (400 MHz, acetone-d6) δ
11.15 (br s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.70 (br d, J = 8.0 Hz,
1
AcOH was coevaporated with toluene. H NMR (400 MHz,
acetone-d6) δ 11.01 (br s, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.21-
7.11 (m, 2H), 7.07 (d, J = 7.7 Hz, 1H), 4.23 (s, 2H), 2.63 (s, 3H),
2.34 (s, 3H).
Methyl 4-[(1S)-1-({[4-(3-Chlorobenzyl)-2,5-dimethyl-3-thienyl]-
carbonyl}amino)ethyl]benzoate (25a). The acid 20a (97 mg, 0.345
mmol), (1S)-1-[4-(methoxycarbonyl)phenyl]ethanaminium chlo-
ride (22)16 (78 mg, 0.363 mmol), HATU (138 mg, 0.363 mmol),
and DIEA (139 μL, 0.795 mmol) were reacted in DMF (1 mL)
under the conditions described above for the preparation of
23a. The crude product was purified by flash chromatography

2236 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Blouin et al.
1H), 7.51-7.37 (m, 6H), 5.24 (m, 1H), 4.08 (d, JAB = 15.7
Hz, 1H), 4.02 (d, JAB = 15.7 Hz, 1H), 2.42 (s, 3H), 2.33
(s, 3H), 1.45 (d, J = 7.1Hz, 3H). HRMS calcd for C24H21F3NO3S
[(M - H)-], 460.1200; found, 460.1194. Anal. (C24H22F3NO3S)
C, H, N.
3-Bromo-2,5-dimethyl-4-[4-(trifluoromethyl)benzyl]thiophene
(18d). Triethylsilane (5.11 mL, 32.0 mmol) and TFA (6.14 mL,
80.0 mmol) were successively added to a solution of 16d (2.92 g,
8.00 mmol) in CH2Cl2 (18 mL) under the conditions described
above for the preparation of 18a. The crude product was
purified by chromatography using the CombiFlash system
(EtOAc/hexane 0:100 to 10:90, 28 min) to afford 18d as a
colorless liquid (2.36 g, 84%). H NMR (400 MHz, acetone-
d6) δ 7.63 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 4.07 (s,
2H), 2.41 (s, 3H), 2.35 (s, 3H).
2,5-Dimethyl-4-[4-(trifluoromethyl)benzyl]thiophene-3-car-
boxylic Acid (20d). A solution of 18d (2.34 g, 6.70 mmol) in THF
(30 mL) was treated with n-BuLi (2.5 M in hexane, 2.68 mL, 6.70
mmol) and CO2 gas under the conditions described above for the
preparation of 20a. The crude material was triturated in EtOAc/
hexane 10:90, collected by filtration, rinsed with the same
solvent followed by hexane, and dried to afford pure 20d as an
off-white solid (1.11 g, 53%). 1H NMR (400 MHz, acetone-d6) δ
11.01 (br s, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.0 Hz,
2H), 4.32 (s, 2H), 2.64 (s, 3H), 2.34 (s, 3H).
Methyl 4-{(1S)-1-[({2,5-Dimethyl-4-[4-(trifluoromethyl)-
benzyl]-3-thienyl}carbonyl)amino]ethyl}benzoate (25d). The acid
20d (250 mg, 0.795 mmol), (1S)-1-[4-(methoxycarbonyl)-
phenyl]ethanaminium chloride (22)16 (172 mg, 0.795 mmol),
HATU (302 mg, 0.795 mmol), and DIEA (316 μL, 1.83 mmol)
were reacted in DMF (5 mL) under the conditions described
above for the preparation of 23a. The precipitate formed in the
NaHCO3 solution was collected by filtration, successively rinsed
with water, EtOH, and hexane, and dried to provide 25d as a
white solid (332 mg, 88%). 1H NMR (400 MHz, acetone-d6) δ
7.93 (d, J = 8.2 Hz, 2H), 7.65 (br d, J = 8.2 Hz, 1H), 7.48 (m,
4H), 7.29 (d, J = 8.0 Hz, 2H), 5.24 (m, 1H), 4.08 (d, JAB = 15.5
Hz, 1H), 3.98 (d, JAB = 15.5 Hz, 1H), 3.88 (s, 3H), 2.42 (s, 3H),
2.33 (s, 3H), 1.46 (d, J = 7.1 Hz, 3H).
(4-Bromo-2,5-dimethyl-3-thienyl)(4-chlorophenyl)methanol
(16c). A solution of 3,4-dibromo-2,5-dimethylthiophene26 (14)
(1.00 g, 3.70 mmol) in THF (15 mL) was treated with n-BuLi
(1.6 M in hexane, 2.32 mL, 3.70 mmol) and 4-chlorobenzaldehyde
(521 mg, 3.70 mmol) under the conditions described above for the
preparation of 15a. The crude product was purified by flash
chromatography (EtOAc/hexane 10:90 to 25:75) to afford 16c
1
1
(975 mg, 79%). H NMR (400 MHz, CDCl3) δ 7.32 (s, 4H),
6.05 (d, J = 6.0 Hz, 1H), 2.48 (d, J = 6.0 Hz, 1H), 2.36 (s, 3H),
2.34 (s, 3H).
3-Bromo-4-(4-chlorobenzyl)-2,5-dimethylthiophene (18c). Tri-
ethylsilane (1.88 mL, 11.8 mmol) and TFA (2.26 mL, 29.4
mmol) were successively added to a solution of 16c (975 mg,
2.94 mmol) in CH2Cl2 (5 mL) under the conditions described
above for the preparation of 18a. The crude product was
purified by flash chromatography (hexane) to afford 18c as a
white solid (835 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 7.25
(d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 3.91 (s, 2H), 2.39
(s, 3H), 2.35 (s, 3H).
4-(4-Chlorobenzyl)-2,5-dimethylthiophene-3-carboxylic Acid
(20c). A solution of 18c (824 mg, 2.61 mmol) in a mixture of
THF (5 mL) and Et2O (10 mL) was treated with n-BuLi (1.6 M in
hexane, 1.63 mL, 2.61 mmol) and CO2 gas under the conditions
described above for the preparation of 20a. Pure 20c was
obtained as a light-yellow solid without further purification
(472 mg, 64%). 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 8.0
Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 4.18 (s, 2H), 2.70 (s, 3H), 2.32
(s, 3H).
Methyl 4-[(1S)-1-({[4-(4-Chlorobenzyl)-2,5-dimethyl-3-thienyl]-
carbonyl}amino)ethyl]benzoate (25c). The acid 20c (472 mg, 1.68
mmol), (1S)-1-[4-(methoxycarbonyl)phenyl]ethanaminium chlo-
ride (22)16 (363 mg, 1.68 mmol), HATU (639 mg, 1.68 mmol),
and DIEA (675 μL, 3.87 mmol) were reacted in DMF (10 mL)
under the conditions described above for the preparation of 23a
(an additional 2 mL of DMF needed to be added and the
mixture was allowed to reach room temperature for homo-
geneity). The crude product was purified by flash chromatog-
raphy (MeOH/CH2Cl2 0:100 to 2:98) to provide 25c as a white
solid (540 mg, 73%). 1H NMR (400 MHz, CDCl3) δ 7.97 (d, J =
8.2 Hz, 2H), 7.20-7.17 (m, 4H), 6.98 (d, J = 8.2 Hz, 2H), 5.58
(br d, J = 8.0 Hz, 1H), 5.18 (m, 1H), 3.97-3.88 (m, 5H), 2.43 (s,
3H), 2.32 (s, 3H), 1.38 (d, J = 8.0 Hz, 3H).
4-[(1S)-1-({[4-(4-Chlorobenzyl)-2,5-dimethyl-3-thienyl]carbonyl}-
amino)ethyl]benzoic Acid (28c).The ester 25c (540 mg, 1.22 mmol) in
THF (12 mL) and MeOH (4 mL) was treated with LiOH (aq 0.5 M,
7.33 mL, 3.67 mmol) for 2 h under the conditions described above
for the preparation of 27a. Pure 28c was obtained as a white solid
without further purification (520 mg, 99%). 1H NMR (400 MHz,
DMSO-d6) δ12.83 (s, 1H), 8.61 (d, J= 8.2 Hz, 1H), 7.86 (d, J=8.1
Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 7.02 (d,
J = 8.2 Hz, 2H), 5.09 (m, 1H), 3.87 (d, JAB = 15.3 Hz, 1H), 3.79 (d,
JAB = 15.2 Hz, 1H), 2.34 (s, 3H), 2.29 (s, 3H), 1.34 (d, J = 7.0 Hz,
3H). HRMS calcd for C23H21ClNO3S [(M - H)-], 426.0936;
found, 426.0930.
4-{(1S)-1-[({2,5-Dimethyl-4-[4-(trifluoromethyl)benzyl]-3-thienyl}-
carbonyl)amino]ethyl}benzoic acid (28d). The ester 25d (327 mg, 0.688
mmol) in THF (13 mL) and MeOH (6.5 mL) was treated with LiOH
(aq 1 M, 2.06 mL, 2.06 mmol) for 17 h under the conditions
described above for the preparation of 27a. The crude product was
purified by chromatography using the CombiFlash system (EtOH/
CHCl3 10:90) to afford 28d as an off-white solid (185 mg, 58%).
1H NMR (400 MHz, DMSO-d6) δ 12.81 (br s, 1H), 8.61 (d, J = 8.2
Hz, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.38
(d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 5.09 (m, 1H), 3.99 (d,
JAB = 15.3 Hz, 1H), 3.89 (d, JAB = 15.3 Hz, 1H), 2.35 (s, 3H), 2.32
(s, 3H), 1.32 (d, J = 7.0 Hz, 3H). HRMS calcd for C24H21F3NO3S
[(M - H)-], 460.1200; found, 460.1192. Anal. (C24H22F3NO3S)
C, H, N.
4-(1-Aminocyclopropyl)benzonitrile (30). To a solution of 1,4-
dicyanobenzene (29) (3.30 g, 25.8 mmol) in CH2Cl2 (130 mL)
was added Ti(OiPr)4 (7.56 mL, 25.8 mmol) followed by EtMgBr
(3 M in Et2O, 15.5 mL, 46.4 mmol) dropwise (exothermic, gas
evolution occurred after one equivalent of reagent added), and
the mixture was stirred at room temperature for 45 min.
BF3 Et2O (5.71 mL, 46.4 mmol) was added and the mixture
3
was stirred at the same temperature for 2 h, quenched with
NH4Cl and 1 N HCl, and separated. The aqueous layer was
washed once with Et2O, and the pH was adjusted to 9-10 with
10 N NaOH (precipitate formation). The mixture was filtered
through celite and the cake washed with water and EtOAc. The
layers were separated and the aqueous phase extracted with
EtOAc. The organic layers were combined, washed with brine,
dried (Na2SO4), filtered, and concentrated to afford 30 as a
viscous oil which solidified at -20 ꢀC. The crude was used
directly without further purification. 1H NMR (400 MHz,
CDCl3) δ 7.60 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H),
1.26-1.18 (m, 2H), 1.10-1.05 (m, 2H).
(4-Bromo-2,5-dimethyl-3-thienyl)[4-(trifluoromethyl)phenyl]-
methanol (16d). A solution of 3,4-dibromo-2,5-dimethylthio-
phene26 (14) (3.00 g, 11.1 mmol) in THF (50 mL) was treated
with n-BuLi (2.5 M in hexane, 4.44 mL, 11.1 mmol) and
4-(trifluoromethyl)benzaldehyde (1.48 mL, 11.1 mmol) under
the conditions described above for the preparation of 15a. The
crude product was purified by chromatography using the Com-
biFlash system (EtOAc/hexane 2.98 to 15:85, 26 min) to afford
16d as a yellow oil (2.93 g, 72%). 1H NMR (400 MHz, acetone-
d6) δ 7.69 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 6.16 (d,
J = 4.4 Hz, 1H), 5.15 (d, J = 4.2 Hz, 1H), 2.34 (s, 6H).
1-(4-Carboxyphenyl)cyclopropanaminium Chloride (31). A
mixture of 30 (576 mg, 3.64 mmol) and 6 N HCl (12 mL) was
heated to the reflux temperature for 40 h, cooled to room

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2237
temperature, and concentrated to dryness to afford 31 as a beige
solid. The crude was used without further purification.
(30) (144 mg, 0.910 mmol), HATU (346 mg, 0.910 mmol), and
DIEA (362 μL, 2.09 mmol) were reacted in DMF (5 mL) under
the conditions described above for the preparation of 23a. The
crude product was purified by chromatography using the Com-
biFlash system (EtOAc/CHCl3 2:98 to 5:95 to 10:90, 27 min)
Methyl 4-(1-Aminocyclopropyl)benzoate (32). Aqueous HCl
(12 N, 2 drops) was added to a suspension of 31 (3.64 mmol) in
MeOH (5 mL). The resulting mixture was heated to the reflux
temperature for 16 h, cooled to room temperature, and con-
centrated to dryness. The residue was partitioned between
EtOAc and 5% aqueous NaHCO3. The organic layer was
washed with brine, dried (Na2SO4), filtered, and concentrated
to afford 32 as an orange oil (472 mg, 68%). The crude was used
1
to provide 35 as a pale-yellow solid (179 mg, 43%). H NMR
(400 MHz, acetone-d6) δ 8.01 (s, 1H), 7.58 (m, 4H), 7.34-7.28
(m, 4H), 4.11 (s, 2H), 2.48 (s, 3H), 2.33 (s, 3H), 1.35-1.29 (m,
2H), 1.29-1.22 (m, 2H).
2,5-Dimethyl-N-{1-[4-(2H-tetrazol-5-yl)phenyl]cyclopropyl}-
4-[4-(trifluoromethyl)benzyl]thiophene-3-carboxamide (36). To a
suspension of 35 (174 mg, 0.383 mmol) in toluene (2.5 mL) was
added azidotributyltin (317 μL, 1.15 mmol), and the mixture
was heated at the reflux temperature. After 20 h, the solution
then obtained was allowed to cool to room temperature and
AcOH (365 μL) was added. The heterogeneous mixture was
stirred for 4 h, and the precipitated solid was collected by
filtration, successsively rinsed with toluene and hexane, and
then dried to afford 36 as an off-white solid (170 mg, 89%). 1H
NMR (400 MHz, DMSO-d6) δ 8.86 (s, 1H), 7.86 (d, J = 8.2 Hz,
2H), 7.58 (d, J = 8.0 Hz, 2H), 7.29-7.23 (m, 4H), 4.01 (s, 2H),
2.42 (s, 3H), 2.29 (s, 3H), 1.28-1.21 (m, 2H), 1.08-1.03 (m,
2H).). HRMS calcd for C25H22F3N5OS [(M - H)-], 496.1424;
found, 496.1420.
1
without further purification. H NMR (400 MHz, CDCl3) δ
7.99 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 3.92 (s, 3H),
1.20-1.15 (m, 2H), 1.10-1.05 (m, 2H).
Methyl 4-{1-[({2,5-Dimethyl-4-[3-(trifluoromethyl)benzyl]-3-
thienyl}carbonyl)amino]cyclopropyl}benzoate (33b). The acid
20b (299 mg, 0.952 mmol), methyl 4-(1-aminocyclopro-
pyl)benzoate (32) (182 mg, 0.952 mmol), HATU (362 mg,
0.952 mmol), and DIEA (379 μL, 2.19 mmol) were reacted in
DMF (6 mL) under the conditions described above for the
preparation of 23a. The crude product was purified by chroma-
tography using the CombiFlash system (EtOAc/CHCl3 2:98 to
5:95 to 10:90, 30 min) to provide 33b as an off-white solid (226
1
mg, 49%). H NMR (400 MHz, acetone-d6) δ 8.03 (br s, 1H),
7.82 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 7.7 Hz, 1H), 7.49-7.43 (m,
2H), 7.40 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 8.3 Hz, 2H), 4.12 (s,
2H), 3.86 (s, 3H), 2.49 (s, 3H), 2.31 (s, 3H), 1.32-1.25 (m, 2H),
1.24-1.18 (m, 2H).
Acknowledgment. We thank the Comparative Medicine
groupatMerckFrosst for theirhelp withthe pharmacokinetic
studies.
4-{1-[({2,5-Dimethyl-4-[3-(trifluoromethyl)benzyl]-3-thienyl}-
carbonyl)amino]cyclopropyl}benzoic Acid (34b). The ester 33b
(223 mg, 0.457 mmol) in THF (8.5 mL) and MeOH (4.2 mL) was
treated with LiOH (aq 1 M, 1.37 mL, 1.37 mmol) for 18 h under
the conditions described above for the preparation of 27a. The
crude material was triturated in EtOAc, collected by filtration,
rinsed with EtOAc/hexane 25:75 followed by hexane, and dried
to afford pure 34b as a white solid (186 mg, 86%). 1H NMR (400
MHz, acetone-d6) δ 12.15 (br s, 1H), 8.29 (s, 1H), 7.85 (d, J = 8.3
Hz, 2H), 7.53 (d, J = 7.7 Hz, 1H), 7.50-7.43 (m, 2H), 7.41 (d, J =
7.8 Hz, 1H), 7.27 (d, J = 8.3 Hz, 2H), 4.12 (s, 2H), 2.49 (s, 3H),
2.31 (s, 3H), 1.31-1.24 (m, 2H), 1.24-1.18 (m, 2H). HRMS calcd
for C25H21F3NO3S [(M - H)-], 472.1200; found, 472.1193.
Methyl 4-{1-[({2,5-Dimethyl-4-[4-(trifluoromethyl)benzyl]-3-
thienyl}carbonyl)amino]cyclopropyl}benzoate (33d). The acid
20d (275 mg, 0.875 mmol), methyl 4-(1-aminocyclopropyl)-
benzoate (32) (167 mg, 0.873 mmol), HATU (333 mg, 0.875
mmol), and DIEA (348 μL, 2.01 mmol) were reacted in DMF (6
mL) under the conditions described above for the preparation of
23a. The crude product was purified by chromatography using
the CombiFlash system (EtOAc/CHCl3 2:98 to 5:95 to 10:90, 30
min) to provide 33d as a white solid (176 mg, 41%). 1H NMR
(400 MHz, acetone-d6) δ 7.98 (br s, 1H), 7.83 (d, J = 8.3 Hz,
2H), 7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.27 (d,
J = 8.3 Hz, 2H), 4.11 (s, 2H), 3.86 (s, 3H), 2.48 (s, 3H), 2.32 (s,
3H), 1.32-1.26 (m, 2H), 1.26-1.20 (m, 2H).
4-{1-[({2,5-Dimethyl-4-[4-(trifluoromethyl)benzyl]-3-thienyl}-
carbonyl)amino]cyclopropyl}benzoic Acid (34d). The ester 33d
(172 mg, 0.353 mmol) in THF (7 mL) and MeOH (3.5 mL) was
treated with LiOH (aq 1 M, 1.06 mL, 1.06 mmol) for 17 h under
the conditions described above for the preparation of 27a. The
crude material was triturated in EtOH/hexane 10:90, collected
by filtration, rinsed with the same solvent followed by hexane,
and dried to afford 34d as a white solid (134 mg, 80%). 1H NMR
(400 MHz, DMSO-d6) δ 12.75 (s, 1H), 8.84 (s, 1H), 7.73 (d, J =
8.1 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H),
7.08 (d, J = 8.1 Hz, 2H), 4.02 (s, 2H), 2.41 (s, 3H), 2.29 (s, 3H),
1.28-1.18 (m, 2H), 1.14-1.00 (m, 2H). HRMS calcd for
C25H21F3NO3S [(M - H)-], 472.1200; found, 472.1192. Anal.
(C25H22F3NO3S) H, N. C: calcd, 63.41; found, 62.90.
Supporting Information Available: Synthetic procedures for
the preparation of test compounds 38, 39, and 40, combustion
analysis for compounds 12, 27a, 28b, 28d, 34d, and 40, and
HPLC purity data for compounds 12, 26a, 27a, 28a-d, 34b, 34d,
36, 38, 39, and 40. This material is available free of charge via the
Internet at http://pubs.acs.org.
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