Discovery of highly selective and orally active lysophosphatidic acid receptor-1 antagonists with potent activity on human lung fibroblasts

Article abstract of DOI:10.1021/jm301022v

Lysophosphatidic acid is a class of bioactive phospholipid that mediates most of its biological effects through LPA receptors, of which six isoforms have been identified. The recent results from LPA1 knockout mice suggested that blocking LPA1 signaling could provide a potential novel approach for the treatment of idiopathic pulmonary fibrosis. Here, we report the design and synthesis of pyrazole- and triazole-derived carbamates as LPA1-selective and LPA1/3 dual antagonists. In particular, compound 2, the most selective LPA1 antagonist reported, inhibited proliferation and contraction of normal human lung fibroblasts (NHLF) following LPA stimulation. Oral dosing of compound 2 to mice resulted in a dose-dependent reduction of plasma histamine levels in a murine LPA challenge model. Furthermore, we applied our novel antagonists as chemistry probes and investigated the contribution of LPA1/2/3 in mediating the pro-fibrotic responses. Our results suggest LPA1 as the major receptor subtype mediating LPA-induced proliferation and contraction of NHLF.

Full text of DOI:10.1021/jm301022v

Article
pubs.acs.org/jmc
Discovery of Highly Selective and Orally Active Lysophosphatidic
Acid Receptor‑1 Antagonists with Potent Activity on Human Lung
Fibroblasts
Yimin Qian,*,† Matthew Hamilton,† Achyutharao Sidduri,† Stephen Gabriel,† Yonglin Ren,‡ Ruoqi Peng,‡
Rama Kondru,† Arjun Narayanan,† Terry Truitt,§ Rachid Hamid,§ Yun Chen,§ Lin Zhang,∥
Adrian J. Fretland,⊥ Ruben Alvarez Sanchez,⊥ Kung-Ching Chang,‡ Matthew Lucas,†
Ryan C. Schoenfeld,† Dramane Laine,† Maria E. Fuentes,‡ Christopher S. Stevenson,‡ and David C. Budd‡
‡
§
∥
†Discovery Chemistry, Discovery Inflammation and Respiratory Diseases, Discovery Technology, Pharmaceutical and Analytical
⊥
Research, and Drug Metabolism and Pharmacokinetics, Small Molecule Research, Pharmaceutical Research and Early Drug
Development, Hoffmann-La Roche, 340 Kingsland Street, Nutley, New Jersey 07110, United States
ABSTRACT: Lysophosphatidic acid is a class of bioactive phospholipid that
mediates most of its biological effects through LPA receptors, of which six isoforms
have been identified. The recent results from LPA1 knockout mice suggested that
blocking LPA1 signaling could provide a potential novel approach for the treatment
of idiopathic pulmonary fibrosis. Here, we report the design and synthesis of
pyrazole- and triazole-derived carbamates as LPA1-selective and LPA1/3 dual
antagonists. In particular, compound 2, the most selective LPA1 antagonist reported,
inhibited proliferation and contraction of normal human lung fibroblasts (NHLF) following LPA stimulation. Oral dosing of
compound 2 to mice resulted in a dose-dependent reduction of plasma histamine levels in a murine LPA challenge model.
Furthermore, we applied our novel antagonists as chemistry probes and investigated the contribution of LPA1/2/3 in mediating
the pro-fibrotic responses. Our results suggest LPA1 as the major receptor subtype mediating LPA-induced proliferation and
contraction of NHLF.
Lysophosphatidic acid (LPA) is a class of biologically active
phospholipids produced from lysophosphatidyl choline (LPC)
catalyzed by the enzyme autotaxin.3 LPA exerts a wide range of
cellular responses, such as calcium mobilization, cell prolifer-
ation, cell transformation, and chemotaxis through a family of
membrane bound GPCRs.4 There are at least six LPA receptors
(LPAR1−6) identified and characterized, with LPA1/2/3 from
the EDG family sharing a relatively high homology.5,6 LPA has
been demonstrated to induce the proliferation of lung
fibroblasts,7 to promote the differentiation of lung fibroblasts
to myofibroblasts,8 and to augment fibroblast-mediated
contraction of released three-dimensional collagen gels9
underscoring the importance of this pathway in mediating
profibrotic responses of lung fibroblasts in vitro. Using an in vivo
murine unilateral ureteral obstruction (UUO) model to study
renal fibrosis, expression of LPA1 was significantly up-regulated,
while LPA3 was down-regulated, with no change in expression
of LPA2 or LPA4 reported.10 In the in vivo model of murine
bleomycin-induced pulmonary fibrosis, LPA levels were
increased in bronchoalveolar lavage fluid (BALF) following
lung injury.11 In support of a role of the LPA pathway in
mediating lung fibrosis in response to bleomycin challenge,
LPA1 knockout mice displayed a significant reduction in
fibroblast recruitment, reduced vascular leakage, and increased
INTRODUCTION
■
Idiopathic pulmonary fibrosis (IPF) represents the most severe
form of interstitial pneumonias with a typical survival time of
between three to five years after diagnosis. In addition, the lack
of pharmaceutical treatments capable of altering the natural
course of the disease underlies the exceptionally high unmet
medical need and underscores the urgent necessity of
identifying and developing novel efficacious treatment options.
Although the exact molecular mechanisms of IPF are
incompletely understood, it is recognized that the pathogenesis
of pulmonary fibrosis is characterized by excessive extracellular
matrix (ECM) protein deposition into lung alveolar inter-
stitium leading to irreversible scarring, loss of function of the
capillary alveolar unit, impaired gas exchange, and ultimately
respiratory failure and death. Numerous pathways have been
implicated in driving these diverse processes, highlighting the
complexity of the pathophysiological mechanisms driving
interstitial lung fibrosis.1,2 The paucity of approved therapies
that demonstrate robust efficacy for IPF is most likely due to
the involvement of multiple pathways and mechanisms that
ultimately contribute to irreversible lung scarring. The lung
myofibroblast represents the central effector cell in pulmonary
fibrosis by virtue of the ability of this cell type to synthesize and
secrete extracellular matrix proteins.2 Thus, pathways that act to
induce the accumulation of interstitial lung myofibroblasts or
promote their synthetic, profibrotic properties represent
attractive pharmaceutical targets for the treatment of IPF.
Received: July 13, 2012
Published: August 15, 2012
© 2012 American Chemical Society
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Figure 1. Structures of LPAR antagonists.
a
Scheme 1. Preparation of Compounds 3−10
a
Reaction conditions: (a) (i) Pd(PPh3)4 (cat), Cs2CO3, DMF, 80 °C; (ii) LiOH (aq), THF, 60 °C; (b) diphenylphosphoryl azide, triethylamine,
toluene, 85 °C; (c) X-Phos (cat), Pd(OAc)2 (cat), K3PO4, toluene/water, 95 °C; (d) triphosgene, triethylamine, dichloromethane, 85 °C; (e) THF/
LiOH (aq), rt.
survival following bleomycin challenge compared to wild-type
animals.11 Additional murine models of pulmonary fibrosis such
as the radiation-induced model of lung fibrosis report the
possible involvement of LPA1 and LPA3 in driving the fibrotic
responses.12 In IPF patients, LPA levels were elevated in BALF,
and LPA1 was identified as the predominant LPA receptor
expressed in lung fibroblasts and was responsible for mediating
enhanced migration of these cells.11 Similarly, in our own
studies we have also detected the expression of LPA1, LPA2,
LPA3, LPA4, and LPA6 using quantitative PCR with the
predominant isoform being LPA1 in normal human lung
fibroblasts (NHLFs) and lung fibroblasts isolated from IPF
patients (unpublished data). Taken together, this preclinical
and clinical data suggests that targeting the LPA1 receptor
could provide a novel therapeutic approach to treat IPF.
Several LPAR antagonists have been reported in the
literature.13−16 These antagonists have been classified as lipid-
like molecules, such as nonhydrolyzable LPA analogues, and
non lipid-like small molecules. The first non lipid-like LPAR
antagonist, Ki16425, was reported as a dual LPA1/3
antagonist.17 On the basis of the structure of Ki16425, biphenyl
substituted isoxazole analogues AM095 and AM966 were
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a
Scheme 2. Preparation of Compounds 11−20
a
Reaction conditions: (a) 4-hydroxyphenylboronic acid, Pd(PPh3)4 (cat), K2CO3, DMF; (b) (CF3SO2)2O, triethylamine, dichloromethane, −78 °C
to rt; (c) bis(pinacolato)diboron, KOAc, PdCl2(dppf) (cat), dioxane, 90 °C; (d) X-Phos (cat), Pd(OAc)2 (cat), K3PO4, toluene/water, 100 °C; (e)
LiOH (aq), THF, rt; (f) triphosgene, triethylamine, dichloromethane, 85 °C; (g) NaOH (aq), ethanol, THF, rt.
reported as orally active, selective LPA1 antagonists.18−20
Recently, LPA2 signaling has also been implicated in LPA-
induced αvβ6 integrin-mediated TGF-β activation.21 LPA2-
selective antagonists have also been reported (compound 1 in
Figure 1).22
isocyanate. For the preparation of compound 10, 4-bromo-1-
methypyrazole-5-carboxylic acid was first converted to
carbamate 45, which was further coupled with arylboronate
46 followed by saponification.
The preparation of compounds 11−20 is described in
Scheme 2. The required biphenylboronic acid esters 47 and 48
were synthesized in three steps from the corresponding
arylbromide. Compound 11 was obtained from the coupling
of 47 with 45 followed by saponification. Using the same
synthetic sequence, the cyclopropanecarboxylic acid analogues
12−20 were prepared as shown in Scheme 2.
The synthesis of N-aryltriazole analogues is described in
Scheme 3. The reaction of arylazide with substituted alkyne
gave triazole derivatives. The desired triazole was confirmed by
NOE analysis. Following the sequence of saponification,
Curtius rearrangement, biaryl coupling, and the final
saponification, the acetic acid derivative (compound 21),
cyclopropanecarboxylic acid derivatives (2, 22−28, 31−32),
and N-acylsulfonamide 29 were synthesized. The preparation of
compound 30 was through the conversion of the intermediate
cyanide to the corresponding tetrazole using methods
previously described in the literature.23
In order to identify novel LPA1-selective antagonists, we
undertook a bioisostere approach and discovered pyrazole and
triazole-derived carboxylic acids as LPA1-selective and LPA1/3
dual antagonists. In particular, compound 2 (Figure 1) to our
knowledge represents the most selective LPA1 antagonist so far
reported. Compound 2 demonstrated dose-dependent oral
efficacy in blocking LPA-induced histamine release in mice.
Furthermore, we applied our novel LPAR antagonists as
chemical probes to study the contribution of LPAR isoforms in
mediating the pro-fibrotic responses of normal human lung
fibroblasts (NHLFs) in response to LPA stimulation. Our
results clearly suggest LPA1, but not LPA2 or LPA3, is the
major receptor mediating LPA-stimulated proliferation and
contraction in NHLFs. Herein we report the design, synthesis,
and biological studies of a novel series of LPA1 antagonists
(compounds 2−43) from the substituted pyrazole and triazole
chemical class.
Scheme 4 describes the synthesis of 4-N-aryl-3-amino-4H-
[1,2,4]triazole analogue 33. 4-Bromoaniline was converted to 2-
methyl-1-arylisothiourea through a sequence of thioisocyanate
formation, thiourea formation, and then methylation of the
thiourea. After displacement of the methylthio group by
hydrazine and subsequent ring formation with formic acid, 4-
N-aryl-3-amino-4H-[1,2,4]triazole 52 was formed. Conversion
of the arylbromide to the corresponding biphenyl derivative
followed by carbamate formation and final saponification gave
compound 33.
CHEMISTRY
■
Compounds 3−10 were prepared as described in Scheme 1.
The commercially available 4-bromo-1-methyl-1H-pyrazole-5-
carboxylic acid methyl ester was reacted with an arylboronic
acid under Suzuki coupling conditions. Following saponifica-
tion and Curtius rearrangement, compounds 3−7 were
obtained. Alternatively, aminopyrazole 44 was reacted with an
arylboronic acid under Suzuki coupling conditions. The
resulting 5-amino-4-aryl-1-methylpyrazole was converted to
the corresponding carbamate 8−9 through the intermediate
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a
Scheme 3. Synthesis of N-Aryltriazole Compounds 2 and 21−32
a
Reaction conditions: (a) sodium azide, Cu(OAc)2 (cat), methanol, rt; (b) toluene, sealed vessel, 150 °C, 4.5 h; (c) (i) LiOH (aq), THF, rt; (ii)
diphenylphosphoryl azide, triethylamine, toluene, 65 °C; (d) S-Phos (cat), Pd(OAc)2 (cat), K3PO4, toluene/water, 100 °C, 4 h; (e) NaOH (aq),
THF, ethanol, rt; (f) bis(pinacolato)diboron, KOAc, PdCl2(dppf) (cat), dioxane, 90 °C; (g) TMSN3, n-Bu2SnO (2 equiv), 100 °C, 15 h.
Finally, the synthesis of N-methyltriazole analogues 34−43 is
described in Scheme 5. The required N-methyltriazole was
formed through the reaction of an arylacetylene derivative with
trimethylsilymethylazide. The structures of the two regio-
isomers were assigned by NOE analysis. Following the
deprotection of the trimethylsilyl group and ester saponifica-
tion, 1N-methyl-4-aryl-[1,2,3]-triazole-5-carboxylic acid and
1N-methyl-5-aryl-[1,2,3]-triazole-4-carboxylic acid were ob-
tained. Conversion of the carboxylic acid to compounds 34−
42 was carried out using the same method described in Scheme
3. For the preparation of the cyclohexane acetic acid analogue
43, 4-iodocyclohexane acetic acid ethyl ester was coupled with
an arylbromide through the formation of an alkyl zinc
intermediate. The 1,4-substitutions of cyclohexane in 43
compose a mixture of cis/trans isomers.
RESULTS AND DISCUSSION
■
The design of novel bioisosteres has been practiced in drug
discovery to optimize drug−target interactions.24 We envi-
sioned that aminopyrazole and aminotriazole could function as
bioisosteres of the aminoisoxazole in Ki16425 and AM095. In
particular, these novel heterocyclic bioisosteres could provide
unique selectivity toward LPAR isoforms. To this end, we
investigated the potential of N-methyl-5-aminopyrazole as a
bioisostere of 4-amino-3-methylisoxazole. Compounds pre-
pared in Scheme 1 were tested in a fluorometric imaging plate
reader (FLIPR) assay using an engineered Chem-1 cell line
overexpressing the human LPA1 or LPA3 receptors. The
natural ligand LPA (18:1) was used to stimulate calcium
mobilization at a final concentration of 1 μM. To prevent false
positives in the FLIPR assay, compounds were also tested in
wild-type Chem-1 cell lines without LPAR overexpression and
stimulated with ionomycin. LPA1 and LPA3 antagonist activity
is expressed as IC50, the compound concentration to inhibit
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a
Scheme 4. Synthesis of 4-N-Aryl-[1,2,4]triazole Derivative 33
a
Reaction conditions: (a) thiophosgene, CaCO3, dichloromethane; (b) NH3/THF; (c) MeI, methanol, rt; (d) hydrazine monohydrate; (e) formic
acid, 120 °C, 3.5 h; (f) PdCl2(dppf) (cat), Na2CO3 (aq), dioxane, 100 °C, 24 h; (g) LiHMDS, THF; (h) LiOH (aq), THF.
a
Scheme 5. Synthesis of Compounds 34−43
a
Reaction conditions: (a) benzene, reflux, 4 h; (b) TBAF, THF, 0 °C; (c) LiOH (aq), THF, rt; (d) diphenylphosphoryl azide, triethylamine,
toluene, 80 °C; (e) X-Phos (cat), Pd(OAc)2 (cat), K3PO4, toluene/water, 100 °C; (f) NaOH (aq), THF, ethanol, rt; (g) activated zinc, THF; (h)
Pd(OAc)2 (cat), S-Phos (cat), THF; (i) Pd/C, H2; (j) diphenylphosphoryl azide, (R)-phenylethanol, triethylamine, 80 °C; (k) NaOH (aq), THF,
ethanol; (l) PdCl2(dppf) (cat), DPPF (cat), DMF, Na2CO3 (aq), 80 °C.
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50% activity of the intracellular calcium responses following
stimulation of the cells with the natural ligand agonist, LPA
(18:1). As indicated in Table 1, the carboxylic acid is not
LPA has been reported to act as a potent mitogenic factor on
NHLFs.7 In order to assess the effects of LPA on the
proliferation of NHLFs, we measured the incorporation of
nucleoside bromodeoxyuridine (BrdU) into the newly
synthesized DNA of NHLFs following stimulation with LPA
(18:1). Figure 2A demonstrates that LPA enhanced the
proliferation of NHLFs in a concentration-dependent manner
(EC50 = 12 μM). Since LPA1/2/3 belong to the same EDG
family and they share significant structural homology,25 we
applied our selective LPAR antagonists as chemical tools to
determine which receptor isoforms were responsible for the
NHLF proliferation mediated by LPA (18:1). For NHLF
proliferation assays, NHLFs were stimulated with 15 μM LPA
(18:1). Compound 1, a potent LPA2 antagonist (IC50 = 17
nM),22 showed no LPA1 or LPA3 antagonist activity in our
FLIPR assay. As displayed in Figure 2B, compound 1 did not
reach 50% inhibition even at high compound concentrations
(10 μM). Compound 8, the (R)-enantiomer with LPA1 activity
in our FLIPR assay (LPA1 IC50 = 0.64 μM), inhibited LPA-
Table 1. SAR of the N-Methylpyrazole Derived Non-
Carboxylic Acid LPA1 Antagonists 3−10
(R) or
(S)
LPA1 IC
LPA3 IC
a50
a50
compd
R1
R2
(μM)
(μM)
3
H
H
(R)
17.5 (65%@
>30
b
30)
4
5
6
4-OCH3
4-phenyl
H
H
H
(R)
(R)
>30
>30
>30
>30
>30
2-Cl (R)/(S) 3.59 (>97%@
b
30)
induced NHLF proliferation more than 50% at 1 μM (IC50
=
7
8
4-F
2-F
2-F
2-Cl (R)/(S) 3.97 (>97%@
>30
>30
b
30)
0.81 μM) and reached complete inhibition at higher
concentrations, while the inactive (S)-enantiomer, compound
9 (IC50 > 30 μM in the LPA1 FLIPR assay), showed
significantly less potency (Figure 2B). Taking the combined
data from Figure 2A and B, our data strongly suggest that
LPA1, and not LPA2, plays a critical role in mediating NHLF
proliferation in response to LPA (18:1) addition.
2-Cl (R)
0.644 (>97%@
b
30)
9
2-Cl (S)
>30
>30
>30
>30
10
4-
H
(R)
CH2COOH
a
Mean value from at least three FLIPR assay IC50 determinations; the
b
average CV of the assay was 15%. % inhibition at the indicated
Despite noncarboxylic acid 8 demonstrating reasonable
potency in both FLIPR and NHLF proliferation assays, the
microsomal instability of this compound (mouse liver micro-
somal clearance of 89 mL/min/kg and human liver microsomal
clearance of 22 mL/min/kg) prevented us using this for in vivo
studies. After unsuccessful attempts to identify more potent
noncarboxylic acid LPA1 antagonists in this chemical class
(data not shown), we investigated carboxylic acid analogues of
biphenyl substituted aminopyrazoles. Compounds 11−20 were
studied in the LPA1/3 FLIPR assay (Table 2).
Although direct attachment of an acetic acid moiety at the 4-
position of the phenyl group (compound 10, Table 1)
abolished LPA1 activity, and substituting phenyl with a
biphenyl resulted in an inactive compound (compound 5,
Table 1), adding an acetic acid to the 4-position of the biphenyl
(compound 11, Table 2) led to a very potent LPA1 antagonist
with high selectivity against LPA3 (>850-fold). Replacing the
acetic acid with cyclopropanecarboxylic acid gave similar LPA1
concentration.
required for LPA1 inhibition (3, 6, 7, and 8). The 2-chloro
substitution at the R2 position of the carbamate significantly
improved the potency from a partial antagonist (compound 3)
to a full antagonist (compound 6). Interestingly, the same
carbamate moiety was present in both Ki16425 and AM966.
Substitution at the 4-position of the phenyl ring (R1 in Table
1) with a methoxy or phenyl group completely abolished LPA1
inhibition potency (4 and 5). While the 4-fluorine substitution
did not change the potency, the 2-fluorine substitution
improved activity by 2- to 3-fold (compare 6 with 7 and 8).
We also observed a stereochemical preference in favor of the
(R)-enantiomer (compare 8 with 9), which was consistent with
the structure of AM095. When an acetic acid moiety was
attached at the 4-position (compound 10), inhibition of LPA
receptor activity was completely lost.
Figure 2. Inhibition of LPA-induced NHLF proliferation by LPA1 and LPA2 antagonists. (A) LPA concentration-dependently increases NHLF
proliferation. (B) Effects of compounds 1, 8, and 9 on LPA (18:1) (15 μM)-induced NHLF proliferation.
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LPA1 than compounds from the corresponding pyrazole series
(compare Table 2 with Table 3). Substitution on the phenyl
ring of the carbamate or replacing the phenyl group with alkyl
groups led to reduced antiproliferative potency (compounds
22−28). In comparison with Ki16425 and AM095, compound
2 showed much improved antiproliferative activity (Figure 4A).
The carboxylic acid isosteres 29 and 30 maintained LPA1
potency but were less active in blocking proliferation (compare
29 and 30 with 2 in Table 3). For the heterocyclic triazole core,
deletion of the methyl group or substitution with an ethyl
group was well tolerated (31 and 32). Interestingly, unlike the
N-aryl-[1,2,3]triazole 32, the N-aryl-[1,2,4]triazole 33 was
significantly less active. The observed differences between the
two heterocycles can be explained by the stable conformation
analysis as illustrated in Scheme 6. It is likely that the NH group
in compound 33 exists as the less active conformation due to
the unfavorable electronic repulsion. The similar effects of
conformational preference due to dipole−dipole interaction on
biological activity were reported in GSK3 inhibitors.26
To support our conformational analysis, we investigated the
differences in the energetic landscapes of the triazole-carbamate
torsion angles of 2, 32, and 33 through gas-phase density
functional theory (DFT) calculations at the B3LYP/6-31G**
level. The energy and torsion angle plots revealed that both 32
and 2 have energetic minima at 0° and 40°, respectively, near
the orientation shown on the left of Scheme 6 (Figure 3). In
contrast, the less active analogue 33 shows a minimum near
100°, where the plane of the carbamate is approximately
perpendicular to the plane of the triazole. The difference in
energies for 33 between the minimum energy conformation
and the conformations with 0° and 40° torsion angles are 4.36
and 3.57 kcal/mol, respectively (Figure 3). These calculations
support the hypothesis that differences in conformational
preference underlie the differences in activity of 32 and 33.
Finally, we investigated 1N-methyl-4-aryl-[1,2,3]triazole
derivatives. As listed in Table 4, unlike N-aryl-[1,2,3]triazole
21, the acetic acid analogue 34 exhibited potent inhibition of
LPA1 in the FLIPR assays with good selectivity over LPA3.
However, the cyclopropane carboxylic acid analogues displayed
much higher potency in inhibiting NHLF proliferation induced
by LPA (18:1) (compare 34 with 35 and 36). Fluorine
substitution improved the LPA1 selectivity by 10-fold over
LPA3 without affecting the LPA1 potency (compare 35 with
36). The nonbiaryl acetic acid analogue 43 not only maintained
LPA1 inhibitory potency but also displayed high inhibitory
activity in blocking LPA-induced NHLF proliferation. The
regio-isomeric 1N-methyl-5-aryl-[1,2,3]triazole derivative 40
showed significantly less inhibitory activity toward LPA1 or
LPA3 in the FLIPR assays. This can also be explained by the
unfavorable dipole−dipole interaction that causes the less active
conformation as illustrated in Scheme 6.
Table 2. SAR of the N-Methylpyrazole Derived Carboxylic
Acid LPA1 Antagonists 11−20
(R) or
(S)
LPA1 IC
a50
LPA3 IC
a50
BrdU IC
b50
compd
R
(μM)
0.035
(μM)
>30
(μM)
11
12
13
14
15
16
phenyl
phenyl
phenyl
(R)
(R)
(R)
0.047
c
d
65%@30
0.021
>30
ND
8.34
5.80
8.26
0.478
0.0026
0.0045
0.005
2-F-phenyl (R)
0.024
4-F-phenyl (R)/(S)
0.057
3-CF3-
phenyl
(R)
0.041
0.037
c
c
17
18
19
ethyl
(R)
(R)
0.038
0.106
0.176
0% @ 30
9% @ 30
0.048
0.03
isopropyl
13.7% @
0.192
c
30
c
c
d
20
0% @ 30
0% @ 30
ND
a
Mean value from at least three FLIPR assay IC50 determinations.
b
NHLF proliferation induced by LPA (18:1) (15 μM) and determined
c
by BrdU incorporation. % inhibition at the indicated concentration.
d
Not determined.
potency but significantly improved antiproliferation activity
(compare 11 with 13). The carboxylic acid was also found to be
crucial for LPA1 inhibition (compare 12 with 13). The
improved inhibitory potency in the NHLF proliferation assay
observed with the cyclopropanecarboxylic acid prompted us to
further study this chemical class. As shown in Table 2, an aryl
carbamate is not necessary to maintain LPA1 antagonist
potency (R group in Table 2). However, substituting aryl with
alkyl groups decreased the inhibitory activity of the series in the
NHLF proliferation assay (compare 13 with 17 and 18).
Introduction of polar groups, such as oxetane, caused complete
loss of antagonist activity (compare 19 with 20). Trifluor-
omethyl substitution at the meta-position significantly increased
inhibitory potency in the LPA3 FLIPR assay (17-fold) with
little impact on the LPA1 FLIPR assay (compare 13 with 16).
Interestingly, the LPA1/3 dual antagonist 16 was 10-fold less
potent than the LPA1-selective antagonist 13 in the NHLF
proliferation assay.
To our surprise, the acetic acid derivative of N-aryltriazole
(compound 21) showed significantly reduced LPA1 antagonist
activity in the FLIPR assays (compare 21 with 11). However,
the corresponding cyclopropanecarboxylic acid analogue
(compound 2) displayed very potent and highly selective
inhibitory activity toward LPA1, with little inhibition on LPA3
even at very high concentrations (Table 3). To our knowledge,
compound 2 is the most selective nonlipid LPA1 antagonist so
far reported in the literature. It appears that compounds from
the N-aryltriazole chemical class were much more selective for
The most interesting observations were obtained using the
LPA1/3 dual antagonists. The trifluoromethyl substitution
increased LPA3 inhibition potency by 17-fold without affecting
LPA1 inhibitory activity (compare 38 with 35 in Table 4),
which is consistent with the data obtained with the
corresponding pyrazole analogues (compare 13 and 16 in
Table 2). The same trend was observed with N-acylsulfonamide
analogues (compare 41 with 42 in Table 4). More interestingly,
the LPA1/3 dual antagonist was significantly less active than
the LPA1-selective antagonist in inhibiting NHLF proliferation
mediated by LPA (18:1) despite demonstrating comparable
LPA1 inhibitory potency (compare 42 with 41). This
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Table 3. SAR of N-Aryltriazole Derived LPA1 Antagonists 2 and 21−33
a,
c
a,
c
b
compd
R
(R) or (S)
LPA1 IC50 (μM)
LPA3 IC50 (μM)
BrdU IC50 (μM)
21
2
phenyl
(R)
41% @ 30
0.0252
0.0345
0.112
3% @ 30
20% @ 30
42% @ 30
55% @ 30
6.86
ND
phenyl
(R)
0.004
0.021
0.077
1.20
22
23
24
25
26
27
28
29
30
31
32
33
2-F-phenyl
2-CF3-phenyl
3-CF3-phenyl
isopropyl
ethyl
(R)
(R)
(R)
0.174
(R)
0.217
17% @ 30
0% @ 30
26% @ 30
0% @ 30
7% @ 30
28% @ 30
63% @ 30
23% @ 30
17% @ 30
0.060
0.178
0.189
1.62
(R)
0.398
cyclobutyl
gem-dimethyl
phenyl
(R)/(S)
0.161
0.985
(R)
(R)
(R)
(R)
(R)
0.032
0.060
0.105
0.016
0.040
phenyl
0.023
phenyl
0.042
phenyl
0.036
d
phenyl
11% @ 30
ND
a
b
c
Mean value from at least three FLIPR assay IC50 determinations. NHLF proliferation induced by LPA (18:1) (15 μM). % inhibition at the
d
indicated concentration. Not determined.
Scheme 6. Stable Conformation Analysis of 32 and 33
observation may be due to the differential roles of individual
LPAR isoforms that effect cellular responses to LPA such as
migration and colony formation in soft agar as has been
observed in transfected B103 cells.27 Interestingly, in these
studies, LPA1 and LPA3 exhibited opposing effects in B103 cell
migration and colony formation in soft agar in response to LPA
challenge.27 It should be noted that in NHLFs we can detect
the expression of both LPA1 and LPA3 by quantitative PCR
(data not shown). Figure 4B shows the concentration-
dependent inhibition of LPA (18:1)-induced NHLF prolifer-
ation by LPA1 and LPA1/3 dual antagonists. Taking the results
from Tables 1−4 and Figures 2 and 4, we conclude that in
Figure 3. Energy as a function of the triazole-carbamate torsion angle
for the [1,2,3]-triazole compound 32 (magenta circles), its active 4-
methyl analogue 2 (cyan squares), and the less active [1,2,4]-triazole
compound 33 (green diamonds). A torsion angle of 0° or 360°
corresponds to the conformations drawn on the left side of Scheme 6,
while a torsion angle of 180° corresponds to the conformations drawn
on the right side of Scheme 6.
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Table 4. SAR of N-Methyltriazole Derived LPA1 Antagonists 34−43
a,
c
a,
c
b
compd
R
R1
(R) or (S)
LPA1 IC50 (μM)
LPA3 IC50 (μM)
BrdU IC50 (μM)
34
35
36
37
38
39
40
41
42
43
phenyl
(R)
(R)
(R)
(R)
(R)
(S)
(R)
(R)
(R)
(R)
0.048
0.018
0.018
0.070
0.017
2.65
21.94
0.06
phenyl
OH
OH
OH
OH
OH
OH
2.29
0.003
0.002
0.020
0.074
phenyl
27.35
isopropyl
3-CF3-phenyl
3-CF3-phenyl
phenyl
24% @ 30
0.132
d
6.30
ND
d
12% @ 30
0.019
0.024
0.058
10% @ 30
2.31
ND
phenyl
CH3SO2NH
CH3SO2NH
0.004
0.331
0.007
3-CF3-phenyl
phenyl
0.065
18.6
a
b
c
Mean value from at least three FLIPR assay IC50 determinations. NHLF proliferation induced by LPA (18:1) (15 μM). % inhibition at the
d
indicated concentration. Not determined.
NHLFs, LPA (18:1)-induced proliferation is mediated mainly
through LPA1 and not LPA2 or LPA3 signaling.
LPA has been reported to act as a contractile agent on
NHLFs using the three-dimensional collagen gel contraction
assay.9 Additional studies using lung fibroblasts obtained from
the lung of IPF patients suggests that these cells may exhibit
elevated expression of contractile proteins and to demonstrate
enhanced contraction in three-dimensional collagen gels.28 We
used NHLFs cultured in three-dimensional collagen gels to
assess the effect of LPA (18:1) on NHLF contraction. As
shown in Figure 5A, LPA concentration-dependently induced
contraction of collagen gel matrices containing cultured
NHLFs. At concentrations as low as 1 μM, LPA was able to
decrease the gel size, with the maximum effect at 30 μM. We
next studied the most selective LPA1 antagonist (compound 2)
and the literature LPA2-selective antagonist (compound 1).
Following LPA stimulation (15 μM), the LPA2 antagonist
showed no detectable inhibition of the collagen gel contraction,
while the LPA1-selective antagonist concentration-dependently
inhibited the contraction as shown in Figure 5B. To further
determine the relationship between NHLF contraction and
proliferation induced by LPA, we evaluated LPA1 and LPA1/3
dual antagonists (compound 41 and 42). As shown in Figure
5C, the LPA1/3 dual antagonist 42 demonstrated significantly
less activity in preventing NHLF contraction than LPA1-
selective antagonist 41. Interestingly, this trend correlated well
with LPA-induced NHLF proliferation (Figures 4B and 5C).
Therefore, using our novel LPAR antagonists as probes, we
have pharmacologically dissected the role of LPA1/2/3
function in mediating LPA-stimulated NHLF proliferation
and contraction, processes critical in the progression of lung
fibrosis. Table 5 lists the IC50 determinations of selected potent
LPA1 antagonists in inhibiting NHLF contraction in response
to LPA (18:1).
Figure 4. Inhibition of LPA (18:1)-induced NHLF proliferation by
LPA1 and LPA1/3 dual antagonists. (A) Concentration-dependent
inhibition of LPA (18:1) (15 μM)-stimulated NHLF proliferation by
2, AM095, and Ki16425. (B) Effects of LPA1 and LPA1/3 dual
antagonists (41 and 42) on LPA (18:1) (15 μM)-stimulated NHLF
proliferation.
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Figure 5. LPA-mediated contraction of NHLFs assessed in three-dimensional collagen gels. (A) LPA (18:1) concentration-dependently increased
NHLF contraction. (B) Photo image of the effects of LPAR inhibitors on 15 μM LPA (18:1)-mediated contraction. (C) Effects of LPA1 and LPA1/
3 dual antagonists (41 and 42) on 15 μM LPA (18:1)-mediated NHLF contraction.
intravenous LPA injection, the LPA-induced histamine level
was significantly blocked (Figure 6B). A clear PK/PD
relationship was demonstrated by the correlation between the
levels of compound 2 and LPA-induced histamine concen-
trations in plasma (Figure 6C). Although AM095 almost
completely blocked histamine release (100 mg/kg), analysis of
plasma samples revealed more than 65-fold higher concen-
trations of AM095 than compound 2 (100 mg/kg). The ability
of compound 2 to block histamine release at much lower
plasma concentration suggests that further improvement of
pharmacokinetic properties of this chemical class could lower
the effective dose.
IPF represents a disease of enormous unmet medical need
due to the lack of effective treatment options and the dismal
prognosis associated with the disease. Preclinical and clinical
data exist that support targeting LPA1 for the treatment of
organ fibrosis including IPF due to the role of this receptor in
mediating the profibrotic effects of LPA in fibroblasts.7,8,11
More recently, it has been reported that LPA acting through
the LPA1 receptor isoform may induce apoptosis of human
bronchial epithelial cells suggesting that LPA1 may promote
lung fibrosis in cell types other than pulmonary fibroblasts.29 In
this work, we have used our novel LPA1, LPA1/3 dual, and
literature LPA2 antagonists as chemical probes to pharmaco-
Table 5. IC50 Determinations of Selected Potent LPA1
Antagonists in Inhibiting NHLF Contraction in Response to
15 μM LPA (18:1)
a
compd
inhibition of contraction IC50 (μM)
AM095
0.15
0.16
0.39
0.02
0.10
0.02
0.34
0.02
0.22
Ki16415
34
35
2
13
38
36
43
a
NHLF contraction induced by LPA (18:1) @15 μM.
The unique LPA1 selectivity determined for compound 2
prompted us to evaluate this molecule to inhibit LPA-
stimulated histamine release in the mouse, an acute PK/PD
model to study LPA1 antagonist activity reported in the
literature.19 As shown in Figure 6A, LPA dose-dependently
caused an acute histamine increase in the plasma following
intravenous injection. When mice were orally dosed with
compound 2 (100 mg/kg, aqueous suspension) prior to
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inhibition of LPA-induced NHLF proliferation and the
inhibition of LPA-induced NHLF contraction by our novel
LPA1 antagonists further underscore that LPA1 represents a
potentially attractive therapeutic target for the treatment of IPF.
EXPERIMENTAL SECTION
■
All reactions were carried out under an argon atmosphere. Solvents
were purchased from commercial sources and used without further
drying. 1H NMR spectra were recorded with Mercury 300 and
Unityplus 400 MHz spectrometers. All compounds were analyzed by
LC/MS (liquid chromatography/mass spectrometry) using a Waters
ZQ mass detector and Waters LC system. Ionization was generally
achieved via electron spray (ES). The LC fraction detection consisted
of both a diode array detector and an evaporative light scattering
detector, and all tested compounds had purity greater than 98%.
[4-(2-Fluoro-phenyl)-2-methyl-2H-pyrazol-3-yl]-carbamic
Acid (R)-1-(2-Chloro-phenyl)-ethyl Ester (8). 4-Bromo-1-methyl-
1H-pyrazol-5-amine (800 mg, 4.55 mmol), 2-fluorophenylboronic acid
(890 mg, 6.36 mmol), X-Phos (217 mg, 0.45 mmol), palladium acetate
(51 mg, 0.227 mmol), and potassium phosphate tribasic (1.93 g, 9.09
mmol) were combined in 12 mL of toluene. Degassed water (4 mL)
was added, and argon was bubbled through for 3 min. The reaction
tube was sealed, and the mixture was stirred at 95 °C overnight.
Solvents were evaporated, and the residue was extracted with ethyl
acetate and water. The organic layer was washed with brine and dried.
Solvents were evaporated, and the residue was purified by flash column
chromatography (ethyl acetate containing 3% methanol in hexanes
10% to 80%) to give 4-(2-fluorophenyl)-2-methyl-2H-pyrazole-3-
1
amine as a brown oil (649 mg, 74.7% yield). H NMR (DMSO-d6) δ
ppm 3.60 (s, 3H), 5.30 (br s, 2H), 7.14−7.25 (m, 3H), 7.30 (d, J = 2.5
Hz, 1H), 7.39−7.52 (m, 1H); LC/MS calcd for C10H10FN3 (m/e)
191.0; obsd, 192.0 (M + H, ES+).
4-(2-Fluorophenyl)-2-methyl-2H-pyrazol-3-amine (110 mg, 0.575
mmol) and triphosgene (222 mg, 0.748 mmol) were combined with
dichloromethane (4 mL) and toluene (6 mL). The reaction tube was
sealed. The mixture was stirred in an ice bath, and triethylamine (0.7
mL) was added. The mixture was stirred at 85 °C for 20 min, and 1-
(2-chlorophenyl)ethanol (117 mg, 0.748 mmol) in 1 mL of toluene
was added. The mixture was stirred at 90 °C for 1 h. Solvents were
evaporated, and the residue was extracted with ethyl acetate and water.
The organic layer was dried and concentrated. The residue was
purified by flash column chromatography (ethyl acetate in hexanes 0%
to 50%) to give [4-(2-fluoro-phenyl)-2-methyl-2H-pyrazol-3-yl]-
carbamic acid 1-(2-chloro-phenyl)-ethyl ester as an amorphous
Figure 6. LPA-induced histamine release in C57BL/6 mice. (A) LPA
(intravenous injection) dose-dependently increased plasma histamine
levels. (B) Dose-dependent inhibition of LPA-mediated plasma
histamine levels by orally dosed compound 2. (C) Correlation of
histamine levels with compound 2 concentrations in plasma.
Significance is denoted as follows: * p < 0.05, **p < 0.01, and ***p
< 0.001.
1
powder (134 mg, 62.3% yield). H NMR (DMSO-d6) δ ppm 1.06−
1.28 (m, 1H), 1.53 (br d, J = 5.6 Hz, 2H), 3.65 (br s, 3H), 5.83−5.98
(m, 1H), 6.83−7.50 (m, 7H), 7.56 (d, J = 6.8 Hz, 1H), 7.65 (br s, 1H),
9.66 (s, 1H); LC/MS calcd for C19H17ClFN3O2 (m/e) 373.0; obsd,
372.0 (M − H, ES−). This pure racemic material was separated by
supercritical fluid chromatography using a chiral WHELKO column
(10% to 65% methanol in CO2, 70 mL/min). The second fraction
(longer retention time) was concentrated to give a pure enantiomer as
compound 8.
logically dissect the function of LPA1/2/3 in driving LPA-
mediated profibrotic responses in NHLFs. Our data suggests
that the LPA1 receptor is the predominant receptor for
mediating LPA-induced NHLF proliferation and contraction,
critical cellular responses implicated in the progression of IPF.
Since the first publication of the isoxazole-derived LPAR
antagonist, Ki16425 in 2003, the majority of nonlipid LPA1
antagonists disclosed in patent applications or published in the
literature are still based on the isoxazole core structure.30,31 By
taking bioisostere approaches, we discovered pyrazole and
triazole-derived LPA1 selective and LPA1/3 dual antagonists.
Compound 2 demonstrated the highest LPA1 selectivity and
attenuated LPA-induced NHLF proliferation and contraction
with high potency. Oral dosing of compound 2 in mice caused
a dose-dependent reduction in serum histamine levels induced
following intravenous LPA stimulation. The carboxylic acid-
derived LPA1 antagonists from our pyrazole and triazole
chemical class showed no liability to CYP, hERG, and other in
vitro predictive toxicity assays. The correlation between the
[4-(2-Fluoro-phenyl)-2-methyl-2H-pyrazol-3-yl]-carbamic
Acid (S)-1-(2-Chloro-phenyl)-ethyl Ester (9). The first fraction
from the chiral SFC separation of [4-(2-fluoro-phenyl)-2-methyl-2H-
pyrazol-3-yl]-carbamic acid 1-(2-chloro-phenyl)-ethyl ester as de-
scribed for the preparation of 8 gave a pure enantiomer as compound
9.
2-Methyl-4-phenyl-2H-pyrazol-3-yl-carbamic Acid (R)-1-
Phenyl-ethyl Ester (3). Methyl 4-bromo-2-methyl-2H-pyrazole-3-
carboxylate (438.1 mg, 2.0 mmol), phenylboronic acid (244 mg, 2.0
mmol), and cesium carbonate (1.3 g, 4.0 mmol) were dissolved in 10
mL of DMF, and the solution was degassed with argon. To this
mixture was added Pd(PPh3)4 (139 mg, 0.12 mmol). The mixture was
stirred at 80 °C for 12 h. The resulting mixture was cooled to room
temperature and filtered. The solid was rinsed with THF. The filtrate
was concentrated and purified by ISCO flash column chromatography
(0% to 25% ethyl acetate in hexanes) to give an oily material as methyl
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2-methyl-4-phenyl-2H-pyrazole-3-carboxylate (388.6 mg, 89.8% yield).
1H NMR (CDCl3) δ ppm 3.77 (s, 3H), 4.21 (s, 3H), 7.31−7.36 (m,
1H), 7.37−7.42 (m, 4H), 7.52 (s, 1H); LC/MS calcd for C12H12N2O2
216.0; obsd, 217.0 (M + H, ES+).
2H-pyrazol-3-yl-carbamic acid (R)-1-phenyl-ethyl ester (45). LRMS
calcd for C13H14BrN3O2 (m/e) 324.0; obsd, 323.0 (M − H, ES−).
4-Bromo-2-methyl-2H-pyrazol-3-yl-carbamic acid (R)-1-phenyl-
ethyl ester (263.5 mg, 0.61 mmol), ethyl 1-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-phenyl)-acetate (354 mg, 1.22 mmol), X-
Phos (116 mg, 0.244 mmol), and palladium acetate (27.4 mg, 0.122
mmol) were dissolved in toluene (6 mL), and potassium phosphate
tribasic (518 mg, 2.44 mmol) in degassed water (1.5 mL) was added.
The mixture was degassed for 5 min and sealed. The mixture was
stirred at 100 °C for 4 h and then extracted with ethyl acetate and
water. The organic layer was washed with brine and dried. Solvents
were evaporated, and the residue was purified by ISCO flash column
chromatography using ethyl acetate in hexanes (0% to 50%) to give a
waxy pale yellow material as {4-[1-methyl-5-((R)-1-phenyl-ethoxycar-
bonylamino)-1H-pyrazol-4-yl]-phenyl}-acetic acid ethyl ester (134 mg,
54% yield). LC/MS calcd for C23H25N3O4 (m/e) 407.0; obsd, 408.0
(M + H, ES+).
{4-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-pyrazol-
4-yl]-phenyl}-acetic acid ethyl ester (134 mg, 0.33 mmol) was
dissolved in THF (4 mL), and aqueous lithium hydroxide solution
(0.5N, 1 mL) was added. The reaction was stirred for 6 h. Solvents
were evaporated, and the residue was treated with water (15 mL) to
give a clear solution followed by the addition of 1 N hydrochloric acid
(0.55 mL). The white solid was filtered and rinsed with water and
dried in air to give {4-[1-methyl-5-((R)-1-phenyl-ethoxycarbonylami-
no)-1H-pyrazol-4-yl]-phenyl}-acetic acid (90 mg, 72% yield). 1H
NMR (DMSO-d6) δ ppm 1.48 (br s, 3H), 3.48 (s, 2H), 3.54 (s, 3H),
5.70 (d, J = 5.6 Hz, 1H), 7.06−7.42 (m, 9H), 7.67 (s, 1H), 9.48 (br s,
1H), 12.28 (br s, 1H); LC/MS calcd for C21H21N3O4 (m/e) 379.0;
obsd, 380.0 (M + H, ES+).
Methyl 2-methyl-4-phenyl-2H-pyrazole-3-carboxylate (388.6 mg,
1.8 mmol) was dissolved in THF (8 mL), and 0.5 N LiOH solution
(4 mL) was added. The mixture was stirred at 60 °C for 2 h and then
concentrated. The residue was dissolved in water (30 mL) and filtered.
The filtrate was neutralized with 1 N hydrochloric acid, and the white
precipitate was filtered and dried in a vacuum oven at 60 °C overnight
to provide 2-methyl-4-phenyl-2H-pyrazole-3-carboxylic acid (338.5
1
mg, 93.1% yield). H NMR (DMSO-d6) δ ppm 4.06 (s, 3H), 7.25−
7.42 (m, 5H), 7.60 (s, 1H), 13.42 (s, 1H); LC/MS calcd for
C11H10N2O2 202.0; obsd, 201.0 (M − H, ES−).
2-Methyl-4-phenyl-2H-pyrazole-3-carboxylic acid (100 mg, 0.495
mmol), (R)-1-phenylethanol (60.4 mg, 0.495 mmol), DPPA (136 mg,
0.495 mmol), and TEA (100 mg, 0.989 mmol) were mixed with 3 mL
of toluene. The mixture was stirred at 80 °C for 1 h. Solvents were
evaporated, and the residue was purified by ISCO flash column
chromatography (0% to 55% ethyl acetate in hexanes) to give 2-
methyl-4-phenyl-2H-pyrazol-3-yl-carbamic acid (R)-1-phenyl-ethyl
1
ester as a white powder (106 mg, 66.7% yield). H NMR (DMSO-
d6) δ ppm 1.13−1.29 (br, 1 H), 1.52 (d, J = 5.8 Hz, 2 H), 3.60 (s, 3H),
5.58−5.82 (br m, 1H), 6.92−7.53 (m, 10H), 7.74 (s, 1H), 9.55 (s,
1H); LC/MS calcd for C19H19N3O2 321.0; obsd, 320.0 (M − H, ES−).
Compounds 4 to 7 were prepared using the same procedure as that
for the preparation of compound 3.
[4-(4-Methoxy-phenyl)-2-methyl-2H-pyrazol-3-yl]-carbamic
1
Acid (R)-1-Phenyl-ethyl Ester (4). H NMR (DMSO-d6) δ ppm
1.15−1.30 (br, 1H), 1.54 (d, J = 5.8 Hz, 2 H), 3.60 (br s, 3H), 3.75 (s,
3H), 5.76 (d, J = 6.1 Hz, 1H), 6.87 (d, J = 7.6 Hz, 2H), 6.97−7.25 (m,
1H), 7.28−7.51 (m, 6H), 7.67 (s, 1H), 9.48 (br s, 1H); LC/MS calcd
for C25H23N3O2 (m/e) 397; obsd, 398.0 (M + H, ES+); LC/MS calcd
for C20H21N3O3 (m/e) 351; obsd, 350.0 (M − H, ES−).
{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
pyrazol-4-yl]-biphenyl-4-yl}-acetic Acid (11). Ethyl 2-(4-bromo-
phenyl)-acetate (2.43 g, 10 mmol), 4-hydroxyphenylboronic acid (1.65
g, 12 mmol), Pd(PPh3)4 (693 mg, 0.6 mmol), and potassium
carbonate (2.76 g, 20 mmol) were combined in 14 mL of dry DMF.
The mixture was bubbled with nitrogen and sealed. The mixture was
stirred at 85 °C for 15 h. Solvents were evaporated, and the residue
was extracted with ethyl acetate and water. The organic layer was
washed with brine and dried over sodium sulfate. Solvents were
evaporated, and the residue was purified by flash column
chromatography (5% to 50% ethyl acetate in hexanes). The pure
fraction was combined and concentrated. The residue was dissolved in
ethyl acetate (4 mL), and hot hexanes were added. The white solid was
filtered and dried to give 4′-hydroxy-biphenyl-4-yl-acetic acid ethyl
(4-Biphenyl-4-yl-2-methyl-2H-pyrazol-3-yl)-carbamic Acid
1
(R)-1-Phenyl-ethyl Ester (5). H NMR (DMSO-d6) δ ppm 1.12−
1.30 (br, 1H), 1.57 (d, J = 5.8 Hz, 2 H), 3.64 (br s, 3H), 5.79 (d, J =
5.8 Hz, 1H), 7.29−7.51 (m, 8H), 7.52−7.58 (m, 2H), 7.59−7.65 (m,
2H), 7.68 (d, J = 7.6 Hz, 2H), 7.83 (s, 1H), 9.63 (br s, 1H); LC/MS
calcd for C25H23N3O2 (m/e) 397; obsd, 398.0 (M + H, ES+).
(2-Methyl-4-phenyl-2H-pyrazol-3-yl)-carbamic Acid 1-(2-
Chloro-phenyl)-ethyl Ester (6). 2-Methyl-4-phenyl-2H-pyrazole-3-
carboxylic acid (60 mg, 0.297 mmol), 1-(2-chlorophenyl)ethanol (46.5
mg, 0.297 mmol), DPPA (81.7 mg, 0.297 mmol), and triethylamine
(0.09 mL) were combined in 2.5 mL of toluene. The mixture was
stirred at 85 °C for 3 h. Solvents were evaporated, and the residue was
purified by flash column chromatography (0% to 50% ethyl acetate in
hexanes) to give (2-methyl-4-phenyl-2H-pyrazol-3-yl)-carbamic acid 1-
(2-chloro-phenyl)-ethyl ester as a white fluffy solid (40 mg, 38% yield).
1H NMR (DMSO-d6) δ ppm 1.15−1.28 (br, 1H), 1.55 (d, J = 5.8 Hz,
1
ester (1.68 g, 65.6% yield). H NMR (CDCl3) δ ppm 1.29 (t, J = 7.1
Hz, 3H), 3.66 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 4.95 (br. s., 1 H), 6.87
(d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.47 (m, 4H); LC/MS
calcd for C16H16O3 (m/e) 256.0; obsd, 257.1 (M + H, ES+).
4′-Hydroxy-biphenyl-4-yl-acetic acid ethyl ester (641 mg, 2.5
mmol) was dissolved in dichloromethane (15 mL). To this solution
was added trifluoromethanesulfonic anhydride (706 mg, 2.5 mmol) at
−78 °C. Triethylamine (0.35 mL, 2.5 mmol) was added. The mixture
was stirred at −78 °C for 10 min and warmed to room temperature.
After 30 min, the mixture was extracted with water and methylene
chloride. The organic layer was washed with diluted hydrochloric acid
and concentrated sodium bicarbonate solution. Solvents were
evaporated, and the residue was treated with hexanes. The gray
crystalline material was filtered to give 4′-trifluoromethanesulfonyloxy-
2H), 3.54−3.78 (m, 3H), 5.82−6.10 (m, 1H), 7.23 (d, J = 6.8 Hz,
1H), 7.28−7.54 (m, 7H), 7.59 (d, J = 6.8 Hz, 1H), 7.76 (br s, 1H),
9.68 (s, 1H). LC/MS calcd for C19H18ClN3O2 (m/e) 355.0; obsd,
356.0 (M + H, ES+).
[4-(4-Fluoro-phenyl)-2-methyl-2H-pyrazol-3-yl]-carbamic
1
Acid 1-(2-Chloro-phenyl)-ethyl Ester (7). H NMR (DMSO-d6) δ
ppm 1.15−1.30 (br, 1H), 1.56 (br d, J = 5.3 Hz, 2H), 3.62 (br s, 3H),
5.86−6.06 (m, 1H), 6.81−7.27 (m, 3H), 7.31−7.65 (m, 5H), 7.76 (br
s, 1H), 9.65 (s, 1H); LC/MS calcd for C19H17ClFN3O2 (m/e) 373.0;
obsd, 374.0 (M + H, ES+).
1
biphenyl-4-yl-acetic acid ethyl ester (812 mg, 83.6% yield). H NMR
(CDCl3) δ ppm 1.29 (t, J = 7.1 Hz, 3H), 3.68 (s, 2H), 4.19 (q, J = 7.1
Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H), 7.53 (d, J
= 8.0 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H).
{4-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-Pyr-
azol-4-yl]-phenyl}-acetic Acid (10). 4-Bromo-2-methyl-2H-pyra-
zole-3-carboxylic acid (787.2 mg, 3.84 mmol), DPPA (1.16 g, 4.22
mmol), (R)-1-phenylethanol (491 mg, 4.02 mmol), and TEA (1.10
mL, 7.68 mmol) were combined in 15 mL of toluene to give a clear
solution. The mixture was heated to 80 °C and stirred for 1 h. Solvents
were evaporated, and the residue was extracted with ethyl acetate and
sodium bicarbonate solution. The organic layer was dried and
4′-Trifluoromethanesulfonyloxy-biphenyl-4-yl-acetic acid ethyl ester
(800 mg, 2.06 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-
dioxaborolane) (628 mg, 2.47 mmol), potassium acetate (607 mg,
6.18 mmol), and Pd(dppf)Cl2 (90.4 mg, 0.124 mmol) were combined
in dry dioxane (15 mL). The mixture was stirred with argon bubbled
through for 5 min. The mixture was heated to 90 °C and stirred for 4
h. The resulting material was filtered through a layer of silica gel and
rinsed with ethyl acetate. Solvents were evaporated, and the residue
1
evaporated to give an oily material (1.38 g). H NMR of the crude
material indicated 75% of the desired compound as 4-bromo-2-methyl-
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was purified by flash column chromatography (0% to 30% ethyl
acetate in hexanes) to give [4′-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-biphenyl-4-yl]-acetic acid ethyl ester as a white
solid (728 mg, 96.5% yield). 1H NMR (CDCl3) δ ppm 1.28 (t, J = 7.1
Hz, 3H), 1.37 (s, 12H), 3.67 (s, 2H), 4.18 (q, J = 7.1 Hz, 2H), 7.37 (d,
J = 8.1 Hz, 2H), 7.60 (dd, J = 8.0, 6.4 Hz, 4H), 7.88 (d, J = 8.1 Hz,
2H).
4.14 (q, J = 7.1 Hz, 2H), 5.93 (m, 1H), 6.25 (br s, 1H), 7.35−7.48 (m,
9H), 7.53−7.64 (m, 4H), 7.72 (s, 1H); LC/MS calcd for C31H31N3O4
(m/e) 509.0; obsd, 510.0 (M + H, ES+).
1-{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
pyrazol-4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid (13).
1-{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-pyrazol-
4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid ethyl ester (80 mg,
0.157 mmol) was dissolved in 4 mL of THF, and lithium hydroxide
solution (0.5N, 2 mL) was added. The mixture was stirred at room
temperature for 5 min. Methanol (1 mL) was added to give a clear
solution. The mixture was stirred at 65 °C for 5 h and then at 30 °C
overnight. Solvents were evaporated, and the residue was dissolved in
water (12 mL). Hydrochloric acid (1 N, 1.3 mL) was added, and the
solid was filtered to give a white solid (65 mg). LC/MS indicated 85%
purity with the major impurity as the cleavage of carbamate. This solid
was dissolved in acetonitrile/methylene chloride (containing 5%
methanol) and purified by flash column chromatography (methanol in
dichloromethane 0% to 5%) to give a white solid as 1-{4′-[1-methyl-5-
((R)-1-phenyl-ethoxycarbonylamino)-1H-pyrazol-4-yl]-biphenyl-4-yl}-
cyclopropanecarboxylic acid (45 mg, 59.5% yield). 1H NMR (DMSO-
d6) δ ppm 1.19−1.25 (m, 2H), 1.49−1.57 (m, 5H), 3.64 (s, 3H),
5.65−5.83 (m, 1H), 6.95−7.25 (br m, 1H), 7.27−7.48 (m, 6H), 7.49−
7.67 (m, 6H), 7.82 (br s, 1H), 9.25 (br s, 0.2H), 9.57 (s, 0.8H), 12.35
(s, 1H); LC/MS calcd for C29H27N3O4 (m/e) 481.0; obsd, 482.1 (M +
H, ES+).
In a 50 mL round-bottom flask, palladium acetate (12.5 mg, 0.055
mmol), X-Phos (52.9 mg, 0.11 mmol), and potassium phosphate
tribasic (236 mg, 1.11 mmol) were mixed in 0.5 mL of degassed water
and 1 mL of toluene and stirred for 1 min. Then 4-bromo-2-methyl-
2H-pyrazol-3-yl-carbamic acid (R)-1-phenyl-ethyl ester (180 mg, 0.55
mmol) in 2 mL of toluene was added followed by the addition of [4′-
(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-biphenyl-4-yl]-acetic
acid ethyl ester (203 mg, 0.55 mmol) and 1 mL of toluene. The
mixture was degassed with argon and sealed. The mixture was stirred
at 95 °C overnight. The resulting mixture was extracted with ethyl
acetate and water. The organic layer was dried and evaporated. The
residue was purified by flash column chromatography (ethyl acetate
with 5% methanol in hexanes) to give {4′-[1-methyl-5-((R)-1-phenyl-
ethoxycarbonylamino)-1H-pyrazol-4-yl]-biphenyl-4-yl}-acetic acid
ethyl ester (78.0 mg, 29.1% yield) as an amorphous material. LC/
MS calcd for C29H29N3O4 (m/e) 483.0; obsd, 484.1 (M + H, ES+).
This material was dissolved in 6 mL of THF, and lithium hydroxide
solution (1 mL, 0.5 N) was added followed by 0.2 mL of methanol.
The mixture was stirred at room temperature for 4 h and concentrated.
Warm water (35 mL) was added, and the mixture was filtered. The
filtrate was acidified with 1 N hydrochloric acid (0.6 mL) and filtered.
The solid was dried under vacuum overnight to give a pale yellow solid
as {4′-[1-methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-pyrazol-
4-yl]-biphenyl-4-yl}-acetic acid (30 mg, 40.8% yield). 1H NMR
(DMSO-d6) δ ppm 1.15−1.32 (br, 1H), 1.56 (d, J = 5.8 Hz, 2H), 3.62
(s, 2H), 3.63 (s, 3H), 5.66−5.83 (br m, 1H), 7.03 (br, 0.5H), 7.18 (br,
0.5H), 7.28−7.38 (m, 3H), 7.38−7.48 (m, 3H), 7.49−7.56 (m, 1H),
7.57−7.68 (m, 5H), 7.81 (s, 1H), 9.62 (s, 1H), 12.38 (s, 1H); LC/MS
calcd for C27H25N3O4 (m/e) 455.0; obsd, 456.0 (M + H, ES+).
1-{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
pyrazol-4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid
Ethyl Ester (12). Ethyl 1-(4′-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-biphenyl-4-yl)-cyclopropanecarboxylate (100 mg, 0.255
mmol), 4-bromo-1-methyl-1H-pyrazol-5-amine (67.3 mg, 0.38
mmol), X-Phos (36.5 mg, 0.077 mol), potassium phosphate tribasic
(162 mg, 0.76 mmol), and palladium acetate (8.6 mg, 0.038 mmol)
were mixed in 4 mL of toluene. The mixture was stirred, and degassed
water (0.8 mL) was added. The mixture was degassed with nitrogen
and sealed. The mixture was stirred at 100 °C overnight and then
extracted with ethyl acetate and water. Solvents were evaporated, and
the residue was purified by flash column chromatography (methanol in
methylene chloride 0% to 10%) to give a pale gray solid as 1-[4′-(5-
amino-1-methyl-1H-pyrazol-4-yl)-biphenyl-4-yl]-cyclopropanecarbox-
Compounds 14−20 were prepared with the same method described
for the preparation of compound 13.
(R)-1-{4′-[5-((1-(2-Fluorophenyl)ethoxy)carbonylamino)-1-
methyl-1H-pyrazol- 4-yl]-biphenyl-4-yl}-cyclopropanecarbox-
ylic Acid (14). 1H NMR (DMSO-d6) δ ppm 12.37 (br. s., 1H),
9.67 (s, 1H), 7.80 (s, 2H), 7.45−7.70 (m, 8H), 7.39 (d, 2H), 7.19−
7.33 (m, 1H), 5.97 (d, J = 5.7 Hz, 1H), 3.62 (s, 3H), 1.57 (m, 2H),
1.38−1.52 (m, 2H), 1.22 (s, 1H), 1.15 (d, 2H); LC/MS calcd for
C29H26FN3O4 (m/e) 499.0; obsd, 500.1 (M + H, ES+).
1-{4′-[5-((1-(4-Fluorophenyl)ethoxy)carbonylamino)-1-
methyl-1H-pyrazol- 4-yl]-biphenyl-4-yl}-cyclopropanecarbox-
ylic Acid (15). 1H NMR (DMSO-d6) δ ppm 12.36 (br. s., 1H),
9.62 (br. s., 1H), 7.82 (s, 1H), 7.60 (d, J = 8.1 Hz, 4H), 7.51 (d, J = 7.8
Hz, 3H), 7.35−7.44 (m, 2H), 7.26 (t, J = 7.8 Hz, 2H), 6.94−7.18 (m,
1H), 5.79 (d, J = 5.8 Hz, 1H), 3.68 (br. s., 3H), 1.56 (d, J = 5.8 Hz,
2H), 1.42−1.51 (m, 2H), 1.21−1.33 (br.m, 1H), 1.11−1.21 (m, 2H);
LC/MS calcd for C29H26FN3O4 (m/e) 499.0; obsd, 498.0 (M − H,
ES−).
(R)-1-{4′-[1-Methyl-5-((1-(3-(trifluoromethyl)phenyl)ethoxy)-
carbonylamino)-1H-pyrazol- 4-yl]-biphenyl-4-yl}-cyclopropa-
1
necarboxylic Acid (16). H NMR (DMSO-d6) δ ppm 12.34 (br.
s., 1H), 9.71 (br. s., 1H), 7.45−7.85 (m, 10H), 7.39 (d, J = 8.3 Hz,
3H), 5.87 (d, J = 6.0 Hz, 1H), 3.65 (s, 3H), 1.57 (d, J = 6.0 Hz, 2H),
1.39−1.51 (m, 2H), 1.22 (br. s., 1H), 1.09−1.19 (m, 2H); LC/MS
calcd for C30H26F3N3O4 (m/e) 549.0; obsd, 550.1 (M + H, ES+).
(R)-1-{4′-[5-(sec-Butoxycarbonylamino)-1-methyl-1H-pyra-
1
1
ylic acid ethyl ester (48 mg, 52.1% yield). H NMR (CDCl3) δ ppm
zol- 4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (17). H
1.19−1.25 (m, 5H), 1.64 (m, 2H), 3.77 (s, 3H), 3.81(br s, 2H), 4.13
(q, J = 7.1 Hz, 2H), 7.44 (t, J = 8.8 Hz, 4H), 7.53 (s, 1H), 7.56 (d, J =
8.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H); LC/MS calcd for C22H23N3O2
(m/e) 361.0; obsd, 362.1 (M + H, ES+).
NMR (DMSO-d6) δ ppm 12.32 (br. s., 1H), 9.38 (br. s., 1H), 7.80 (s,
1H), 7.50−7.69 (m, 6H), 7.38 (d, J = 8.3 Hz, 2H), 4.69 (br. s., 1H),
3.65 (s, 3H), 1.58 (br. s., 2H), 1.33−1.52 (m, J = 2.6 Hz, 2H), 1.22
(br. s., 3H), 1.14 (m, J = 2.6 Hz, 2H), 0.91 (br. s., 3H); LC/MS calcd
for C25H27N3O4 (m/e) 433.0; obsd, 434.1 (M + H, ES+).
(R)-1-{4′-[5-(1,2-Dimethyl-propoxycarbonylamino)-1-meth-
yl-1H-pyrazol- 4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic
Acid (18). 1H NMR (DMSO-d6) δ ppm 12.34 (br. s., 1H), 9.37
(br. s., 1H), 7.81 (s, 1H), 7.49−7.68 (m, 6H), 7.38 (d, J = 8.3 Hz, 2H),
4.58 (br. s., 1H), 3.65 (s, 3H), 1.82 (br. s., 1H), 1.44 (br. s., 2H),
1.03−1.30 (m, 5H), 0.92 (br. s., 6H); LC/MS calcd for C26H29N3O4
(m/e) 447.0; obsd, 448.2 (M + H, ES+).
1-{4′-[5-(Cyclobutoxycarbonylamino)-1-methyl-1H-pyrazol-
4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (19). 1H
NMR (Methanol-d4) δ ppm 7.77 (s, 1H), 7.52−7.72 (m, 5H), 7.44
(d, J = 8.3 Hz, 2H), 5.01 (br. s., 1H), 3.79 (s, 3H), 2.40 (br.s., 2H),
2.05−2.29 (m, 2H), 1.78−1.94 (m, 1H), 1.46−1.76 (m, 3H), 1.12−
1.42 (m, 2H); LC/MS calcd for C25H25N3O4 (m/e) 431.0; obsd, 432.0
(M + H, ES+).
1-[4′-(5-Amino-1-methyl-1H-pyrazol-4-yl)-biphenyl-4-yl]-cyclopro-
panecarboxylic acid ethyl ester (100 mg, 0.277 mmol) and triphosgene
(123 mg, 0.415 mmol) were dissolved in methylene chloride (2 mL)
to give a solution. Toluene (6 mL) was added, and the mixture was
stirred followed by the addition of TEA (0.16 mL). The mixture was
sealed and stirred at 90 °C for 10 min. (R)-(+)-1-Phenylethanol (68
mg, 0.553 mmol) in toluene (2 mL) was added. The mixture was
stirred at 85 °C for 2 h. This material was extracted with ethyl acetate
and ammonium chloride solution. The organic layer was dried over
sodium sulfate and filtered. Solvents were evaporated, and the residue
was purified by flash column chromatography (0% to 60% ethyl
acetate in hexanes) to give a gray powder as 1-{4′-[1-methyl-5-((R)-1-
phenyl-ethoxycarbonylamino)-1H-pyrazol-4-yl]-biphenyl-4-yl}-cyclo-
1
propanecarboxylic acid ethyl ester (101 mg, 71.6% yield). H NMR
(CDCl3) δ ppm 1.20−1.34 (m, 6H), 1.49−1.75 (m, 4H), 3.80 (s, 3H),
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1-{4′-[1-Methyl-5-((oxetan-3-yloxy)carbonylamino)-1H-pyra-
vial was sealed, heated in an oil bath at 100 °C for 4 h, and cooled to
room temperature overnight. The reaction was diluted with ethyl
acetate (50 mL) and water (100 mL), filtered, and rinsed with water
(30 mL) and ethyl acetate (50 mL). The filtrate was separated and the
organic layer was dried over MgSO4, filtered, concentrated, dissolved
in minimal dichloromethane, and purified by flash column
chromatography (0% to 50% ethyl acetate in hexanes) to give 1-{4′-
[4-methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-[1,2,3]triazol-1-yl]-
biphenyl-4-yl}-cyclopropanecarboxylic acid methyl ester (2.65 g,
71.4% yield) as a white solid. LC/MS calcd for C29H28N4O4 (m/e)
496; obsd, 497 (M + H, ES+).
1
zol- 4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (20). H
NMR (DMSO-d6) δ ppm 12.33 (br. s., 1H), 7.93 (s, 1H), 7.66−7.73
(m, J = 8.3 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.53−7.59 (m, J = 8.1
Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H), 5.41 (br. s., 1H), 4.85−4.94 (m,
1H), 3.90 (t, 1H), 3.66−3.83 (m, 6H), 3.57 (br. d., 1H), 1.42−1.62
(m, 2H), 1.13−1.22 (m, 2H); LC/MS calcd for C24H23N3O5 (m/e)
433.0; obsd, 434.1 (M + H, ES+).
1-{4′-[4-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-
[1,2,3]triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid
(2). In a 350 mL reaction vial, 4-bromo-phenylboronic acid (21.17 g,
105 mmol), sodium azide (10.3 g, 158 mmol), and copper(II) acetate
(1.91 g, 10.5 mmol) were combined with methanol (200 mL) to give a
brown suspension. The reaction was stirred at room temperature open
to the atmosphere for 23 h. The resulting mixture was concentrated,
diluted with ethyl ether/hexanes, and washed with water, saturated
ammonium chloride, and ammonium hydroxide solution. The organic
layer was dried over MgSO4 and stored in the refrigerator overnight.
The crude material was warmed to room temperature, filtered,
concentrated to give a red/yellow oil which was dissolved in hexanes
(20 mL), and purified by flash column chromatography eluted with
hexanes to obtain 1-azido-4-bromo-benzene (19.5 g, 93.4% yield) as a
yellow oil. LC/MS calcd for C6H4BrN3 (m/e) 197/199; obsd, 170/
172 (M-N2+H, ES+).
In a 350 mL reaction vial, 1-azido-4-bromo-benzene (10 g, 50.5
mmol) and methyl but-2-ynoate (5.45 g, 5.56 mL, 55.5 mmol) were
combined with toluene (106 mL) to give a yellow suspension. The vial
was sealed and heated in an oil bath at 150 °C for 4.5 h. The mixture
was cooled and stored at room temperature. The reaction was filtered,
and the solid was washed with toluene and ethyl acetate (3 × 15 mL).
The filtrate was concentrated, dissolved in minimal dichloromethane,
and purified by flash column chromatography (0% to 50% ethyl
acetate in hexanes) to give 3-(4-bromo-phenyl)-5-methyl-3H-[1,2,3]-
triazole-4-carboxylic acid methyl ester (4.5 g, 30.1% yield) as a light
brown solid. LC/MS calcd for C11H10BrN3O2 (m/e) 295/297; obsd,
296/298 (M + H, ES+).
{4′-[4-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-[1,2,3]-
triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid methyl ester
(2.65 g, 5.34 mmol) was combined with THF (50 mL) to give a
yellow solution. To this was added LiOH (1.28 g, 53.4 mmol)
dissolved in water (12.5 mL, heated to partially dissolve). The reaction
flask was sealed and heated in an oil bath at 60 °C for 5 h. The
reaction was cooled to room temperature overnight. The mixture was
diluted with water (100 mL), concentrated, diluted with more water
(500 mL), and acidified with 1 N HCl. The resulting precipitate was
filtered, washed with water and hexanes, and dried. The crude product
(2.8 g) was purified by reverse phase chromatography (C18 Silicycle
120 g, 60 mL/min, 20−100% acetonitrile/H2O) to give 1.65 g of a
white solid. This solid was crystallized from acetonitrile to provide
pure compound 2 (1.48 g, 57.5% yield). LC/MS calcd for C28H26N4O4
1
(m/e) 482; obsd, 483 (M + H, ES+); H NMR (DMSO-d6) δ 12.40
(br. s., 1H), 9.69 (br. s., 1H), 7.83 (d, J = 7.0 Hz, 2H), 7.67 (d, J = 8.3
Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.5 Hz, 2H), 7.00−7.42
(m, 5H), 5.71 (br. s., 1H), 2.18 (s, 3H), 1.29−1.69 (m, 5H), 1.13−
1.26 (m, 2H).
{4′-[4-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-[1,2,3]-
triazol-1-yl]-biphenyl-4-yl}-acetic Acid (21). In a 350 mL reaction
vial, ethyl 2-(4-bromophenyl)acetate (25 g, 103 mmol), bis-
(pinacolato)diboron (31.3 g, 123 mmol), and potassium acetate
(20.2 g, 206 mmol) were combined with 1,4-dioxane (190 mL) to give
a white suspension. The mixture was purged with nitrogen for 5 min,
PdCl2(dppf) (4.2 g, 5.74 mmol) was added, and the vial was sealed
and heated in an oil bath at 80 °C for 3 h. The reaction was filtered,
rinsed with ethyl ether, concentrated, diluted with water (500 mL),
and extracted with ethyl ether (2 × 300 mL), and the organic layers
were washed with brine (250 mL). The ethyl ether layers were
combined, dried over MgSO4, filtered, and concentrated as red oil.
The crude material was purified by flash column chromatography (0%
to 20% EtOAc in hexanes) to give [4-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-phenyl]-acetic acid ethyl ester (25.14 g, 84.2%
yield) as an oil. LC/MS calcd for C16H23BO4 (m/e) 290; obsd, 291 (M
+ H, ES+).
To a 1 L round-bottom flask containing 3-(4-bromo-phenyl)-5-
methyl-3H-[1,2,3]triazole-4-carboxylic acid methyl ester (4.5 g, 15.2
mmol) dissolved in THF (200 mL) was added LiOH (2.77 g, 120
mmol) dissolved in water (75 mL, with heat). The solution was stirred
at room temperature for 16 h. The reaction was concentrated, diluted
with water, and extracted with ethyl ether. The aqueous layer was
acidified with 1 N HCl, and the resulting precipitate was filtered,
washed with water and hexanes, and dried over an in-house vacuum to
provide 3-(4-bromo-phenyl)-5-methyl-3H-[1,2,3]triazole-4-carboxylic
acid (4.25 g, 99% yield) as an off-white solid. LC/MS calcd for
C10H8N3O2 (m/e) 281/283; obsd, 282/284 (M + H, ES+).
In a 350 mL reaction vial, 3-(4-bromo-phenyl)-5-methyl-3H-
[1,2,3]triazole-4-carboxylic acid (3.6 g, 12.8 mmol), (R)-1-phenyl-
ethanol (3.04 g, 3 mL, 24.9 mmol), and triethylamine (3.27 g, 4.5 mL,
32.3 mmol) were combined with toluene (100 mL) to give a yellow
solution. To this solution was added diphenylphosphoryl azide (8.94 g,
7 mL, 32.5 mmol). The mixture was heated in an oil bath at 65 °C for
2 h and cooled to room temperature overnight. The reaction was
concentrated as a yellow viscous oil, diluted with dichloromethane, and
purified by flash column chromatography (0−50% ethyl acetate in
hexanes) to give [3-(4-bromo-phenyl)-5-methyl-3H-[1,2,3]triazol-4-
yl]-carbamic acid (R)-1-phenyl-ethyl ester (4.07 g, 79.5% yield) as a
white solid. LC/MS calcd for C18H17BrN4O2 (m/e) 400/402; obsd,
401/403 (M + H, ES+).
In a 350 mL vial, 1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-
yl)-phenyl]-cyclopropanecarboxylic acid methyl ester (2.49 g, 8.22
mmol, prepared from 1-(4-bromophenyl)-cyclopropanecarboxylic acid
methyl ester), [3-(4-bromo-phenyl)-5-methyl-3H-[1,2,3]triazol-4-yl]-
carbamic acid (R)-1-phenyl-ethyl ester (3.0 g, 7.48 mmol), 2-
dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (S-Phos) (921 mg,
2.24 mmol), and palladium(II) acetate (252 mg, 1.12 mmol) were
combined with toluene (120 mL) to give a light yellow solution. To
this was added tripotassium phosphate (4.76 g, 22.4 mmol) dissolved
in water (30.0 mL). The mixture was bubbled with nitrogen, and the
In a 20 mL vial, [4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-
phenyl]-acetic acid ethyl ester (79.5 mg, 0.274 mmol), [1-(4-bromo-
phenyl)-5-methyl-1H-[1,2,3]triazol-4-yl]-carbamic acid (R)-1-phenyl-
ethyl ester (100 mg, 0.249 mmol), tripotassium phosphate (159 mg,
0.748 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (S-
Phos) (30.7 mg, 0.0748 mmol), and palladium(II) acetate (8.4 mg,
0.037 mmol) were combined with toluene (2 mL) and water (0.5 mL)
(previously purged with nitrogen for 20 min) to give a light yellow
suspension. The mixture was bubbled with nitrogen, and the vial was
sealed, heated in a dry block at 100 °C for 6 h, and cooled to room
temperature overnight. The reaction was diluted with ethyl acetate (50
mL) and washed with water (50 mL) and brine. The aqueous layers
were extracted with ethyl acetate (50 mL). The organic layers were
combined, dried over MgSO4, filtered, concentrated, dissolved in
minimal dichloromethane, and purified by flash column chromatog-
raphy (0% to 60% ethyl acetate in hexanes) to give {4′-[4-methyl-5-
((R)-1-phenyl-ethoxycarbonylamino)-[1,2,3]triazol-1-yl]-biphenyl-4-
yl}-acetic acid ethyl ester (40 mg, 33.1% yield) as a colorless waxy
solid. LC/MS calcd for C28H28N4O4 (m/e) 484; obsd, 485 (M + H,
ES+).
In a 200 mL round-bottomed flask, {4′-[4-methyl-5-((R)-1-phenyl-
ethoxycarbonylamino)-[1,2,3]triazol-1-yl]-biphenyl-4-yl}-acetic acid
ethyl ester (34 mg, 0.0702 mmol) was combined with THF (2 mL)
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Journal of Medicinal Chemistry
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to give a yellow solution. To this was dripped in LiOH (16.8 mg, 0.702
mmol) dissolved in water (0.5 mL, heated to partially dissolve). The
mixture was stirred for 11 h. The reaction was diluted with water, and
acidified with 1 N HCl. The resulting precipitate was filtered, washed
with water and hexanes, and dried to provide compound 21 as an off-
white solid. LC/MS calcd for C26H24N4O4 (me) 456; obsd, 457 (M +
(R)-1-Phenyl-ethyl-1-(4′-(1-(1H-tetrazol-5-yl)cyclopropyl)-
biphenyl-4-yl)-4-methyl-1H-1,2,3-triazol-5-ylcarbamate (30).
To a mixture of (R)-1-phenylethyl 4-methyl-1-(4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-1,2,3-triazol-5-ylcarbamate
(485 mg, 1.08 mmol), 1-(4-bromophenyl)cyclopropanecarbonitrile
(360 mg, 1.62 mmol), palladium(II) acetate (36.4 mg, 0.16 mmol), 2-
dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (133 mg, 0.33 mmol),
and potassium phosphate tribasic (689 mg, 3.25 mmol) in a vial were
added toluene (9 mL) and water (2.0 mL) at room temperature under
nitrogen atmosphere. The vial was sealed, and the resulting light
brown suspension was heated to 105 °C and stirred for 3 h. The
reaction mixture was cooled and diluted with water. The organic
compound was extracted into ethyl acetate (2 × 50 mL), and the
combined extracts were washed with brine solution and dried over
anhydrous MgSO4. Filtration and concentration gave the crude
residue, which was purified by flash column chromatography eluting
with 0−100% ethyl acetate in hexanes to give (R)-1-phenyl-ethyl-1-
(4′-(1-cyanocyclopropyl)biphenyl-4-yl)-4-methyl-1H-1,2,3-triazol-5-yl-
carbamate (190 mg, 38% yield) as a white solid. LC/MS calcd for
C28H25N5O2 (m/e) 463; obsd, 464.8 (M + H, ES+).
1
H, ES+); H NMR (DMSO-d6) δ ppm 12.42 (br. s., 1H), 9.19−9.80
(m, 1H), 7.83 (d, J = 6.5 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.57 (d, J
= 7.3 Hz, 2H), 7.08−7.47 (m, 7H), 5.69 (br. s., 1H), 3.65 (s, 2H), 2.16
(s, 3H), 1.13−1.64 (m, 3H).
Compounds 22−29 and 31−32 were prepared with the same
method described for the preparation of compound 2.
1-(4′-{5-[(R)-1-(2-Fluoro-phenyl)-ethoxycarbonylamino]-4-
methyl-[1,2,3]triazol-1-yl}-biphenyl-4-yl)-cyclopropanecarbox-
1
ylic Acid (22). H NMR (DMSO-d6) δ ppm 12.39 (br. s., 1H), 9.74
(br. s., 1H), 7.84 (d, J = 6.5 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.58 (d,
J = 8.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 6.69−7.42 (m, 4H), 5.89 (br.
s., 1H), 2.17 (br. s., 3H), 1.26−1.74 (m, 5H), 1.14−1.24 (m, 2H); LC/
MS calcd for C28H25FN4O4 (m/e) 500; obsd, 501 (M + H, ES+).
1-(4′-{4-Methyl-5-[(R)-1-(2-trifluoromethyl-phenyl)-ethoxy-
carbonylamino]-[1,2,3]triazol-1-yl}-biphenyl-4-yl)-cyclopropa-
To a solution of (R)-1-phenylethyl-1-(4′-(1-cyanocyclopropyl)-
biphenyl-4-yl)-4-methyl-1H-1,2,3-triazol-5-ylcarbamate (50 mg, 0.11
mmol) in toluene (5 mL) were added di-n-butyltin oxide (5.37 mg,
0.22 mmol) and azidotrimethylsilane (12.4 mg, 14.3 μL, 0.11 mmol) at
room temperature under nitrogen atmosphere. The resulting cloudy
solution was heated to 100 °C and stirred for 15 h. The mixture was
cooled to room temperature, poured into brine solution, and extracted
with ethyl acetate. The organic layer was washed with brine solution
and dried over anhydrous MgSO4. Filtration and concentration gave
the crude product which was purified by flash column chromatography
eluting with 0−100% ethyl acetate in hexanes and 10% methanol in
dichloromethane to give compound 30 as a white solid (25 mg, 46%
yield). LC/MS calcd for C28H26N8O2 (m/e) 506; obsd, 507.1 (M + H,
1
necarboxylic Acid (23). H NMR (DMSO-d6) δ ppm 12.40 (br. s.,
1H), 9.77 (br. s., 1H), 7.83 (d, J = 7.3 Hz, 2H), 7.63−7.78 (m, 5H),
7.39−7.62 (m, 5H), 5.96 (br. s., 1H), 2.15 (br. s., 3H), 1.50 (d, J = 2.3
Hz, 5H), 1.20 (d, J = 2.0 Hz, 2H); LC/MS calcd for C29H25F3N4O4
(m/e) 550; obsd, 551 (M + H, ES+).
1-(4′-{4-Methyl-5-[(R)-1-(3-trifluoromethyl-phenyl)-ethoxy-
carbonylamino]-[1,2,3]triazol-1-yl}-biphenyl-4-yl)-cyclopropa-
1
necarboxylic Acid (24). H NMR (DMSO-d6) δ ppm 12.39 (br. s.,
1H), 9.47 (br. s., 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.64 (dd, J = 18.7, 8.2
Hz, 4H), 7.46 (d, J = 8.3 Hz, 2H), 4.67 (br. s., 1H), 2.36 (br. s., 1H),
2.20 (s, 3H), 1.54−2.02 (m, 6H), 1.43−1.53 (m, 2H), 1.17−1.31 (m,
2H), 1.05 (br. s., 3H); LC/MS calcd for C26H28N4O4 (m/e) 460; obsd,
461 (M + H, ES+).
1-{4′-[5-((R)-1,2-Dimethyl-propoxycarbonylamino)-4-meth-
yl-[1,2,3]triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic
Acid (25). 1H NMR (DMSO-d6) δ ppm 12.37 (br. s., 1H), 9.43 (br. s.,
1H), 7.88 (d, J = 8.0 Hz, 2H), 7.57−7.73 (m, 4H), 7.46 (d, J = 8.3 Hz,
2H), 4.50 (br. s., 1H), 2.21 (s, 3H), 1.71 (br. s., 1H), 1.40−1.57 (m,
2H), 1.00−1.32 (m, 5H), 0.84 (br. s., 6H); LC/MS calcd for
C25H28N4O4 (m/e) 448; obsd, (M + H, ES+).
1
ES+); H NMR (DMSO-d6) δ ppm 16.08 (br. s., 1H), 9.20−9.84 (m,
1H), 7.85 (d, J = 7.0 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H), 7.52−7.65 (m,
2H), 7.46 (d, J = 8.3 Hz, 2H), 7.34 (br. s., 5H), 5.52−5.84 (m, 1H),
2.17 (s, 3H), 1.51−1.63 (m, 4H), 1.15−1.35 (m, 3H).
1-{4′-[4-Ethyl-5-((R)-1-phenyl-ethoxycarbonylamino)-[1,2,3]-
triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (31).
1H NMR (DMSO-d6) δ ppm 12.40 (br. s., 1H), 9.66 (br. s., 1H), 7.83
(d, J = 6.5 Hz, 2H), 7.67 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 7.3 Hz, 2H),
7.47 (d, J = 8.0 Hz, 2H), 7.34 (br. s., 5H), 5.71 (br. s., 1H), 2.56 (d, J =
7.5 Hz, 2H), 1.36−1.60 (m, 5H), 1.16−1.23 (m, 5H); LC/MS calcd
for C29H28N4O4 (m/e) 496; obsd, 497.1 (M + H, ES+).
1-{4′-[5-((R)-sec-Butoxycarbonylamino)-4-methyl-[1,2,3]-
triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid (26).
1H NMR (DMSO-d6) δ ppm 12.37 (br. s., 1H), 9.44 (br. s., 1H), 7.88
(d, J = 8.5 Hz, 2H), 7.56−7.72 (m, 4H), 7.46 (d, J = 8.3 Hz, 2H), 4.61
(br. s., 1H), 2.21 (s, 3H), 1.38−1.66 (m, 4H), 1.03−1.34 (m, 5H),
0.85 (dd, J = 10.7, 6.9 Hz, 3H); LC/MS calcd for C24H26N4O4 (m/e)
434; obsd, 435 (M + H, ES+).
1-{4′-[5-(1-Cyclobutyl-ethoxycarbonylamino)-4-methyl-
[1,2,3]triazol-1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid
(27). 1H NMR (DMSO-d6) δ ppm 12.39 (br. s., 1H), 9.47 (br. s., 1H),
7.88 (d, J = 7.8 Hz, 2H), 7.64 (dd, J = 18.7, 8.2 Hz, 4H), 7.46 (d, J =
8.3 Hz, 2H), 4.67 (br. s., 1H), 2.36 (br. s., 1H), 2.20 (s, 3H), 1.54−
2.02 (m, 6H), 1.43−1.53 (m, 2H), 1.17−1.31 (m, 2H), 1.05 (br. s.,
3H); LC/MS calcd for C26H28N4O4 (m/e) 460; obsd, 461 (M + H,
ES+).
1-[4′-(5-tert-Butoxycarbonylamino-4-methyl-[1,2,3]triazol-
1-yl)-biphenyl-4-yl]-cyclopropanecarboxylic Acid (28). 1H
NMR (DMSO-d6) δ ppm 12.40 (br. s., 1H), 9.24 (br. s., 1H), 7.90
(d, J = 8.0 Hz, 2H), 7.58−7.71 (m, 4H), 7.46 (d, J = 8.3 Hz, 2H), 2.20
(s, 3H), 1.16−1.55 (m, 13H); LC/MS calcd for C24H26N4O4 (m/e)
434; obsd, 435 (M + H, ES+).
1-{4′-[5-((R)-1-Phenyl-ethoxycarbonylamino)-[1,2,3]triazol-
1-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (32). 1H
NMR (DMSO-d6) δ ppm 12.38 (br. s., 1H), 10.04 (br. s., 1H), 7.87
(d, J = 8.3 Hz, 2H), 7.82 (s, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.62 (d, J =
8.3 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.17−7.41 (m, 5H), 5.74 (d, J =
5.8 Hz, 1H), 1.34−1.62 (m, 5H), 1.21 (d, J = 2.5 Hz, 2H); LC/MS
calcd for C27H24N4O4 (m/e) 468; obsd, 469 (M + H, ES+).
1-{4′-[3-((R)-1-Phenyl-ethoxycarbonylamino)-[1,2,4]triazol-
4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid (33). In a 250
mL round-bottomed flask, calcium carbonate (6.11 g, 61.0 mmol) and
4-bromoaniline (5 g, 29.1 mmol) were combined with dichloro-
methane (25 mL) and water (25.0 mL) to give a light brown
suspension. The reaction mixture was cooled to 0 °C, and
thiophosgene (3.68 g, 2.45 mL, 32.0 mmol) was added dropwise
over 4 min. The reaction was stirred at 0 °C for 30 min then at 25 °C
for 19 h. The reaction mixture was filtered through Celite, and the
filter cake was washed with dichloromethane. The aqueous layer was
back-extracted with dichloromethane (1 × 25 mL). The organic layers
were combined, washed with water (1 × 25 mL) and saturated NaCl
(1 × 20 mL), dried over Na2SO4, and concentrated in vacuo. The light
brown solid was dried under vacuum to give 1-bromo-4-
{3-[4′-(1-Methanesulfonylaminocarbonyl-cyclopropyl)-bi-
phenyl-4-yl]-5-methyl-3H-[1,2,3]triazol-4-yl}-carbamic Acid
1
(R)-1-Phenyl-ethyl Ester (29). H NMR (DMSO-d6) δ ppm 11.24
1
(br. s., 1H), 9.68 (br. s., 1H), 7.85 (d, J = 7.0 Hz, 2H), 7.72 (d, J = 8.3
Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.34 (br. s.,
5H), 5.70 (br. s., 1H), 3.23 (s, 3H), 2.17 (s, 3H), 1.29−1.73 (m, 5H),
1.23 (br. s., 2H); LC/MS calcd for C29H29N5O5S (m/e) 559; obsd,
560 (M + H, ES+).
isothiocyanatobenzene (5.43 g, 87%). H NMR (DMSO-d6) δ ppm
7.55−7.74 (m, 2H), 7.28−7.50 (m, 2H).
In a 500 mL round-bottomed flask, 1-bromo-4-isothiocyanatoben-
zene (1.5 g, 7.01 mmol) was combined with 0.4 M ammonia in THF
(52.5 mL, 21.0 mmol) to give a yellow solution. The reaction was
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stirred at 25 °C overnight. The crude reaction mixture was
concentrated in vacuo to afford 1-(4-bromophenyl)thiourea (1.63 g,
quantitative) as a light brown solid. (M + H)+ = 230.9/233.0 (m/e).
In a 250 mL round-bottomed flask, 1-(4-bromophenyl)thiourea
(1.62 g, 7.01 mmol) was combined with methanol (50 mL) to give a
light brown suspension. Methyl iodide (1.09 g, 482 μL, 7.71 mmol)
was added, and the reaction mixture was stirred at 25 °C for 17 h. The
crude reaction mixture was concentrated in vacuo to yield 1-(4-
bromophenyl)-2-methyl-isothiourea hydroiodide (2.61 g, 100%) as a
light brown powder. The material was used without further
purification.
In a 250 mL round-bottomed flask, 1-(4-bromophenyl)-2-methyl-
isothiourea hydroiodide (2.61 g, 7.00 mmol) was combined with water
(10 mL) and ethanol (10.0 mL) to give a light brown solution.
Hydrazine monohydrate (525 mg, 509 μL, 10.5 mmol) was added and
the reaction was stirred at 25 °C for 20 h. The crude reaction mixture
was concentrated in vacuo to about half volume, and silver nitrate (1.19
g, 7.00 mmol) was added with vigorous stirring. The gray/brown solid
was filtered through Celite, and the filter cake was washed twice with
boiling water. The filtrate was concentrated in vacuo to give a yellow
oil. The oily material was dried under vacuum with slight heating (2.27
g). This material (2.27 g, 7.77 mmol) and formic acid (715 mg, 596
μL, 15.5 mmol) were combined to give a yellow solution. The reaction
mixture was heated to 120 °C for 3.5 h. The reaction was cooled and
neutralized with 3 M NaOH. The mixture was diluted with 150 mL of
dichloromethane and stirred vigorously. The insoluble solid was
filtered, and the phases were separated. The organic phase was dried
over Na2SO4 and filtered. The filtered solid was combined with the
dried organic phase and concentrated in vacuo. The residue was taken
up in refluxing ethanol and filtered hot to remove a small amount of
white insoluble solid. The light brown filtrate was stripped to a tan
powder and dried under vacuum to afford 4-(4-bromophenyl)-4H-
1,2,4-triazol-3-amine (1.665 g, 90%). (M + H)+ = 239.0/240.9 (m/e).
1H NMR (DMSO-d6) δ ppm 8.20 (s, 1H), 7.66−7.81 (m, 2H), 7.34−
7.54 (m, 2H), 5.86 (s, 2H).
In a 20 mL sealed tube, 4-(4-bromophenyl)-4H-1,2,4-triazol-3-
amine (349 mg, 1.46 mmol), 4-(1-(methoxycarbonyl)cyclopropyl)-
phenylboronic acid (450 mg, 2.04 mmol) and 2 M Na2CO3 (2.19 mL,
4.38 mmol) were combined with dioxane (6 mL) to give a light yellow
suspension. PdCl2(dppf) (95.4 mg, 117 μmol) was added, and the
reaction was purged with argon. The reaction mixture was sealed and
heated to 100 °C for 24 h under argon. The reaction was cooled and
diluted with ethyl acetate and water. The mixture was filtered, and the
filtrate was washed with water and brine. The organic layer was dried
over Na2SO4, combined with the filtered solid, and concentrated in
vacuo. Celite was added to the residue, and the mixture was triturated
with refluxing methanol. The mixture was filtered, and the filter cake
was washed twice with refluxing methanol. The filtrate was stripped in
vacuo, and the crude material was purified by flash chromatography
(0% to 10% methanol in dichloromethane) to afford methyl 1-(4′-(3-
amino-4H-1,2,4-triazol-4-yl)biphenyl-4-yl)cyclopropanecarboxylate
(257 mg, 53%) as a light brown powder. (M + H)+ = 335.1 (m/e). 1H
NMR (DMSO-d6) δ ppm 8.24 (s, 1H), 7.79−7.86 (m, 2H), 7.62−7.70
(m, 2H), 7.53−7.60 (m, 2H), 7.41−7.49 (m, 2H), 5.86 (s, 2H), 3.58
(s, 3H), 1.42−1.61 (m, 2H), 1.16−1.35 (m, 2H).
In a 250 mL round-bottomed flask, (R)-1-phenylethanol (2.01 g,
16.5 mmol) and carbonyl diimidazole (2.67 g, 16.5 mmol) were
combined with ethyl acetate (40 mL) to give a colorless solution. The
reaction mixture was refluxed for 20 h under argon, cooled, and diluted
with ethyl acetate. The mixture was washed with water (2 × 40 mL)
and saturated NaCl (1 × 20 mL), dried over Na2SO4, and
concentrated in vacuo. The material crystallized upon standing to
afford (R)-1-phenylethyl 1H-imidazole-1-carboxylate (3.42 g, 96%) as
off white needles. 1H NMR (DMSO-d6) δ ppm 8.42 (s, 1H), 7.65 (dd,
J = 1.8, 1.3 Hz, 1H), 7.45−7.54 (m, 2H), 7.22−7.45 (m, 3H), 7.09
(dd, J = 1.6, 0.9 Hz, 1H), 6.05 (q, J = 6.6 Hz, 1H), 1.66 (d, J = 6.6 Hz,
3H).
suspension. LiHMDS (1 M) in THF (447 μL, 447 μmol) was added,
and the brown solution was stirred at 25 °C under argon for 15 min.
(R)-1-phenylethyl 1H-imidazole-1-carboxylate (112 mg, 516 μmol) in
1 mL of THF was added, and the reaction mixture was stirred for 15
min at 25 °C. The reaction was quenched with water and diluted with
10% methanol in dichloromethane. Sodium sulfate was added, and the
mixture was filtered through Celite, and the brown filtrate was
concentrated in vacuo. The crude material was purified by flash column
chromatography (0% to 10% methanol in dichloromethane) to afford
1-{4′-[3-((R)-1-phenyl-ethoxycarbonylamino)-[1,2,4]triazol-4-yl]-bi-
phenyl-4-yl}-cyclopropanecarboxylic acid methyl ester (85 mg, 51%)
as an off white solid. (M + H)+ = 483.1 (m/e). 1H NMR (DMSO-d6) δ
ppm 10.01 (s, 1H), 8.87 (s, 1H), 7.76−7.93 (m, 2H), 7.58−7.75 (m,
2H), 7.38−7.57 (m, 4H), 7.12−7.38 (m, 5H), 5.62 (d, J = 6.8 Hz,
1H), 3.58 (s, 3H), 1.47−1.60 (m, 2H), 1.34 (d, J = 5.6 Hz, 2H), 1.15−
1.31 (m, 3H). This material (110 mg, 228 μmol) was combined with
THF (5 mL) and methanol (1 mL) to give a yellow solution. LiOH (1
M) (2 mL, 2.00 mmol) was added, and the reaction was stirred at 25
°C for 17 h. The crude reaction mixture was concentrated in vacuo,
acidified with 1 M HCl, and diluted with ethyl acetate. The organic
layer was washed with water (1 × 15 mL) and saturated NaCl (1 × 15
mL), dried over Na2SO4 and concentrated in vacuo. The crude
material was purified by flash column chromatography (0% to 10%
methanol in dichloromethane) to afford compound 33 (86 mg, 80%)
as a white solid. 1H NMR (DMSO-d6) δ ppm 12.39 (br. s., 1H), 10.01
(br. s., 1H), 8.87 (br. s., 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.3
Hz, 2H), 7.39−7.59 (m, 4H), 7.10−7.39 (m, 5H), 5.62 (d, J = 6.3 Hz,
1H), 1.45−1.54 (m, 2H), 1.40 (br. s., 1H), 1.09−1.37 (m, 4H); LC/
MS calcd for C27H24N4O4 (m/e) 468; obsd, 469 (M + H, ES+).
{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
[1,2,3]triazol-4-yl]-biphenyl-4-yl}-acetic Acid (34). LDA solution
(2 M) in THF (20.71 mL, 41.436 mmol) was added to a stirred
solution of 1-bromo-4-ethynyl-benzene (3 g, 16.57 mmol) in dry THF
(40 mL) at −70 °C and stirred for 30 min. Ethyl chloroformate (11.81
mL, 74.58 mmol) was added, and the mixture was allowed to warm to
ambient temperature. Stirring was continued for 2 h. The mixture was
cooled and quenched with saturated NH4Cl solution. THF was
evaporated under reduced pressure, and the aqueous layer was
extracted with ethyl acetate. The organic layer was dried over Na2SO4,
concentrated, and purified by flash column chromatography using
ethyl acetate and hexane as eluting solvent to give (4-bromo-phenyl)-
propyonic acid ethyl ester (2.9 g, 69.13% yield) as a light yellow liquid.
1
GC-MS (M + H)+ 253 (m/e); H NMR (CDCl3) δ ppm 1.32 (t, J =
7.0 Hz, 3H). 4.28 (q, J = 7.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.51
(d, J = 8.4 Hz, 2H).
To a stirred solution of (4-bromo-phenyl)-propynoic acid ethyl
ester (2.5 g, 9.881 mmol) in benzene (4 mL), was added
trimethylsilylmethyl azide (5.108 g, 39.52 mmol). The reaction
mixture was refluxed for 4 h, then cooled to rt and concentrated
under reduced pressure. Crude mass was purified by normal silica gel
column chromatography using ethyl acetate−hexane as eluting solvent
to get two major fractions. One fraction is 5-(4-bromo-phenyl)-3-
trimethylsilanylmethyl-3H-[1,2,3] triazole-4-carboxylic acid ethyl ester
(1.7 g, 45%) as a light yellow liquid. LC-MS (M + H)+ 382 (m/e); 1H
NMR (DMSO-d6) δ ppm 0.10 (s, 9H), 1.21 (t, J = 7.2 Hz, 3H), 4.31
1
(m, 4H), 7.66 (s, 4H). The regio-chemistry was assigned by the H-
NOE study. The other major fraction gave 5-(4-bromo-phenyl)-1-
trimethylsilanylmethyl-1H-[1,2,3] triazole-4-carboxylic acid ethyl ester
(1.8 g, 47.7% yield) as a light yellow liquid. LC-MS (M + H)+ 382 (m/
e); 1H NMR (DMSO-d6) δ ppm 0.02 (s, 9H), 1.13 (t, J = 7.2 Hz, 3H),
3.76 (s, 2H), 4.16 (q, J = 7.2 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.74
(d, J = 8.4 Hz, 2H). The regio-chemistry was also assigned by the 1H-
NOE study.
To a stirred solution of 5-(4-bromo-phenyl)-3-trimethylsilanyl-
methyl-3H-[1,2,3] triazole-4-carboxylic acid ethyl ester (1.9 g, 4.974
mmol) in THF (40 mL) was added water (0.18 mL, 9.948 mmol) and
cooled to 0 °C. Then TBAF (1 M) solution in THF (5.9 mL, 5.9
mmol) was added, and the mixture was stirred at 0 °C for 10 min.
Volatiles were distilled off, and crude mass was purified by normal
silica gel column chromatography using ethyl acetate−hexane as
In a 250 mL round-bottomed flask, methyl 1-(4′-(3-amino-4H-
1,2,4-triazol-4-yl)biphenyl-4-yl)cyclopropanecarboxylate (115 mg, 344
μmol) was combined with THF (6 mL) to give a light brown
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eluting solvent to get 5-(4-bromo-phenyl)-3-methyl-3H-[1,2,3]
triazole-4-carboxylic acid ethyl ester (0.7 g, 45.4% yield) as an off
The organic layer was washed with brine and dried. Solvents were
evaporated, and the residue was purified by flash column
chromatography (ethyl acetate in hexanes 10% to 70%) to give 1-
{4′-[1-methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-[1,2,3]-
triazol-4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid methyl ester
as a pale yellow solid (370 mg, 52.8% yield). LC/MS calcd for
C29H28N4O4 (m/e) 496.0; obsd, 497.0 (M + H); 1H NMR (CDCl3) δ
ppm 1.25 (m, 3H), 1.66 (m, 4H), 3.67 (s, 3H), 3.93 (s, 3H), 5.91 (m,
1H), 6.44 (br, 1H), 7.29−7.40 (m, 5H), 7.44 (d, J = 8.1 Hz, 2H), 7.57
(d, J = 8.3 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.78 (br d, J = 6.6 Hz,
2H). This material (50 mg) was dissolved in 1 mL of THF and 1 mL
of ethanol. To this mixture was added 1 N sodium hydroxide solution
(1 mL). The clear solution was stirred at room temperature for 12 h.
Solvents were evaporated, and the residue was treated with 2 N
hydrochloric acid (1.4 mL). The solid was filtered and rinsed with
water and dried in air to give compound 35 (47.5 mg, 97.8% yield).
LC/MS calcd for C28H26N4O4 (m/e) 482.0; obsd, 483.0 (M + H); 1H
NMR (DMSO-d6) δ ppm 1.14−1.24 (m, 3H), 1.49 (m, 2H), 1.59 (m,
2H), 3.86 (s, 3H), 5.80 (m, 1H), 7.28−7.50 (m, 7H), 7.63 (d, J = 8.1
Hz, 2H), 7.71 (m, 2H), 7.80 (d, J = 7.6 Hz, 2H), 9.95 and 9.62 (br s,
1H), 12.35 (s, 1H).
1
white solid. LC-MS (M + H)+ 310 (m/e); H NMR (DMSO-d6) δ
ppm 1.23 (t, J = 7.0 Hz, 3H), 4.27 (s, 3H), 4.31 (q, J = 7.0 Hz, 2H),
7.68 (s, 4H). 5-(4-Bromo-phenyl)-3-methyl-3H-[1,2,3] triazole-4-
carboxylic acid ethyl ester (1.2 g, 3.87 mmol) was dissolved in THF
(15 mL), and lithium hydroxide solution (0.5 N, 10 mL) was added.
The mixture was stirred at room temperature for 3 h. The mixture was
concentrated, and the residue was dissolved in water (20 mL) and
filtered. The filtrate was acidified with 2 N hydrochloric acid (3 mL).
The white solid was filtered and dried to give 5-(4-bromo-phenyl)-3-
methyl-3H-[1,2,3] triazole-4-carboxylic acid as a white solid (1.03 g,
94.4% yield). Mp 209−210 °C; LC-MS calcd for C10H8BrN3O2 (m/e)
1
283.0; obsd, 284.0 (M + H); H NMR (DMSO-d6) δ ppm 4.26 (s,
3H), 7.66−7.72 (m, 4H), 14.20 (br s, 1H).
5-(4-Bromophenyl)-3-methyl-3H-[1,2,3]triazole-4-carboxylic acid
(500 mg, 1.77 mmol), (R)-1-phenylethanol (260 mg, 2.13 mmol),
DPPA (537 mg, 1.95 mmol), and TEA (179 mg, 1.7 mmol) were
combined in toluene (10 mL). The mixture was stirred at 90 °C for 2
h, and solvents were evaporated. The residue was extracted with ethyl
acetate and water. The organic layer was washed with sodium
bicarbonate solution and dried. Solvents were evaporated, and the
residue was purified by flash column chromatography (ethyl acetate in
hexanes 0% to 50%) to give [5-(4-bromo-phenyl)-3-methyl-3H-
[1,2,3]triazol-4-yl]-carbamic acid (R)-1-phenyl-ethyl ester as an
amorphous powder (440 mg, 61.9% yield). LC/MS calcd for
C18H17BrN4O2 (m/e) 402; obsd, 402.9 (M + H); 1H NMR
(DMSO-d6) δ ppm 1.55 (br, 3H), 3.83 (s, 3H), 5.76 (br s, 1H),
7.20−7.50 (m, 5H), 7.57−7.70 (m, 4H), 9.95 (br s, 1H).
Compounds 36−39 were prepared with the same method described
in the preparation of compound 35.
1-{3′-Fluoro-4′-[1-methyl-5-((R)-1- phenyl-ethoxycarbonyla-
mino)-1H-[1,2,3]triazole-4-yl]-biphenyl-4-yl}-cyclopropanecar-
boxylic Acid (36). LC/MS calcd for C28H25FN4O4 (m/e) 500.0;
obsd, 501.0 (M + H); 1H NMR (DMSO-d6) δ ppm 12.39 (br. s., 1H),
9.92 (br. s., 1H), 7.64−7.78 (m, 3H), 7.59 (d, J = 7.8 Hz, 2H), 7.37−
7.50 (m, 5H), 7.02−7.37 (m, 2H), 5.74 (br. s., 1H), 3.89 (s, 3H),
1.41−1.60 (m, 4H), 1.09−1.32 (m, 3H).
[5-(4-Bromo-phenyl)-3-methyl-3H-[1,2,3]triazol-4-yl]-carbamic
acid (R)-1-phenyl-ethyl ester (120 mg, 0.30 mmol), ethyl 2-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate (130 mg,
0.45 mmol), X-Phos (43 mg, 0.09 mmol), palladium acetate (10 mg,
0.045 mmol), and potassium phosphate (190 mg, 0.90 mmol) were
combined in 5 mL of toluene. Deionized water (1 mL) was added, and
the mixture was degassed with argon for 2 min. The mixture was
sealed and stirred at 100 °C for 2 h. The mixture was extracted with
ethyl acetate and water, washed with brine, and dried. Solvents were
evaporated, and the residue was purified by flash column
chromatography (0% to 70% ethyl acetate in hexanes) to give {4′-
[1-methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-[1,2,3]triazol-4-
yl]-biphenyl-4-yl}-acetic acid ethyl ester as an amorphous powder (70
mg, 48.3% yield). LC/MS calcd for C28H28N4O4 (m/e) 484.0; obsd,
1-{4′-[5-((R)-1,2-Dimethyl-propoxycarbonylamino)-1-meth-
yl-1H-[1,2,3]triazol-4-yl]-biphenyl-4-yl}-cyclopropanecarbox-
1
ylic Acid (37). H NMR (DMSO-d6) δ ppm 0.52 (br, 1H), 0.88 (br,
5H), 1.04−1.25 (m, 5H), 1.35−1.43 (m, 2H), 1.62−1.85 (br, 1H),
3.80 (s, 3H), 4.55 (br, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.3
Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H), 9.34 and
9.67 (br s, 1H), 12.30 (br s, 1H); LC/MS calcd for C25H28N4O4 (m/e)
448.0; obsd, 449.2 (M + H).
1-(4′-{1-Methyl-5-[(R)-1-(3-trifluoromethyl-phenyl)-ethoxy-
carbonylamino]-1H-[1,2,3]triazol-4-yl}-biphenyl-4-yl)-cyclo-
propanecarboxylic Acid (38). This compound was obtained from
the chiral separation of the racemate by super critical fluid
chromatography on Waters/Berger Multigram II using a Whelk-O1
(R,R)-column (3 × 25 cm) eluted with 50% isopropanol in CO2 at 70
mL/min (detection at 220 nM, 100 bar backpressure, and 35 °C
oven). The second fraction gave compound 38 as a white solid. LC/
1
485.1 (M + H); H NMR (CDCl3) δ ppm 1.31 (t, J = 7.2 Hz, 3H),
1.53−1.76 (m, 3H), 3.69 (s, 2H), 3.95 (s, 3H), 4.21 (q, J = 7.2 Hz,
2H), 5.93 (m, 1H), 6.40 (br s, 1H), 7.31−7.49 (m, 7H), 7.56−7.66
(m, 4H), 7.80 (d, J-6.8 Hz, 2H).
1
MS calcd for C29H25F3N4O4 (m/e) 550.0; obsd, 551.0 (M + H); H
NMR (DMSO-d6) δ ppm 1.13−1.29 (m, 3H), 1.44−1.52 (m, 2H),
1.60 (br d, 2H), 3.86 (s, 3H), 5.75−5.95 (br m, 1H), 7.43 (d, J = 8.1
Hz, 2.5H), 7.61 (d, J = 8.1 Hz, 2.5H), 7.69 (br m, 3.5H), 7.79 (d, J =
7.6 Hz, 3.5H), 9.65 and 10.05 (br s, 1H), 12.35 (s, 1H).
1-(4′-{1-Methyl-5-[(S)-1-(3-trifluoromethyl-phenyl)-ethoxy-
carbonylamino]-1H-[1,2,3]triazol-4-yl}-biphenyl-4-yl)-cyclo-
propanecarboxylic Acid (39). The first fraction from the chiral
separation (conditions described in the preparation of 38) gave
compound 39 as a white solid.
{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-[1,2,3]-
triazol-4-yl]-biphenyl-4-yl}-acetic acid ethyl ester (60 mg, 0.124
mmol) was dissolved in 3 mL of THF, and lithium hydroxide solution
(0.5 N, 1.0 mL) was added. The mixture was stirred at room
temperature for 3 h, concentrated, and dissolved in water (8 mL). The
clear solution was treated with hydrochloric acid (1 N, 0.6 mL). The
mixture was filtered, and the white solid was dried to give compound
34 (51 mg, 90.2% yield). LC/MS calcd for C26H24N4O4 (m/e) 456.0;
1
obsd, 457.0 (M + H); H NMR (DMSO-d6) δ ppm 1.12−1.32 (m,
1-{4′-[3-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-3H-
[1,2,3]triazol-4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid
(40). This compound was prepared using the same method as that
described for the preparation of compound 35, except that 5-(4-
bromo-phenyl)-1-methyl-1H-[1,2,3] triazole-4-carboxylic acid was
used. LC/MS calcd for C28H26N4O4 (m/e) 482.0; obsd, 483.0 (M +
1H), 1.59 (br, 2H), 3.64 (s, 2H), 3.89 (s, 3H), 5.80 (br m, 1H), 6.96−
7.54 (m, 7H), 7.61−7.74 (m, 4H), 7.80 (d, J = 7.6 Hz, 2H), 9.54 and
9.95 (br s, 1H), 12.38 (br s, 1H).
1-{4′-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
[1,2,3]triazol-4-yl]-biphenyl-4-yl}-cyclopropanecarboxylic Acid
(35). [5-(4-Bromo-phenyl)-3-methyl-3H-[1,2,3]triazol-4-yl]-carbamic
acid (R)-1-phenyl-ethyl ester (566 mg, 1.41 mmol), methyl 1-(4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
cyclopropanecarboxylate (511 mg, 1.69 mmol), X-Phos (134 mg, 0.28
mmol), palladium acetate (31.7 mg, 0.14 mmol), and potassium
phosphate (898 mg, 4.23 mmol) were combined in toluene (12 mL),
and degassed water (3 mL) was added. The mixture was degassed and
sealed. The mixture was stirred at 95 °C for 3 h and cooled to room
temperature. The mixture was extracted with ethyl acetate and water.
1
H); H NMR (DMSO-d6) δ ppm 1.15−1.30 (m, 2H), 1.32−1.62 (m,
5H), 4.05 (s, 3H), 5.69 (br m, 1H), 7.20−7.40 (br m, 5H), 7.46 (d, J =
8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.66 (d, J = 8.1 Hz, 2H), 7.77
(d, J = 7.6 Hz, 2H), 9.34 (br s, 1H), 12.39 (s, 1H).
{5-[4′-(1-Methanesulfonylaminocarbonyl-cyclopropyl)-bi-
phenyl-4-yl]-3-methyl-3H-[1,2,3]triazol-4-yl}-carbamic acid (R)-
1-phenyl-ethyl Ester (41). In a 100 mL round-bottomed flask, 1-(4-
bromo-phenyl)-cyclopropanecarboxylic acid (4 g, 16.6 mmol) was
combined with dichloromethane (15 mL) and 3 drops of DMF to give
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Article
a white suspension. To this was added dropwise a clear solution of
oxalyl chloride (6.96 g, 4.8 mL, 54.8 mmol) dissolved in dichloro-
methane (6 mL). After 10 min, the mixture became clear, and the
reaction was stirred at room temperature for 2 h. The reaction was
concentrated, diluted with toluene and hexanes, concentrated, and
stored in a freezer overnight. In a 200 mL round-bottomed flask, NaH
(60% mineral dispersion, 876 mg, 36.5 mmol) was washed with
hexanes, and the resulting solid was diluted with DMF (6 mL) to give
a white suspension. The suspension was cooled in an ice bath, and
methanesulfonamide (3.16 g, 33.2 mmol) dissolved in DMF (6 mL)
was added dropwise under nitrogen. After addition (5 min), the ice
bath was removed, and the reaction was warmed to room temperature
overnight. The reaction was cooled in an ice bath, the acid chloride
previously prepared and dissolved in DMF (6 mL) was added
dropwise, and the reaction was warmed to room temperature
overnight. The reaction was diluted with 0.2 N HCl (200 mL) and
extracted with ethyl acetate (2 × 100 mL). The organic layers were
washed with brine, combined, dried over MgSO4, and concentrated.
The crude material was dissolved in minimal dichloromethane and
purified by flash column chromatography (0% to 60% ethyl acetate in
hexanes, 0.5% acetic acid) to give N-[1-(4-bromo-phenyl)-cyclo-
propanecarbonyl]-methanesulfonamide (2.74 g, 51.9% yield) as a
white solid. LC/MS calcd for C11H12BrNO3S (m/e) 317/319; obsd,
318/320 (M + H, ES+). This material (2.71 g, 8.52 mmol) was
combined with bis-pinacolatodiboron (3.24 g, 12.8 mmol), potassium
acetate (2.51 g, 25.6 mmol), and 1,4-dioxane (63.8 mL) to give a white
suspension. The mixture was purged with nitrogen for 20 min, and
then PdCl2(dppf)CH2Cl2 (701 mg, 859 μmol) was added. The vial
was sealed and heated in an oil bath at 80 °C for 16 h. The reaction
was diluted with ethyl acetate (150 mL), filtered, and rinsed with 0.2
M HCl (200 mL) and ethyl acetate (50 mL). The combined filtrate
was mixed vigorously, filtered, and separated. The aqueous layer was
extracted once with EtOAc (150 mL). The organic layers were washed
with brine, combined, dried over MgSO4, filtered, concentrated, and
dried from dichloromethane/hexanes to give a brown solid (4 g). The
crude material was supported on Celite and purified by flash column
chromatography (0 to 60% EtOAc in hexanes, 0.5% AcOH) to give N-
{1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-cyclopro-
panecarbonyl}-methanesulfonamide (2.75 g, 88.4% yield) as a white
solid. LC/MS calcd for C17H24BNO5S (m/e) 365; obsd, 366 (M + H,
ES+).
1.47−1.53 (m, 2H), 1.20 (d, J = 1.8 Hz, 2H); LC/MS calcd for
C30H28F3N5O5S (m/e) 627; obsd, 628 (M + H, ES+).
(4-{4-[1-Methyl-5-((R)-1-phenyl-ethoxycarbonylamino)-1H-
[1,2,3]triazol-4-yl]-phenyl}-cyclohexyl)-acetic Acid (43). To a
suspension of 5-(4-bromophenyl)-3-methyl-3H-[1,2,3]triazole-4-car-
boxylic acid (209 mg, 0.74 mmol) and sodium bicarbonate (187 mg,
2.22 mmol) in DMF (10 mL) was added an excess of benzyl bromide
(380 mg, 264 μL, 2.22 mmol) at room temperature under nitrogen
atmosphere. The resulting colorless reaction mixture was stirred for 15
h and then diluted with water and extracted with ethyl acetate (2 × 70
mL). The combined extracts were washed with brine solution and
dried over anhydrous MgSO4. Filtration and concentration gave the
crude colorless oil which was purified by column chromatography
eluting with ethyl acetate in hexanes (0−60%) to give 5-(4-
bromophenyl)-3-methyl-3H-[1,2,3]triazole-4-carboxylic acid benzyl
ester as a viscous oil (275 mg, 99% yield). LC/MS calcd for
C17H14BrN3O2 (m/e) 373; obsd, 373.8 (M + H, ES+).
To a mixture of 2-(4-hydroxycyclohexyl)acetic acid ethyl ester (3 g,
16.1 mmol), iodine (6.13 g, 24.2 mmol), imidazole (1.64 g, 24.2
mmol), and triphenylphosphine (6.34 g, 24.2 mmol) was added
dichloromethane (100 mL) at room temperature under nitrogen
atmosphere. The resulting brown suspension was stirred for 15 h, and
the solvent was removed under vacuum. The residue was extracted
with ethyl acetate, washed twice with a solution of water and methanol
(3:1), and then washed with brine solution. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated to give the
crude residue, which was purified by column chromatography eluting
with ethyl acetate in hexanes (0−50%) to give 2-(4-iodocyclohexyl)-
acetic acid ethyl ester (3.39 g, 71.1% yield) as a viscous light yellow oil.
1H NMR of this product indicated that it contained ∼30−40% of
elimination product (olefin), which was not separable on TLC.
To a 3-neck 50 mL round-bottom flask, equipped with an additional
funnel and thermometer, was added zinc dust (490 mg, 7.5 mmol) at
room temperature under nitrogen atmosphere. THF (2 mL) was
added to cover the zinc dust followed by the addition of 1,2-
dibromoethane (60.6 mg, 27.8 μL, 0.322 mmol). The mixture was
heated with a heat gun until evolution of ethylene gas ceased. The
suspension was cooled to room temperature, chlorotrimethylsilane
(35.0 mg, 40.8 μL, 0.322 mmol) was added, and the mixture was
stirred for 15 min at room temperature. Then, a solution of 2-(4-
iodocyclohexyl)acetic acid ethyl ester (740 mg, 2.5 mmol) in THF (2
and 1 mL for rinsing) was added dropwise for 5 min. After addition,
the reaction mixture was heated to 60 °C in an oil bath, and stirred for
3 h. The excess zinc dust was allowed to settle (15 h) to give a
colorless solution. In another 2-neck 25 mL round-bottom flask,
palladium(II) acetate (18.1 mg, 0.081 mmol) and 2-dicyclohexylphos-
phino-2′,6′-dimethoxybiphenyl (66.2 mg, 0.162 mmol) were charged,
and the flask was purged with nitrogen gas. Then, THF (1 mL) was
added, and the resulting light brown suspension was stirred for 5 min
before the addition of a solution of 5-(4-bromophenyl)-3-methyl-3H-
[1,2,3]triazole-4-carboxylic acid benzyl ester (120 mg, 0.322 mmol) in
THF (3 mL) at room temperature under nitrogen atmosphere. The
above prepared colorless zinc solution was added to this mixture. The
mixture was heated to 60 °C and stirred for 5 h. The reaction was
cooled to room temperature and diluted with saturated ammonium
chloride solution and ethyl acetate. The organic extracts were washed
with brine and dried over anhydrous MgSO4. Filtration and
evaporation of solvents gave a crude light yellow residue, which was
purified by column chromatography eluting with ethyl acetate in
hexanes (0−100%) to provide 5-[4-(4-ethoxycarbonylmethyl-cyclo-
hexyl)-phenyl]-3-methyl-3H-[1,2,3]triazole-4-carboxylic acid benzyl
ester (55 mg, 37.0% yield) as a light brown oil. LC/MS calcd for
C27H31N3O4 (m/e) 461; obsd, 462.1 (M + H, ES+). This material (51
mg, 0.11 mmol) and 10% Pd/C (58.8 mg, 0.552 mmol) were
combined with ethyl acetate (5 mL) at room temperature and
hydrogenated with a hydrogen balloon overnight. The reaction
mixture was filtered, and the charcoal was washed with hot ethyl
acetate (50 mL), THF (25 mL), CH3CN (75 mL), and ethanol (25
mL). The filtrate was concentrated, and the residue was dried under
high vacuum to obtain 5-[4-(4-ethoxycarbonylmethyl-cyclohexyl)-
In a 20 mL vial, [5-(4-bromo-phenyl)-3-methyl-3H-[1,2,3]triazol-4-
yl]-carbamic acid (R)-1-phenyl-ethyl ester (110.3 mg, 275 μmol), N-
{1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-cyclopro-
panecarbonyl}-methanesulfonamide (112 mg, 307 μmol), DPPF (38
mg, 68.5 μmol), and PdCl2(dppf)CH2Cl2 (39 mg, 47.8 μmol) were
combined with DMF (5 mL), and the mixture was bubbled with
nitrogen for 20 min to give a light brown/red solution. To this was
added 2 N Na2CO3 (550 μL, 1.1 mmol), and the red mixture had
nitrogen bubbled through for 1 min. The vial was sealed, placed in a
dry block, and heated at 80 °C for 4 h. The reaction was diluted with
ethyl acetate (75 mL) and 0.1 M HCl (100 mL), and the layers were
separated. The aqueous layer was extracted with ethyl acetate (75 mL).
The organic layers were washed with brine, dried over MgSO4, filtered,
and concentrated to give a crude material (311 mg). The crude
material was dissolved in minimal dichloromethane and purified by
flash column chromatography (0% to 100% EtOAc in hexanes, 0.5%
AcOH) to provide compound 41 (98.5 mg, 64% yield). LC/MS calcd
1
for C29H29N5O5S (m/e) 559; obsd, 560 (M + H, ES+); H NMR
(DMSO-d6) δ ppm 11.16 (br. s., 1H), 9.93 (br. s., 1H), 7.80 (d, J = 7.8
Hz, 2H), 7.62−7.76 (m, 4H), 7.06−7.57 (m, 7H), 5.65−5.93 (m, 1H),
3.85 (s, 3H), 3.20 (s, 3H), 1.38−1.76 (m, 5H), 1.20 (br. s., 2H).
{5-[4′-(1-Methanesulfonylaminocarbonyl-cyclopropyl)-bi-
phenyl-4-yl]-3-methyl-3H-[1,2,3]triazol-4-yl}-carbamic Acid
(R)-1-(3-trifluoromethyl-phenyl)-ethyl Ester (42). This com-
pound was prepared with the same method described in the
1
preparation of 41. H NMR (DMSO-d6) δ ppm 11.16 (br. s., 1H),
9.47−10.15 (m, 1H), 7.53−7.94 (m, 10H), 7.41 (d, J = 8.3 Hz, 2H),
5.89 (br. s., 1H), 3.86 (br. s., 3H), 3.20 (s, 3H), 1.60 (br. s., 3H),
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phenyl]-3-methyl-3H-[1,2,3]triazole-4-carboxylic acid (40 mg, 97.5%
yield) as a white solid. LC/MS calcd for C20H25N3O4 (m/e) 371; obsd,
372.3 (M + H, ES+). This material was converted to the desired
carbamate followed by saponification (2 steps) to afford compound
vehicle or LPAR antagonists for 1 h at the concentrations indicated
and were then stimulated with LPA (18:1) at the indicated
concentration for 48 h. Proliferation was assessed by determining
the incorporation of BrdU using a BrdU proliferation assay kit
(Calbiochem, San Diego, CA, USA) in accordance with the
manufacturer’s protocol. The degree of BrdU incorporation was
assessed using an EnVision Multilabel Reader (Perkin-Elmer Inc., San
Jose, CA, USA).
Three-Dimensional Collagen Gel Contraction Assay. Lung
fibroblast contraction was assessed using a commercially available
three-dimensional collagen gel contraction kit (Cell Biolabs, San
Diego, CA, USA) and performed according to the manufacturer’s
instructions. Briefly, NHLFs (2.5 × 105 cells/well) were mixed 1:4
with a collagen gel lattice mixture using the volumes indicated in the
manufacturer’s instructions and 0.5 mL plated per well in a 24-well
plate, which was incubated for 2 h at 37 °C to allow gel
polymerization. Following gel polymerization, 1 mL of basal media
containing 0.1% (v/v) FBS, 0.1% (v/v) fatty acid-free BSA, and either
vehicle or LPAR antagonists was added to each well. The gels were
detached, and then vehicle or LPA (18:1) at the concentrations
indicated was added. Then, 18 h later the images of the contracted gel
discs were obtained and quantitation performed by assessing the
weight of the gel discs.
1
43. H NMR (DMSO-d6) δ ppm 12.04 (br. s., 1H), 9.85 (br. s., 1H),
7.62 (d, J = 7.3 Hz, 2H), 6.84−7.54 (m, 7H), 5.78 (br. s., 1H), 3.83 (s,
3H), 2.16 (d, J = 6.8 Hz, 2H), 1.65−1.93 (m, 5H), 1.39−1.64 (m,
5H), 1.01−1.35 (m, 3H). LC/MS calcd for C26H30N4O4 (m/e) 462;
obsd, 463.3 (M + H, ES+).
Calculations of Torsion Angle Preferences. Truncated models of
compounds 32, 33, and 2 were built using Maestro version 9.1
(Schrodinger, LLC, New York, NY). Specifically, the biaryl
substitutions linked to the triazole were truncated to contain only a
single phenyl group. All potential energies were calculated using Jaguar
(Schrodinger, LLC, New York, NY) using the Density Functional
Theory (DFT) method with the B3LYP hybrid functional and the 6-
31G** basis set. The torsion angle was defined using the atom at the
4-position (in 32 and 2) or the 2-position (in 33) of the triazole, the
carbon of the triazole bonded to the carbamate, the nitrogen of the
carbamate, and the carbonyl carbon of the carbamate. The triazole-
carbamate torsion angle was scanned in 20° increments, with up to 50
rounds of geometry optimization of the remaining degrees of freedom
at each torsion angle.
LPA-Induced Histamine Release in Mice. Male C57BL6/J mice
(7−9 weeks old) were purchased from the Jackson Laboratory (Bar
Harbor, ME) and housed under pathogen-free conditions with food
and water ad libitum. All animal care and experimental procedures
were approved by the Roche Animal Care and Use Committee, which
is a facility accredited by the American Association for the
Accreditation of Laboratory Animal Care (AAALAC).
For dose−response studies, mice received intravenous LPA
challenge (0−1000 μg/mouse in 0.1% fatty acid-free bovine serum
albumin) and then were sacrificed 3 min after LPA administration with
4% isoflurane. Blood was collected into EDTA-coated tubes,
centrifuged 10,000g for 10 min at 4 °C, and frozen on dry ice. The
plasma was stored at −80 °C before analysis. Histamine concen-
trations were measured by ELISA (Oxford Biomedical Research)
based on the manufacturer’s instructions. Data are presented as the
mean SEM of n = 5−6 mice.
For the compound efficacy studies, mice received compound 2 (1−
100 mg/kg), the LPA1R antagonist AM095 (100 mg/kg) as a positive
control, or vehicle per os (p.o.) 2 h prior to the intravenous
administration of LPA (100 μg/mouse). Histamine levels in the
plasma were determined as described above. The plasma concen-
trations of compound 2 were analyzed by liquid chromatography/mass
spectrometry. Data are expressed as the mean SEM of n = 5−8 mice.
Significance (relative to the LPA vehicle control) was determined
using a one-way ANOVA and Dunnet’s posthoc test. The relationship
between plasma histamine and compound 2 concentrations was
determined using a Spearman’s rank correlation coefficient.
LPA1/3 Calcium Flux Assay Using a Fluorometric Imaging Plate
Reader (FLIPR Assay). The ChemiScreen Calcium-optimized stable
cell lines containing the human recombinant LPA1 or LPA3
Lysophospholipid receptors along with the parental line were
purchased from Chemicon International, Inc./Millipore. One day
prior to assay, cells were harvested and seeded at 10,000 cells per well
into 384 well black/clear tissue culture treated plates. On the day of
the assay, a calcium loading dye (Calcium-4, Molecular Devices) in
Hank’s Balanced Salt Solution containing 20 mM HEPES and 2.5 mM
probenecid was added to each well, and plates were then incubated for
30 min at 37 °C. After incubation, 20 μL of test compound in assay
buffer with 2.2% DMSO was transferred to the cell plate by the FLIPR.
Compound addition was monitored by the FLIPR to detect any
agonist activity of the compounds. Plates were then incubated for 30
min at room temperature protected from light. After incubation, plates
were returned to the FLIPR, and 20 μL of 4.5 μM LPA (18:1) or
ionomycin (for parental line) was added to the cell plates. The
fluorescence was continuously monitored before, during, and after
sample addition for a total elapsed time of 100 s. Responses (increase
in peak fluorescence) in each well following agonist addition is
determined. The initial fluorescence reading from each well, prior to
ligand stimulation, was used as zero baseline value for the data from
that well. The responses are expressed as % inhibition of the buffer
control. The IC50 value, defined as the concentration of a compound
that is required for 50% inhibition of the buffer control, was calculated
by fitting the percent inhibition data for 10 concentrations to a
sigmoidal dose−response (4 parameter logistic) model using the
Genedata Condoseo program [ model 205, F(x) = (A + (B − A)/
(1+((C/x)D)))].
Cell Culture of NHLFs. NHLFs obtained from healthy, nonsmoking
donors, (Lonza Rockland Inc., Rockland, ME, USA) were grown at 37
°C in a humidified 5% CO2 atmosphere in complete medium (FGM
fibroblast basal medium supplemented with 0.5 mL of recombinant
human fibroblast growth factor-B, 0.5 mL of insulin, 0.5 mL of
gentamicin sulfate amphotericin-B, and 2% (v/v) fetal bovine serum
(FBS) according to the manufacturer’s instructions). Where indicated,
NHLFs were serum-starved in basal medium (FGM fibroblast basal
medium supplemented with 0.5 mL of recombinant human fibroblast
growth factor-B, 0.5 mL of insulin, 0.5 mL of gentamicin sulfate
amphotericin-B, 0.1% (v/v) FBS, and 0.1% (v/v) fatty acid-free BSA).
For all experiments, NHLFs were used between passages 5 and 9.
Assessment of Proliferation Using BrdU Incorporation. NHLFs
were seeded in BioCoat Collagen I 96-well clear black plates (BD
Biosciences, Franklin Lakes, NJ, USA) at a density of 5 × 103 cells/
well and incubated overnight in complete medium after which the cells
were starved with basal media containing 0.1% (v/v) FBS and 0.1%
(v/v) fatty acid-free BSA for 24 h. The cells were incubated with
AUTHOR INFORMATION
Corresponding Author
*Phone: (973)235-3240. E-mail: yimin.qian@roche.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Paul Gillespie, Karen Lackey, and Satwant Narula for
constructive discussion, Ted Lambros for SFC chiral separation,
1
and Gino Sasso for the H NMR NOE study.
ABBREVIATIONS USED
■
LPA, lysophosphatidic acid; LPAR, lysophosphatidic acid
receptor; LPA1, lysophosphatidic acid receptor-1; IPF,
idiopathic pulmonary fibrosis; ECM, extracellular matrix;
BALF, bronchoalveolar lavage fluid; UUO, unilateral ureteral
7938
dx.doi.org/10.1021/jm301022v | J. Med. Chem. 2012, 55, 7920−7939

Journal of Medicinal Chemistry
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obstruction; NHLF, normal human lung fibroblast; BrdU,
bromodeoxyuridine; FLIPR, fluorometric imaging plate reader
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