Discovery of isoquinolinone indole acetic acids as antagonists of chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) for the treatment of allergic inflammatory diseases

Article abstract of DOI:10.1021/jm401509e

Previously we reported the discovery of CRA-898 (1), a diazine indole acetic acid containing CRTH2 antagonist. This compound had good in vitro and in vivo potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. H

Full text of DOI:10.1021/jm401509e

Article
pubs.acs.org/jmc
Discovery of Isoquinolinone Indole Acetic Acids as Antagonists of
Chemoattractant Receptor Homologous Molecule Expressed on Th2
Cells (CRTH2) for the Treatment of Allergic Inflammatory Diseases
Neelu Kaila,* Bruce Follows, Louis Leung, Jennifer Thomason, Adrian Huang, Alessandro Moretto,
Kristin Janz, Michael Lowe, Tarek S. Mansour, Cedric Hubeau, Karen Page, Paul Morgan, Susan Fish,
Xin Xu, Cara Williams, and Eddine Saiah
Pfizer Research and Development, Cambridge, Massachusetts 02140, United States
S
* Supporting Information
ABSTRACT: Previously we reported the discovery of CRA-898 (1), a diazine indole acetic acid
containing CRTH2 antagonist. This compound had good in vitro and in vivo potency, low rates of
metabolism, moderate permeability, and good oral bioavailability in rodents. However, it showed low oral
exposure in nonrodent safety species (dogs and monkeys). In the current paper, we wish to report our
efforts to understand and improve the poor PK in nonrodents and development of a new isoquinolinone
subseries that led to identification of a new development candidate, CRA-680 (44). This compound was
efficacious in both a house dust mouse model of allergic lung inflammation (40 mg/kg qd) as well as a
guinea pig allergen challenge model of lung inflammation (20 mg/kg bid).
(OC000459), and Array Biopharma (ARRY-502). Although no
beneficial effect was observed with AZD1981 in patients with
moderate to severe COPD,7 clinical data from Oxagen looks
promising. Their orally active CRTH2 antagonist, OC000459,
when dosed at 200 mg twice daily for 16 days, showed a 25%
reduction in the late asthmatic response area under the curve as
measured by forced expiratory volume in 1 s (FEV1) to inhaled
allergen change.8 Array BioPharma also observed positive results
from a phase 2 trial; their oral CRTH2 antagonist, ARRY-502,
improved FEV1 by 3.9% versus placebo in mild to moderate
persistent allergic asthma when dosed at 200 mg twice daily for
4 weeks, achieving statistical significance (P = 0.02).9 These results
have stimulated interest in discovering potent, selective, and orally
active CRTH2 antagonists for the treatment of asthma.
Indomethacin was identified as a CRTH2 receptor agonist
soon after the discovery of the CRTH2 receptor.10 After
indomethacin was discovered, a number of bicyclic indole acetic
acids and related analogues were disclosed as potent CRTH2
antagonists. Over the years, aryl acetic acid is a common
pharmacophore for a large number of CRTH2 antagonists
reported in literature.6,11 We recently reported discovery of a
new class of CRTH2 antagonists, the pyridazine linker
containing indole acetic acids.6 We had started from an initial
phthalazinone indole acetic acid hit, which had good potency but
poor permeability, metabolic stability, and PK. Optimization led
to CRA-898 (1), a compound with good potency, low rates of
metabolism, moderate permeability, and good oral bioavailability
in rodents. It was efficacious in mouse models of contact
hypersensitivity (1 mg/kg bid) and house dust allergen induced
INTRODUCTION
■
Arachidonic acid (AA) is released upon catalytic action of
cytosolic phospholipase A2 (cPLA2) on membrane phospholi-
pids, which in turn is initiated by activation of mast cells.1,2 Once
the AA cascade is triggered, several metabolites are produced.
PGD2 is a downstream metabolite and is a major prostanoid; it
binds to three different receptors: thromboxane type prostanoid
receptor (TP), the PGD2 receptor (DP), and the chemo-
attractant receptor homologous molecule expressed on Th2 cells
(CRTH2). CRTH2 is a promiscuous receptor and in addition to
PGD2 binds to several of its metabolites.3 It is a seven
transmembrane Gi coupled receptor expressed on Th2 cells,
eosinophils, basophils, and monocytes. Signaling through
CRTH2 results in an inhibition of cAMP, resulting in T cell,
eosinophil, basophil, and monocyte chemotaxis as well as
stimulation of Th2 T cell cytokine production. Activation of
CRTH2 contributes to Th2/eosinophilic type responses associated
with allergy and asthma. A comparison of CRTH2 expression with
chemotactic behavior of eosinophils in healthy and atopic
individuals revealed higher mRNA and protein levels of CRTH2
in eosinophils from atopic subjects compared with those in healthy
controls. In agreement with the increased number of CRTH2
receptors in eosinophils from atopic individuals, chemotaxis that
is induced by PGD2 was significantly higher in these cells.4
There is also evidence that genetic alterations of CRTH2
are linked to the severity of allergic asthma.5 These findings
indicate that higher levels of CRTH2 expression and increased
responsiveness to its ligand PGD2 might be the biological basis
for its association with asthma.
CRTH2 antagonists have been moved to clinical trials for
treatment of allergic rhinitis, asthma, and COPD.6 Recent
clinical data has come from AstraZeneca (AZD1981), Oxagen
Received: September 27, 2013
Published: February 10, 2014
© 2014 American Chemical Society
1299
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of Pyridazine Linker Analogues
Scheme 2. Synthesis of {3-[(1-Benzyl-6-oxo-1,6-dihydropyridin-3-yl)methyl]-5-fluoro-2-methyl-1H-indol- 1-yl}acetic Acid
Table 1. PK Properties of Compound 1
a
a
a
b
b
species
iv Cl (mL/min/kg)
t1/2
h
Vss (L/kg)
AUC0−inf (h·kg·ng/mL/mg)
Cmax (ng/mL)
bioavailability (%)
rat
12
20
3
1.6
0.4
0.4
5066
678
772
716
246
37
8
dog
2.3
2.3
monkey
21
1154
12
a
b
Intravenous dose, 2 mg/kg. Oral dose, 10 mg/kg.
lung inflammation (20 mg/kg qd) when dosed orally. However,
compound 1 showed low oral exposure in nonrodent safety
species (dogs and monkeys) (Table 1). In the current paper,
we wish to report our efforts to understand and improve the poor
PK in nonrodents and to describe the development of a new
isoquinolinone subseries that led to identification of a new
development candidate, 44.
CHEMISTRY
■
N-Benzyl Pyridazine Linker Analogues. The pyridazine
linker compounds (4, 6, 8, 10, and 12, Scheme 1) were prepared
by reductive alkylation of methyl 2-(5-fluoro-2-methyl-1H-indol-
1-yl)acetate (I-1A) with 1-benzyl-6-oxo-1,6-dihydropyridazine-
3-carbaldehyde (I-2)6 using triethyl silane/TFA mixture as the
reducing agent (Scheme 1).12 The resulting intermediate I-3
1300
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
N-benzyl pyridone linker analogue 3 was obtained by hydrolysis
of I-6 (Scheme 2). To obtain the substituted N-benzylated
pyridone linker analogues (5, 7, 9, 11, 13, and 14, Scheme 3), a
procedure by Bowman et al.13 was used, I-7 was reacted with
benzyl bromides activated by sodium iodide, and the products
(I-8) were hydrolyzed with base.
Scheme 3. Synthesis of Substituted N-Benzyl Pyridone Linker
Analogues
N-Alkyl Pyridone Linker Analogues. Synthesis of the alkyl
pyridone linker analogues was less straightforward (Scheme 4);
alkylation of I-7, using alkyl halides, was not successful because
the alkyl halides were not electrophilic enough to react with the
pyridyl nitrogen. Therefore, the methoxy group was cleaved
using TMSI, and the intermediate I-9 thus obtained was reacted
with I-1A to give I-10. Amide I-10 was treated with alkyl halide
and a base to give I-11. The latter were hydrolyzed to get the
N-alkylated pyridone linker analogues (16, 18, 23). Small
amounts of O-alkylated side products were also observed.
N-Benzyl Pyridones. The direct linked N-benzylated
pyridones (19−22, 34, 35, 38 Scheme 5) were prepared by
benzylation of the methoxy pyridine I-14 followed by hydrolysis
of the methyl ester of intermediate I-15. Intermediates I-14 were
acquired by palladium mediated coupling of 3-bromo indoles
(I-13) with 6-methoxypyridin-3-ylboronic acid. Intermediates
I-13 were obtained in 2 steps from 5-fluoro-2-methyl indole; first
bromination, followed by reaction with methylbromo acetate. A
similar Scheme 7 was followed to access 32 and 33, except tert-
butyl ester (I-18−I-20) was used as a protecting group instead of
methyl ester because better yields were obtained with the former.
The 2-methyl propionic acids (43a and 43b Scheme 8) were
prepared by benzylation of I-22 followed by hydrolysis of the
methyl ester I-23. I-22 were acquired via I-21 by following a
procedure similar to Scheme 5.
was debenzylated using aluminum chloride. In this reaction, the
methyl ester was hydrolyzed as well. The resulting pyridazine
carboxylic acid (I-4) was alkylated using substituted benzyl
bromides to give the dibenzylated intermediates I-5. Base
hydrolysis of benzyl esters gave the final products.
N-Benzyl Pyridone Linker Analogues. To obtain the N-
benzylated pyridone linker analogues, reductive alkylation was
done either with 1-benzyl-6-oxo-1,6-dihydropyridine-3-carbal-
dehyde (Scheme 2) or 6-methoxy pyridine carbaldehyde
(Scheme 3) to give intermediates I-6 or I-7, respectively. The
N-Alkyl Pyridones. To access the N-alkylated pyridones
(24−31, and 36, Scheme 6), the methyl ether in I-14 was
removed using acid and the product I-16 was alkylated with alkyl
bromides using potassium carbonate to give I-17, which was
hydrolyzed to give the final products. A minor amount of
O-alkylated analogue 37 was isolated alongside of N-alkylated
Scheme 4. Synthesis of N-Alkyl Pyridone Linker Analogues
1301
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 5. Synthesis of Substituted N-Benzyl Pyridone Analogues
analogue 36. The reaction conditions for synthesis of I-16
required some optimization; conventional heating for 48 h gave
moderate yield, and heating in the microwave improved the
yields.
couplings were unsuccessful, giving in most cases the reduced
form of the indole ring as the major side product.
In a third approach to a synthetic route to the benzyl
isoquinolinone (48), coupling of 3-boronic ester indole with 4-
bromoquinolinone was tried (Scheme 9C). This route examined
the effect of switching the boronic ester and the aryl halide
components of the Suzuki reaction. The literature revealed that
most of the Suzuki reactions with 3-boronic acid indoles require
an electron withdrawing group at the indole nitrogen such as a
sulfonamide. Using a sulfonamide protecting group on the
nitrogen atom of the indole (Scheme 9C; step 1), the 3-
bromoindole was converted to the 3-boronic ester indole, giving
a separable mixture of the desired product and the reduced form
of the indole as a side product (ratio 2:3). Unfortunately,
coupling of the boronic ester with 4-bromo isoquinolinone did
not give the desired product. On the basis of the observations
above, we suspected that 4-bromoisoquinolinone may be a poor
partner in the Suzuki coupling reactions. The coupling described
in Scheme 7 was different in that the halide coupling partner
was aromatic. On the basis of this observation, a benzyl group
N-Benzyl Isoquinolinone. Initially, the synthesis of the
isoquinolinone analogues was tried using the same approach
taken for the synthesis of the pyridone analogues shown in
Scheme 5. Following this sequence, 4-bromo-methoxyisoquino-
line was converted to the pinocol boronic ester and subsequently
coupled to 3-bromoindole in low but acceptable yields (28% and
45%, Scheme 9A). No reaction was observed, however, when the
methoxyisoquinoline analogue was subjected to sodium iodide
and benzylbromide. The ring system in this example seems to be
more stable than the methoxypyridine (I-14) example described
above in Scheme 5. In fact, when the methoxyisoquinoline was
subjected to conditions typically used to cleave arylmethoxy
groups, such as HBr in acetic acid, no reaction took place.
Scheme 9B illustrates another unsuccessful attempt, where
isoquinolinone-4-boronic acid (or its N-benzyl derivative) was
directly coupled to the indole core. Unfortunately, these
1302
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 6. Synthesis of N-Alkyl Pyridone Analogues
was used as a protecting group on the oxygen during the Suzuki
coupling (Scheme 9D). The O-benzyl starting material was
prepared via Mitsunobu reaction from 4-bromoisoquinolin-
1(2H)-one.14 Coupling of the boronic ester with 3-bromoindole
using PdCl2(dppf)-CH2Cl2 provided the desired product in 56%,
a much improved yield in the coupling step. A variety of
conditions were tried to cleave benzyl groups, but none were
successful. The entire sequence was also run using a PMB
protecting group. Cleavage should have occurred easily with
either DDQ or TFA, however, neither condition worked.
Scheme 10A illustrates direct coupling of isoquinoline to the
indole core, followed by a benzylation/oxidation protocol to
convert the isoquinoline to an benzylisoquinolinone as described
in literature.15 The benzylation step proceeded as expected,
however, when the intermediate obtained from the benzylation
was treated with the oxidant, K3Fe(CN)6, overoxidation took
place to give unwanted side products. By changing the protecting
group to BOC on the indole nitrogen, the oxidation step took
place cleanly to afford the desired benzylisoquinolinone product
(I-26, Scheme 10B). In the last three steps of the route, the BOC
group was cleaved with TFA and elaborated to the carboxylic side
chain to give a 37% overall yield in the 5-step transformation.
N-Alkyl Isoquinolinones. To access the N-alkyl isoquino-
linone analogues (44−47 Scheme 10C), the alkyl isoquinolinone
boronic ester (I-29) was coupled with 3-iodo indole (I-30). I-29
was prepared in 2 steps from 2-alkylisoquinolin-1(2H)-one
(I-27) via the 4-iodo-2-alkyllisoquinolin-1(2H)-one (I-28).
1303
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 7. Synthesis of N-Benzyl 5-Chloro- and 5-H Pyridones
Scheme 8. Synthesis of 2-Methyl Propionoc Acid Pyridone Analogues
of I-34 with indole I-30, followed by hydrolysis, gave final
products (49−52).
Saturated Isoquinolinones. The saturated isoquinolinones
(Scheme 11) were synthesized using a similar approach as the
alkyl isoquinolinones (Scheme 10C). The required boronic
esters (I-34) were prepared in 3 steps (via I-32 and I-33) from
the tetrahydroisoquinolin-1(2H)-one I-31. The latter was
prepared by platinum oxide reduction of the commercially
available isoquinolin-1(2H)-one.16 Palladium catalyzed coupling
RESULTS AND DISCUSSION
■
Compound 1, our first development track candidate, showed
good potency in cell based, binding and whole blood assays.
This compound exhibited a good PK profile in rodents and was
1304
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 9. Synthesis of N-Benzyl Isoquinolinone
efficacious in mouse models of contact hypersensitivity and
house dust allergen induced lung inflammation when dosed orally.
However, compound 1 showed low oral exposure in nonrodent
safety species (dogs and monkey). PK properties of 1 in preclinical
animal species are shown in Table 1. A large interspecies variability
was observed in plasma clearance (relative to hepatic blood flow)
in preclinical animal species (dog > monkey > rat). This was an
unexpected result because earlier studies with cyropreserved
hepatocytes from each of the species had shown low intrinsic
metabolic clearance (<5 μL/min/106 cells). Further studies were
1305
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 10. (A) Synthesis of N-Benzyl Isoquinolinone, (B) Synthesis of N-Benzyl Isoquinolinone, (C) Synthesis of N-Alkyl
Isoquinolinone
performed to examine alternative clearance pathways (e.g., renal,
biliary) in addition to metabolic clearance.
Biliary excretion studies in bile duct cannulated rats showed
that following a single iv dose of 5 mg/kg of 1, the average plasma
clearance was 14 mL/min/kg, with about 27% of the parent drug
recovered in bile and none in urine over a period of 24 h. These
results suggest that metabolism is the major elimination pathway
in rat with biliary excretion of unchanged drug a relatively modest
clearance pathway. Nevertheless, a different picture was observed
in dogs. A single intravenous dose of 5 mg/kg of 1 was administered,
1306
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Scheme 11. Synthesis of Saturated Isoquinolinone
and quantification of unchanged drug and drug-related products in
urine and feces over a 24 h period was assessed by 19F-NMR and
LC/MS. A majority of the administered dose (>90%) was recovered
as unchanged drug in feces. These results suggest that in dogs, unlike
rats, fecal excretion potentially via biliary excretion represents a
major clearance pathway of 1.
To further characterize the hepatobiliary disposition of 1,
sandwich-cultured hepatocytes from rat and human were used as
an in vitro model. Sandwich-cultured hepatocytes form func-
tional bile canalicular networks and maintain the expression and
function of key uptake and efflux transporters relative to
hepatocytes in vivo. In rat, the in vitro intrinsic biliary clearance
was determined to be low (<2.6 μL/min/μg) and seemed to be
consistent with the low in vivo biliary clearance (3.8 mL/min/kg).
The in vitro intrinsic biliary clearance was also determined to be low
(<1 μL/min/μg) in human sandwich-cultured hepatocytes.
However, it should be kept in mind that the use of sandwich-
cultured hepatocytes to accurately predict biliary clearance in
humans remains to be fully established. As a result, despite the low
predicted human clearance, the decision was made to identify a
different development candidate with improved PK properties in
nonrodent species.
A cAMP cell based competitive immunoassay (hCRTH2
FRET) using Eu3+ cryptate-labeled anti-cAMP and d2-labeled
cAMP was used as a primary in vitro screen. Additionally, three
secondary assays were used to evaluate compounds: (a) A
basophil chemotaxis assay to examine CRTH2 mediated cell
migration. This assay was run in a 96-transwell format with
human CRTH2+ basophils, derived from IL-3 treated CD34+
progenitor cells, in the presence of 10 nM PGD2. An initial set of
compounds were tested in the chemotaxis assay, and a very good
correlation was observed between the FRET and chemotaxis
assays (Figure 2A). (b) A human CRTH2 competitive binding
assay with tritium labeled PGD2. A good correlation was
observed between the binding assay and the FRET assay,
confirming that the FRET potency was coming from direct
binding to hCRTH2 (Figure 2B). Because a good correlation was
established between the FRET versus chemotaxis and FRET
versus binding assays, only selected compounds were tested in
these assays. (c) A human whole blood eosinophil shape change
assay (hEOS). Eosinophil shape change is a consequence of
cytoskeleton reorganization and is a prerequisite for leukocyte
movement, transmigration, and activation, and it can be
quantified by flow cytometry through changes in forward light
scatter. In this assay, human whole blood was stimulated with
PGD2 and analyzed by flow cytometry. Eosinophils were
Additional experiments were conducted to further investigate
the difference in biliary clearance observed in rats (low) and dogs
(high). We reasoned that 1 may have different hepatobiliary
uptake or efflux in different species. Permeability of 1 was
measured in the Caco-2 cell line at reduced pH 6.5. Compound 1
exhibited reasonable permeability (Papp 7.1 × 10−6 cm/s) across
Caco-2 monolayers, suggesting that the brush-border (microvilli)
region of the jejunum segment of the small intestine (where the pH
is slightly acidic) may be a predominant site of absorption. Results
from initial studies using Caco-2 cell monolayers and selective P-gp
(verapamil) or MRP2 (MK-571) inhibitors indicated that MRP2
and to a lesser extent P-gp was involved in the efflux of 1.
Because MRP2 seemed to be largely responsible for the efflux
of 1 in Caco-2 monolayers, membrane vesicles prepared from Sf9
cells expressing Mrp2/MRP2 from rats, dogs, monkeys, or
humans were used to further assess potential species differences
in biliary efflux of 1. Figure 1 shows the uptake of 1 into inside-out
membrane vesicles. The uptake in dog vesicles was found to be 3−4-
fold higher than that in rat, monkey, and human, suggesting species
differences in transporter-mediated efflux.
Figure 1. Uptake of 1 into Sf9 cells expressing Mrp2/MRP2.
1307
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Figure 2. (A) Plot of log IC50 in the FRET and basophil chemotaxis assay. (B) Plot of log IC50 in the FRET and binding assay.
selected based on their autofluorescence in the FL2 channel.
Shape change was estimated based on mean forward light scatter
values. This assay was used as a secondary assay and could
potentially be used as a biomarker in in vivo models and clinical
trials. A good correlation was seen between the FRET and hEOS
(free) assay (Figure 3). Potency in the FRET assay (IC50 ≤ 50 nM)
and good chemical properties were used as filters to advance
compounds to the hEOS assay.
Our initial strategy to improve the permeability and oral
bioavailability of the diazine class of compounds was to lower the
TPSA. One of the nitrogen atoms in the diazine was replaced
with carbon giving pyridine indoles. Table 2 shows comparison
Table 2. Potency in the FRET Assay for Diazine and Pyridone
Indoles
compd
X
R
FRET IC50 (nM)
2
N
Bn
10
11
3
3
CH
N
Bn
4
2-F
2-F
3-F
3-F
4-F
4-F
5
CH
N
8
6
8
7
CH
N
4
8
6
9
CH
N
3
10
11
12
13
2,6-diF
5
CH
N
2,6-diF
7
2,5-diF
2
CH
N
2,5-diF
8
1 (CRA-898)
2,4-DiF
2,4-DiF
CH2CF3
CH2CF3
(CH2)3CF3
(CH2)3CF3
2
a
14
CH
N
4
Figure 3. Plot of log IC50 in the FRET and hEOS (free) assay.
*Calculated human plasma protein binding used to determine hEOS
(free) values
15
16
17
18
18
52
11
150
CH
N
CH
a
Ligands for the receptors in the prostaglandin family show
some degree of cross-reactivity, and there is potential for small
molecule inhibitors to do the same. Therefore, selectivity of these
compounds against two other prostaglandin receptors (DP1 and
TXB2) was investigated using assays described previously.6
Representative examples from the series were tested and found
to be greater than thousand-fold selective for CRTH2. Early
compounds were also tested for affinity to the mouse CRTH2
receptor. A good correlation was seen between the human and
murine CRTH2. This result is of importance for the feasibilityof in
vivo pharmacological studies in mouse models. Compounds were
tested in these assays before advancing to in vivo models.
PK rat (iv 2 mg/kg, PO 10 mpk) Cl = 32 mL/min/kg; F = 4%.
of the diazine and pyridine indoles synthesized. In general, the
N-benzyl pyridones showed improved IC50s but comparable
LipE to the diazine indoles. The N-alkyl pyridones showed loss in
potency (15 versus 16 and 17 versus 18). Compound 14 was
evaluated in in vivo rat pharmacokinetic studies. The compound
had high clearance and poor bioavailability (Table 2). In an effort
to address the high clearance of these compounds, the meta-
bolically labile methylene group was removed. The direct linked
pyridine compounds (Table 3) showed comparable potency to
1308
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
addition of a methyl group at the methylene next to the
carboxylic acid reduced potency (43A, 43B).
Table 3. Potency in the FRET Assay for Pyridone Indoles
One of the pyridone analogues, 19, was taken forward for rat
pharmacokinetic studies. The compound had moderate
clearance and poor bioavailability (Table 3). So far, the
N-benzyl pyridones had shown poor rat PK properties (14, 19),
therefore we decided to select an N-alkyl pyridone (16) for
evaluation in in vivo rat pharmacokinetic studies (Table 3). This
compound showed low clearance and improved bioavailability. A
quick calculation using an in-house model showed that the predicted
kinetic solubility at pH 6.5 in PBS buffer for the alkyl pyridone
(16, solubility = 240 μg/mL) was higher than the benzyl pyridones
(19, solubility = 63 μg/mL; 14, solubility = 17 μg/mL), which could
result in improved absorption leading to higher bioavailability
observed with 16. Regrettably, thus far the alkyl pyridones had
shown low potency (16, 18, 23−29). A plot of cLogP and FRET
IC50s (Figure 4) for the pyridones showed that the optimal range
for clogP to achieve a potency less than 20 nM in the FRET assay is
3.5 and 5. We decided to pursue the isoqinolinone series to
modulate the lipophilicity of the N-alkyl pyridones. The N-alkyl
isoquinolinone (44−47) showed 10−20-fold improvement in
potency against CRTH2 in the FRET assay with improved or
comparable LipE (Table 6). The N-benzyl isoqinolinone (48) had
similar potency but lower LipE compared to the pyridone. The good
potency of the N-alkyl isoquinolinones was confirmed in the human
whole blood hEOS assay, and these compounds were then advanced
to in vivo studies. Additional ways to modulate the lipophilicity of
this scaffold include saturating the ring adjacent to the isoquinoline
ring. However, these compounds (49−52) showed loss in potency.
compd
n
R
FRET IC50 (nM)
3
1
0
1
0
1
0
1
0
1
0
1
0
Bn
Bn
11
5
a
19
14
20
9
2,4-DiF
2,4-DiF
4-F
5
17
3
21
11
22
23
24
4-F
6
2,6-diF
2,6-diF
i-Pr
7
6
137
70
52
84
i-Pr
b
16
CH2CF3
CH2CF3
25
a
b
PK rat (iv 2 mg/kg, PO 10 mpk) Cl = 15 mL/min/kg; F < 10%. PK
rat (iv 2 mg/kg, PO 10 mpk) Cl = 7 mL/min/kg; F 20%.
Table 4. SAR at the 5-Position
IN VIVO STUDIES
■
We utilized an oxazolone induced contact hypersensitivity
(CHS) model in the mouse as a primary in vivo screen. In this
model, mice were sensitized with oxazolone on their shaved
abdomen and then challenged on day 5 by painting their ears
with oxazolone. On the day of challenge, the test compound was
administered and inhibition of ear swelling was determined as a
measure of efficacy. A 40% reduction in ear swelling was observed
with CRTH2 knockout mice. Selected compounds were tested
in this model at 3 mg/kg po b.i.d. Figure 5 shows results for
compounds 44−47. All compounds showed reduction in ear
swelling in the oxazolone CHS model at 3 mg/kg po bid.
Compound 47 was not taken further due to its physicochemical
properties. Compounds 44, 45, and 46 were progressed into in
vivo rat pharmacokinetic studies. To establish pharmacokinetic
profiles, compounds were dosed intravenously in rats at 3 mg/kg
and orally at 10 mg/kg. Compounds 44 (Table 7) and 45
(Cl = 20 mL/min/kg; F = 35%) showed moderate clearance and
oral bioavailability, whereas compound 46 had moderate clearance
(35 mL/min/kg) but poor oral bioavailability (<5%). Compound
45 had poor oral exposure in guinea pigs, and because our efficacy
model was the guinea pig allergen induced pulmonary
inflammation model, it would have been very challenging to
dose compound 45. On the other hand, the po exposure in guinea
pigs was acceptable for compound 44. Similarly, its PK profile was
acceptable in dog and monkey (Table 7). Therefore, this
compound was selected for evaluation in efficacy models.
compd
R1
Cl
R
FRET IC50 (nM)
26
24
27
28
25
29
30
31
32
19
33
34
22
35
i-Pr
195
70
F
i-Pr
H
i-Pr
556
148
84
Cl
F
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
Bn
H
711
1120
2000
10
Me
OMe
Cl
F
Bn
5
H
Bn
42
Cl
F
2,6-DiF
2,6-DiF
2,6-DiF
8
7
H
21
linker pyridines. The SAR of this series was further expanded.
Substitution at the 5 position of the indole was studied for the
N-alkyl and N-benzyl subseries (Table 4). Substituents at the
5-position had an important effect on activity. In both subseries,
chloro (26) or fluoro (24) substitution at the 5 position improved
potency. Polar groups like methoxy had a detrimental effect on
activity (31).
Transferring the substituent from the nitrogen to the oxygen
in the pyridone ring was not tolerated. Greater then 40-fold
loss in potency was observed (Table 5). Conversion of the acid
to amide (39), substituted amide (40 and 41), and aliphatic
sulfonamide (42) all resulted in loss of potency. Likewise,
First, we sought to evaluate the potential of compound 44 to
modulate house dust mite (HDM)-induced allergic airway
disease in mice (Figure 6). This model has been previously
described6 and uses dexamethasone (Dex) intraperitoneally at
1 mg/kg (qd) as a positive control. Airway inflammation was
almost completely abrogated with Dex. Compound 44 was
1309
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Table 5. SAR of the Pyridone Indoles
administered orally at 40 mg/kg (qd), and the eosinophilic
component of HDM-induced allergic airway disease was
significantly decreased. These anti-inflammatory effects of 44
treatment on airway neutrophilia were observed along with
significantly decreased total BAL cellularity as well as significantly
decreased BAL counts of eosinophils and lymphocytes.
However, BAL macrophage numbers were unaffected by 44
treatment. Next, 44 was tested in a guinea pig allergen challenge
model; guinea pig airway allergen challenge models have been
used routinely in preclinical development of numerous
molecules targeted towards asthma and are recognized to elicit
robust allergic pulmonary inflammation that consists primarily of
eosinophils. Guinea pigs were sensitized by IP injection with
100 μg of ovalbumin (OVA) emulsified in 100 mg of alum on
days 1 and 5. Animals were then challenged via the airways
with an aerosol of 0.01% OVA for 60 min on day 15. Compound
was dosed at 20 mg/kg bid 1 h prior to challenge and 6 h post
Figure 4. Plot of cLogP versus FRET IC50 (nM).
Table 6. SAR of Isoquinolinone Indoles
FRET IC50
(nM)
FRET IC50
(nM)
hEOS IC50
(nM)
FRET IC50
(nM)
compd
R
LipE compd
R1
LipE
compd
R2
24
25
36
i-Pr
70
84
16
4.01
3.97
4.53
44
45
46
47
48
i-Pr
6
4
4.46
4.55
4.41
4.04
3.52
8
19
3
49
50
51
i-Pr
107
1000
123
CH2CF3
CH2CF3
(CH2)3CF3
CH2C(CH3)3
Bn
CH2CF3
(CH2)3CF3
1
(CH2)3CF3
3
18
19
Bn
5
4.9
14
52
Bn
192
1310
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
of Ascaris induced airway bronchoconstriction and AHR
(Figure 8A).18 Compound 44, when dosed at 3 mg/kg iv bid
the day prior to challenge and then again 1 h prior to challenge,
completely blocked the late phase allergic airway bronchocon-
striction (LAR). The animals were also given a fourth dose of 44,
8 h post challenge, to evaluate attenuation of airway hyper-
responsiveness (AHR) to aerosolized carbachol that was
measured the day following allergen challenge. Using this dosing
regimen, 44 (3 mg/kg iv) completely blocked the allergen
induced AHR to carbachol. 44 was also evaluated for efficacy
following subchronic administration at 1 mg/kg iv qd for 7 days
prior to challenge. Using this dosing regimen, the compound
completely blocked the LAR and AHR. Following measurement of
AHR, three bronchi from each sheep were lavaged and cell counts
determined. 44 reduced BAL inflammation at this dose (Figure 8B).
Preliminary studies also point to a role for CRTH2 in driving
impaired airway mucociliary clearance after allergen challenge.
Tracheal mucus velocity (TMV), a marker of airway mucociliary
clearance, was measured in sheep by a technique described
previously.6 Administration of 44 at 3 mg/kg iv resulted in a
more rapid return to baseline levels with a TMV of approx 70% of
baseline at 4 h and a TMV of 98% of baseline at 8 h compared to
an 8 h TMV of 47% of baseline with vehicle treated animals
(Figure 8C). Five sheep were used for these studies.
Figure 5. In vivo efficacy in CHS model.
challenge on day 15. Animals were sacrificed on day 16 and
bronchoalveolar cavities lavaged with 2 × 10 mL of warm PBS.
Total BAL cell counts and cell differentials were quantified as
described previously.17 Compound 44 significantly attenuated
total BAL cellularity, and BAL eosinophil, numbers 24 h post
OVA challenge (Figure 7). The efficacious doses in the CHS,
HDM, and OVA models were different; however, the exposures
at these doses were within 2-fold (exposures were predicted
based on pharmacokinetic studies).
Compound 44 was further profiled in safety and selectivity
studies (Table 8). The potential for clinical metabolic drug−drug
interactions was considered low based on in vitro CYP inhibition
results (CYP1A2, 2A6, 2B6, 2C19, 2D6, and 3A4 IC50s >100 μM,
CYP2C9 IC50 = 55 μM). Compound was clean in the AMES assay.
To explore efficacy in a nonrodent species and to determine
effects on lung function, 44 was evaluated in a sheep model
Table 7. PK Properties of 44
a
a
a
b
b
species
iv Cl (mL/min/kg)
t1/2
h
Vss (L/kg)
AUC0−inf (h·kg·ng/mL/mg)
Cmax (ng/mL)
bioavailability (%)
rat
18
2.7
3.4
4.1
1.2
1676
9459
253
2011
2967
20
32
69
dog
5.6
0.7
monkey
6.1
0.95
19675
a
b
Intravenous dose, 2 mg/kg. Oral dose, 10 mg/kg.
Figure 6. Modulation of HDM-induced allergic airway disease in BALB/c mice with 44 and dexamethasone (Dex). Total cellularity and eosinophil,
macrophage, lymphocyte, and neutrophil cell numbers in BAL samples collected 72 h post-HDM challenge.
1311
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Figure 7. Modulation of OVA-induced allergic lung inflammation in the guinea pig: total cellularity and eosinophils, in BAL samples collected 24 h post-
OVA challenge. Solid bars are OVA sensitized, PBS challenged animals; hatched bars are OVA sensitized OVA challenged animals that received vehicle
orally 1 h prior to challenge and 6 h post challenge (1 mL of 0.5 Tween 80 in FlavoRX Cherry blast syrup) N = 15 animals; gray bars are animals that have
been treated with dexamethasone orally at 30 mg/kg 1 h prior to challenge. N = 9 animals; open bars are animals that have been treated with compound
44 orally at 20 mg/kg 1 h prior to challenge and 6 h post challenge. N = 14 animals. P values as determined by student’s unpaired t test compared with
vehicle treated animals are provided above the relevant bars.
Figure 8. (A) Administration of compound 44 at 3 mg/kg iv in a sheep model of allergic airway bronchoconstriction and AHR. (i) While compound 44
(pink line) did not attenuate EAR as determined by increases in airway resistance above baseline, it completely ablated the Ascaris suum allergen induced
LAR compared to untreated historical control values observed after allergen challenge in the same animals. (ii) Compound 44 blocks AHR to
aerosolized carbachol (pink bar) as determined by an increase in PC400 values (the cumulative carbachol breath units that increased specific lung
resistance by 400% (PC400) following allergen challenge compared to historical untreated control values in the same animals (blue bar). (B)
Compound 44 attenuates Ascaris induced BAL fluid inflammation at 1 mg/kg iv qd for 7 days in sheep. (C) Reduction in mucocilliary clearance in sheep
with 44 at 3 mpk iv.
No hERG inhibition was seen at 33 μM. Greater than 800-fold sel-
ectivity was seen against DP1 and TP receptors and in the Novascreen
and nuclear receptor panel. On the basis of these results and in vivo
efficacy, 44 (CRA-680) was advanced to development track.
In summary, we started with 1, a compound with good in vitro
and in vivo potency, good oral bioavailability in rodents, but low
oralexposureinnonrodent species (dogs and monkeys) (Table1).
Key medicinal chemistry optimization changes included decreasing
polar surface area and eliminating metabolically labile methylene
group. This led to a new development candidate 44 with
comparable potency to 1, moderate bioavailability in rats, and
improved PK in dogs and monkeys (Table 7).
1312
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
Table 8. Overall Profile of Compound 44
hCRTH2 FRET
(IC50 nM)
hCRTH2 binding
(IC50 nM)
basophil chemotaxis
(IC50 nM)
hEOS
(IC50 nM)
mCRTH2 binding
(IC50 nM)
DP1 FRET
(IC50 nM)
TXA2 binding
(IC50 nM)
Caco2 permeability at
(pH 6) cm/s
8
1.4
1.2
8
4
16000
>1000
2.7 × 106
layers were washed with water and brine and dried over MgSO4. Filtration
and concentration in vacuo gave a tan solid. The crude material was purified
by silica gel chromatography.
EXPERIMENTAL SECTION
■
Chemistry. Reactions were run using commercially available starting
materials and solvents without further purification. Proton NMR spectra
were recorded at 300 MHz on a Varian Gemini 2000 or at 400 MHz
on a Bruker AV-400 spectrometer using TMS (δ 0.0) as a reference. The
microwave procedures were carried out with a Biotage microwave.
Preparative HPLC was run using a Waters reverse phase prep HPLC
with Xterra C18 5 μM, 30 mm × 100 mm column. The flow rate was
40 mL/min and mobile phase A was water, mobile phase B was CH3CN,
and triethylamine was used as a modifier. Purity in H2O−CH3CN
was determined using an Agilent 1100 HPLC instrument, and all
compounds analyzed were >95% pure.
Pyridazine Linker Analogue. To a 50 mL round-bottom flask under an
atmosphere of nitrogen was added I-5 (0.06 mmol, 1.0 equiv) and 20 mL of
methanol. To this was added 0.12 mL of 5.0 N NaOH. The resulting
mixture was allowed to stir for 16 h at room temperature. Mixture was
poured into 100 mL of 1.2 N HCl and extracted with three 50 mL portions
of ethyl acetate. The combined organic layers were washed with water
and brine and dried over MgSO4. Filtration and concentration in vacuo
gave crude material which was purified by reverse phase HPLC.
The isolated material was then suspended in acetonitrile/water, frozen,
and lyophilized to give the desired product (the yields have been reported
over three steps).
(3-{[1-(2-Fluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]methyl}-
5-fluoro-2- methyl-1H-indol-1-yl)acetic Acid (4). General procedure A
was followed to make this compound in 33% yield. 1H NMR
(400 MHz, DMSO-d6) δ 13.03 (br s, 1H), 7.32−7.40 (m, 2H), 7.11−
7.25 (m, 5H), 6.84−6.90 (m, 2H), 5.28 (s, 2H), 4.93 (s, 2H), 3.93 (s, 2H),
2.28 (s, 3H).
Biology. All procedures performed on these animals were in
accordance with regulations and established guidelines and were
reviewed and approved by an Institutional Animal Care and Use
Committee or through an ethical review process.
General Procedure A (Scheme 1). Methyl 2-(5-fluoro-2-methyl-
1H-indol-1-yl)acetate (I-1A). In a 500 mL 2-necked round-bottomed
flask, 5-fluoro-2-methylindole (5.00 g) was taken up with 100 mL
anhydrous DMF, under nitrogen. NaH (1.69 g of a 60 wt % suspension
in mineral oil, 1.01 g, 42.2 mmol) was added in small aliquots, and the
mixture allowed to stir at room temperature for 30 min. Methyl
bromoacetate (3.9 mL, 6.5 g, 42 mmol) was added all at once by syringe,
and the reaction was allowed to stir overnight. It was then quenched by
addition of 20 mL of brine, via syringe, and partitioned between 400 mL
each ethyl acetate and brine. The aqueous layer was extracted with
additional ethyl acetate (2×), and the combined organic extracts washed
with brine (3×), dried over anhydrous magnesium sulfate, filtered,
evaporated, and purified by flash chromatography over silica gel (2−20%
ethyl acetate in hexanes) to give a white solid that gradually turned pink
over several days (5.39 g, 69% yield).
Methyl 2-(3-((1-benzyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl)-5-
fluoro-2-methyl-1H-indol-1-yl)acetate (I-3). To a 250 mL round-bottom
flask under an atmosphere of nitrogen was added I-1A (3.26 g, 14.77
mmol, 1.0 equiv), 1-benzyl-6-oxo-1,6-dihydropyridazine-3-carbaldehyde
(I-2) (3.46g, 16.24 mmol, 1.1 equiv), and 100 mL of anhydrous
methylene chloride. The resulting solution was cooled to 0 °C in an
ice/water bath, and triethylsilane (8.26 mL, 51.69 mmol, 3.5 equiv)
and trifluoroacetic acid (3.3 mL, 44.30 mmol, 3.0 equiv) were added
dropwise. The mixture was allowed to warm to room temperature and
then stirred for 24 h. The mixture was then poured into satd NaHCO3(aq)
and the aqueous layer extracted with two 50 mL portions of methylene
chloride. The combined organic layers were then washed with water and
brine and dried over magnesium sulfate. Filtration and removal of solvent
in vacuo gave the crude material, which was then purified by silica gel
chromatography to give a white solid (2.66g, 43%)
2-(5-Fluoro-2-methyl-3-((6-oxo-1,6-dihydropyridazin-3-yl)methyl)-
1H-indol-1-yl)acetic acid (I-4). To a 5 mL microwave reaction vessel
was added I-3 (0.197g, 0.47 mmol, 1.0 equiv), aluminum trichloride
(0.375g, 2.84 mmol, 6.0 equiv), and 5 mL of toluene. The vessel was
sealed and the mixture heated to 140 °C in a microwave reactor for 1 h. The
mixture was then poured into 50 mL of water and extracted with three
50 mL portions of ethyl acetate. The combined organic layers were washed
with water and brine then dried over MgSO4. Filtration and concentration
in vacuo gave the crude product which was taken to the next step.
2-(3-((1-Substituted-6-oxo-1,6-dihydropyridazin-3-yl)methyl)-5-fluoro-
2-methyl-1H-indol-1-yl)ester I-5. To a 100 mL round-bottom flask under a
nitrogen atmosphere was added intermediate I-4 (0.069 g, 0.22 mmol,
1.0 equiv), selected bromide (0.67 mmol, 3.0 equiv), potassium carbonate
(0.123g, 0.89 mmol, 4.0 equiv), and 40 mL of DMF. The resulting
suspension was heated to 85 °C for 16 h. The mixture was then allowed to
cool to room temperature and then poured into 200 mL of water. This was
extracted with three 50 mL portions of ethyl acetate. The combined organic
(3-{[1-(3-Fluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]methyl}-
5-fluoro-2- methyl-1H-indol-1-yl)acetic Acid (6). General procedure A
was followed to make this compound in 23.1% yield. 1H NMR
(400 MHz, DMSO-d6) δ 7.31−7.40 (m, 2H), 7.06−7.19 (m, 5H), 6.82−
6.89 (m, 2H), 5.24 (s, 2H), 4.87 (s, 2H), 3.96 (s, 2H), 2.30 (s, 3H).
(3-{[1-(4-Fluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]methyl}-
5-fluoro-2- methyl-1H-indol-1-yl)acetic Acid (8). General procedure A
was followed to make this compound in 21.2% yield. 1H NMR
(400 MHz, DMSO-d6) δ 7.23−7.33 (m, 3H), 7.02−7.12 (m, 4H), 6.74−
6.84 (m, 2H), 5.14 (s, 2H), 4.85 (s, 2H), 3.88 (s, 2H), 2.23 (s, 3H).
(3-{[1-(2,6-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]-
methyl}-5-fluoro-2- methyl-1H-indol-1-yl)acetic Acid (10). General
1
procedure A was followed to make this compound in 28% yield. H
NMR (400 MHz, DMSO-d6) δ 13.13 (br s, 1H), 7.52 (tt, J = 6.66,
8.37 Hz, 1H), 7.38 (dd, J = 4.42, 8.97 Hz, 1H), 7.12−7.22 (m, 3H), 7.09
(dd, J = 2.53, 9.85 Hz, 1H), 6.86−6.95 (m, 2H), 5.36 (s, 2H), 4.95
(s, 2H), 3.89 (s, 2H), 2.26 (s, 3H).
(3-{[1-(2,5-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-yl]-
methyl}-5-fluoro-2- methyl-1H-indol-1-yl)acetic Acid (12). General
1
procedure A was followed to make this compound in 22% yield. H
NMR (400 MHz, DMSO-d6) δ 7.16−7.38 (m, 4H), 7.12 (dd, J = 2.53,
9.85 Hz, 1H), 7.02 (ddd, J = 3.28, 5.62, 8.78 Hz, 1H), 6.83−6.91
(m, 2H), 5.28 (s, 2H), 4.92 (s, 2H), 3.94 (s, 2H), 2.30 (s, 3H).
{3-[(1-Benzyl-6-oxo-1,6-dihydropyridin-3-yl)methyl]-5-fluoro-2-
methyl-1H-indol-1-yl}acetic Acid (3) (Scheme 2). The procedure
described above for I-3 was followed, reacting 1-benzyl-6-oxo-1,6-dihydro-
pyridine-3-carbaldehyde19 with I-1A to give methyl 2-(3-((1-benzyl-6-oxo-
1,6-dihydropyridin-3-yl)methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetate
(I-6). To a 100 mL round-bottom flask was added intermediate I-6 (0.335g,
0.80 mmol, 1.0 equiv) and 20 mL of THF. To this was added a solution of
lithium hydroxide (0.096g, 4.0 mmol, 5.0 equiv) in 10 mL of water. To the
resulting biphasic mixture was added methanol dropwise until a single layer
formed. The resulting solution was stirred for 2 h at room temperature. It
was then poured into 1.2 N HCl(aq), and the aqueous layer was extracted
with three 50 mL portions of ethyl acetate. The combined organic layers
were washed with water and brine and dried over magnesium sulfate.
Filtration and removal of solvent in vacuo gave the crude material. This was
purified by reverse phase HPLC and the isolated product lyophilized to give
1
a powder (0.161g, 51% yield.). H NMR (400 MHz, DMSO-d6) δ 7.74
(d, J = 2.02 Hz, 1H), 7.22−7.37 (m, 6H), 7.19 (td, J = 2.53, 9.35 Hz, 2H),
6.86 (td, J = 2.65, 9.28 Hz, 1H), 6.31 (d, J = 9.35 Hz, 1H), 5.05 (s, 2H), 4.92
(s, 2H), 3.72 (s, 2H), 2.30 (s, 3H).
General Procedure B (Scheme 3). Methyl 2-(5-fluoro-3-
((6-methoxypyridin-3-yl)methyl)-2-methyl-1H-indol-1-yl)acetate (I-7).
1313
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
I-1A (1.00 g, 4.52 mmol) was reacted with 6-methoxy-3-pyridinecarbox-
aldehyde (0.620 g, 4.52 mmol) using the procedure for intermediate I-3
to give I-7 (0.861 g, 56% yield).
2-(3-((1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (14). Intermedi-
ate I-8−6. Methyl 2-(3-((1-(2,4-difluorobenzyl)-6-oxo-1,6-dihydropyridin-
3-yl)methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetate. General procedure
B was followed to make I-8−6 in 56% yield.
General procedure B was followed to make 14 from I-8−6 in 44%
yield. 1H NMR (DMSO-d6) δ 7.63 (d, J = 2.5 Hz, 1H), 7.16−7.29 (m,
4H), 7.13 (dd, J = 9.9, 2.5 Hz, 1H), 6.98−7.06 (m, J = 8.5, 8.5, 2.6, 0.9
Hz, 1H), 6.82 (td, J = 9.2, 2.5 Hz, 1H), 6.31 (d, J = 9.3 Hz, 1H), 5.05 (s,
2H), 4.70 (s, 2H), 3.72 (s, 2H), 2.29 (s, 3H).
General Procedure C (Scheme 4). 6-Oxo-1,6-dihydropyridine-3-
carbaldehyde (I-9). In a flame-dried 250 mL 2-necked round-bottomed
flask fitted with a condenser, 6-methoxy-3-pyridinecarboxaldehyde
(3.43 g, 24.9 mmol) was taken up in 30 mL of anhydrous dichloro-
methane under nitrogen. Iodotrimethylsilane (5.0 g, 25 mmol) was added
by syringe. The mixture was stirred at room temperature for 2.5 h then
refluxed for 1.5 h. After cooling to room temperature, 4.1 mL of methanol
was added by syringe. The solvent was evaporated, and the residue purified
by flash chromatography over silica gel (7−60% acetone in dichloro-
methane) to give pure product (2.67 g, 87% yield).
Methyl 2-(5-fluoro-2-methyl-3-((6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-1H-indol-1-yl)acetate (I-10). The procedure described above
for I-7 was followed, reacting I-1A (2.52 g, 11.4 mmol) with
intermediate I-9 (1.40 g, 11.4 mmol) in the presence of triethylsilane
(5.1 mL, 3.7 g, 32 mmol) and trifluoroacetic acid (1.8 mL, 2.6 g, 23 mmol).
Because of the relative insolubility of the product, the workup was modified
as follows: the cooled reaction mixture was partitioned between saturated
sodium bicarbonate and dichloromethane and the organic layer washed
with water and evaporated (product was already starting to precipitate out,
so drying over magnesium sulfate and filtering was concluded to be a bad
idea). The crude product was purified by recrystallization from acetonitrile,
collecting two crops of pure product (2.82 g, 76% yield).
Methyl 2-(3-((1-alkyl-6-oxo-1,6-dihydropyridin-3-yl)methyl)-5-fluo-
ro-2-methyl-1H-indol-1-yl)acetate (I-11). In a 100 mL round-bottomed
flask under nitrogen, I-10 (1.75 mmol) and cesium carbonate or
potassium carbonate (8.8 mmol) were taken up in 25 mL of anhydrous
DMF and selected alkyl halide (8.8 mmol) was added. The mixture was
heated to 55−85 °C until almost complete conversion to product had
occurred. The cooled reaction mixture was poured into 250 mL of water
and extracted into ethyl acetate; the combined organic extracts were
washed with brine, dried over anhydrous magnesium sulfate, filtered,
evaporated, and purified by flash chromatography over silica gel to give
pure product.
Alkyl Pyridone Linker Analogue. The procedure described above
for pyridone linker analogue (in Scheme 4) was followed, reacting
I-11 (0.764 mmol) with lithium hydroxide monohydrate (0.642 g,
15.3 mmol). The crude product was purified by recrystallization.
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihy-
dropyridin-3-yl)methyl-1H-indol-1-yl)acetic Acid (16). Methyl 2-(5-
fluoro-2-methyl-3-((6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyridin-
3-yl)methyl-1H-indol-1-yl)acetate (I-11−1). General procedure C was
followed reacting I-10 with and 1,1,1-trifluoro-2-iodoethane to make
I-11−1 in 44% yield.
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(4,4,4-trifluorobutyl)-1,6-dihy-
dropyridin-3-yl)methyl)-1H-indol-1-yl)acetic Acid (18). Methyl
2-(5-fluoro-2-methyl-3-((6-oxo-1-(4,4,4-trifluorobutyl)-1,6-dihydropyri-
din-3-yl)methyl)-1H-indol-1-yl)acetic acid (I-11−2). General procedure C
was followed reacting I-10 with 4-bromo-1,1,1-trifluorobutane to make
I-11−2 in 38% yield.
General procedure C was followed to make 18 from I-11−2 in 40%
yield. 1H NMR (DMSO-d6) δ 13.00 (br s, 1H), 7.62 (d, J = 2.0 Hz, 1H),
7.35 (dd, J = 9.0, 4.4 Hz, 1H), 7.22 (dd, J = 10.1, 2.5 Hz, 1H), 7.17 (dd,
J = 9.3, 2.5 Hz, 1H), 6.86 (td, J = 9.1, 2.5 Hz, 1H), 6.29 (d, J = 9.3 Hz, 1H),
4.95 (s, 2H), 3.90 (t, J = 6.9 Hz, 2H), 3.73 (s, 2H), 2.32 (s, 3H), 2.16−2.31
(m, 2H), 1.84 (quin, J = 7.6 Hz, 2H).
Methyl 2-(3-((1-substituted benzyl-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetate I-8. Intermediate I-7
(0.954 mmol) and sodium iodide (0.286 g, 1.91 mmol) were taken up in
10 mL of anhydrous acetonitrile, and selected bromide (1.9 mmol) was
added. The mixture was refluxed overnight then poured into a mixture of
50 mL each brine and 5% sodium thiosulfate and extracted into ethyl
acetate (2×). The combined organic extracts were washed with brine,
dried over anhydrous magnesium sulfate, filtered, evaporated, and
purified by flash chromatography over silica gel.
Pyridone Linker Analogue. Intermediate I-8 (0.513 mmol) was
taken up in 15 mL of methanol and 5 mL of tetrahydrofuran. A solution
of lithium hydroxide monohydrate (0.430 g, 10.3 mmol) in 5 mL of
water was added, and the reaction stirred at room temperature for
55 min until LC-MS analysis showed complete hydrolysis of the ester.
The reaction mixture was then acidified with concentrated hydrochloric
acid, extracted into ethyl acetate (3×), washed with brine, dried over
anhydrous magnesium sulfate, filtered, evaporated, and purified by
preparative HPLC (water/acetonitrile with 0.1% formic acid).
2-(5-Fluoro-3-((1-(2-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-2-methyl-1H-indol-1-yl)acetic Acid (5). Methyl 2-(5-fluoro-3-
((1-(2-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)methyl)-2-methyl-
1H-indol-1-yl)acetate (I-8−1). General procedure B was followed to
make I-8−1 from I-7 and 2-fluorobenzyl bromide in 66% yield.
General procedure A was followed to make 5 from I-8−1 in 39%
yield. 1H NMR (DMSO-d6) δ 12.99 (br s, 1H), 7.67 (d, J = 2.0 Hz, 1H),
7.30−7.38 (m, 2H), 7.16−7.24 (m, 3H), 7.05−7.16 (m, 2H), 6.86 (td,
J = 9.1, 2.5 Hz, 1H), 6.32 (d, J = 9.3 Hz, 1H), 5.09 (s, 2H), 4.94 (s, 2H),
3.74 (s, 2H), 2.30 (s, 3H).
2-(5-Fluoro-3-((1-(3-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-2-methyl-1H-indol-1-yl)acetic Acid. (7). Methyl 2-(5-fluoro-
3-((1-(3-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)methyl)-2-methyl-
1H-indol-1-yl)acetate (I-8−2). General procedure B was followed to make
I-8−2 from I-7 and 3-fluorobenzyl bromide in 64% yield.
General procedure A was followed to make 7 from I-8−2 in 39% yield.
1H NMR (DMSO-d6) δ 7.76 (d, J = 2.0 Hz, 1H), 7.31−7.40 (m, 2H), 7.19
(dd, J = 9.5, 2.7 Hz, 2H), 7.05−7.14 (m, 3H), 6.86 (td, J = 9.2, 2.5 Hz, 1H),
6.33 (d, J = 9.1 Hz, 1H), 5.06 (s, 2H), 4.94 (s, 2H), 3.73 (s, 2H), 2.31 (s, 3H).
2-(5-Fluoro-3-((1-(4-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-2-methyl-1H-indol-1-yl)acetic Acid (9). Methyl 2-(5-fluoro-3-
((1-(4-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)methyl)-2-methyl-
1H-indol-1-yl)acetate (I-8−3). General procedure B was followed to
make I-8−3 from I-7 and 4-fluorobenzyl bromide in 54% yield.
General procedure B was followed to make 9 from I-8−3 in 55%
yield. 1H NMR (DMSO-d6) δ 12.97 (br s, 1H), 7.73 (d, J = 2.0 Hz, 1H),
7.30−7.37 (m, 3H), 7.09−7.20 (m, 4H), 6.86 (td, J = 9.1, 2.5 Hz, 1H), 6.31
(d, J = 9.3 Hz, 1H), 5.03 (s, 2H), 4.93 (s, 2H), 3.72 (s, 2H), 2.30 (s, 3H).
2-(3-((1-(2,6-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (11). Methyl
2-(3-((1-(2,6-difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)methyl)-
5-fluoro-2-methyl-1H-indol-1-yl)acetate (I-8−4) General procedure B
was followed to make I-8−4 from I-7 and 2,6-difluorobenzyl bromide in
72% yield.
General procedure B was followed to make 11 from I-8−4 in 60%
yield. 1H NMR (DMSO-d6) δ 12.98 (br s, 1H), 7.56 (s, 1H), 7.40 (tt, J =
8.3, 6.6 Hz, 1H), 7.34 (dd, J = 8.8, 4.3 Hz, 1H), 7.16 (td, J = 9.3, 2.5 Hz,
2H), 7.01−7.09 (m, 2H), 6.86 (td, J = 9.2, 2.7 Hz, 1H), 6.25 (d, J = 9.3
Hz, 1H), 5.07 (s, 2H), 4.94 (s, 2H), 3.74 (s, 2H), 2.29 (s, 3H).
2-(3-((1-(2,5-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (13). Methyl
2-(3-((1-(2,5-difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)methyl)-5-fluoro-
2-methyl-1H-indol-1-yl)acetate (I-8−5). General procedure B was followed to
make I-8−5 from I-7 and 2,5-difluorobenzyl bromide in 68% yield.
General procedure B was followed to make 13 from I-8−5 in 37%
yield. 1H NMR (DMSO-d6) δ 13.27 (s, 1H), 7.69 (d, J = 1.8 Hz, 1H),
7.14−7.36 (m, 5H), 6.80−6.91 (m, 2H), 6.33 (d, J = 9.3 Hz, 1H), 5.07
(s, 2H), 4.90 (s, 2H), 3.74 (s, 2H), 2.31 (s, 3H).
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(4,4,4-trifluorobutyl)-1,6-dihy-
dropyridazin-3-yl)methyl)-1H-indol-1-yl)acetic Acid (15). The proce-
dure described in Scheme 5A of ref 6 was followed to make this compound
1
in 63% yield. H NMR (400 MHz, DMSO-d6) δ 7.35 (dd, J = 4.29,
8.84 Hz, 1H), 7.23 (dd, J = 2.53, 9.85 Hz, 1H), 7.14 (d, J = 9.60 Hz, 1H),
1314
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
6.88 (td, J = 2.40, 9.16 Hz, 1H), 6.81 (d, J = 9.35 Hz, 1H), 4.90 (s, 2H),
3.95 (s, 2H), 3.91 (s, 2H), 2.34 (s, 3H), 0.95 (s, 9H).
General procedure D was followed to make 20 from I-15A-2 in 76%
yield. 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, 1H), 7.57 (dd, J = 2.65,
9.22 Hz, 1H), 7.23−7.43 (m, 3H), 7.05−7.15 (m, 2H), 6.89 (td, J = 2.53,
9.22 Hz, 1H), 6.53 (d, J = 9.35 Hz, 1H), 5.21 (s, 2H), 4.56 (br s, 2H),
2.30 (s, 3H).
2-(5-Fluoro-3-(1-(4-fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
2-methyl-1H-indol-1-yl)acetic Acid (21). Methyl 2-(5-fluoro-3-(1-(4-
fluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-1H-indol-1-yl)-
acetate (I-15A-3). General procedure D was followed to make I-15A-3
from I-14A and 4-fluorobenzyl bromide in 42% yield.
General procedure D was followed to make 21 from I-15A-3 in 76%
yield. 1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 2.02 Hz, 1H), 7.56
(dd, J = 2.65, 9.22 Hz, 1H), 7.43−7.50 (m, 3H), 7.17−7.22 (m, 2H),
7.12 (dd, J = 2.27, 9.85 Hz, 1H), 6.97 (td, J = 2.65, 9.16 Hz, 1H), 6.55 (d,
1H), 5.19 (s, 2H), 5.17 (s, 2H), 3.70 (s, 3H), 2.31 (s, 3H).
2-(3-(1-(2,6-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)acetic Acid (22). Methyl 2-(3-(1-(2,6-
difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-methyl-1H-
indol-1-yl)acetate (I-15A-4). General procedure D was followed to
make I-15A-4 from I-14A and 2,6-difluorobenzyl bromide in 53% yield.
General procedure D was followed to make 22 from I-15A-4 in 78%
yield. 1H NMR (400 MHz, DMSO-d6) δ 13.16 (br s, 1H), 7.81 (s, 1H),
7.55 (dd, J = 2.53, 9.35 Hz, 1H), 7.37−7.51 (m, 2H), 7.07−7.19 (m,
3H), 6.97 (td, J = 2.53, 9.09 Hz, 1H), 6.46 (d, J = 9.35 Hz, 1H), 5.24 (s,
2H), 5.03 (s, 2H), 2.33 (s, 3H).
General Procedure E (Scheme 6). Methyl 2-(5-halo-2-methyl-3-
(6-oxo-1,6-dihydropyridin-3-yl)-1H-indol-1-yl)acetate (I-16). To a
microwave vessel was added I-14 (2.25 mmol), MeOH (10 mL), and
concentrated HCl (0.5 mL, 16.5 mmol). The reaction was heated at
125 °C for 60 min in the microwave. The solvent was removed under
reduced pressure, and the crude material was purified by silica gel
column chromatography.
Methyl 2-(5-halo-3-(1-alkyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-
1H-indol-1-yl)acetate I-17. To a microwave vessel was added I-16
(1 mmol), potassium carbonate (0.7 g, 5 mmol), and DMF (12 mL).
Then selected bromide (2 mmol) was added and the vessel sealed. The
reaction was heated at 65 °C for 20 h on an oil bath. The reaction was
diluted with EtOAc (100 mL) and washed with brine (3 × 100 mL). The
organic layer was dried (MgSO4) and filtered. The solvent was removed
under reduced pressure, and the crude material was purified by silica gel
column chromatography
Alkyl Pyridone Analogue. The method described for pyridone linker
analogue in Scheme 4 was used. The crude material was purified by HPLC.
2-(5-Fluoro-3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-2-
methyl-1H-indol-1-yl)acetic Acid (24). Methyl 2-(5-fluoro-2-methyl-3-
(6-oxo-1,6-dihydropyridin-3-yl)-1H-indol-1-yl)acetate (I-16A). Gener-
al procedure E was followed to make I-16A from I-14A in 75% yield.
Methyl 2-(5-fluoro-3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-2-
methyl-1H-indol-1-yl)acetate (I-17A-1). General procedure E was
followed to make I-17A-1 from I-16A and 2-bromo propane in 64% yield.
General procedure E was followed to make 24 from I-17A-1 in 70%
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (d, J = 6.3 Hz, 6 H),
2.35 (s, 3 H), 5.05 (s, 2 H), 5.24−5.35 (m, 1 H), 6.85 (d, J = 8.3 Hz, 1 H),
6.97 (td, J = 9.1, 2.5 Hz, 1 H), 7.16 (dd, J = 9.9, 2.5 Hz, 1 H), 7.47 (dd, J =
8.8, 4.5 Hz, 1 H), 7.75 (dd, J = 8.5, 2.4 Hz, 1 H), 8.19 (d, J = 2.5 Hz, 1 H),
13.11 (br s, 1 H).
(5-Fluoro-2-methyl-3-{[6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydro-
pyridazin-3- yl]methyl}-1H-indol-1-yl)acetic Acid (17). The procedure
described in Scheme 5A of ref 6 was followed to make this compound in
37% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.27 (dd, J = 4.55, 8.84 Hz,
1H), 7.10−7.18 (m, 2H), 6.76−6.87 (m, 2H), 4.77−4.90 (m, 4H), 3.89
(s, 2H), 2.26 (s, 3H).
General Procedure D (Scheme 5). 3-bromo-5-halo-2-methyl-1H-
indole (I-12). To a round-bottom flask containing 5-halo-2-methyl-1H-
indole (15.3 mmol) in DMF (50 mL) was added bromine (0.8 mL,
15.3 mmol). The reaction was stirred at room temperature for 30 min
and diluted with EtOAc (400 mL). The solution was washed with H2O
(3 × 250 mL), 0.1 M Na2S2O3 (75 mL), and brine (75 mL). The organic
layer was dried (MgSO4) and filtered. The solvent was removed under
reduced pressure to give the desired product.
Methyl 2-(3-bromo-5-halo-2-methyl-1H-indol-1-yl)acetate (I-13).
To a round-bottom flask containing I-12 (19 mmol), K2CO3 (10.5 g,
76 mmol) in DMF (250 mL) was added methylbromoacetate (3.5 mL,
38 mmol). The reaction was heated to 85 °C for 3 h and cooled to room
temperature. The reaction was diluted with EtOAc (500 mL) and
washed with H2O (3 × 200 mL) and brine (100 mL). The organic layer
was dried (MgSO4) and filtered. The solvent was removed under
reduced pressure to give the product.
Methyl 2-(5-halo-3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-
yl)acetate (I-14). To a microwave vessel was added I-13 (1.3 mmol),
6-methoxypyridin-3-ylboronic acid (0.47 g, 3.1 mmol), PdCl2(dppf)-
CH2Cl2 (49 mg, 0.07 mmol), and Cs2CO3 (2.0 g, 6.1 mmol) in DME
(10.3 mL). The contents were degassed with nitrogen and sealed. The
reaction was heated at 150 °C for 20 min in the microwave. The reaction
was diluted with EtOAc (250 mL) and washed with H2O (3 × 100 mL).
The organic layer was dried (MgSO4) and filtered. The solvent was
removed under reduced pressure, and the crude material was purified by
silica gel column chromatography.
Methyl 2-(3-(1-substituted benzyl-6-oxo-1,6-dihydropyridin-3-yl)-5-
halo-2-methyl-1H-indol-1-yl)acetate (I-15). To a flask containing I-14
(1.1 mmol) in CH3CN (6 mL) was added sodium iodide (321 mg,
2.1 mmol) and selected bromide (2.1 mmol). The reaction was heated
to reflux for 15 h and cooled to room temperature. The contents of the
reaction were poured into 0.1 M Na2S2O3 (50 mL) and brine (25 mL)
and extracted with EtOAc (3 × 50 mL). The organic layer was dried
(MgSO4) and filtered. The solvent was removed under reduced
pressure, and the crude material was purified by silica gel column
chromatography.
Pyridone Analogue. The method described for pyridone linker
analogue in Scheme 4 was used. The crude material was purified by
chromatography.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-methyl-
1H-indol-1-yl)acetic Acid (19). 3-Bromo-5-fluoro-2-methyl-1H-indole
(I-12A). General procedure D was followed to give the desired product.
Methyl 2-(3-bromo-5-fluoro-2-methyl-1H-indol-1-yl)acetate
(I-13A). General procedure D was followed to give I-13A from I-12A in
37% yield (2 steps).
Methyl 2-(5-fluoro-3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-
yl)acetate (I-14A). General procedure D was followed to give I-14A
from I-13A in 56% yield.
Methyl 2-(3-(1-benzyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-
methyl-1H-indol-1-yl)acetate (I-15A-1). General procedure D was
followed to give I-15A-1 from I-14A and benzyl bromide in 95% yield.
General procedure D was followed to make 19 from I-15A-1 in 48%
yield. 1H NMR (400 MHz, DMSO-d6) δ 13.10 (br s, 1H), 7.84 (d, J =
2.02 Hz, 1H), 7.56 (dd, J = 2.65, 9.22 Hz, 1H), 7.45 (dd, J = 4.29, 8.84
Hz, 1H), 7.27−7.41 (m, 5H), 7.10 (dd, J = 2.53, 9.85 Hz, 1H), 6.95
(td, J = 2.53, 9.09 Hz, 1H), 6.55 (d, J = 9.09 Hz, 1H), 5.21 (s, 2H), 5.03
(s, 2H), 2.30 (s, 3H).
2-(3-(1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)acetic Acid (20). Methyl 2-(3-(1-(2,4-
difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-methyl-1H-
indol-1-yl)acetate (I-15A-2). General procedure D was followed to
make I-15A-2 from I-14A and 2,4-difluorobenzyl bromide in 61% yield.
2-(5-Fluoro-2-methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihy-
dropyridin-3-yl)-1H-indol-1-yl)acetic Acid (25). Methyl 2-(5-fluoro-2-
methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyridin-3-yl)-1H-
indol-1-yl)acetate (I-17A-2). General procedure E was followed to
make I-17A-2 from I-16A in 24% yield.
General procedure E was followed to make 24 from I-17A-2 in 70%
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 4.97 (q, J =
9.3 Hz, 2 H), 5.05 (s, 2 H), 6.61 (d, J = 9.3 Hz, 1 H), 6.98 (td, J = 9.2, 2.3
Hz, 1 H), 7.18 (dd, J = 9.9, 2.5 Hz, 1 H), 7.47 (dd, J = 9.0, 4.4 Hz, 1 H),
7.64 (dd, J = 9.6, 2.5 Hz, 1 H), 7.75 (d, J = 2.0 Hz, 1 H), 13.12 (s, 1 H).
2-(5-Fluoro-3-(1-isopropyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-
methyl-1H-indol-1-yl)acetic Acid (23). Methyl 2-(5-fluoro-3-((1-
isopropyl-6-oxo-1,6-dihydropyridin-3-yl)methyl)-2-methyl-1H-indol-
1-yl)acetate (I-11−3). General procedure C was followed reacting I-10
1315
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
with and 2-bromopropane to make I-11−3. Crude product was taken
forward.
(d, J = 8.6 Hz, 2 H), 7.65 (dd, J = 9.5, 2.7 Hz, 1 H), 7.74 (d, J = 2.3 Hz,
1 H), 13.08 (br s, 1 H)
General procedure C was followed to make 23 from I-11−3 in 68%
2-(2,5-Dimethyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyri-
din-3-yl)-1H-indol-1-yl)acetic Acid (30). 3-Bromo-2,5-dimethyl-1H-
indole (I-12D). Intermediate I-12D was prepared according to the
method described for I-12A.
1
yield. H NMR (400 MHz, DMSO-d6) δ 7.72 (d, J = 9.60 Hz, 1H),
7.49−7.55 (m, 2H), 7.43−7.48 (m, 2H), 6.97−7.06 (m, 2H), 5.22−5.30
(m, 1H), 5.09 (s, 2H), 1.36 (d, J = 6.57 Hz, 6H).
Methyl 2-(3-bromo-2,5-dimethyl-1H-indol-1-yl)acetate (I-12D).
Intermediate I-13D was prepared according to the method described
for I-13A. Yield 76% (2 steps).
Methyl 2-(3-(6-methoxypyridin-3-yl)-2,5-dimethyl-1H-indol-1-yl)-
acetate (I-14D). Intermediate I-14D was prepared according to the
method described for I-14A. Yield 66%.
Methyl 2-(2,5-dimethyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-1H-
indol-1-yl)acetate (I-16D). Intermediate I-16D was prepared according
to the method described for I-16A. Yield 78%.
Methyl 2-(2,5-dimethyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihy-
dropyridin-3-yl)-1H-indol-1-yl)acetate (I-17D). Intermediate I-17D
was prepared according to the method described for intermediate I-17A.
Yield 38%.
2-(5-Chloro-3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-2-
methyl-1H-indol-1-yl)acetic Acid (26). 3-Bromo-5-chloro-2-methyl-
1H-indole (I-12B). Intermediate I-12B was prepared according to the
method described for I-12-A and the crude product carried forward.
Methyl 2-(3-bromo-5-chloro-2-methyl-1H-indol-1-yl)acetate (I-13B).
Intermediate I-13B was prepared according to the method described for
intermediate I-13A. 63% yield (2 steps).
Methyl 2-(5-chloro-3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-yl)-
acetate (I-14B). Intermediate I-14B was prepared according to the method
described for intermediate I-14A. 81% yield.
Methyl 2-(5-chloro-2-methyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-1H-
indol-1-yl)acetate (I-16B). Intermediate I-16B was prepared according
to the method described for I-16A. 63% yield.
Methyl 2-(5-chloro-3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-
2-methyl-1H-indol-1-yl)acetate (I-17B-1). Intermediate I-17B-1 was
prepared according to the method described for intermediate I-17A.
62% yield.
The title compound was prepared according to the method described
for 24. 66% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.34 (d, J =
6.1 Hz, 6 H), 2.36 (s, 3 H), 5.07 (s, 2 H), 5.19−5.36 (m, 1 H), 6.86 (d, J =
8.3 Hz, 1 H), 7.14 (dd, J = 8.6, 2.0 Hz, 1 H), 7.40 (d, J = 2.0 Hz, 1 H),
7.50 (d, J = 8.8 Hz, 1 H), 7.76 (dd, J = 8.3, 2.5 Hz, 1 H), 8.19 (d, J = 2.5
Hz, 1 H), 13.13 (s, 1 H).
The title compound was prepared from I-17A according to the
method described for 24. Yield 72%. 1H NMR (400 MHz, DMSO-d6) δ
ppm 2.32 (s, 3 H), 2.36 (s, 3 H), 4.87−5.05 (m, 4 H), 6.61 (d, J = 9.3 Hz,
1 H), 6.95 (dd, J = 8.5, 1.4 Hz, 1 H), 7.22 (s, 1 H), 7.31 (d, J = 8.3 Hz,
1 H), 7.64 (dd, J = 9.5, 2.7 Hz, 1 H), 7.71 (d, J = 2.3 Hz, 1 H), 13.02
(br s, 1 H).
2-(5-Methoxy-2-methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihy-
dropyridin-3-yl)-1H-indol-1-yl)acetic Acid (31). 3-Bromo-5-methoxy-
2-methyl-1H-indole (I-12E). Intermediate I-12E was prepared
according to the method described for I-12A.
Methyl 2-(3-bromo-5-methoxy-2-methyl-1H-indol-1-yl)acetate (I-13E).
Intermediate I-13E was prepared according to the method described for
I-13a. Yield 92% (2 steps).
Methyl 2-(5-methoxy-3-(6-methoxypyridin-3-yl)-2-methyl-1H-
indol-1-yl)acetate (I-14E). Intermediate I-14E was prepared according
to the method described for I-14A. Yield 54%.
Methyl 2-(5-methoxy-2-methyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-
1H-indol-1-yl)acetate (I-16E). Intermediate I-16E was prepared
according to the method described for intermediate I-16D. Yield 46%.
Methyl 2-(5-methoxy-2-methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-
1,6-dihydropyridin-3-yl)-1H-indol-1-yl)acetate (I-17E). Intermediate
I-17E was prepared according to the method described for I-17A. The
product was carried on to the next step without purification.
The title compound was prepared from I-17E according to the
method described for 24. Yield 17% for 2 steps. 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.32 (s, 3 H), 3.74 (s, 3 H), 4.89−5.02 (m, 4 H), 6.61
(d, J = 9.3 Hz, 1 H), 6.77 (dd, J = 8.8, 2.3 Hz, 1 H), 6.91 (d, J = 2.3 Hz,
1 H), 7.34 (d, J = 8.6 Hz, 1 H), 7.64 (dd, J = 9.5, 2.7 Hz, 1 H), 7.73
(d, J = 2.5 Hz, 1 H), 13.01 (br s, 1 H)
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridin-3-yl)-5-chloro-2-methyl-
1H-indol-1-yl)acetic Acid (32). tert-Butyl 2-(3-bromo-5-chloro-2-
methyl-1H-indol-1-yl)acetate (I-18A). Intermediate I-18A was pre-
pared according to the method described for I-13A reacting I-12B with
tert-butyl bromo acetate. Yield 81% (2 steps).
tert-Butyl 2-(5-chloro-3-(6-methoxypyridin-3-yl)-2-methyl-1H-
indol-1-yl)acetate (I-19A). Intermediate I-19A was prepared according
to the method described for I-14A. Yield 42%.
2-(3-(1-Isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-1H-
indol-1-yl)acetic Acid (27). 3-Bromo-2-methyl-1H-indole (I-12C).
Intermediate I-12C was prepared according to the method described
for intermediate I-12A.
Methyl 2-(3-bromo-2-methyl-1H-indol-1-yl)acetate (I-13C). Inter-
mediate I-13-C was prepared according to the method described for
intermediate I-13-A. 80% yield. (2 steps).
Methyl 2-(3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-yl)-
acetate (I-14C). Intermediate I-14C was prepared according to the
method described for intermediate I-14A. 88% yield.
Methyl 2-(2-methyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-1H-indol-1-yl)-
acetate (I-16C). Intermediate I-16C was prepared according to the
method described for intermediate I-16A. 75% yield.
Methyl 2-(3-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-
1H-indol-1-yl)acetate (I-17C-1). Intermediate I-17C-1 was prepared
according to the method described for intermediate I-17A.
The title compound was prepared from I-17C-1 according to the
method described for 24. 44% yield. 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.34 (d, J = 6.3 Hz, 6 H), 2.36 (s, 3 H), 5.04 (s, 2 H), 5.23−5.35
(m, 1 H), 6.86 (d, J = 8.3 Hz, 1 H), 7.05 (t, J = 7.5 Hz, 1 H), 7.13 (td, J =
7.6, 1.1 Hz, 1 H), 7.45 (t, J = 8.0 Hz, 1 H), 7.76 (dd, J = 8.6, 2.5 Hz, 1 H),
8.20 (d, J = 2.5 Hz, 1 H), 13.08 (br s, 1 H).
2-(5-Chloro-2-methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihy-
dropyridin-3-yl)-1H-indol-1-yl)acetic Acid (28). Methyl 2-(5-chloro-2-
methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyridin-3-yl)-1H-
indol-1-yl)acetate (I-17-B2). Intermediate I-17B-2 was prepared
according to the method described for I-17A. 33% yield.
The title compound was prepared according to the method described
for 24. 58% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H),
4.97 (q, J = 9.2 Hz, 2 H), 5.06 (s, 2 H), 6.61 (d, J = 9.3 Hz, 1 H), 7.14 (dd,
J = 8.6, 2.0 Hz, 1 H), 7.43 (d, J = 2.3 Hz, 1 H), 7.50 (d, J = 8.6 Hz, 1 H),
7.64 (dd, J = 9.3, 2.5 Hz, 1 H), 7.77 (d, J = 1.5 Hz, 1 H), 13.14 (s, 1 H).
2-(2-Methyl-3-(6-oxo-1-(2,2,2-trifluoroethyl)-1,6-dihydropyridin-
3-yl)-1H-indol-1-yl)acetic Acid (29). Methyl 2-(2-methyl-3-(6-oxo-1-
(2,2,2-trifluoroethyl)-1,6-dihydropyridin-3-yl)-1H-indol-1-yl)acetate
(I-17C-2). Intermediate I-17C-2 was prepared according to the method
described for I-17A. 50% yield.
The title compound was prepared from I-17C-2 according to the
method described for 24. 60% yield. 1H NMR (400 MHz, DMSO-d6)
δ ppm 2.35 (s, 3 H), 4.97 (q, J = 9.3 Hz, 2 H), 5.04 (s, 2 H), 6.61 (d, J =
9.3 Hz, 1 H), 7.06 (t, J = 7.6 Hz, 1 H), 7.13 (t, J = 8.0 Hz, 1 H), 7.44
tert-Butyl 2-(3-(1-benzyl-6-oxo-1,6-dihydropyridin-3-yl)-5-chloro-2-
methyl-1H-indol-1-yl)acetate I-20A-1. Intermediate I-20A-1 was
prepared according to the method described for I-15A. Yield 52%.
The title compound was prepared according to this method. A round-
bottom flask containing I-20A-1 (0.7 mmol) was treated with TFA
(4 mL) at room temperature for 30 min. The solution was concentrated
under reduced pressure, and the crude material was purified by HPLC to
1
give 32 in 99% yield. H NMR (400 MHz, DMSO-d6) δ 13.12 (br s,
1H), 7.86 (d, J = 2.02 Hz, 1H), 7.55 (dd, J = 2.53, 9.35 Hz, 1H), 7.48 (d,
J = 8.59 Hz, 1H), 7.27−7.43 (m, 6H), 7.12 (dd, J = 2.15, 8.72 Hz, 1H),
6.55 (d, J = 9.35 Hz, 1H), 5.21 (s, 2H), 5.04 (s, 2H), 2.31 (s, 3H).
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-1H-indol-
1-yl)acetic Acid (33). tert-Butyl 2-(3-bromo-2-methyl-1H-indol-1-yl)-
acetate (I-18B). Intermediate I-18B was prepared according to the
1316
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
method described for I-13A reacting I-12C with tert-butyl bromo
acetate. Yield 86% (2 steps).
tert-Butyl 2-(3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-yl)-
acetate (I-19B). Intermediate I-19B was prepared according to the
method described for I-14A.
0.52 mmol), and pyrrolidine (43 uL, 0.52 mmol) in DMF (5 mL) was
added DIEA (122 μL, 0.7 mmol) to give a orange solution. This was
stirred at room temp for 18 h until complete by TLC and LC/MS.
Aqueous workup followed by prep HPLC purification and lyophilization
afforded the title compound as a white solid. 56% yield. 1H NMR (400
MHz, DMSO-d6) δ ppm 1.76−1.87 (m, 2 H), 1.91−2.03 (m, 2 H), 2.29
(s, 3 H), 3.31−3.36 (m, 2 H), 3.65 (t, J = 6.8 Hz, 2 H), 5.06 (s, 2 H), 5.30
(s, 2 H), 6.55 (d, J = 9.3 Hz, 1 H), 6.93 (td, J = 9.2, 2.7 Hz, 1 H), 7.05 (td,
J = 6.3, 1.5 Hz, 1 H), 7.15 (dd, J = 9.9, 2.5 Hz, 1 H), 7.17−7.26 (m, 1 H),
7.32−7.46 (m, 2 H), 7.60 (dd, J =9.2, 2.7Hz, 1 H), 7.81(d, J = 2.5 Hz, 1 H).
2-(3-(1-(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)-N,N-dimethylacetamide (40). The title
compound was prepared according to the method described for 39.
60% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.27 (s, 3 H), 2.86 (s,
3 H), 3.16 (s, 3 H), 5.15 (s, 2 H), 5.30 (s, 2 H), 6.55 (d, J = 9.3 Hz, 1 H),
6.92 (td, J = 9.2, 2.5 Hz, 1 H), 7.05 (td, J = 6.3, 1.5 Hz, 1 H), 7.14 (dd, J =
10.0, 2.4 Hz, 1 H), 7.17−7.26 (m, 1 H), 7.32−7.44 (m, 2 H), 7.60
(dd, J = 9.2, 2.7 Hz, 1 H), 7.80 (d, J = 2.5 Hz, 1 H).
2-(3-(1-(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)acetamide (41). The title compound was
prepared according to the method described for 39. 75% yield. 1H NMR
(400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 4.81 (s, 2 H), 5.30 (s, 2 H),
6.55 (d, J = 9.3 Hz, 1 H), 6.96 (td, J = 9.2, 2.5 Hz, 1 H), 7.02−7.09 (m, 1
H), 7.15 (dd, J = 10.0, 2.4 Hz, 1 H), 7.18−7.25 (m, J = 8.1, 8.1, 5.1, 1.5
Hz, 1 H), 7.28 (s, 1 H), 7.34−7.43 (m, 2 H), 7.60 (dd, J = 9.3, 2.5 Hz,
2 H), 7.80 (d, J = 2.5 Hz, 1 H).
2-(3-(1-(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)-N-(methylsulfonyl)acetamide (42). The
title compound was prepared according to the method described for 39.
58% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.33 (s, 3 H), 3.27
(s, 3 H), 5.05 (s, 2 H), 5.30 (s, 2 H), 6.55 (d, J = 9.3 Hz, 1 H), 6.92−7.09
(m, 2 H), 7.11−7.27 (m, 2 H), 7.33−7.49 (m, 2 H), 7.60 (dd, J = 9.3,
2.5 Hz, 1 H), 7.83 (d, J = 2.3 Hz, 1 H).
(R)-2-(3-(1-(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-
fluoro-2-methyl-1H-indol-1-yl)propanoic Acid (43a) and (S)-2-(3-(1-
(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-
methyl-1H-indol-1-yl)propanoic Acid (43b). Methyl 2-(3-bromo-5-
fluoro-2-methyl-1H-indol-1-yl)propanoate (I-21). Intermediate I-21
was prepared according to the method described for I-13. 36% yield.
Methyl 2-(5-fluoro-3-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-1-
yl)propanoate (I-22). Intermediate I-22 was prepared according to the
method described for I-14. 41% yield.
tert-Butyl 2-(3-(1-benzyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-
1H-indol-1-yl)acetate(I-20B-1). Intermediate I-20B-1 was prepared
according to the method described for I-15A. Yield 65%.
The title compound was prepared from I-20B-1 according to the
method described for 32 (Yield 56%). 1H NMR (400 MHz, DMSO-d6)
δ 13.15 (s, 1H), 7.81 (d, J = 2.53 Hz, 1H), 7.57 (dd, J = 2.53, 9.35 Hz,
1H), 7.34−7.44 (m, 6H), 7.31 (dd, J = 3.16, 5.43 Hz, 1H), 7.07−7.15
(m, 1H), 6.97−7.06 (m, 1H), 6.55 (d, J = 9.35 Hz, 1H), 5.21 (s, 2H),
4.96 (s, 2H), 2.31 (s, 3H).
2-(5-Chloro-3-(1-(2,6-difluorobenzyl)-6-oxo-1,6-dihydropyridin-
3-yl)-2-methyl-1H-indol-1-yl)acetic Acid (34). Methyl 2-(5-chloro-3-
(1-(2,6-difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-1H-
indol-1-yl)acetate (I-15B-1). General procedure D was followed to
make I-15B-1 from I-14B, and the product was carried on to the next
step without purification.
General procedure D was followed to make 34 from I-15B-1 in 63%
yield for 2 steps. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.33 (s, 3 H),
5.05 (s, 2 H), 5.25 (s, 2 H), 6.47 (d, J = 9.3 Hz, 1 H), 7.07−7.17 (m, 3 H),
7.38 (d, J = 2.0 Hz, 1 H), 7.39−7.48 (m, 1 H), 7.49 (d, J = 8.3 Hz, 1 H),
7.55 (dd, J = 9.3, 2.5 Hz, 1 H), 7.82 (s, 1 H), 13.14 (br s, 1 H)
2-(3-(1-(2,6-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-2-
methyl-1H-indol-1-yl)acetic Acid (35). Methyl 2-(3-(1-(2,6-difluor-
obenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-2-methyl-1H-indol-1-yl)-
acetate (I-15C-1). General procedure D was followed to make I-15C-1
from I-15B in 68% yield.
General procedure D was followed to make 35 from I-15C-1 in 76%
1
yield. H NMR (400 MHz, DMSO-d6) δ ppm 2.34 (s, 3 H), 5.02
(s, 2 H), 5.24 (s, 2 H), 6.47 (d, J = 9.3 Hz, 1 H), 7.05 (t, J = 7.5 Hz, 1 H),
7.08−7.18 (m, 3 H), 7.35−7.50 (m, 3 H), 7.56 (dd, J = 9.3, 2.5 Hz, 1 H),
7.78 (s, 1 H), 13.07 (br s, 1 H).
2-(5-Fluoro-2-methyl-3-(6-oxo-1-(4,4,4-trifluorobutyl)-1,6-dihy-
dropyridin-3-yl)-1H-indol-1-yl)acetic Acid (36) and 2-(5-Fluoro-2-
methyl-3-(6-(4,4,4-trifluorobutoxy)pyridin-3-yl)-1H-indol-1-yl)acetic
Acid (37). Methyl 2-(5-fluoro-2-methyl-3-(6-oxo-1-(4,4,4-trifluorobu-
tyl)-1,6-dihydropyridin-3-yl)-1H-indol-1-yl)acetate (I-17A-3) and
methyl 2-(5-fluoro-2-methyl-3-(6-(4,4,4-trifluorobutoxy)pyridin-3-yl)-
1H-indol-1-yl)acetate (I-17A-3B). General procedure E was followed to
make I-17A-3 and I-17A-3B from I-16A, and the mixture was carried on
to the next step without purification.
Methyl 2-(3-(1-(2,3-difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-
5-fluoro-2-methyl-1H-indol-1-yl)propanoate (I-23). Intermediate 23
was prepared according to the method described for I-15. Purification by
flash chromatography afforded the product in a 75% yield.
General procedure E was followed to make 36 and 37 from mixture of
I-17A-3 and I-17A-3B. The mixture was separated by column
chromatography.
The title compound was prepared from I-23 according to the method
described for pyridine analogue Scheme 5. 15% yield for 43a. 1H NMR
(400 MHz, DMSO-d6) δ ppm 1.62 (d, J = 7.1 Hz, 3 H), 2.38 (s, 3 H),
5.29 (s, 2 H), 5.44 (q, J = 6.3 Hz, 1 H), 6.54 (d, J = 9.3 Hz, 1 H), 6.95 (td,
J = 9.0, 2.3 Hz, 1 H), 7.05 (t, J = 6.9 Hz, 1 H), 7.14 (dd, J = 9.9, 2.3 Hz,
1 H), 7.17−7.27 (m, 1 H), 7.29−7.46 (m, 2 H), 7.60 (dd, J = 9.3, 2.3 Hz,
1 H), 7.84 (d, J = 1.8 Hz, 1 H), 13.20 (br s, 1 H). 15% yield for 43b. 1H
NMR (400 MHz, DMSO-d6) δ ppm 1.62 (d, J = 7.3 Hz, 3 H), 2.38 (s, 3
H), 5.28 (s, 2 H), 5.46 (q, J = 7.2 Hz, 1 H), 6.54 (d, J = 9.1 Hz, 1 H), 6.96
(td, J = 9.2, 2.7 Hz, 1 H), 7.02−7.09 (m, 1 H), 7.14 (dd, J = 9.9, 2.5 Hz,
1 H), 7.21 (qd, J = 8.1, 5.1, 1.5 Hz, 1 H), 7.30−7.44 (m, 2 H), 7.60
(dd, J = 9.2, 2.7 Hz, 1 H), 7.83 (d, J = 2.5 Hz, 1 H), 13.13 (br s, 1 H).
2-(3-(2-Benzyl-1-oxo-1,2-dihydroisoquinolin-4-yl)-5-fluoro-2-
methyl-1H-indol-1-yl)acetic Acid (48) (Scheme 10B). tert-Butyl 3-
bromo-5-fluoro-2-methyl-1H-indole-1-carboxylate (I-24). To a 1000 mL
round-bottom flask containing 5-fluoro-2-methyl-1H-indole (5.66 g,
38 mmol) and DMF (127 mL) was added bromine (2.0 mL, 38 mmol).
After 15 min, the reaction was diluted with EtOAc (800 mL) and washed
with water (500 mL) and brine (500 mL) and dried (MgSO4). The
suspension was filtered and concentrated. The residue was dissolved in
THF (381 mL) and treated with BOC2 (8.3 g, 38 mmol) and DMAP
(232 mg, 1.9 mmol). After 3 h, the reaction was concentrated to remove
the THF. The residue was diluted with EtOAc (500 mL) and washed
with water (250 mL) and brine (250 mL). The organic layer was dried
36 in 70% yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.87−2.01
(m, 2 H), 2.23−2.42 (m, 5 H), 4.05 (t, J = 7.3 Hz, 2 H), 5.02 (s, 2 H),
6.51 (d, J = 9.1 Hz, 1 H), 6.95 (td, J = 9.2, 2.5 Hz, 1 H), 7.18 (dd, J = 9.9,
2.5 Hz, 1 H), 7.44 (dd, J = 8.8, 4.3 Hz, 1 H), 7.54 (dd, J = 9.2, 2.7 Hz, 1
H), 7.75 (d, J = 2.3 Hz, 1 H), 13.24 (br s, 1 H).
37 in 70% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 1.77 Hz,
1H), 7.79 (dd, J = 2.53, 8.59 Hz, 1H), 7.46 (dd, J = 4.29, 8.84 Hz, 1H),
7.15 (dd, J = 2.53, 9.85 Hz, 1H), 6.92−7.00 (m, 2H), 5.02 (s, 2H), 4.37
(t, J = 6.44 Hz, 2H), 2.38−2.45 (m, 2H), 2.35 (s, 3H), 1.94−2.03 (m, 2H).
2-(3-(1-(2,3-Difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-flu-
oro-2-methyl-1H-indol-1-yl)acetic Acid (38). Methyl 2-(3-(1-(2,3-
difluorobenzyl)-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-methyl-1H-
indol-1-yl)acetate (I-15A-5). General procedure D was followed to
make I-15A-5 from I-14A and 2,3-difluorobenzyl bromide in 78% yield.
General procedure D was followed to make 38 from I-15A-5 in 82%
yield. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.32 (s, 3 H), 4.85 (s, 2
H), 5.29 (s, 2 H), 6.54 (d, J = 9.1 Hz, 1 H), 6.92 (td, J = 9.2, 2.5 Hz, 1 H),
7.05 (td, J = 7.1, 1.5 Hz, 1 H), 7.14 (dd, J = 10.0, 2.4 Hz, 1 H), 7.17−7.25
(m, 1 H), 7.33−7.45 (m, 2 H), 7.60 (dd, J = 9.2, 2.7 Hz, 1 H), 7.81 (d, J =
2.5 Hz, 1 H).
1-(2,3-Difluorobenzyl)-5-(5-fluoro-2-methyl-1-(2-oxo-2-(pyrroli-
din-1-yl)ethyl)-1H-indol-3-yl)pyridin-2(1H)-one (39). To a round-
bottom flask containing 38 (200 mg, 0.469 mmol), BOP (228 mg,
1317
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
(MgSO4), filtered, and concentrated. The crude material was purified by
Biotage to give a beige solid (12.45 g, 75% yield).
2-Alkyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolin-
1(2H)-one (I-29). In a 500 mL round-bottomed flask was bis(pinacolato)-
diboron (5.68 g, 22.35 mmol) and I-28 (22.35 mmol), acetic acid, and
potassium acetate (6.58 g, 67.1 mmol) in DMSO (112 mL) to give a dark-
orange solution. The reaction was purged with nitrogen gas, and then
PdCl2(dppf)-CH2Cl2 adduct (1.095 g, 1.341 mmol) was added. The reaction
was heated at 80 °C for 18 h and then cooled to room temperature. The
reaction mixture was diluted with EtOAc (800 mL) and washed with water
(3 × 300 mL) and saturated NaCl (1 ×300 mL). The organic layer was dried
(MgSO4), filtered and concentrated. The residue was purified via Biotage.
Methyl 2-(5-fluoro-3-iodo-2-methyl-1H-indol-1-yl)acetate (I-30).
To a 500 mL round-bottomed flask under an atmosphere of nitrogen
was added 5-fluoro-3-iodo-2-methyl-1H-indole (8.3 g, 30.2 mmol),
methyl 2-bromoacetate (11.10 mL, 121 mmol), and potassium
carbonate (20.85 g, 151 mmol) in 150 mL of DMF to give a brown
suspension. This was heated to 90 °C and allowed to stir for 16 h. The
mixture was cooled to room temperature and poured into 800 mL of
water. This was extracted with three 250 mL portions of ethyl acetate.
The combined organic layers were washed with water and brine then
dried over MgSO4. Filtration and concentration in vacuo gave the crude
material which was purified by silica gel chromatography (6−50%
EtOAc/Hex; 340 g SNAP column) to give a tan solid (8.08 g, 77% yield).
To a 500 mL round-bottomed flask under an atmosphere of nitrogen
was added 5-fluoro-3-iodo-2-methyl-1H-indole (8.3 g, 30.2 mmol),
methyl 2-bromoacetate (11.10 mL, 121 mmol), and potassium carbonate
(20.85 g, 151 mmol) in 150 mL of DMF to give a brown suspension. This
was heated to 90 °C and allowed to stir for 16 h. The mixture was cooled
to room temperature and poured into 800 mL of water. Thiswas extracted
with three 250 mL portions of ethyl acetate. The combined organic layers
were washed with water and brine then dried over MgSO4. Filtration and
concentration in vacuo gave the crude material which was purified by silica
gel chromatography to give I-30 as a tan solid (8.08 g, 77% yield).
Alkyl Isoquinolinone. To a 75 mL sealed vessel was added I-29
(18.65 mmol), methyl 2-(5-fluoro-3-iodo-2-methyl-1H-indol-1-yl)-
acetate (9.71 g, 28.0 mmol, I-30), potassium phosphate tribasic
monohydrate (7.92 g, 37.3 mmol), and 2-dicyclohexylphosphino-
2′,6′-dimethoxy-1,1′-biphenyl (0.765 g, 1.865 mmol) in butan-1-ol
(133 mL) and water (53.3 mL) to give a suspension. The reaction
was purged with nitrogen. The catalyst, palladium(II) acetate (0.209 g,
0.932 mmol), was added, and the vessel was sealed. The tube was heated
at 100 °C in an oil bath for 18 h. To the aqueous layer was added 1 N
HCl until the solution was acidic by litmus test. The solution was stirred
rigorously for 5 min and allowed to partition into two layers. The
aqueous layer was decanted using a pipet. The organic layer was filtered
through filter paper to remove the catalyst and then concentrated under
reduced pressure to remove the nBuOH. The material was dissolved in
MeOH (80 mL) and THF (80 mL) and treated with 50 mL of 1N aqueous
NaOH. After stirring for 20 min, the organic layer was removed under
reduced pressure and the aqueous layer was rendered acidic by addition of
1N HCl (50 mL). The product was extracted with EtOAc (3 × 200 mL).
The organic layer was dried (MgSO4), filtered, and concentrated to give the
crude material. The crude material was purified to give the final product.
2-(5-Fluoro-3-(2-isopropyl-1-oxo-1,2-dihydroisoquinolin-4-yl)-2-
methyl-1H-indol-1-yl)acetic Acid (44). 2-Isopropylisoquinolin-1(2H)-
one (I-27−1). General procedure F was followed to give I-27−1 from
isoquinolin-1(2H)-one and 2-iodopropane in 26% yield.
tert-Butyl 5-fluoro-3-(isoquinolin-4-yl)-2-methyl-1H-indole-1-car-
boxylate (I-25). To a microwave vial containing I-24 (1.03 g, 3.1
mmol), isoquinolin-4-ylboronic acid (1.02 g, 3.1 mmol), Na2CO3
(640 mg, 6.1 mmol), and THF−H2O (20 mL, 1:1) was added
Pd(PPh3)4 (200 mg, 0.15 mmol). The vessel was sealed and heated at
150 °C for 15 min. The reaction was filtered through filter paper and
diluted with EtOAc (200 mL) and water (100 mL). The organic layer
was dried (MgSO4), filtered, and concentrated. The crude material was
purified by silica gel chromatography, eluting with an EtOAc/hexane
gradient to give a red solid (1.18g, 30% yield).
tert-Butyl 3-(2-benzyl-1-oxo-1,2-dihydroisoquinolin-4-yl)-5-fluoro-2-
methyl-1H-indole-1-carboxylate (I-26). To a flask containing I-25 (159
mg, 0.42 mmol) in CH3CN (4.5 mL) was added benzyl iodide (100 mg,
0.46 mmol). The reaction was heated at reflux for 3 h. The solution was
cooled and diluted with EtOAc (100 mL). The organic layer was washed
with water (50 mL) and brine (50 mL). The organic layer was dried
(MgSO4), filtered, and concentrated to give an amber oil. To the amber
oil was added hexane (∼5−10 mL), and the suspension was agitated and
stirred until a powdery light-brown solid appeared. The hexane was
decanted, and the solid was dried in the vacuum oven. To the material
was added water (2 mL) and THF (2 mL). An aqueous solution of KOH
(1.8 M, 1.68 mmol, 0.93 mL) was added immediately, followed by
addition of a solution of K3Fe(CN)6 (415 mg, 1.26 mmol) in water
(0.5 M, 2.5 mL). DMF was slowly added to the mixture until the
reaction appeared to be homogeneous. The reaction was diluted with
EtOAc (50 mL) and washed with water (50 mL). The eluent was
concentrated, and the product was taken into the next step without
further purification (91 mg, 45% yield).
Compound 48. To a flask containing I-26 (202 mg, 0.42 mmol) was
added TFA (2 mL). The reaction was stirred for 20 min and then
concentrated. The residue was dissolved in EtOAc (50 mL) and washed
with H2O (25 mL). The organic layer was dried (MgSO4), filtered, and
concentrated. The crude material was dissolved in DMF (4 mL) and
treated with methylbromoacetate (116 μL, 1.3 mmol) and potassium
carbonate (232 mg, 1.7 mmol). The reaction was heated to 90 °C for 4 h
and cooled to room temperature. The solution was diluted with EtOAc
(75 mL) and washed with water (3 × 50 mL). The organic layer was
concentrated to remove the EtOAc. The residue was dissolved in THF−
MeOH−H2O (15 mL, 1:1:1) and treated with 1 M NaOH (2 mL). The
reaction was stirred for 2−3 h and then concentrated to remove the
volatile solvent. The aqueous layer was made acidic by addition of 1 M
HCl. The product was extracted with EtOAc (3 × 15 mL) and purified
1
by reverse phase HPLC to give a beige solid, yield 25%. H NMR
(400 MHz, MeOD) δ 8.47 (dd, 1H), 7.52−7.68 (m, 2H), 7.25−7.44
(m, 8H), 6.90 (td, J = 2.65, 9.16 Hz, 1H), 6.71 (dd, J = 2.53, 9.60 Hz,
1H), 5.20−5.45 (m, 2H), 4.96 (s, 2H), 2.21 (s, 3H).
General Procedure F (Scheme 10C). 2-Alkylisoquinolin-1(2H)-
one (I-27). In a 1000 mL round-bottom flask was isoquinolin-1(2H)-
one (14.27 g, 98 mmol), selected alkylhalide (98 mmol), and cesium
carbonate (32.0 g, 98 mmol) in DMF (328 mL) to give a pale-yellow
suspension. The reaction was heated to 50 °C in an oil bath for 3 h. The
reaction mixture was diluted with ethyl acetate (600 mL). The organic
layer was washed with water (4 × 250 mL) and dried (MgSO4). The
suspension was filtered, and the solvent was removed under reduced
pressure. A solution of the material in CH2Cl2 and MeOH was added to
a Biotage samplet. The samplet was then put in a chamber and evacuated
to remove the excess solvent. Purification using a Biotage column
yielded 2-alkylisoquinolin-1(2H)-one.
4-Iodo-2-isopropylisoquinolin-1(2H)-one (I-28−1). General proce-
dure F was followed to give I-28−1 from I-27−1 in 44% yield.
2-Isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
isoquinolin-1(2H)-one (I-29−1). General procedure F was followed to
give I-29−1 from I-28−1 in 83% yield.
4-Iodo-2-alkylisoquinolin-1(2H)-one (I-28). To a cooled 500 mL
round-bottomed flask of Et2O (103 mL) containing a suspension of
silver trifluoromethanesulfonate (13.19 g, 51.3 mmol), potassium
hydroxide (2.88 g, 51.3 mmol), and I-27 (51.3 mmol) was added
iodine (13.03 g, 51.3 mmol) at 0 °C to give a cloudy suspension. After
2 h, the suspension was diluted with diethylether (200 mL) and filtered to
remove the silver. The organic layer was washed with 0.1 M sodium
thiosulfate (200 mL) and brine (200 mL) and dried (MgSO4). The solution
was filtered and concentrated. The residue was purified via Biotage.
General procedure F was followed to convert I-29−1 to 44 in 74%
yield. 1H NMR (400 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.32−8.40 (m,
1H), 7.63 (td, J = 1.39, 7.64 Hz, 1H), 7.48−7.56 (m, 2H), 7.42 (s, 1H),
7.22 (s, 1H), 6.97 (td, J = 2.65, 9.16 Hz, 1H), 6.81 (dd, J = 2.53, 9.85 Hz,
1H), 5.22−5.36 (m, 1H), 5.10 (d, J = 2.53 Hz, 2H), 2.22 (s, 3H), 1.39
(dd, J = 3.79, 6.82 Hz, 6H).
2-(5-Fluoro-2-methyl-3-(2-neopentyl-1-oxo-1,2-dihydroisoquino-
lin-4-yl)-1H-indol-1-yl)acetic Acid (45). 2-(2,2,2-Trifluoroethyl)-
isoquinolin-1(2H)-one (I-27−2). General procedure F was followed
1318
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
to give I-27−2 from isoquinolin-1(2H)-one and 1,1,1-trifluoro-2-
iodoethane, 97% yield.
3 × 100 mL portions of ethyl acetate. The combined organic layers were
washed with water and brine and then dried over MgSO4 and filtered.
The resulting solution was concentrated, and the residue was purified by
silica gel chromatography to give the desired product.
4-Iodo-2-(2,2,2-trifluoroethyl)isoquinolin-1(2H)-one (I-28−2). Bis-
(pyridine)iodonium tetrafluoroborate (5.67 g, 15.25 mmol) was
dissolved in dry dichloromethane (69.3 mL) and added slowly to I-
27−2 (3.15 g, 13.87 mmol) and trifluoromethanesulfonic acid (2.71 mL,
30.5 mmol) in CH2Cl2. After completion of the reaction by TLC, the
reaction was quenched by the addition of 0.1 M sodium thiosulfate
(∼100 mL) and washed with saturated NaCl (250 mL). The organic
layer was isolated, dried (MgSO4), filtered, and concentrated to give a
dark-orange solid. The solid was purified to give an orange solid (3.9 g;
80% yield).
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2,2,2-trifluoroethyl)-
isoquinolin-1(2H)-one (I-29−2). General procedure F was followed to
give I-29−2 from I-28−2, 69% yield.
General procedure F was followed to convert I-29−2 to 45, yield
34%. 1H NMR (400 MHz, DMSO-d6) δ 13.13 (s, 1H), 8.34−8.41 (m,
1H), 7.71 (ddd, J = 1.52, 7.14, 8.27 Hz, 1H), 7.60 (ddd, J = 1.26, 7.07,
8.08 Hz, 1H), 7.53 (dd, J = 4.29, 9.09 Hz, 1H), 7.48 (s, 1H), 7.26 (d, J =
7.33 Hz, 1H), 6.98 (td, J = 2.53, 9.22 Hz, 1H), 6.83 (dd, J = 2.40, 9.73 Hz,
1H), 5.12 (s, 2H), 4.93−5.09 (m, 2H), 2.23 (s, 3H).
2-(5-Fluoro-3-(1-oxo-2-(4,4,4-trifluorobutyl)-1,2-dihydroisoquino-
lin-4-yl)-1H-indol-1-yl)acetic Acid (46). 2-(4,4,4-Trifluorobutyl)-
isoquinolin-1(2H)-one (I-27−3). General procedure F was followed
to give I-27−3 from isoquinolin-1(2H)-one and 4-bromo-1,1,1-
trifluorobutane, 93% yield.
4-Iodo-2-(4,4,4-trifluorobutyl)isoquinolin-1(2H)-one (I-28−3).
General procedure F was followed to give I-28−3 from I-27−3 in
58% yield.
4-Iodo-2-substituted-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-
33). To a 50 mL round-bottomed flask under an atmosphere of
nitrogen was added intermediate I-32 (1.694 mmol) and trifluor-
omethanesulfonic acid (0.331 mL, 3.73 mmol) in 20 mL of
dichloromethane to give a tan solution. Dipyridinium-1-yliodate(I),
BF4− (0.693 g, 1.863 mmol) was added slowly as a solution in 5 mL of
dichloromethane, and the resulting solution was allowed to stir 1 h. The
reaction was then quenched with 100 mL of 0.1 M sodium thiosulfate.
The organic layer was washed with brine and dried over MgSO4 and
then filtered and concentrated in vacuo to give an orange solid. The
residue was purified by silica gel chromatography to give the desired
product.
2-Substituted-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-34). To a 50 mL round-
bottomed flask under an atmosphere of nitrogen was added
4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.401 g,
1.578 mmol), intermediate I-33 (1.435 mmol), and potassium acetate
(0.422 g, 4.30 mmol) in 25 mL of DMSO to give an orange solution.
PdCl2(dppf)-CH2Cl2 adduct (0.070 g, 0.086 mmol) was added. The
reaction was heated to 80 °C and allowed to stir for 16 h. The mixture was
then poured into 100 mL of water and extracted with three 50 mL portions
of ethyl acetate. The combined organic layers were washed with water and
brine, dried over magnesium sulfate, filtered, and concentrated. The residue
was purified by silica gel to give the desired product.
Saturated Isoquinolinone. To a 5 mL microwave vessel was added
intermediate I-30 (0.571 g, 1.645 mmol), intermediate I-34 (0.823 mmol),
and potassium phosphate tribasic monohydrate (0.349 g, 1.645 mmol) in
4 mL of butan-1-ol and 1.6 mL water to give a tan suspension. 2-Dicyclo-
hexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl (0.034 g, 0.082 mmol) was
added. The vial was purged with nitrogen, and palladium(II) acetate
(9.24 mg, 0.041 mmol) was added. The vessel was sealed and heated to
100 °C in an oil bath and allowed to stir for 16 h. The reaction mixture was
then poured into 50 mL of water extracted with three 25 mL portions of
ethyl acetate. The combined organic layers were washed with water and
brine. The organic was dried over MgSO4, filtered, and concentrated in
vacuo. The residue was redissolved into 10 mL of THF. To this was added
lithium hydroxide (0.073 g, 3.06 mmol) dissolved into 5 mL of water.
Methanol was added until solution was monophasic. The mixture was
stirred for 30 min and was then poured into 100 mL of 1.2 N HCl. The
aqueous layer was extracted with three 50 mL portions of ethyl acetate. The
combined organic layers were washed with water and brine. The organic
was dried MgSO4, filtered, and concentrated. The crude material was then
purified.
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4,4,4-
trifluorobutyl)isoquinolin-1(2H)-one (I-29−3). General procedure F
was followed to give I-29−3 from I-28−3, 71% yield.
General procedure F was followed to convert I-29−3 to 46, yield
64%. 1H NMR (400 MHz, DMSO-d6) δ 13.09 (br s, 1H), 8.31−8.39 (m,
1H), 7.61−7.68 (m, 1H), 7.50−7.58 (m, 2H), 7.50 (s, 1H), 7.21 (d, J =
7.58 Hz, 1H), 6.96 (td, J = 2.53, 9.22 Hz, 1H), 6.86 (dd, J = 2.53, 9.60 Hz,
1H), 5.10 (s, 2H), 4.07−4.18 (m, 2H), 2.35 (d, J = 11.37 Hz, 2H), 2.22
(s, 3H), 1.98 (d, J = 7.07 Hz, 2H).
2-(5-Fluoro-3-(2-neopentyl-1-oxo-1,2-dihydroisoquinolin-4-yl)-
1H-indol-1-yl)acetic Acid (47). 2-Neopentylisoquinolin-1(2H)-one (I-
27−4). General procedure F was followed to give I-27−3 from
isoquinolin-1(2H)-one and 1-bromo-2,2-dimethylpropane, yield 57%.
4-Iodo-2-neopentylisoquinolin-1(2H)-one (I-28−4). Intermediate I-
28−4 was prepared according to the method described for intermediate
I-28−3, 73% yield.
2-Neopentyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
isoquinolin-1(2H)-one (I-29−4). General procedure F was followed to
give I-29−4 from I-28−4, 50% yield.
2-(5-Fluoro-3-(2-isopropyl-1-oxo-1,2,5,6,7,8-hexahydroisoquino-
lin-4-yl)-2-methyl-1H-indol-1-yl)acetic Acid (49). 2-Isopropyl-5,6,7,8-
tetrahydroisoquinolin-1(2H)-one (I-32−1). General procedure G was
followed to give I-32−1 from I-31 and 2-iodopropane, yield 25%.
4-Iodo-2-isopropyl-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-33−1).
General procedure G was followed to give I-33−1 from I-32−1, yield 85%.
2-Isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6,7,8-
tetrahydroisoquinolin-1(2H)-one (I-34−1). General procedure G was
followed to give I-34−1 from I-33−1, 57% yield.
General procedure F was followed to convert I-29−4 to 47, yield
47%. 1H NMR (400 MHz, DMSO-d6) δ 13.11 (br s, 1H), 8.35 (dd, J =
1.01, 8.08 Hz, 1H), 7.63 (td, J = 1.52, 7.58 Hz, 1H), 7.47−7.56 (m, 2H),
7.35 (s, 1H), 7.19 (d, J = 7.58 Hz, 1H), 6.96 (td, J = 2.65, 9.16 Hz, 1H),
6.80 (dd, J = 2.53, 9.60 Hz, 1H), 5.10 (s, 2H), 3.85−4.07 (m, 2H), 2.22
(s, 3H), 0.99 (s, 9H).
General Procedure G. 5,6,7,8-Tetrahydroisoquinolin-1(2H)-one
(intermediate I-31). To a 100 mL round-bottomed flask under an atmosphere
of nitrogen was added isoquinolin-1(2H)-one (0.5 g, 3.44 mmol) in
20 mL of acetic acid to give a colorless solution. Platinum(IV) oxide
(0.235 g, 1.033 mmol) was added. Hydrogen gas was sparged through the
solution via a needle attached to a balloon for 10 min. The reaction was
then allowed to stir for 16 h under one atmosphere of nitrogen. The catalyst
was filtered offand the acetic acid removed by azeotroping with hexane. The
residue was purified by silica gel chromatography to give the desired product
as a white solid (0.100g, 20% yield).
General procedure G was followed to convert I-34−1 to 49 yield
1
23%. H NMR (400 MHz, DMSO-d6) δ 1.30 (d, 6 H) 1.41−1.61
(m, 2 H) 1.68 (quin, J = 5.9 Hz, 2 H) 2.01−2.25 (m, 5 H) 2.44 (d, J = 5.6
Hz, 2 H) 5.02 (d, J = 1.0 Hz, 2 H) 5.14 (quin, J = 6.8 Hz, 1 H) 6.84−6.97
(m, 2 H) 7.29 (s, 1 H) 7.43 (dd, J = 9.0, 4.4 Hz, 1 H) 13.07 (br s, 1 H).
2-(5-Fluoro-2-methyl-3-(1-oxo-2-(2,2,2-trifluoroethyl)-1,2,5,6,7,8-
hexahydroisoquinolin-4-yl)-1H-indol-1-yl)acetic Acid (50). 2-(2,2,2-
Trifluoroethyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-32−2).
General procedure G was followed to give I-32−2 from I-31 and
1,1,1-trifluoro-2-iodoethane, 66% yield.
2-Substituted-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-32). To a
20 mL microwave vial was added intermediate I-31 (1.01 g, 6.77 mmol)
and cesium carbonate (4.41 g, 13.54 mmol) in 10 mL of DMF to give a
white suspension. Selected halide (20.31 mmol) was added. The vial was
sealed and the mixture heated to 50 °C. The reaction was allowed to stir
16 h. The mixture was then poured into water and extracted with
4-Iodo-2-(2,2,2-trifluoroethyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-
one (I-33−2). General procedure G was followed to give I-33−2 from
I-32−2, 19% yield.
1319
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2,2,2-trifluor-
oethyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-34−2). General
procedure G was followed to give I-34−2 from I-33−2, 61% yield.
General procedure G was followed to convert I-34−2 to 50, 25%
yield. 1H NMR (400 MHz, DMSO-d6) δ 1.45−1.63 (m, 2 H) 1.64−1.75
(m, 2 H) 2.04−2.28 (m, 5 H) 2.39−2.53 (m, 2 H) 4.78−4.98 (m, 2 H)
5.03 (d, J = 1.5 Hz, 2 H) 6.85−6.97 (m, 2 H) 7.37 (s, 1 H) 7.45 (dd, J =
8.8, 4.3 Hz, 1 H) 13.09 (br s, 1 H).
2-(5-Fluoro-2-methyl-3-(1-oxo-2-(4,4,4-trifluorobutyl)-1,2,5,6,7,8-
hexahydroisoquinolin-4-yl)-1H-indol-1-yl)acetic Acid (51). 2-(4,4,4-
Trifluorobutyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-32−3).
General procedure G was followed to give I-32−3 from I-31 and
4-bromo-1,1,1-trifluorobutane, 53% yield.
temperature, d2-cAMP and europium-labeled antibody supplied with the
Hi-Range Assay Reagent kit (Cisbio, Bedford, MA) were separately diluted
into the supplier-provided cell lysis buffer, and each was added to the assay
plate, as per manufacturer’s directions. Data was collected on the Envision
plate reader (Perkin-Elmer, Waltham, MA) using λex, 340 nm, and λem,
615/665. Samples containing cells and DMSO alone were included as a
control in order to measure the maximal signal.
Human Eosinophil Shape Change Assay (hEOS). Peripheral
blood from healthy human donors, prescreened for having greater
than 3% eosinophils, was collected into sodium heparin tubes. Blood
was incubated with compound for 10 min at room temperature
and then activated with 50 nM PGD2 for 10 min at 37 °C. The
reaction was stopped by transferring the plates to ice and adding 1%
paraformaldehyde fixative. The samples were then transferred to
ammonium chloride lysis solution, mixed gently, and incubated on ice
for 40 min. Eosinophil shape change was analyzed by flow cytometry
using a FACSCalibur outfitted with a 96-well autosampler. Individual
cell populations were separated based on their forward and side scatter
properties. Gross cellular toxicity was noted as a reduced or abnormal
forward/side scatter pattern compared to untreated or placebo treated
blood cell populations. Granulocytes were gated and assessed for
autofluorescence in the FL1 and FL2 channels. Eosinophils were
isolated based on their higher autofluorescence in both channels. Finally,
shape change was measured by alterations in the mean forward scatter of
the eosinophils in response to PGD2.
4-Iodo-2-(4,4,4-trifluorobutyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-
one (I-33−3). General procedure G was followed to give I-33−2 from
I-32−2 in 21% yield.
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(4,4,4-trifluoro-
butyl)-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-34−3). General
procedure G was followed to give I-34−3 from I-33−3, 61% yield.
General procedure G was followed to convert I-34−3 to 51, 33%
yield. 1H NMR (400 MHz, DMSO-d6) δ 1.42−1.60 (m, 2 H) 1.61−1.74
(m, 2 H) 1.90 (quin, J = 7.6 Hz, 2 H) 2.04−2.23 (m, 5 H) 2.23−2.36 (m,
2 H) 2.40−2.45 (m, 2 H) 3.89−4.03 (m, 2 H) 5.02 (d, J = 1.3 Hz, 2 H)
6.84−6.97 (m, 2 H) 7.38 (s, 1 H) 7.43 (dd, J = 8.7, 4.4 Hz, 1 H) 13.08
(br s, 1 H).
2-(3-(2-Benzyl-1-oxo-1,2,5,6,7,8-hexahydroisoquinolin-4-yl)-5-
fluoro-2-methyl-1H-indol-1-yl)acetic Acid (52). 2-Benzyl-5,6,7,8-
tetrahydroisoquinolin-1(2H)-one (I-32−4). General procedure G was
followed to give I-32−4 from I-31 and benzyl bromide, 73% yield.
2-Benzyl-4-iodo-5,6,7,8-tetrahydroisoquinolin-1(2H)-one (I-33−4).
General procedure G was followed to give I-33−4 from I-32−4, 53%
yield.
2-Benzyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6,7,8-tetra-
hydroisoquinolin-1(2H)-one (I-34−4). General procedure G was
followed to give I-34−4 from I-33−4, 71% yield.
General procedure G was followed to convert I-34−4 to 52, 33%
yield. 1H NMR (400 MHz, DMSO-d6) δ 1.47−1.66 (m, 2 H) 1.68−1.80
(m, 2 H) 2.05−2.31 (m, 5 H) 2.42−2.68 (m, 2 H) 5.07 (s, 2 H) 5.20 (d,
J = 4.8 Hz, 2 H) 6.90 (dd, J = 9.6, 2.3 Hz, 1 H) 6.98 (td, J = 9.1, 2.5 Hz,
1 H) 7.29−7.37 (m, 1 H) 7.37−7.44 (m, 4 H) 7.49 (dd, J = 8.7, 4.4 Hz,
1 H) 7.54 (s, 1 H).
Membrane Vesicles and Sandwich-Cultured Hepatocytes
Studies. Membrane vesicles prepared from Mrp2/MRP2 expressing
Sf9 insect cells were purchased from Invitrogen (Carlsbad, CA).
Incubations were performed according to the vendor’s recommended
procedures using a substrate concentration of 10 μM, 50 μg protein/
incubation, and an incubation time of 5 min at 37 °C in the presence or
absence of ATP and an ATP generating system. MK571 (250 μM), a
Mrp2/MRP2 inhibitor, was included in separate incubations. Net
uptake activity was obtained by subtracting activity in the presence of
ATP from that in the presence of AMP. Rat and human sandwich-
cultured hepatocytes were purchased from Qualyst (Durham, NC), and
incubations were performed according to the vendor’s recommended
procedures using substrate concentrations of 1−50 μM (rat) or 1−10 μM
(human). The amount of substrate excreted into the bile canalicular
network was calculated as the difference between the amount of substrate
accumulated in the hepatocytes when incubations were conducted in the
presence or absence of Ca2+. In vitro intrinsic biliary clearance was calculated
by the amount of substrate excreted in the bile divided by the concentration
of substrate present with time.
Contact Hypersensitivity Mouse Ear Model (CHS). Inflamma-
tion was measured in the ear skin of mice. One week prior to challenge,
mice were sensitized on the shaved abdomen by applying 100 μL of 2%
oxazolone made in 100% ethanol. Five days later, baseline ear thickness
measurements were taken on the left and right ears using a Mitutoyo
micrometer. Compound was given orally prior to challenge. One hour
after treatment, the left ears of the mice were challenged topically with
10 μL of 2% oxazolone on the left ear, and as a negative control 10 μL of
100% ethanol on the right ear. Mice received another dose of compound
orally 7 h post challenge. The following day (24 h) ear swelling was
measured again with the micrometer, and the swelling was expressed
postchallenge−prechallenge thickness (Δ) (Student’s unpaired t test
used for statistical analysis).
House Dust Mite Model. Mouse model of house dust mite-induced
allergic airway disease: Age-matched 6−12 week old female BALB/c
mice were obtained from Taconic Farms (Hudson, NY). All in vivo
experiments were performed in accordance with animal use protocols
approved by Pfizer’s Institutional Animal Care and Use Committee. On
day 0, 7, and 14 of the protocol, mice were anesthetized with isoflurane
and received house dust mite extract from Dermatophagoides
pteronyssinus (HDM, Greer Laboratories, Lenoir, NC). Briefly, 100 μg
HDM extract in 40 μL of saline were instilled intratracheally using a
100 μL Hamilton glass syringe terminated with a 1.5 in. polyethylene
catheter mounted on a 30G needle. The catheter was introduced
through the animal’s vocal cords into the trachea using an otoscope
terminated with a 2 mm speculum. When indicated, mice were treated
(qd) either intraperitoneally with dexamethasone (1 mg/kg) or perorally
with compound 44 (40 mg/kg). All mice were euthanized by CO2
asphyxiation on day 17 of the protocol to collect bronchoalveolar lavage
(BAL) fluid and various tissue samples for analysis. BAL collection was
performed as previously described.17 Because systemic inflammation is
minimal in this model, no blood samples were collected.
Statistical Analysis. Results represent the mean SEM. Statistical
significance for all results was determined using a Mann−Whitney U
test. Results were considered significant for P values <0.05.
hCRTH2 FRET Assay. The cAMP TR-FRET assay was performed by
incubating a dilution series of compound concentrations with 30000
cells per assay well of CHO-K1 cells expressing the recombinant human
CRTH2 receptor (Euroscreen, Belgium). Incubations were performed
at room temperature for 30 min in the presence of 10 uM forskolin and
10 nM PGD2. The assay buffer used contained 25 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES), pH 7.4, 124 mM sodium
chloride, 5 mM potassium chloride, 1.45 mM calcium chloride, 1.25 mM
magnesium chloride, 1.25 mM potassium dihydrogen phosphate, 13.3 mM
glucose, and 0.5 g/L BSA. Following the 30 min incubation at room
Guinea Pig Allergen Challenge Model. Female Hartley guinea
pigs between the weight range of 375 and 425 g (5−7 weeks) were
obtained from Charles River . On days 1 and 5, guinea pigs were
anesthetized with isoflurane inhalation prior to receiving a 1 mL
intraperitoneal injection of 100 μg of OVA (Ovalbumin; grade V,
minimum 98% agarose gel electrophoresis; batch no. 087K7004; A5503-
50G; Sigma) with 100 mg of aluminum hydroxide (reagent grade; batch
no. 09712JE; Sigma no. 239186-500G) in PBS. On day 15, animals were
exposed to an aerosol of 0.01% OVA allergen in PBS for 60 min. Animals
were dosed per os with test articles 1 h prior to and 6 h post OVA
1320
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
allergen challenge. To facilitate oral dosing, animals were anesthetized
with isoflurane inhalation. An infant feeding tube was then placed into
the stomach, and compounds were dosed in a 1 mL volume in 0.5%
Tween made up in Cherry Blast FlavoRX syrup. All animals were
euthanized by pentabarbitol overdose on day 16 of the protocol in order
to collect bronchoalveolar lavage (BAL) fluid and various tissue samples
for analysis. For BAL fluid collection lungs were lavaged with 2 × 10 mL
of PBS. Recovered total cell numbers were pooled and counted using a
hemocytometer. Evans Blue exclusion was used to assess cell viability. In
terms of cell viability, there were no differences between vehicle treated
and compound treated animals. Cell differentials were quantified from
cells smears (prepared by spinning a 100 μL aliquot (106 cells/mL) in a
Cytospin (Shandon) centrifuge) that were fixed using a solution of
methanol for 10 s and stained with buffered eosin (10 s) and methylene
blue/Azur (5 s). A total of 500 cells per smear were counted by light
microscopy under oil immersion (×1000).
structure, properties, and functions in leukocytes. Prostaglandins,
Leukotrienes Essent. Fatty Acids 2003, 69, 169−177. (c) Kostenis, E.;
Ulven, T. Emerging roles of DP and CRTH2 in allergic inflammation.
Trends Mol. Med. 2006, 12, 148−158. Epub. (d) Cosmi, L.; Annunziato,
F.; Galli, G.; Manetti, R.; Maggi, E.; Romagnani, S. CRTH2: marker for
the detection of human Th2 and Tc2 cells. Adv. Exp. Med. Biol. 2001,
495, 25−29.
(4) Hirai, H.; Tanaka, K.; Yoshie, O.; Ogawa, K.; Kenmotsu, K.;
Takamori, Y.; Ichimasa, M.; Sugamura, K.; Nakamura, M.; Takano, S.
Prostaglandin D2 Selectively Induces Chemotaxis in T Helper Type 2
Cells, Eosinophils, and Basophils via Seven-Transmembrane Receptor
CRTH2. J. Exp. Med. 2001, 193, 255−262.
(5) Huang, J.-L.; Gao, P.-S.; Mathias, R. A.; Yao, T.-C.; Chen, L.-C.;
Kuo, M.-L.; Hsu, S.-C.; Plunkett, B.; Togias, A.; Barnes, K. C. Sequence
variants of the gene encoding chemoattractant receptor expressed on
Th2 cells (CRTH2) are associated with asthma and differentially
influence mRNA stability. Hum. Mol. Genet. 2004, 13, 2691−2697.
(6) Kaila, N.; Huang, A.; Moretto, A.; Follows, B.; Janz, K.; Lowe, M.;
Thomason, J.; Mansour, T. S.; Hubeau, C.; Page, K.; Morgan, P.; Fish,
S.; Xu, X.; Williams, C.; Saiah, E. Diazine Indole Acetic Acids as Potent,
Selective, and Orally Bioavailable Antagonists of Chemoattractant
Receptor Homologous Molecule Expressed on Th2 Cells (CRTH2) for
the Treatment of Allergic Inflammatory Diseases. J. Med. Chem. 2012,
55, 5088−5109.
ASSOCIATED CONTENT
* Supporting Information
Analytical data for intermediates. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: (617) 665-5626. E-mail: neelu.kaila@pfizer.com.
■
(7) Snell, N.; Foster, M.; Vestbo, J. Efficacy and safety of AZD1981, a
CRTH2 receptor antagonist, in patients with moderate to severe
COPD. Respir. Med. 2013, 107, 1722−1730.
(8) Singh, D.; Cadden, P.; Hunter, M.; Collins, L. P.; Perkins, M.;
Pettipher, R.; Townsend, E.; Vinall, S.; O’Connor, B. Inhibition of the
asthmatic allergen challenge response by the CRTH2 antagonist
OC000459. Eur. Respir. J. 2013, 41, 46−52.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Seonghee Park, Aram Oganesian, Jianyao Wang, and
Robert Espina for the in vitro and in vivo ADME studies and
Professor William Abraham from the University of Miami for
sheep studies. We would also like to thank the DAC group for
analytical data.
(9) Arry-502/CRTH2 Antagonist. http://www.arraybiopharma.com/
ProductPipeline/Inflamation/CRTh2.asp (accessed December 20, 2013).
(10) Hirai, H.; Tanaka, K.; Takano, S.; Ichimasa, M.; Nakamura, M.;
Nagata, K. Cutting edge: agonistic effect of indomethacin on a
prostaglandin D2 receptor, CRTH2. J. Immunol. 2002, 168, 981−985.
(11) (a) Fretz, H.; Valdenaire, A.; Pothier, J.; Hilpert, K.; Gnerre, C.;
Peter, O.; Leroy, X.; Riederer, M. A. Identification of 2-(2-(1-
Naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)-
acetic Acid (Setipiprant/ACT-129968), a Potent, Selective, and Orally
Bioavailable Chemoattractant Receptor-Homologous Molecule Ex-
pressed on Th2 Cells (CRTH2) Antagonist. J. Med. Chem. 2013, 56,
4899−4911. (b) TumeyL. N.CRTH2 antagonists. In Anti-Inflammatory
Drug Discovery; RSC Drug Discovery Series, Vol. 26; Royal Society of
Chemistry: London, 2012; pp 104−134 (c) Valdenaire, A.; Pothier, J.;
Renneberg, D.; Riederer, M. A.; Peter, O.; Leroy, X.; Gnerre, C.; Fretz,
H. Evolution of novel tricyclic CRTh2 receptor antagonists from a (E)-
2-cyano-3-(1H-indol-3-yl)acrylamide scaffold. Bioorg. Med. Chem. Lett.
2013, 23, 944−948. (d) Andres, M.; Bravo, M.; Buil, M. A.; Calbet, M.;
Castro, J.; Domenech, T.; Eichhorn, P.; Ferrer, M.; Gomez, E.; Lehner,
M. D.; Moreno, I.; Roberts, R. S.; Sevilla, S. 2-(1H-Pyrazol-4-yl)acetic
acids as CRTh2 antagonists. Bioorg. Med. Chem. Lett. 2013, 23, 3349−
3353. (e) Lamers, C.; Flesch, D.; Schubert-Zsilavecz, M.; Merk, D.
Novel prostaglandin receptor modulators: a patent review (2002−
2012), part I: Non-EP receptor modulators. Expert Opin. Ther. Pat.
2013, 23, 47−77. (f) Sandham, D. A.; Arnold, N.; Aschauer, H.; Bala, K.;
Barker, L.; Brown, L.; Brown, Z.; Budd, D.; Cox, B.; Docx, C.; Dubois,
G.; Duggan, N.; England, K.; Everatt, B.; Furegati, M.; Hall, E.; Kalthoff,
F.; King, A.; Leblanc, C. J.; Manini, J.; Meingassner, J.; Profit, R.;
Schmidt, A.; Simmons, J.; Sohal, B.; Stringer, R.; Thomas, M.; Turner, K.
L.; Walker, C.; Watson, S. J.; Westwick, J.; Willis, J.; Williams, G.;
Wilson, C. Discovery and characterization of NVP-QAV680, a potent
and selective CRTh2 receptor antagonist suitable for clinical testing in
allergic diseases. Bioorg. Med. Chem. 2013, 21, 6582−6591.
ABBREVIATIONS USED
■
CRTH2, chemoattractant receptor homologous molecule
expressed on Th2 cells; PGD2, prostaglandin D2; cPLA2,
cytosolic phospholipase A2; TP, thromboxane type prostanoid
receptor; DP, the PGD2 receptor; TXB2, 11-dehydro-
thromboxane B2; FEV1, forced expiratory volume in 1 s; AHR,
airway hyperresponsiveness; LAR, late airway response; EAR,
early airway response; FRET, fluorescence resonance energy
transfer; BOP, benzotriazol-1-yloxytris(dimethylamino) phos-
phonium hexafluorophosphate; Papp, apparent permeability
coefficient; pH, potential hydrogen; nM, nanomolar; HDM,
house dust mite; BAL, bronchoalveolar lavage; MgSO4,
magnesium sulfate; HCl, hydrochloric acid; SEM, standard
error of the mean; P-gp, P-glycoprotein 1; MRP, multidrug
resistant protein; LipE, lipophillic efficiency
REFERENCES
■
(1) (a) Haeggstrom, J. Z.; Rinaldo-Matthis, A.; Wheelock, C. E.;
Wetterholm, A. Advances in eicosanoid research, novel therapeutic
implications. Biochem. Biophys. Res. Commun. 2010, 21, 135−139.
(2) Castellani, M. L.; Felaco, M.; Pandolfi, F.; Salini, V.; De Amicis, D.;
Orso, C.; Vecchiet, J.; Tete, S.; Ciampoli, C.; Conti, F.; Cerulli, G.;
Caraffa, A.; Antinolfi, P.; Cuccurullo, C.; Felaco, P.; Kempuraj, D.;
Boscolo, P.; Sabatino, G.; Shaik, Y. B. Mast cells and arachidonic acid
cascade in inflammation. Eur. J. Inflammation 2009, 7, 131−137.
(3) (a) Nagata, K.; Hirai, H.; Tanaka, K.; Ogawa, K.; Aso, T.;
Sugamura, K.; Nakamura, M.; Takano, S. CRTH2, an orphan receptor of
T-helper-2-cells, is expressed on basophils and eosinophils and responds
to mast cell-derived factor(s). FEBS Lett. 1999, 459, 195−199.
(b) Nagata, K.; Hirai, H. The second PGD(2) receptor CRTH2:
(12) Mahadevan, A.; Sard, H.; Gonzalez, M.; McKew, J. C. A general
method for C3 reductive alkylation of indoles. Tetrahedron Lett. 2003,
44, 4589−4591.
(13) Bowman, W. R.; Bridge, C. F. Synth. Commun. 1999, 29 (22),
4051.
1321
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322

Journal of Medicinal Chemistry
Article
(14) Ferrer, S.; Naughton, D. P.; Parveen, I.; Threadgill, M. D. N- and
O-Alkylation of isoquinolin-1-ones in the Mitsunobu reaction: develop-
ment of potential drug delivery systems. J. Chem. Soc., Perkin Trans. 1
2002, 335−340.
(15) Horning, D. E.; Lacasse, G.; Muchowski, J. M. Can. J. Chem. 1971,
49, 2785−2796.
(16) Peukert, S.; Schwahn, U.; Gussregen, S.; Schreuder, H.;
̈
Hofmeister, A. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors
based on a tetrahydro-1(2H)-isoquinolinone scaffold: synthesis,
biological evaluation and X-ray crystal structure. Synthesis 2005,
1550−1554.
(17) Long, A. J.; Sypek, J. P.; Askew, R.; Fish, S. C.; Mason, L. E.;
Williams, C. M.; Goldman, S. J. Gob-5 contributes to goblet cell
hyperplasia and modulates pulmonary tissue inflammation. Am. J. Respir.
Cell Mol. Biol. 2006, 35, 357−365.
(18) (a) Abraham, W. M.; Ahmed, A.; Sabater, J. R.; Lauredo, I. T.;
Botvinnikova, Y.; Bjercke, R. J.; Hu, X.; Revelle, B. M.; Kogan, T. P.;
Scott, I. L. Selectin blockade prevents antigen-induced late bronchial
responses and airway hyperresponsiveness in allergic sheep. Am. J.
Respir. Crit. Care Med. 1999, 159, 1205−1214. (b) Abraham, W. M.
Modeling of asthma, COPD and cystic fibrosis in sheep. Pulm.
Pharmacol. Ther. 2008, 21, 743−754.
(19) Mellor, B. J.; Murray, P. E.; Thomas, E. J. Aspects of the
Chemistry of Dioxolanes: Synthesis of C-Nucleoside Analogues.
Tetrahedron 1998, 54, 243−256.
1322
dx.doi.org/10.1021/jm401509e | J. Med. Chem. 2014, 57, 1299−1322