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

Article abstract of DOI:10.1021/jm300007n

New classes of CRTH2 antagonists, the pyridazine linker containing indole acetic acids, are described. The initial hit 1 had good potency but poor permeability, metabolic stability, and PK. Initial optimization led to compounds of type 2 with low oxidative metabolism but poor oral bioavailability. Poor permeability was identified as a liability for these compounds. Addition of a linker between the indole and diazine moieties afforded a series with good potency, low rates of metabolism, moderate permeability, and good oral bioavailability in rodents. 32 was identified as the development track candidate. It was potent in cell based, binding, and whole blood assays and exhibited good PK profile. It was efficacious in mouse models of contact hypersensitivity (1 mg/kg b.i.d.) and house dust (20 mg/kg q.d.) when dosed orally. In sheep asthma, administration at 1 mg/kg iv completely blocked the LAR and AHR and attenuated the EAR phase.

Full text of DOI:10.1021/jm300007n

Article
pubs.acs.org/jmc
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
Neelu Kaila,* Adrian Huang, Alessandro Moretto, Bruce Follows, Kristin Janz, Michael Lowe,
Jennifer Thomason, Tarek S. Mansour, Cedric Hubeau, Karen Page, Paul Morgan, Susan Fish, Xin Xu,
Cara Williams, and Eddine Saiah
BioTherapeutics Chemistry, Pfizer Worldwide Medicinal Chemistry, 200 Cambridgepark Drive, Cambridge, Massachusetts 02140,
United States
ABSTRACT: New classes of CRTH2 antagonists, the pyridazine linker containing
indole acetic acids, are described. The initial hit 1 had good potency but poor
permeability, metabolic stability, and PK. Initial optimization led to compounds of
type 2 with low oxidative metabolism but poor oral bioavailability. Poor
permeability was identified as a liability for these compounds. Addition of a linker
between the indole and diazine moieties afforded a series with good potency, low
rates of metabolism, moderate permeability, and good oral bioavailability in
rodents. 32 was identified as the development track candidate. It was potent in cell
based, binding, and whole blood assays and exhibited good PK profile. It was efficacious in mouse models of contact
hypersensitivity (1 mg/kg b.i.d.) and house dust (20 mg/kg q.d.) when dosed orally. In sheep asthma, administration at 1 mg/kg
iv completely blocked the LAR and AHR and attenuated the EAR phase.
linked to increased risk of allergy/asthma. These data strongly
implicate the PGD2/CRTH2 signaling pathway in allergic
disease.5 Several companies have taken CRTH2 antagonists
into clinical trials for treatment of allergic rhinitis, asthma, and
COPD.6 Most advanced are Oxagen and Actelion who have
reported positive results in preliminary phase II studies. The
Oxagen compound shows a significant increase in forced
expiratory volume in 1 s (FEV1) and reduction in IgE and in
sputum eosinophilia at a 100 mg twice daily dose.7 Actelion has
reported a positive proof-of-mechanism study with its orally
active CRTH2 antagonist demonstrating efficacy on the
primary end-point (FEV1) during the late allergic reaction
(3−10 h) after a bronchial allergen challenge.8 Recently
Actelion also reported that its compound met its primary
end-point in seasonal allergic rhinitis. In addition AstraZeneca
has reported successful completion of phase II studies with 4-
(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-
acetic acid (AZD1981) for treatment of COPD.9 Novartis
clinical candidate is being evaluated in two clinical trials for
safety and efficacy in asthma patients.10 Amgen is evaluating a
CRTH2/DP dual inhibitor in a 12-week clinical trial at a once a
day dose of 200 mg and twice daily doses of 100, 25, and 5
mg.11 Actimis is testing its compound in phase I clinical trials.12
Recently, Amira has announced successful completion of
phase 1 trials with a dose proportional pharmacodynamic effect
with two candidates. The first is 2-(2′-((3-benzyl-1-
INTRODUCTION
■
Chemoattractant receptor homologous molecule expressed on
Th2 cells (CRTH2) is a seven transmembrane, Gi coupled
receptor expressed on Th2 cells, eosinophils, basophils, and
monocytes.1 CRTH2 is a rather promiscuous receptor binding
to prostaglandin D2 (PGD2) and several of its metabolites as
well as a number of prostaglandin D (PGD) synthase
independent prostaglandins including 11-dehydrothromboxane
B2 (TXB2), a metabolite of thromboxane A2 (TXA2), and of
prostaglandin F2α (PGF2α). Signaling through CRTH2
attenuates adenyl cyclase (AC) activity through Giα coupled
proteins, resulting in a decline in intracellular cAMP levels and
a rise in intracellular calcium levels that leads to T cell,
eosinophil, basophil, and monocyte chemotaxis as well as
stimulation of TH2 T cell cytokine production. PGD2 is the
major prostanoid produced by mast cells, a key cell type
involved in the pathogenesis of allergic inflammatory diseases.
Following activation of mast cells via IgE/allergen cross-linkage,
cytosolic phospholipase A2 (cPLA2) translocates to the
membrane, leading to the release of arachidonic acid (AA).2,3
AA is further metabolized via cycloxygenase and PGD synthase
to produce PGD2. PGD2 can then bind to either the
prostaglandin D2 receptor DP1 or CRTH2 (prostaglandin
D2 receptor, DP2) to induce multiple proinflammatory
sequelae, resulting in chronic tissue inflammation. Importantly,
blood eosinophilic CRTH2 expression is significantly increased
in atopic dermatitis patients and is directly correlated with
increased clinical severity score.1c,4 Furthermore, single
nucleotide polymorphisms in the CRTH2 gene have been
Received: January 3, 2012
© XXXX American Chemical Society
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Scheme 1. Synthesis of Diazine Indole Acetic Acids
Scheme 2. Synthesis of 5,6-Disubstituted Indoles
ethylureido)methyl)-6-methoxy-4′-(trifluoromethyl)biphenyl-3-
yl)acetic acid (AM211), and the details for the second
compound have not been disclosed.13
clearance in mice. Its in vitro metabolic stability in mouse liver
microsomes was low. In this article we describe the design and
synthesis of novel compounds based on modifications to 1.
Successful clinical results in the past few years have
stimulated interest in discovering potent, selective, and orally
active CRTH2 antagonists for the treatment of asthma.
Arylacetic acid is a common motif for a large number of
CRTH2 antagonists reported in the literature.6,14 Our starting
point was diazine indole acetic acids.15 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. The lead compounds from a limited
set of analogues made in this series showed good potency in the
FRET assay but had high molecular weight and clogP and poor
microsomal stability. The most potent compound in the series
was 1 with IC50 = 2 nM in the FRET assay. A pharmacokinetic
(PK) study of 1 showed poor bioavailability and high total
CHEMISTRY
■
The 3-diazine-substituted indole acetic acids (2−26, 28, 29,
34) were synthesized by the method reported by Pal et al.16
through aluminum chloride induced C−C bond formation
(Scheme 1). Reaction of 2-methylindole (I-1) with dichlor-
odiazine under Friedel−Crafts reaction conditions gave
chlorodiazineindole (I-2), which was converted to indole-N-
acetic acid ester (I-3) upon reaction with bromoacetate. The
latter was converted to the corresponding diazinones (I-4)
using sodium acetate in acetic acid under reflux conditions.
Diversification was then possible by introduction of substituted
benzyl groups in the presence of potassium carbonate in DMF
B
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Scheme 3
Scheme 4
followed by deprotection yielding diazine indole acetic acids 2−
26, 28, 29, and 34. The starting 2-methylindoles (I-1, Scheme
2) for some of the analogues required multistep synthesis. The
5-methylsulfonylindole (for preparation of 17) was prepared by
copper(I) catalyzed coupling reaction of 5-bromoindole with
sulfinic acid salt.17 The synthesis strategy reported by Barluenga
et al. was used to obtain 5,7-disubstituted indoles. The first step
was selective ortho iodination of 2,4-disubstituted anilines. One
of the following two iodinating agents was used: iodine in the
presence of silver sulfate18 or bis(pyridine)iodonium(I)
tetrafluoroborate (IPy2BF4).19 The second step was palla-
dium-catalyzed coupling of the 2-iodoanilines with propyne,
followed by copper iodide mediated nitrogen cyclization onto
alkynes, resulting in 5,7-disubstituted indoles.20
The carboxylic acid modifications were studied on
compound 3 (Scheme 3). Acid functionality was converted to
C
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Scheme 5
D
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Scheme 6
amides (4−7) via BOP mediated coupling. The carboxylic acid
group in 3 was also converted to the corresponding alcohol (9)
through mixed carbonic carboxylic acid anhydride formed in
situ with ethyl chloroformate, followed by reduction with
sodium borohydride. Tetrazole was also used as a surrogate for
carboxylic acid. It was synthesized by addition of sodium azide
to indole acetonitrile (I-9, Scheme 4) using zinc bromide as a
catalyst.
manganese(IV) oxide in refluxing toluene afforded a tandem
reaction where the primary alcohol was first oxidized to the
aldehyde followed by oxidative aromatization to give I-14.
The saturated pyridazine linker compounds (31, 33)
(Scheme 5d) were accessed by reaction of 2-methyl
indoleacetate with N-benzyl-6-oxo-1,4,5,6-tetrahydropyrida-
zine-3-carbaldehyde (I-15). Intermediate I-15 could be
prepared by Swern oxidation of I-18 (Scheme 6). For 33 this
step gave a mixture of I-15 and I-14. The mixture of
intermediates was taken to the next step and then purified as
final products.
In order to introduce a linker between the indole and
phthalazine ring, we started with 2-indoleacetylbenzoic acid (I-
21) and built the phthalazine ring by treatment with hydrazine
(Scheme 7). Reaction of the resulting phthalazinone (I-22)
with benzyl bromide resulted in isolation of only one product,
the 2′-N-benzyl I-23. Next the indole nitrogen was alkylated
with methyl bromoacetate followed by deprotection to give
compound 27.
The pyridazine linker compounds (30, 32, 35−44) were
prepared by reductive alkylation of 2-methyl indoleacetate with
6-oxo-1,6-dihydropyridazine-3-carbaldehyde using triethyl si-
lane/TFA mixture as the reducing agent (Scheme 5).21 The
resulting common intermediate I-11 was derivatized in two
ways: N-alkylation with various benzyl bromides using
potassium or cesium carbonate (Scheme 5a) or Mitsonobu
reaction with substituted benzyl alcohol (Scheme 5b). Since 6-
oxo-1,6-dihydropyridazine-3-carbaldehyde was not readily
available22 for some analogues, the N-substituted pyridazine
carbaldehyde (I-14) was presynthesized and then coupled with
2-methyl indoleacetate (Scheme 5c). Intermediate I-14 were
prepared by the method described by Olsen et al.23 (Scheme
6). Commercially available dimethyl 2-oxopentanedioate was
converted to dihydropyridazinone I-16 using hydrazine in
presence of acid. The nitrogen was then alkylated with
substituted benzyl bromide using potassium carbonate.
Reduction of I-17 using sodium borohydride in a refluxing
mixture of THF and methanol gave the primary alcohol I-18.
The yields in this reaction were very sensitive to the rate of
addition of methanol to the refluxing mixture of sodium
borohydride and I-17 in THF. Also the scale-up efforts of this
reaction resulted in low yields. Treatment of I-18 with activated
RESULTS AND DISCUSSION
■
Preliminary SAR focused on optimization of substitution on the
5-position of indole and 3-position of the diazine in our initial
lead 1.2 This effort led to the identification of 2, which had
lower molecular weight and clogP and overall improved
lipophillic efficiency compared to 1. In addition the
replacement of the metabolically labile phenoxyethyl group in
1 with benzyl resulted in improved microsomal stability (Table
1, Figure 1). However, additional analogues were needed, as the
new lead 2 had poor bioavailability in mouse. Further
investigation showed 2 had low clearance but poor oral
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Scheme 7
Table 1. Comparative Profiles for Compounds 1 and 2
hCRTH2 FRET IC50
(nM)
mouse PK Cl
microsomal stability mouse, t1/2
Caco-2 permeability, Pe × 10−6
compd MW clogP
(mL min−1 kg−1
)
F (%)
(min)
(cm/s)
1
2
522
4.9
5.9
3.5
2
>90
6.9
<1
11
11
0.2
0.3
20
>30
exposure. The in vitro ADME profile of 2 showed good
microsomal stability toward oxidative metabolism in mouse and
human liver microsomes but poor permeability in the Caco-2
cell assay, as expected for a lipophilic and zwitterionic
carboxylic acids. Since the carboxylic acid functionality is part
of the pharmacophore, we tried to find a replacement. Table 2
shows some of our efforts toward this. Replacing the carboxylic
acid with tetrazole (8) was deleterious to CRTH2 activity.
Conversion of the acid to amide (4), substituted amide (5 and
6), and aliphatic sulfonamide (7) all resulted in loss of potency.
Likewise, addition of a methyl group at the methylene next to
the carboxylic acid reduced potency (10). The length of the
spacer between the indole and carboxylic acid was found to be
of crucial importance; a two-carbon chain (11) led to complete
loss of potency. Further structural changes were made to vary
the clogP and pKa of the carboxylic acid, which can modulate
the permeability of these compounds. First, the potency of the
new analogues was measured to determine what structural
variation could be tolerated. At first the substitution on the A
ring of indole was studied (Table 3). Substituents at the 5-
position had an important effect on activity. Hydrophobic
groups (entries 13−16) were preferred, and polar groups
(entries 17, 18, 25) had a detrimental effect on potency.
Substitution at the 7-position reduced activity (entries 19−25).
The size of the substituent at the 7-position was also important;
larger substituents showed the worst activity (14 vs 20 vs 21
F
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model24 was used to calculate the permeability of benzylpyr-
idazineindoles with and without linker. Figure 2a shows that
linker-containing compounds are predicted to have improved
apparent permeability (Papp) and the higher the faster the
compound crosses the cell monolayer. Several linker-containing
analogues were therefore prepared and first evaluated in the
FRET assay. The pyridazine linker compounds were somewhat
more potent than the pyridazines (Figure 2b). Addition of a
linker in the phthalazine series maintained potency (for
example, 26 vs 27, Table 4). The phthalazine linker compounds
were not pursued because of high molecular weight and clogP.
Another approach to increase permeability could be via
improvement of solubility. We hypothesized that reduction of
the diazine ring would disrupt planarity of the indole−diazine
system, resulting in improved solubility. A 10-fold loss in
potency was seen for the saturated phthalazinones (28 vs 29,
Table 4). However, in the linker series saturated compounds
maintained potency (30 vs 31 and 32 vs 33, Table 4). This
SAR study led to identification of the diazine linker series which
was found to be comparable to or a bit more potent than the
pyridazines.
Figure 1. Identification of leads with improved lipophillic efficiency:
plot of hCRTH2 FRET (IC50) and clogP.
Table 2. Carboxylic Acid Replacement
Next, the permeability of these compounds was measured in
the Caco-2 cell line at reduced pH 6.5 to minimize the effect
that the carboxylic acid moiety may have on overall
permeability. Table 5 shows the effect of the linker on
permeability. The pyridazine linker compounds exhibited
acceptable permeability 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. The permeability data
for these compounds suggest that absorption and bioavailability
(after oral administration) should not be limited by
permeability. Compounds 2 and 4 showed 3-fold improvement
in oral exposure in mouse, confirming our original hypothesis
that poor oral exposure was due to poor permeability. A similar
trend was seen for rat PK. Figure 3 shows a plot of oral
exposure in rat and permeability for some of the pyridazines
and linker-pyridazine. It is clear that the linker-pyridazine
compounds have higher permeability and oral exposure
compared to the pyridazines. Representative examples in the
pyridazine linker series exhibited good potency, permeability,
and PK. On the basis of these observations, this series was
prioritized for further creation of analogues.
and 15 vs 22 vs 23 vs 24). None of the new analogues showed
good potency.
Next, addition of a methylene linker between the pyridazine
and indole was investigated. An in-house passive permeability
Table 3. SAR of Substitution on the Indole Ring
For confirmation of potency, compounds potent in the
FRET assay were also tested in a secondary assay. CRTH2
mediated cell migration by our inihibitors was examined in a
basophil chemotaxis assay. 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 4a).
Representative examples were also tested in a hCRTH2
competitive binding assay with tritium labeled PGD2. A good
correlation was observed between binding assay and the FRET
assay, confirming that the FRET potency was coming from
direct binding to hCRTH2 (Figure 4b). Since a good
correlation (within 10-fold) was established between the
FRET and chemotaxis and between FRET and binding assays,
only advanced compounds were tested in these assays.
compd
R1
R2
hCRTH2 FRET IC50 (μM) clogP
pKa
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
Br
Cl
F
H
H
H
H
H
H
H
Br
F
0.134
0.016
0.028
0.023
0.034
0.135
0.104
3.17
4.05
3.90
3.33
3.17
1.71
3.21
1.05
1.05
1.62
3.48
4.05
1.91
1.91
4.15
4.03
4.03
4.02
4.16
4.00
4.07
3.98
3.86
3.88
3.85
3.86
3.83
3.83
Me
MeSO2
OMe
H
>2
Cl
0.090
1.69
Cl
Cl
F
F
0.076
0.245
F
Cl
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
F
MeSO2
F
>1
0.934
MeSO2
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Figure 2. Comparison of calculated permeability (a) and hCRTH2 potency (b) between pyridazine and pyridazine linker series.
these compounds against two other prostaglandin receptors
(DP1 and TXB2) was investigated. For DP1 a cAMP TR-
FRET assay in endogenously expressing NK-92 cells was used.
For TXB2, a receptor binding assay was established.
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Table 4. Antagonist Activity of Saturated Diazines against hCRTH2 in FRET Assay
Table 5. Comparison of Caco-2 Permeability of Pyridazine and Linker Containing Compounds 30 and 32 Compared to 15 and
a
34, Respectively
a
Mouse PK studies showed 3-fold improvements in po exposure for compounds 30 and 32 compared to 15 and 34, respectively.
Representative examples from the series were tested and found
to be greater than 1000-fold selective against 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 feasibility
of in vivo pharmacological studies in mouse models.
Compounds were tested in these assays before advancing to
in vivo models.
series showed a good correlation between the two assays with a
drop-off of less than 10-fold (Table 6). In general these
compounds have high plasma protein binding (>98%) (for
example, compound 32 in an equilibrium dialysis experiment
had a free fraction of 2% in human plasma). Eight compounds
from Table 6 were selected for in vivo evaluation. Compounds
were selected based on permeability, hEOS, and structure
(diversity and structural alerts).
Additionally, a human whole blood eosinophil shape change
assay (hEOS) was developed. Eosinophil shape change is a
consequence of cytoskeleton reorganization predisposing the
leukocyte for cell movement and transmigration and 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
selected based on their autofluorescence in the FL2 channel.
Shape change was estimated based on mean forward light
scatter values. This assay could potentially be used as a
biomarker in in vivo models and clinical trials. Potency in the
FRET assay (IC50 ≥ 50 nM) and good chemical properties
were used as filters to advance compounds to the hEOS assay.
The earlier diazines did not show a correlation between the
FRET and whole blood data. However, the later diazine linker
IN VIVO STUDIES
■
We have utilized an oxazolone induced contact hypersensitivity
(CHS) model in the mouse as a primary in vivo screen. In this
model mice are 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 is
administered and inhibition of ear swelling is determined as a
measure of efficacy. A 50−60% 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 5a shows
results for analogues from the pyridazine linker series. The data
suggest that the compounds need to be very potent (IC50 < 5
nM) in the hEOS assay to be efficacious in the CHS model.
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Figure 3. Plot of permeability and oral exposure in mouse for the pyridiazine linker analogues.
Two compounds, 32 and 31, showed 30% and 34% reduction
in ear swelling in the oxazolone CHS model at 3 mg/kg po
b.i.d., respectively. Both compounds showed moderate Caco-2
permeability and were progressed into in vivo rat pharmaco-
kinetic studies. The compounds had low to moderate clearance
and good in vivo exposure and bioavailability (Table 7).
However, 32 was taken forward because of overall better
profile. A dose response study for 32 in the CHS model gave a
MED of 1 mg/kg (Figure 5b).
Next, we sought to evaluate the potential of compound 32 to
modulate house dust mite (HDM) induced allergic airway
disease in mice (Figure 6). HDM is a natural airborne allergen
that can trigger asthma attacks in susceptible patients. Similar to
asthmatics, mice repeatedly challenged intratracheally with
HDM develop allergic airway disease with prominent
eosinophilia.25 When dexamethasone (Dex) was injected
intraperitoneally at 1 mg/kg (q.d.), airway inflammation was
almost completely abrogated. The total number of inflamma-
tory cells in bronchoalveolar lavage (BAL) samples and BAL
eosinophilia macrophage and lymphocyte numbers were
decreased to baseline levels. The number of neutrophils was
seemingly decreased as well, albeit below statistical significance;
however, it is worth noting that neutrophils represent the
smallest cell population found in BAL samples in this model.
When compound 32 was administered orally at 20 mg/kg
(q.d.), airway inflammation was also largely alleviated. Total
BAL cellularity, BAL eosinophilia, and BAL lymphocyte
numbers were significantly reduced. Conversely, BAL macro-
phage numbers did not significantly change while the subjective
decrease of neutrophil numbers remained below statistical
significance. The microscopic examination of H&E-stained lung
tissue sections from control and treated animals showed that
HDM challenge resulted in bronchovascular and alveolar
inflammation dominated by lymphocytic and histiocytic
infiltrates, with fewer neutrophils and occasional eosinophils.
HDM challenge also resulted in increased intraepithelial mucus
formation along with epithelial/endothelial hyperplasia and
hypertrophy. Treatment with compound 32 substantially
alleviated these pathological features, including the size of
inflammatory infiltrates and the thickening of the respiratory
epithelium and blood vessels.
To explore efficacy in a non-rodent species and to determine
effects on lung function, 32 was evaluated in a sheep model of
Ascaris induced airway bronchoconstriction and AHR (Figure
7a).26 In this model sheep that are naturally sensitized to the
nematode Ascaris are challenged via the airways to induce both
an early phase allergic airway bronchoconstriction (EAR), late
phase allergic airway bronchoconstriction (LAR), and AHR to
aerosolized carbachol. Compound 32, when dosed at 3 mg/kg
iv b.i.d. the day prior to challenge and then again 1 h prior to
challenge, completely blocked the LAR and attenuated the EAR
by approximately 38%. The animals were also given a fourth
dose of 32, 8 h after challenge, to evaluate attenuation of AHR
to aerosolized carbachol that was measured the day following
allergen challenge. By use of this dosing regimen, 32 (3 mg/kg
iv) completely blocked the allergen induced AHR to carbachol.
Conversely, 32 was not efficacious when administered at 1 mg/
kg iv in an identical dosing regimen. 32 was also evaluated for
efficacy following subchronic administration at 1 mg/kg iv q.d.
for 7 days prior to challenge. In this dosing regimen, the
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On the basis of these results and in vivo efficacy, 32 (CRA-898)
was advanced to development track.
CONCLUSION
■
We have discovered a new class of CRTH2 antagonists, the
pyridazine linker containing indole acetic acids. The initial hit 1
had good potency but poor permeability, metabolic stability,
and PK. Careful design and optimization of physicochemical
properties afforded a series with good potency, low rate of
metabolism, moderate permeability, and good oral bioavail-
ability in rodents. 32 was identified as the development track
candidate. It is potent in cell based, binding, and whole blood
assays. 32 exhibits low clearance and good exposure in mouse
and rat. It was efficacious in a mouse model of contact
hypersensitivity with an MED of 1 mg/kg b.i.d. When
administered perorally at 20 mg/kg (q.d.), it alleviated airway
inflammation in the HDM model. In a sheep model of Ascaris
induced allergic airway bronchoconstriction, inflammation, and
AHR, a subchronic administration at 1 mg/kg iv completely
blocked the LAR and AHR and attenuated the EAR phase.
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. Combustion analyses were obtained using a Perkin-Elmer
series II 2400 CHNS/O analyzer. CHN analyses were carried out by
Robertson-Microlit. Where analyses are indicated by symbols of the
elements, analytical results obtained for those elements were within 0.4
of the theoretical values. Low resolution mass spectra were obtained
using a Micromass platform electrospray ionization quadrupole mass
spectrometer. High resolution mass spectra were obtained using a
Bruker APEXIII Fourier transform ion cyclotron resonance (FT-ICR)
mass spectrometer equipped with an actively shielded 7 T super-
conducting magnet (Magnex Scientific, Ltd., U.K.) and an external
Bruker APOLLO electrospray ionization (ESI) source. The microwave
procedures were carried out with a Biotage microwave. Preparative
HPLC was run using a Waters reverse phase preparative HPLC
instrument with an Xterra C18 5 μm, 30 mm × 100 mm column. The
flow rate was 40 mL/min. Mobile phase A was water. Mobile phase B
was acetonitrile, and triethylamine was used as a modifier. Purity in
two solvent systems [water−acetonitrile (method 1) and water−
methanol (method 2)] was determined using an Agilent 1100 HPLC
instrument, and all compounds analyzed were >95% pure. The indoles
used were either commercially available or prepared by general
procedure B.
General Procedure A (Scheme 1). Intermediate I-1b. In a two-
necked 500 mL round-bottomed flask, under nitrogen, 2-methylindole
(5.00 g, 38.1 mmol) was taken up in 100 mL of anhydrous DMF.
Sodium hydride (1.83 g of a 60 wt % mineral oil suspension, 1.10 g,
45.7 mmol) was added and the reaction mixture stirred for 30 min.
Appropriate bromide (46 mmol) was then added by syringe, and the
mixture was stirred overnight. It was then quenched with 10 mL of
saturated ammonium chloride solution and partitioned between 400
mL each of ethyl acetate and brine. The aqueous layer was extracted
with an additional 100 mL of ethyl acetate. The combined organic
extracts were washed with brine (3×), dried over MgSO4, filtered, and
evaporated. The crude product was purified by flash chromatography
over silica gel
Figure 4. (a) Plot of log(IC50) in the FRET and basophil chemotaxis
assay. (b) Plot of log(IC50) in the FRET and binding assay.
compound completely blocked the LAR and AHR. Interest-
ingly, similar to the results observed at 3 mg/kg, 32 also
attenuated the EAR (by approximately 20%). Following
measurement of AHR, three bronchi from each sheep were
lavaged and cell counts determined. 32 reduced BAL
inflammation at this dose (Figure 7b).
Preliminary studies also point to a role for CRTH2 in
impaired airway mucociliary clearance after allergen challenge.
Tracheal mucus velocity (TMV), a marker of airway
mucociliary clearance, is measured in sheep by a technique in
which radiopaque disks are insufflated into the trachea (Figure
7c). The cephalad-axial velocities of the individual disks are
then recorded and calculated. Following allergen challenge the
TMV of the beads was reduced to approximately 50% of
baseline TMV at 4 h after challenge; TMV remained attenuated
for up to 24 h after allergen challenge. Administration of 32 at 3
mg/kg iv resulted in a more rapid return to baseline levels with
a TMV of approximately 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. Five sheep were used for
these studies.
Compound 32 was further profiled in safety and selectivity
studies (Table 8). On the basis of CYP inhibition data, there
were no drug−drug interaction concerns (tested against six
isoforms 3A4, 2D6, 2C9, 2C19, 2C8, 2B6, 1A2, 2A6, with IC50
ranging from 16 to 100 uM). The compound was clean in the
AMES assay. No hERG inhibition was seen at 33 μM. Greater
than 500-fold selectivity was seen against DP1 and TP
receptors and in the Novascreen and nuclear receptor panel.
Intermediate I-2. In a 500 mL round-bottomed flask, the
appropriate indole (I-1a or I-1b, 6.04 mmol) and 1,4-dichloroph-
thalazine or 3,6-dichloropyridazine (6.34 mmol) were taken up in 80
mL of dichloroethane. Aluminum chloride (8.46 mmol) was added,
and the mixture was refluxed overnight under a nitrogen-filled balloon.
In some cases the mixture was stirred in the microwave at 160 °C for 1
h. After cooling slightly, the reaction mixture was poured into a
K
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Table 6. Inhibition of Human Eosinophil Shape Change Induced by PGD2
mixture of ice and 2 M hydrochloric acid. This was stirred until all the
ice had melted, and the layers were separated. The aqueous layer was
extracted with additional dichloroethane, and the combined organic
extracts were washed with brine, dried over anhydrous magnesium
sulfate, filtered, and evaporated to give material of sufficient purity to
be used directly in the next step.
raphy over silica gel. For the methyl/ethyl ester containing compounds
the crude ester was taken up in 3:1 ratio of methanol:/tetrahydrofuran,
and a solution of lithium hydroxide (2 equiv) in water was added. After
the mixture was stirred for 2 h at room temperature, LC−MS analysis
showed complete consumption of starting material. The reaction
mixture was evaporated and purified by flash chromatography over
silica gel.
Intermediate I-3. I-2 (5.94 mmol), potassium carbonate (11.9
mmol), and bromoacetate (12 mmol) were taken up in 30 mL of
DMF in a 250 mL round-bottomed flask and heated at 70 °C
overnight. The reaction mixture was then poured into water, extracted
into ethyl acetate (3×), washed with brine (3×), dried over anhydrous
magnesium sulfate, filtered, and evaporated. The crude product was
purified by flash chromatography over silica gel to give pure product.
Intermediate I-4. In a round-bottomed flask, I-3 was taken up in
100 mL of acetic acid, and 20 mL of 1 M sodium hydroxide was added.
The mixture was heated at 70 °C for 1 h until LC−MS analysis
indicated complete conversion to product. It was then partitioned
between 175 mL each of ethyl acetate and brine, and the aqueous layer
was extracted with additional ethyl acetate. The combined organic
extracts were washed with water (3×) and brine, dried over anhydrous
magnesium sulfate, filtered, evaporated, and azeotroped with toluene
to give pure product
Diazine Indole Acetic Acid. I-4 (0.548 mmol), potassium
carbonate (1.92 mmol), and appropriate benzyl bromide (1.64
mmol) were taken up in 8 mL of DMF and heated at 85 °C for 2.5
h until LC−MS analysis showed complete consumption of starting
material. The mixture was then cooled to room temperature, poured
into 80 mL of water, extracted into ethyl acetate, washed with brine,
dried over anhydrous magnesium sulfate, filtered, and evaporated. For
the tert-butyl ester containing compounds, trifluoroacetic acid was
added to the crude ester, and this reaction mixture was stirred for 1 h,
until LC−MS analysis showed complete disappearance of the ester.
The mixture was then evaporated and purified by flash chromatog-
2-(5-Fluoro-3-(3-(4-fluorobenzyl)-4-oxo-3,4-dihydrophthala-
zin-1-yl)-2-methyl-1H-indol-1-yl)acetic Acid (2). Y = Me. Solid
1
(overall yield, 7%). H NMR (400 MHz, DMSO-d6) δ 13.29 (br s, 1
H), 7.75 (d, J = 9.6 Hz, 1 H), 7.40−7.51 (m, 3 H), 7.30 (dd, J = 10.1,
2.5 Hz, 1 H), 7.16−7.23 (m, 2 H), 7.05 (d, J = 9.6 Hz, 1 H), 6.98 (td, J
= 9.2, 2.4 Hz, 1 H), 5.33 (s, 2 H), 5.01 (s, 2 H), 2.41 (s, 3 H). HRMS:
calcd for C22H17F2N3O3 + H+, 410.131 07; found (ESI, [M + H]+
obsd), 410.1305.
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)acetic Acid (3). Y = t-Bu. Solid (overall yield, 23%).
1H NMR (DMSO-d6) δ: 13.14 (br s, 1H), 8.37−8.43 (m, 1H), 7.82−
7.93 (m, 2H), 7.53−7.58 (m, 1H), 7.48−7.53 (m, 1H), 7.32−7.41 (m,
4H), 7.26−7.32 (m, 1H), 7.11−7.19 (m, 2H), 6.99 (ddd, J = 8.0, 7.0,
0.9 Hz, 1H), 5.35−5.50 (m, 2H), 5.10 (s, 2H), 2.23 (s, 3H). HRMS
(ESI+) calcd for C26H21N3O3 (MH+) 424.1653; found 424.1656
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)propanoic Acid (10). Y = Me. Solid (overall yield,
95.2%). 1H NMR (400 MHz, DMSO-d6) δ 8.37−8.43 (m, 1H), 7.80−
7.93 (m, 2H), 7.49−7.59 (m, 1H), 7.25−7.44 (m, 6H), 7.08−7.16 (m,
2H), 6.94−7.01 (m, 1H), 5.34−5.54 (m, 3H), 2.28 (s, 3H), 1.69 (dd, J
= 6.32, 7.07 Hz, 3H). HRMS: calcd for C27H24N3O3 + H+, 438.1812;
found (ESI, [M + H]+ obsd), 438.1816.
3-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)propanoic Acid (11). Y = Me. Solid (overall yield,
1
69%). H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 8.37−8.41
(m, 1H), 7.80−7.92 (m, 2H), 7.53−7.59 (m, 2H), 7.26−7.40 (m, 5H),
L
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Hz, 1 H), 7.27−7.42 (m, 5 H), 7.51 (d, J = 8.84 Hz, 1 H), 7.57−7.65
(d, J = 2.02 Hz, 1 H), 7.76 (d, J = 9.60 Hz, 1 H).
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-fluoro-2-
methyl-1H-indol-1-yl)acetic Acid (15). Y = Me. Solid (overall
1
yield, 17.4%). H NMR (400 MHz, DMSO-d6) δ 13.17 (br s, 1 H),
7.75 (d, J = 9.9 Hz, 1 H), 7.49 (dd, J = 8.8, 4.5 Hz, 1 H), 7.27−7.41
(m, 6 H), 7.06 (d, J = 9.6 Hz, 1 H), 6.99 (td, J = 9.2, 2.7 Hz, 1 H), 5.35
(s, 2 H), 5.07 (s, 2 H), 2.41 (s, 3 H). HRMS: calcd for C22H18FN3O3 +
H+, 392.140 50; found (ESI, [M + H]+ obsd), 392.1407.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-2,5-dimeth-
yl-1H-indol-1-yl)acetic Acid (16). Y = Me. Solid (overall yield,
15%). 1H NMR (400 MHz, MeOD) δ 7.80 (d, J = 9.6 Hz, 1 H), 7.43−
7.49 (m, 2 H), 7.28−7.41 (m, 4 H), 7.20 (d, J = 8.1 Hz, 1 H), 7.09 (d,
J = 9.6 Hz, 1 H), 6.98−7.04 (m, 1 H), 5.43 (s, 2 H), 4.90 (s, 2 H), 2.44
(s, 3 H), 2.38 (s, 3 H). HRMS: calcd for C23H21N3O3 + H+, 388.165
57; found (ESI, [M + H]+ obsd), 388.1655.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-methyl-5-
(methylsulfonyl)-1H-indol-1-yl)acetic Acid (17). 2-Methyl-5-
(methylsulfonyl)-1H-indole was synthesized from 2-methyl-5-bromo-
1H-indole according to the procedure described by Ma et al.27 Y = Me.
1
Solid (overall yield, 28%). H NMR (400 MHz, MeOD) δ ppm 2.71
(s, 3 H), 3.31 (s, 3 H), 5.28 (s, 2 H), 5.64 (s, 2 H), 7.30 (d, J = 9.60
Hz, 1 H), 7.49−7.55 (m, 1 H), 7.56−7.63 (m, 2 H), 7.72−7.82 (m, 2
H), 7.93−8.01 (m, 2 H), 8.28−8.37 (m, 1 H), 8.60−8.63 (m, 1 H).
HRMS calcd for (C23H21N3O5S + H+), 452.1275; found, 452.1270.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-methoxy-
2-methyl-1H-indol-1-yl)acetic Acid (18). Y = t-Bu. Solid (overall
1
yield, 2.6%). H NMR (DMSO-d6) δ 7.75 (d, J = 9.6 Hz, 1H), 7.26−
7.41 (m, 6H), 7.13 (d, J = 2.5 Hz, 1H), 7.05 (d, J = 9.6 Hz, 1H), 6.75
(dd, J = 8.8, 2.3 Hz, 1H), 5.35 (s, 2H), 4.89 (s, 2H), 3.65 (s, 3H), 2.38
(s, 3H). HRMS (ESI+) calcd for C23H22N3O4 (MH+) 404.1605;
found, 404.1603. Anal. (C23H21N3O4) C, H, N.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-7-bromo-2-
methyl-1H-indol-1-yl)acetic Acid (19). Y = Me. Solid (overall
1
yield, 24%). H NMR (400 MHz, CDCl3) δ ppm 2.29 (s, 3 H), 5.33
(bs, 2 H), 5.35 (s, 2 H), 6.85−6.90 (m, 1 H), 7.11 (d, J = 9.60 Hz, 1
H), 7.21−7.29 (m, 4 H), 7.37−7.41 (m, 2 H), 7.43 (d, J = 9.60 Hz, 2
H). HRMS calcd for (C22H18BrN3O3 + H+), 452.0604; found,
452.0590.
Figure 5. (a) In vivo efficacy in CHS model. (b) MED for compound
32 in CHS model.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-chloro-7-
fluoro-2-methyl-1H-indol-1-yl)acetic Acid (20). Y = Me. Solid
(overall yield, 5%). 1H NMR (DMSO-d6) δ 13.37 (br s, 1H), 7.72 (d, J
= 9.6 Hz, 1H), 7.42 (d, J = 1.3 Hz, 1H), 7.35−7.40 (m, 4H), 7.28−
7.34 (m, 1H), 7.12 (dd, J = 12.4, 1.3 Hz, 1H), 7.07 (d, J = 9.6 Hz, 1H),
5.34 (s, 2H), 5.05 (s, 2H), 2.41 (s, 3H). HRMS (ESI+) calcd for
C22H18ClFN3O3 (MH+) 426.1015; found, 426.1016.
7.10−7.19 (m, 2H), 6.98 (ddd, J = 1.01, 7.01, 7.89 Hz, 1H), 5.34−5.48
(m, 2H), 4.47 (t, J = 7.33 Hz, 2H), 2.77 (t, J = 7.20 Hz, 2H), 2.33 (s,
3H). HRMS: calcd for C27H24N3O3 + H+, 438.1812; found (ESI, [M +
H]+ obsd), 438.1819
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-2-methyl-
1H-indol-1-yl)acetic Acid (12). Y = Me. Solid (overall yield, 26.9%).
1H NMR (400 MHz, DMSO-d6) δ 13.16 (br s, 1 H), 7.76 (d, J = 9.6
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5,7-di-
chloro-2-methyl-1H-indol-1-yl)acetic Acid (21). General proce-
dure A was followed starting with 5,7-dichloro-2-methyl-1H-indole,
which was prepared from 2,4-dichloroaniline according to Scheme
2.18,19 Y = Me. Solid (overall yield, 35%). 1H NMR (400 MHz,
MeOD) δ ppm 2.62 (s, 3 H), 5.53 (s, 2 H), 5.63 (s, 2 H), 7.30 (d, J =
9.60 Hz, 1 H), 7.36 (d, J = 1.77 Hz, 1 H), 7.51−7.55 (m, 1 H), 7.56−
7.62 (m, 2 H), 7.65−7.70 (m, 2 H), 7.71−7.74 (m, 1 H), 7.87 (d, J =
9.60 Hz, 1 H).
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5,7-di-
fluoro-2-methyl-1H-indol-1-yl)acetic Acid (22). General proce-
dure A was followed starting with 5,7-dichloro-2-methyl-1H-indole,
which was prepared from 2,4-dichloroaniline according to Scheme
2.18,19 Y = Me. Solid (overall yield, 24%). 1H NMR (400 MHz,
MeOD) δ ppm 2.61 (s, 3 H), 5.28 (s, 2 H), 5.63 (s, 2 H), 6.93−7.01
Hz, 1 H), 7.62 (d, J = 7.8 Hz, 1 H), 7.46 (d, J = 8.1 Hz, 1 H), 7.28−
7.39 (m, 5 H), 7.12−7.18 (m, 1 H), 7.03−7.09 (m, 2 H), 5.34 (s, 2 H),
5.05 (s, 2 H), 2.41 (s, 3 H). HRMS: calcd for C22H19N3O3 + H+,
374.149 92; found (ESI, [M + H]+ obsd), 374.1498.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-bromo-2-
methyl-1H-indol-1-yl)acetic Acid (13). Y = Me. Solid (overall
1
yield, 48%). H NMR (400 MHz, MeOD) δ ppm 2.31 (s, 3 H), 4.83
(s, 2 H), 5.30 (s, 2 H), 6.95 (d, J = 9.60 Hz, 1 H), 7.17−7.24 (m, 2 H),
7.24−7.31 (m, 3 H), 7.37 (d, J = 8.34 Hz, 2 H), 7.58 (d, J = 9.60 Hz, 1
H), 7.65−7.69 (m, 1 H). HRMS calcd for (C22H18BrN3O3 + H+),
452.0604; found, 452.0603.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-chloro-2-
methyl-1H-indol-1-yl)acetic Acid (14). Solid (overall yield, 50%).
Y = Me. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.42 (s, 3 H), 5.08 (s,
2 H), 5.35 (s, 2 H), 7.06 (d, J = 9.60 Hz, 1 H), 7.16 (dd, J = 8.60, 2.02
Table 7. PK Properties of 32 and 31
a
a
a
b
b
compd
species
iv Cl (mL min−1 kg−1
)
t1/2 (h)
Vss (L/kg)
AUC0−inf (h·kg·ng·mL−1·mg−1
)
Cmax (ng/mL)
bioavailability (%)
31
32
33
rat
13
12
4.3
3
1.7
3568
5066
7526
484
772
28
37
41
rat
1.6
mouse
9
4
1.12
1889
a
b
Intravenous dose, 2 mg/kg. Oral dose, 10 mg/kg.
M
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Figure 6. (a) Modulation of HDM-induced allergic airway disease in BALB/c mice. Total cellularity and eosinophil, macrophage, lymphocyte,
neutrophil cell numbers in BAL samples were collected 72 h after HDM challenge. (b) H&E (hematoxylin and eosin) staining of mouse lung
sections (magnification ×10). P values are given for comparisons between HDM-challenged mice treated with vehicle and HDM-challenged mice
treated with compound 32. Shown are the mean SEM from 8 to 20 mice per group.
(m, 1 H), 7.26−7.35 (m, 2 H), 7.49−7.60 (m, 3 H), 7.63−7.68 (m, 2
H), 7.91 (d, J = 9.60 Hz 1 H). HRMS calcd for (C22H17F2N3O3 + H+),
410.1311; found, 410.1311.
9.60, 2.53 Hz, 1 H), 7.66 (dd, J = 8.84, 2.78 Hz, 1 H), 7.71 (d, J = 9.60
Hz, 1 H).
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-7-fluoro-2-
methyl-5-(methylsulfonyl)-1H-indol-1-yl)acetic Acid (25). Gen-
eral procedure A was followed starting with 7-fluoro-2-methyl-5-
(methylsulfonyl)-1H-indole (I-6-1, general procedure B). Y = t-Bu.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-7-chloro-5-
fluoro-2-methyl-1H-indol-1-yl)acetic Acid (23). General proce-
dure A was followed starting with 7-chloro-5-fluoro-2-methyl-1H-
indole, which was prepared from 2-chloro-4-fluoroaniline according to
Scheme 2 using literature procedure. Y = Me. Solid (overall yield,
1
Solid (overall yield, 3.1%). H NMR (DMSO-d6) δ 13.65 (br s, 1H),
8.10 (d, J = 1.3 Hz, 1H), 7.78 (d, J = 9.9 Hz, 1H), 7.52 (dd, J = 11.9,
1.5 Hz, 1H), 7.42−7.47 (m, 2H), 7.34−7.41 (m, 2H), 7.27−7.33 (m,
1H), 7.12 (d, J = 9.6 Hz, 1H), 5.33 (s, 2H), 5.12 (s, 2H), 3.23 (s, 3H),
2.45 (s, 3H). HRMS (ESI+) calcd for C23H21FN3O5S (MH+)
470.1180; found, 470.1180. Anal. (C23H20FN3O5S) C, H, N.
1
31%). H NMR (400 MHz, MeOD) δ ppm 2.62 (s, 3 H), 5.54 (s, 2
H), 5.63 (s, 2 H), 7.19 (dd, J = 9.09, 2.53 Hz, 1 H), 7.30 (d, J = 9.60
Hz, 1 H), 7.44 (dd, J = 9.35, 2.53 Hz, 1 H), 7.49−7.60 (m, 3 H),
7.63−7.68 (m, 2 H), 7.89 (d, J = 9.60 Hz, 1 H). HRMS calcd for
(C22H17ClFN3O3 + H+), 426.1015; found, 426.1014.
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-fluoro-2-
methyl-7-(methylsulfonyl)-1H-indol-1-yl)acetic Acid (24). Gen-
eral procedure A was followed starting with 7-(methylsulfonyl)-5-
fluoro-2-methyl-1H-indole, which was prepared from 4-fluoro-2-
(methylsulfonyl)aniline according to Scheme 2.14,15 Y = Me. Solid
(overall yield, 24%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.40 (s, 3
H), 3.49 (s, 3 H), 5.37 (s, 2 H), 5.43 (bs, 2 H), 7.10 (d, J = 9.60 Hz, 1
H), 7.30−7.36 (m, 1 H), 7.37 (s, 2 H), 7.38 (s, 2 H), 7.61 (dd, J =
2-(3-(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)-5-chloro-2-
methyl-1H-indol-1-yl)acetic Acid (26). Solid (overall yield, 52%).
1
Y = t-Bu. H NMR (400 MHz, DMSO-d6) δ 13.21 (br s, 1H), 8.38−
8.43 (m, 1H), 7.85−7.94 (m, 2H), 7.56 (d, J = 8.6 Hz, 1H), 7.50−7.54
(m, 1H), 7.27−7.41 (m, 5H), 7.15 (dd, J = 8.7, 2.1 Hz, 1H), 7.11 (d, J
= 2.0 Hz, 1H), 5.32−5.50 (m, 2H), 5.12 (s, 2H), 2.23 (s, 3H).
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-5-fluoro-
2-methyl-1H-indol-1-yl)acetic Acid (28). Y = t-Bu. Solid (overall
1
yield, 34%). H NMR (400 MHz, DMSO-d6) δ 13.20 (br s, 1H),
8.39−8.43 (m, 1H), 7.84−7.94 (m, 2H), 7.51−7.57 (m, 2H), 7.27−
N
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Figure 7. (a) Administration of compound 32 at 3 mg/kg iv in a sheep model of allergic airway bronchoconstriction and AHR: (i, top left)
compound 32 attenuates EAR and blocks LAR; (ii, top right) compound 32 blocks AHR to aerosolized carbachol. (b) Compound 32 attenuates
Ascaris induced BAL fluid eosinophilia at 1 mg/kg iv q.d. for 7 days in sheep. (c) Reduction in mucocilliary clearance in sheep with 32 at 3 mg/kg iv.
O
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Table 8. Overall Profile of Compound 32
IC50 (nM)
IC50 (μM)
hCRTH2 FRET hCRTH2 binding basophil chelmotaxis hEOS mCRTH2 binding DP1 FRET TXA2 binding Caco2 permeability at pH 6 (cm/s)
3
0.8 1.2 19000 560
7.1 × 10−6
3
7
7.41 (m, 5H), 6.99 (td, J = 2.53, 9.22 Hz, 1H), 6.87 (dd, J = 2.53, 9.60
Hz, 1H), 5.43 (s, 2H), 5.11 (s, 2H), 2.23 (s, 3H). HRMS (ESI+) calcd
for C26H21N3O3 (MH+) 442.1562; found, 442.1561.
6.94 (dd, J = 10.9, 1.8 Hz, 1H), 6.21 (ddd, J = 3.4, 1.9, 0.8 Hz, 1H),
2.38 (d, J = 0.8 Hz, 3H).
5-Chloro-7-fluoro-2-methyl-1H-indole (I-1-2). Yield, 52%. H
NMR (DMSO-d6) δ: 12.33 (s, 1H), 7.98−8.02 (m, 1H), 7.89 (d, J =
9.1 Hz, 1H), 7.86 (d, J = 1.5 Hz, 1H), 7.16 (dd, J = 10.9, 1.8 Hz, 1H),
2.65 (s, 3H).
General Procedure C (Scheme 3). Amides of Compound 3.
In a 25 mL round-bottomed flask, compound 3 (0.708 mmol), amine
(2.83 mmol), and BOP (0.344 g, 0.779 mmol) were taken up in 6 mL
of DMF. Base (3.60 mmol) was added, and the mixture was allowed to
stir at room temperature over the weekend. It was then poured into 60
mL of water, and the off-white precipitate was collected, washed three
times with water, dried under vacuum, and purified by flash
chromatography over silica gel to give a solid.
1
2-(3-(1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-
yl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (34). Y = Me.
Solid (overall yield, 23%). 1H NMR (400 MHz, DMSO-d6) δ 2.38 (s,
3 H), 5.05 (s, 3 H), 5.37 (s, 3 H), 6.97 (dt, J = 9.1, 2.5 Hz, 1 H), 7.06
(d, J = 9.6 Hz, 1 H), 7.11 (dt, J = 8.6, 2.5 Hz, 1 H), 7.18 (dd, J = 10.1,
2.5 Hz, 1 H), 7.28 (dt, J = 8.6, 2.5 Hz, 1 H), 7.42−7.55 (m, 2 H), 7.76
(d, J = 9.6 Hz, 1 H). HRMS: calcd for C22H16F3N3O3 + H+, 427.114
38 found (ESI-FTMS, [M + H]1+), 427.113 98.
2-(3-(3-Benzyl-4-oxo-3,4,4a,5,6,7,8,8a-octahydrophthalazin-
1-yl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (29).28 Y =
1
Me. H NMR (400 MHz, DMSO-d6) δ 13.20 (br s, 1H), 7.70−7.66
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)acetamide (4). General procedure C, ammonium
chloride as the amine source, and 4-methylmorpholine as the base
(m, 2H), 7.39−7.35 (m, 4H), 7.27−7.15 (m, 2H), 5.11 (s, 2H), 4.15
(s, 2H), 2.64−2.4 (m, 2H), 2.23 (s, 3H), 1.8−1.6 (m, 1H), 1.55−1.36
(m, 7H).
1
General Procedure B (Scheme 2). Iodoaniline (I-5).15 In a 250
mL round-bottomed flask substituted aniline (19.7 mmol) was taken
up in dichloromethane. Bis(pyridine)iodonium(I) tetrafluoroborate
(29.6 mmol) was added. Then trifluoromethanesulfonic acid (59
mmol) was added slowly via a syringe. LC−MS analysis 5 min after
completion of this addition showed complete conversion to product.
The reaction mixture was quenched with water and then partitioned
between water and dichloromethane, and the aqueous layer was
extracted with additional dichloromethane. The combined organic
extracts were washed with 5% sodium thiosulfate, dried over
anhydrous magnesium sulfate, filtered, evaporated, and purified by
flash chromatography to give iodoaniline (I-6).16
were used. Yield, 51%. H NMR (DMSO-d6) δ 8.37−8.42 (m, 1H),
7.82−7.92 (m, 2H), 7.70 (br s, 1H), 7.60 (dt, J = 7.8, 0.8 Hz, 1H),
7.43−7.47 (m, 1H), 7.26−7.41 (m, 6H), 7.10−7.18 (m, 2H), 6.99
(ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 5.34−5.49 (m, 2H), 4.87 (s, 2H), 2.24
(s, 3H). HRMS (ESI+) calcd for C26H23N4O2 (MH+) 423.1815;
found, 423.1812.
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)-N,N-dimethylacetamide (5). General procedure C,
dimethylamine hydrochloride as the amine source, and 4-methyl-
morpholine as the base were used. Yield, 77%. 1H NMR (DMSO-d6) δ
8.37−8.43 (m, 1H), 7.83−7.92 (m, 2H), 7.57 (dt, J = 7.5, 0.9 Hz, 1H),
7.44−7.49 (m, 1H), 7.32−7.41 (m, 4H), 7.25−7.31 (m, 1H), 7.08−
7.16 (m, 2H), 6.97 (td, J = 7.5, 0.8 Hz, 1H), 5.34−5.50 (m, 2H),
5.15−5.29 (m, 2H), 3.19 (s, 3H), 2.89 (s, 3H), 2.17 (s, 3H). HRMS
(ESI+) calcd for C28H27N4O2 (MH+) 451.2128; found, 451.2133.
2-Benzyl-4-(2-methyl-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-1H-
indol-3-yl)phthalazin-1(2H)-one (6). General procedure C, pyrro-
lidine as the amine source, and 4-methylmorpholine as the base were
In a 350 mL glass pressure vessel with a threaded Teflon cap,
iodoaniline (12.0 mmol), copper iodide (0.16 mmol), and Pd-
(PPh3)2Cl2 (0.144 mmol) were taken up in 165 mL of triethylamine
and cooled to −78 °C. Propyne (48 mmol) was condensed into a
graduated cylinder and added to the reaction vessel. The vessel was
then capped, the cooling bath removed, and the reaction mixture
allowed to stir while warming to room temperature overnight behind a
safety shield. Removal of the triethylamine by evaporation gave a crude
material that was purified by flash chromatography over silica gel to
give pure product.
1
used. Yield, 79%. H NMR (DMSO-d6) δ 8.38−8.42 (m, 1H), 7.82−
7.92 (m, J = 7.3, 7.3, 7.3, 7.3, 1.5 Hz, 2H), 7.55−7.60 (m, 1H), 7.46−
7.51 (m, 1H), 7.32−7.42 (m, 4H), 7.25−7.31 (m, 1H), 7.09−7.15 (m,
2H), 6.94−7.01 (m, 1H), 5.35−5.50 (m, 2H), 5.07−5.20 (m, 2H),
3.68 (t, J = 6.8 Hz, 2H), 3.32−3.38 (m, 2H), 2.19 (s, 3H), 1.98 (quin, J
= 6.8 Hz, 2H), 1.82 (quin, J = 6.8 Hz, 2H). HRMS (ESI+) calcd for
C30H29N4O2 (MH+) 477.2284; found, 477.2287.
5,7-Disubstituted-2-methyl-1H-indole (I-1).16 Propynylaniline
(10.9 mmol) was taken up in 210 mL of anhydrous DMF, and copper
iodide (1.20 mmol) was added. The mixture was refluxed under
nitrogen for 1 h until TLC analysis showed complete conversion to
product. The reaction mixture was then evaporated, and the crude
material was purified by flash chromatography over silica gel to give
pure product.
2-Fluoro-6-iodo-4-(methylsulfonyl)aniline (I-5-1) . Yield, 49%.
1H NMR (DMSO-d6) δ 7.85 (dd, J = 1.8, 1.0 Hz, 1H), 7.56 (dd, J =
10.6, 2.0 Hz, 1H), 6.20 (s, 2H), 3.16 (s, 3H).
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)-N-(methylsulfonyl)acetamide (7). General proce-
dure C, methanesulfonamide as the amine source, and diisopropyle-
thylamine as the base were used. Yield, 7.4%. 1H NMR (DMSO-d6) δ
12.33 (br s, 1H), 8.35−8.46 (m, 1H), 7.82−7.95 (m, J = 7.3, 7.3, 7.3,
7.3, 1.5 Hz, 2H), 7.57 (dt, J = 7.6, 0.9 Hz, 1H), 7.46 (d, J = 8.1 Hz,
1H), 7.32−7.41 (m, 4H), 7.26−7.31 (m, 1H), 7.12−7.19 (m, 2H),
6.98−7.03 (m, 1H), 5.35−5.49 (m, 2H), 5.05 (s, 2H), 3.21 (s, 3H),
2.23 (s, 3H). HRMS (ESI+) calcd for C27H25N4O4S (MH+) 501.1590;
found, 501.1581.
2-Benzyl-4-(1-(2-hydroxyethyl)-2-methyl-1H-indol-3-yl)-
phthalazin-1(2H)-one (9). In a flame-dried two-necked 15 mL
round-bottomed flask, under nitrogen, compound 3 (0.200 g, 0.472
mmol) and triethylamine (66 μL, 48 mg, 0.47 mmol) were taken up in
1.4 mL of anhydrous tetrahydrofuran and cooled to 0 °C with an ice−
water bath. A solution of ethyl chloroformate (45 μL, 51 mg, 0.47
mmol) in 0.3 mL of anhydrous tetrahydrofuran was added dropwise
via syringe. The mixture was stirred for 3 h, at which point sodium
borohydride (36 mg, 0.95 mmol) was added, and the ice bath was
removed. The mixture was stirred for 25 min. LC−MS analysis
showed the presence of the desired product but no mixed anhydride
2-Fluoro-4-(methylsulfonyl)-6-(prop-1-yn-1-yl)aniline (I-6-1).
1
Yield, 92%. H NMR (DMSO-d6) δ: 7.47 (dd, J = 10.9, 2.0 Hz, 1H),
7.43 (d, J = 2.0 Hz, 1H), 6.31 (s, 2H), 3.13 (s, 3H), 2.11 (s, 3H).
7-Fluoro-2-methyl-5-(methylsulfonyl)-1H-indole (I-1-1).
1
Yield, 58%. H NMR (DMSO-d6) δ 12.01 (br s, 1H), 7.88 (d, J =
1.3 Hz, 1H), 7.36 (dd, J = 10.9, 1.5 Hz, 1H), 6.45−6.47 (m, 1H), 3.19
(s, 3H), 2.43 (d, J = 1.0 Hz, 3H).
4-Chloro-2-fluoro-6-iodoaniline (I-5-2). Yield, 91%. H NMR
1
(CDCl3) δ 7.00−7.04 (m, 1H), 6.95 (dd, J = 10.6, 2.3 Hz, 1H), 4.18
(br s, 2H), 2.13 (s, 3H).
4-Chloro-2-fluoro-6-(prop-1-ynyl)aniline (I-6-2). Yield, 88%.
1H NMR (DMSO-d6) δ 11.58 (br s, 1H), 7.29 (d, J = 1.8 Hz, 1H),
P
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Journal of Medicinal Chemistry
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intermediate or acid starting material. The reaction mixture was
partitioned between 5 mL each of ethyl acetate and brine, and the
aqueous layer was extracted with additional ethyl acetate. The
combined organic extracts were washed with 5 mL of brine, dried
over anhydrous magnesium sulfate, filtered, evaporated, and purified
by flash chromatography over silica gel (12−100% ethyl acetate in
hexanes). Additional purification by preparative HPLC (water/
acetonitrile with 0.1% formic acid), followed by lyophilization, gave
2H), 2.37 (s, 3H). HRMS (ESI+) calcd for C26H22N7O (MH+)
448.1880; found, 448.1876.
General Procedure C (Scheme 5). Intermediate I-10. In a 500
mL two-necked round-bottomed flask, 5-substituted-2-methylindole
(33.3 mmol) was taken up 100 mL of anhydrous DMF, under
nitrogen. Sodium hydride (1.69 g of a 60 wt % suspension in mineral
oil, 42.2 mmol) was added in small aliquots, and the mixture was
allowed to stir at room temperature for 30 min. Methyl bromoacetate
(42 mmol) was added all at once by syringe, and the mixture was
allowed to stir overnight. It was then quenched by addition of 20 mL
of brine, via syringe, and partitioned between 400 mL each of ethyl
acetate and brine. The aqueous layer was extracted with additional
ethyl acetate (2×), and the combined organic extracts were washed
with brine (3×), dried over anhydrous magnesium sulfate, filtered,
evaporated, and purified by flash chromatography over silica gel.
Intermediate I-11. To a 500 mL round-bottom flask under an
atmosphere of nitrogen were added I-10 (16.1 mmol), 6-oxo-1,6-
dihydropyridazine-3-carbaldehyde or I-14 or I-15 (16.1 mmol), and
200 mL of anhydrous methylene chloride. The resulting solution was
cooled to 0 °C in an ice−water bath, and triethylsilane (56.4 mmol)
and trifluoroacetic acid (48.3 mmol) were added dropwise. The
mixture was allowed to warm to room temperature and then stirred for
24 h. The mixture was then poured into saturated sodium
bicarbonate(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.
1
pure product (29 mg, 15% yield). H NMR (DMSO-d6) δ 8.36−8.42
(m, 1H), 7.80−7.92 (m, J = 18.4, 7.4, 7.4, 1.4 Hz, 2H), 7.59 (dt, J =
7.8, 0.8 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.32−7.40 (m, 4H), 7.26−
7.31 (m, 1H), 7.09−7.17 (m, 2H), 6.97 (ddd, J = 8.0, 7.1, 0.9 Hz, 1H),
5.34−5.49 (m, 2H), 4.97 (t, J = 5.3 Hz, 1H), 4.30 (t, J = 5.7 Hz, 2H),
3.75 (q, J = 5.2 Hz, 2H), 2.33 (s, 3H). HRMS (ESI+) calcd for
C26H24N3O2 (MH+) 410.1862; found, 410.1872.
4-(1-((2H-Tetrazol-5-yl)methyl)-2-methyl-1H-indol-3-yl)-2-
benzylphthalazin-1(2H)-one (8) (Scheme 4). 1-Chloro-4-(2-
methyl-1H-indol-3-yl)phthalazine (I-2-1). The procedure de-
scribed above for I-2 was followed, reacting 2-methylindole with 1,4-
dichlorophthalazine. Yield, 74%. 1H NMR (DMSO-d6) δ 11.68 (s,
1H), 8.34−8.40 (m, 1H), 8.16 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 8.03−
8.08 (m, 1H), 7.96 (dq, J = 8.3, 0.7 Hz, 1H), 7.45 (dt, J = 8.1, 0.9 Hz,
1H), 7.10−7.19 (m, 2H), 6.99 (ddd, J = 7.9, 7.0, 1.0 Hz, 1H), 2.42 (s,
3H).
2-(3-(4-Chlorophthalazin-1-yl)-2-methyl-1H-indol-1-yl)-
acetonitrile (I-7). The procedure described above for I-3 was
1
followed, reacting I-2-1 with bromoacetonitrile. Yield, 35%. H NMR
(DMSO-d6) δ 8.37−8.42 (m, 1H), 8.18 (ddd, J = 8.3, 7.1, 1.0 Hz, 1H),
8.06 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 7.87−7.91 (m, 1H), 7.74−7.79
(m, 1H), 7.31 (ddd, J = 8.2, 7.1, 1.1 Hz, 1H), 7.20−7.24 (m, 1H),
7.09−7.14 (m, 1H), 5.71 (s, 2H), 2.46 (s, 3H).
2-(3-(4-Hydroxyphthalazin-1-yl)-2-methyl-1H-indol-1-yl)-
acetonitrile (I-8). The procedure described above for I-4 was
followed. Yield, 67%. 1H NMR (DMSO-d6) δ 12.82 (s, 1H), 8.35 (dd,
J = 7.7, 1.4 Hz, 1H), 7.80−7.90 (m, J = 7.4, 7.4, 7.4, 7.4, 1.5 Hz, 2H),
7.70 (d, J = 8.1 Hz, 1H), 7.48 (dd, J = 6.7, 1.4 Hz, 1H), 7.27 (t, J = 8.0
Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.05−7.13 (m, 1H), 5.64 (s, 2H),
2.41 (s, 3H).
Intermediate I-12.17 To a 100 mL round-bottom flask under a
nitrogen atmosphere were added I-11 (6.16 mmol), substituted benzyl
bromide (12.33 mmol), potassium carbonate (18.49 mmol), and 50
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 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.
2-(3-(3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-2-methyl-
1H-indol-1-yl)acetonitrile (I-9). I-8 (0.250 g, 0.795 mmol) was
taken up in 15 mL of DMF, and potassium carbonate (0.385 g, 2.78
mmol) and benzyl bromide (0.28 mL, 0.41 g, 2.4 mmol) were added.
The mixture was heated at 100 °C for 1.5 h until LC−MS analysis
showed complete consumption of starting material. The crude product
was purified by flash chromatography over silica gel (7−60% ethyl
Intermediate I-13. To a mixture of I-11 (1.47 mmol), substituted
benzyl alcohol (2.21 mmol), triphenylphosphine (4.42 mmol), and 8
mL of dry DMF was added diisopropyl diazene-1,2-dicarboxylate (4.42
mmol) at 25 °C. The reaction mixture was stirred at 80 °C for 16 h.
Water (30 mL) and ethyl acetate (50 mL) were added. The organic
layer was washed with brine, dried over magnesium sulfate, and filtered
and the solvent removed in vacuo to give an oil.
Pyridazine Linker (Scheme 5). To a 100 mL round-bottom flask
were added I-12 or I-13 (5.16 mmol) and 20 mL of THF. To this was
added a solution of lithium hydroxide (25.8 mmol) 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 column or reverse phase
HPLC.
1
acetate in hexanes) to give pure product (0.236 g, 73% yield). H
NMR (DMSO-d6) δ 8.38−8.43 (m, 1H), 7.82−7.93 (m, J = 19.1, 7.4,
7.4, 1.5 Hz, 2H), 7.68−7.73 (m, 1H), 7.52−7.57 (m, 1H), 7.32−7.41
(m, 4H), 7.23−7.32 (m, 2H), 7.15−7.20 (m, 1H), 7.04−7.11 (m, 1H),
5.64 (s, 2H), 5.33−5.51 (m, 2H), 2.36 (s, 3H).
Compound 8. In a 10 mL round-bottomed flask, I-9 (0.236 g,
0.583 mmol), zinc bromide (0.131 g, 0.583 mmol), and sodium azide
(42 mg, 0.642 mmol) were taken up in 3 mL of isopropanol and 1.2
mL of water.29 The mixture was refluxed overnight until LC−MS
analysis showed complete conversion to product, and it was cooled to
room temperature. It was partitioned between ethyl acetate and 2 M
hydrochloric acid, and the aqueous layer was extracted with additional
ethyl acetate. The combined organic extracts were evaporated, and the
residue was taken up in 40 mL of 0.25 M NaOH and stirred for 2 h.
Although the reference had suggested that a precipitate of zinc
hydroxide would form, only a faint cloudiness that could not be
removed by filtration was observed. Thus, the filtered (and still
cloudy) solution was acidified with concentrated hydrochloric acid and
the off-white precipitate collected, washed three times with 2 M
hydrochloric acid, and dried under vacuum. It was then purified by
preparative HPLC (water/acetonitrile with 0.1% formic acid) and
lyophilized to give pure product (0.120 g, 46% yield). 1H NMR
(DMSO-d6) δ 8.37−8.42 (m, 1H), 7.81−7.92 (m, J = 18.4, 7.4, 7.4, 1.4
Hz, 2H), 7.55−7.62 (m, 2H), 7.32−7.41 (m, 4H), 7.25−7.31 (m, 1H),
7.10−7.19 (m, 2H), 6.95−7.04 (m, 1H), 5.80 (s, 2H), 5.34−5.49 (m,
General Procedure D (Scheme 6). Methyl 6-Oxo-1,4,5,6-
tetrahydropyridazine-3-carboxylate (I-16). To a 100 mL round-
bottom flask equipped with a condenser was added dimethyl 2-
oxopentanedioate (5.00 g, 28.71, 1.0 equiv), methanol (30 mL, 0.1 M),
hydrazine (0.99 mL, 31.03 mmol, 1.1 equiv), and 7 drops of acetic
acid. The mixture was heated to reflux and stirred under nitrogen for 5
h. The mixture was allowed to cool to room temperature and then was
concentrated in vacuo yielding I-16, methyl 6-oxo-1,4,5,6-tetrahy-
1
dropyridazine-3-carboxylate, as a white solid in quantitative yield. H
NMR (400 MHz, DMSO-d6) δ 11.26 (br s, 1 H), 3.75 (s, 3 H), 2.71−
2.80 (m, 2 H), 2.38−2.47 (m, 2 H).
Intermediate I-17. To a 100 mL round-bottom flask which
contained I-16, methyl 6-oxo-1,4,5,6-tetrahydropyridazine-3-carbox-
ylate (8.01 mmol), were added potassium carbonate (20.02 mmol)
Q
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Journal of Medicinal Chemistry
Article
and DMF (80 mL, 0.1 M). The flask was purged with nitrogen.
Substituted benzyl bromide (16.02 mmol) was added and the mixture
stirred at 90 °C overnight. The mixture was cooled to room
temperature, extracted with ethyl acetate, washed with brine, dried
over MgSO4, and concentrated in vacuo. The resulting material was
purified via silica gel chromatography.
Intermediate I-18. To a 100 mL round-bottom flask equipped
with a condenser were added I-17 (3.05 mmol), THF (12 mL, 0.25
M), methanol (2.4 mL, 1.25 M), and sodium borohydride (3.05
mmol). The mixture was heated to reflux and stirred under nitrogen
for 5 h. The mixture was cooled to room temperature, extracted with
ethyl acetate, washed with brine, dried over MgSO4, and concentrated
in vacuo, resulting in I-18.
Intermediate I-14. To a 100 mL round-bottom flask equipped
with a condenser were added I-18 (0.918 mmol and toluene (20 mL,
0.05 M). Manganese dioxide (13.77 mmol) was added slowly, and the
mixture was heated to reflux and stirred under nitrogen overnight. The
mixture was cooled to room temperature and filtered through Celite,
resulting in I-14.
Intermediate I-15. In a two-necked 100 mL round-bottomed flask
fitted with an addition funnel, under nitrogen, oxalyl chloride (6.4
mmol) was taken up in 11 mL of anhydrous dichloromethane, and the
solution was cooled to −78 °C (dry ice/acetone bath). A solution of
dimethyl sulfoxide (13 mmol) in 3 mL of anhydrous dichloromethane
was added in rapid drops from the addition funnel. The reaction
mixture was stirred for 20 min. Then a solution of I-18 (4.00 mmol) in
3 mL of anhydrous dichloromethane was added over 10 min. The
reaction mixture was now stirred for 1 h at −78 °C. Triethylamine (28
mmol) was added dropwise, and stirring continued for an additional
20 min. Stirring became quite difficult because of the formation of a
thick precipitate. The cooling bath was removed, and the mixture was
allowed to warm to room temperature. Water (20 mL) was added, and
the layers were separated. The aqueous layer was extracted with
additional dichloromethane (2 × 10 mL), and the combined organic
extracts were washed with brine (2 × 10 mL). The dichloromethane
solution was dried over anhydrous magnesium sulfate, filtered, and
evaporated. The residue was taken up in 75 mL of dichloromethane
and washed successively with 20 mL each of 0.5 M hydrochloric acid,
water, 5% sodium carbonate, water, and brine. The solution was then
dried over anhydrous magnesium sulfate, filtered, and evaporated to
give I-15.
{3-[(1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl]-5-flu-
oro-2-methyl-1H-indol-1-yl}acetic Acid (30). Scheme 5a was used.
Solid (overall yield, 5%). 1H NMR (400 MHz, DMSO-d6) δ 13.02 (br
s, 1H), 7.27−7.39 (m, 6H), 7.14−7.21 (m, 2H), 6.82−6.91 (m, 2H),
5.22 (s, 2H), 4.94 (s, 2H), 3.96 (s, 2H), 2.30 (s, 3H)
2-(3-((1-Benzyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)-
methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (31). Gen-
eral procedure C and Scheme 5d were followed using I-15-1 (prepared
using general procedure D). Overall yield, 50%. 1H NMR (DMSO-d6)
δ 12.99 (s, 1H), 7.36 (dd, J = 8.8, 4.5 Hz, 1H), 7.28−7.34 (m, 2H),
7.22−7.28 (m, 3H), 7.14 (dd, J = 9.9, 2.5 Hz, 1H), 6.87 (td, J = 9.2,
2.5 Hz, 1H), 4.94 (s, 2H), 4.84 (s, 2H), 3.65 (s, 2H), 2.26−2.32 (m,
4H), 2.24 (s, 3H). HRMS (ESI+) calcd for C23H23FN3O3 (MH+)
408.1718; found, 408.1714. Anal. (C23H22FN3O3·1/2H2O) C, H, N.
2-(3-((1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-
yl)methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid (32).
Scheme 5a was used. Solid (overall yield, 18%). 1H NMR (400
MHz, DMSO-d6) δ 2.28 (s, 3 H), 3.91 (s, 2 H), 4.94 (s, 2 H), 5.25 (s,
2 H), 6.80−6.91 (m, 2 H), 7.03 (m, J = 8.6, 8.6, 2.6, 1.0 Hz, 1 H), 7.09
(dd, J = 9.7, 2.4 Hz, 1 H), 7.17 (d, J = 9.6 Hz, 1 H), 7.21−7.28 (m, 1
H), 7.28−7.38 (m, 2 H), 13.00 (s, 1 H). HRMS: calcd for
C23H18F3N3O3 + H+, 441.130 03 found (ESI-FTMS, [M + H]1+),
441.130 13.
2-(3-((1-(2,4-Difluorobenzyl)-6-oxo-1,4,5,6-tetrahydropyri-
dazin-3-yl)methyl)-5-fluoro-2-methyl-1H-indol-1-yl)acetic Acid
(33). General procedure C and Scheme 5d were followed using I-15-2
1
(prepared using general procedure D). Overall yield, 62%. H NMR
(DMSO-d6) δ 7.25−7.35 (m, 2H), 7.17−7.24 (m, 1H), 6.97−7.05 (m,
2H), 6.85 (td, J = 9.2, 2.4 Hz, 1H), 4.86 (s, 4H), 3.63 (s, 2H), 2.29 (s,
4H), 2.23 (s, 3H). HRMS (ESI+) calcd for C23H21F3N3O3 (MH+)
444.1530; found, 444.1530.
2-(5-Fluoro-3-((1-(4-(2-hydroxypropan-2-yl)benzyl)-6-oxo-
1,6-dihydropyridazin-3-yl)methyl)-2-methyl-1H-indol-1-yl)-
acetic Acid (35). General procedure C and Scheme 5a were followed
using 2-(4-(bromomethyl)phenyl)propan-2-ol.30 Solid (overall yield,
1
30%). H NMR (400 MHz, MeOD) δ ppm 1.40 (s, 6 H), 2.24 (s, 3
H), 3.92 (s, 2 H), 4.79 (s, 2 H), 5.20 (s, 2 H), 6.71 (d, J = 9.35 Hz, 1
H), 6.71−6.77 (m, 1 H), 6.99 (dd, J = 9.60, 2.27 Hz, 1 H), 7.10 (d, J =
9.35 Hz, 1 H), 7.09−7.13 (m, 1 H), 7.22 (d, J = 8.59 Hz, 2 H), 7.32−
7.36 (m, 2 H). HRMS (ESI+) calcd for C26H26FN3O4 (MH+)
464.1980; found, 464.1981
2-(5-Fluoro-3-((1-((4-fluoropyridin-3-yl)methyl)-6-oxo-1,6-
dihydropyridazin-3-yl)methyl)-2-methyl-1H-indol-1-yl)acetic
Acid (36). General procedure C and Scheme 5b were followed using
2-(3-fluoropyridin-4-yl)methanol (prepared from 3-fluoroisonicotinal-
dehyde).31 Solid (overall yield, 18%). 1H NMR (400 MHz, MeOD) δ
ppm 2.38 (s, 3 H), 4.04 (s, 2 H), 4.84 (s, 2 H), 5.50 (s, 2 H), 6.82−
6.91 (m, 2 H), 7.00−7.05 (m, 1 H), 7.20−7.26 (m, 2 H), 7.29−7.34
(m, 1 H), 8.30−8.34 (m, 1 H), 8.46−8.50 (m, 1 H). HRMS: calcd for
(C22H18F2N4O3 + H+), 425.1420; found, 425.1419.
6-Oxo-1-(2,4,5-trifluorobenzyl)-1,6-dihydropyridazine-3-car-
baldehyde (I-14-1). General procedure D was used. Overall yield,
1
6%. H NMR (400 MHz, CDCl3) δ 9.66 (d, 1 H), 7.70 (dd, J = 9.7,
2.1 Hz, 1 H), 7.03−7.21 (m, 1 H), 6.81−6.99 (m, 2 H), 5.34 (s, 2 H).
6-Oxo-1-(4-(trifluoromethyl)benzyl)-1,6-dihydropyridazine-
3-carbaldehyde (I-14-2). General procedure D was used. Overall
yield, 15%. 1H NMR (400 MHz, CDCl3) δ 5.45 (s, 2 H), 7.00 (dd, J =
9.7, 0.9 Hz, 1 H), 7.53−7.68 (m, 4 H), 7.75 (d, J = 9.6 Hz, 1 H), 9.75
(d, J = 1.0 Hz, 1 H).
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(2,4,5-trifluorobenzyl)-1,6-
dihydropyridazin-3-yl)methyl)-1H-indol-1-yl)acetic Acid (37).
General procedure C and Scheme 5c were followed using I-14-1
1-Benzyl-6-oxo-1,6-dihydropyridazine-3-carbaldehyde (I-
1
14-3). General procedure D was used. Overall yield, 20%. H NMR
1
(400 MHz, CDCl3) δ 5.41 (s, 2 H), 6.97 (dd, J = 9.7, 0.9 Hz, 1 H),
7.29−7.39 (m, 3 H), 7.46 (d, J = 1.5 Hz, 1 H), 7.48 (d, J = 2.0 Hz, 1
H), 7.72 (d, J = 9.6 Hz, 1 H), 9.75 (d, J = 1.0 Hz, 1 H).
1-Benzyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-carbalde-
hyde (I-15-1). General procedure D was used. Overall yield, 36%. 1H
NMR (DMSO-d6) δ 9.42 (s, 1H), 7.19−7.45 (m, 5H), 4.99 (s, 2H),
2.69−2.75 (m, 2H), 2.56−2.63 (m, 2H).
(prepared using general procedure D). Solid (overall yield, 20%). H
NMR (400 MHz, DMSO-d6) δ 7.52−7.60 (m, 1 H), 7.30−7.38 (m, 2
H), 7.18 (d, J = 9.6 Hz, 1 H), 7.03 (dd, J = 10.0, 2.4 Hz, 1 H), 6.82−
6.89 (m, 2 H), 5.24 (s, 2 H), 4.89 (s, 2 H), 3.91 (s, 2 H), 2.29 (s, 3 H).
HRMS: calcd for C23H17F4N3O3 + H+, 460.127 88; found (ESI, [M +
H]+ obsd), 460.1276.
2-(3-((1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl)-5-
1-(2,4-Difluorobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazine-
3-carbaldehyde (I-15-2). General procedure D was used. Overall
yield, 49%. A mixture of I-14-4 (1-(2,4-difluorobenzyl)-6-oxo-1,6-
dihydropyridazine-3-carbaldehyde) and I-15-2 was obtained. No
attempt was made to separate the two at this stage because of stability
chloro-2-methyl-1H-indol-1-yl)acetic Acid (38). Scheme 5c was
1
used. Solid (overall yield, 17%). H NMR (400 MHz, DMSO-d6) δ
2.31 (s, 3 H), 3.98 (s, 2 H), 4.95 (s, 2 H), 5.21 (s, 2 H), 6.85 (d, J =
9.6 Hz, 1 H), 7.05 (dd, J = 8.7, 2.1 Hz, 1 H), 7.16 (d, J = 9.6 Hz, 1 H),
7.24−7.36 (m, 5 H), 7.39 (d, J = 8.8 Hz, 1 H), 7.49 (d, J = 2.3 Hz, 1
H). HRMS: calcd for C23H20ClN3O3 + H+, 421.119 32; found (ESI-
FTMS, [M + H]1+), 421.119 12.
(3-{[1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-
yl]methyl}-2-methyl-1H-indol-1-yl)acetic Acid (39). Scheme 5a
was used. Solid (overall yield, 43%). 1H NMR (400 MHz, DMSO-d6)
δ 12.99 (br s, 1H), 7.21−7.46 (m, 4H), 7.14 (d, J = 9.60 Hz, 1H),
1
concerns. The compounds were purified in the final step. I-14-4: H
NMR (DMSO-d6) δ 9.64 (d, J = 1.0 Hz, 1H), 7.80 (d, J = 9.6 Hz, 1H),
7.43 (td, J = 8.7, 6.6 Hz, 1H), 7.29 (ddd, J = 10.6, 9.3, 2.5 Hz, 1H),
7.05−7.12 (m, 2H), 5.39 (s, 2H). I-15-2: 1H NMR (DMSO-d6) δ 9.40
(s, 1H), 5.00 (s, 2H), 2.69−2.74 (m, 2H), 2.56−2.61 (m, 2H).
1
Aromatic H NMR peaks overlap with those for I-14-4.
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6.96−7.10 (m, 2H), 6.78−6.94 (m, 2H), 5.26 (s, 2H), 4.92 (s, 2H),
3.93 (s, 2H), 2.30 (s, 3H). HRMS: calcd for (C23H19F2N3O3 + H+),
424.1466; found, 424.1455.
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 100 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 desired product (3.59 g, 84%). 1H NMR (400 MHz, DMSO-
d6) δ 2.32 (s, 3 H), 3.56 (s, 2 H), 6.98 (dd, J = 8.5, 2.1 Hz, 1 H), 7.25
(d, J = 8.6 Hz, 1 H), 7.40 (d, J = 2.0 Hz, 1 H), 11.05 (s, 1 H), 12.12 (s,
1 H).
2-(2-(5-Chloro-2-methyl-1H-indol-3-yl)acetyl)benzoic Acid
(I-21). To a 100 mL round-bottom flask under an atmosphere of
nitrogen were added I-20 (3.59 g, 16.13 mmol, 1.0 equiv), phthalic
anhydride (2.39 g, 16.13 mmol, 1.0 equiv), and sodium acetate (7.94 g,
96.81 mmol, 6.0 equiv). Then 40 mL of toluene was added, and the
suspension was sonicated for 5 min. The toluene was then removed in
vacuo and the resulting powder heated neat to 200 °C for 16 h. After
the mixture was cooled to room temperature, the resulting brown solid
was washed with 1.2 N HCl and water and dried. The resulting
material was carried on crude (4.08 g, 77%).
4-((5-Chloro-2-methyl-1H-indol-3-yl)methyl)phthalazin-
1(2H)-one (I-22). To a 500 mL round-bottom flask under an
atmosphere of nitrogen were added I-21 (4.08 g, 12.48 mmol, 1.0
equiv), anhydrous hydrazine (0.78 mL, 24.95 mL, 2.0 equiv), and 250
mL of isopropanol. The resulting mixture was heated to reflux and
allowed to stir for 16 h. The solvent was then removed in vacuo and
the residue purified by silica gel chromatography to give the desired
product as a brown solid (0.755 g, 19%). 1H NMR (400 MHz,
DMSO-d6) δ 2.40 (s, 3 H), 4.32 (s, 2 H), 6.94 (dd, J = 8.5, 2.1 Hz, 1
H), 7.22 (d, J = 8.6 Hz, 1 H), 7.38−7.46 (m, 1 H), 7.75−7.82 (m, 1
H), 7.86 (td, J = 7.6, 1.5 Hz, 1 H), 7.92−7.98 (m, 1 H), 8.24 (dd, J =
7.8, 1.5 Hz, 1 H), 11.06 (s, 1 H), 12.52 (s, 1 H).
2-Benzyl-4-((5-chloro-2-methyl-1H-indol-3-yl)methyl)-
phthalazin-1(2H)-one (I-23). To a 100 mL round-bottom flask
under a nitrogen atmosphere were added I-22 (0.755 g, 2.34 mmol,
1.0 equiv), benzyl bromide (0.56 mL, 4.67 mmol, 2.0 equiv),
potassium carbonate (0.806 g, 5.84 mmol, 2.5 equiv), and 50 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 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 to give a white solid (0.200 g,
21%).
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(pyridin-3-ylmethyl)-1,6-
dihydropyridazin-3-yl)methyl)-1H-indol-1-yl)acetic Acid (40).
Scheme 5a was used. Solid (overall yield, 11.7%). 1H NMR (400 MHz,
DMSO-d6) δ 8.57 (d, J = 2.3 Hz, 1 H), 8.50 (dd, J = 4.9, 1.6 Hz, 1 H),
7.69 (dt, J = 7.8, 2.1 Hz, 1 H), 7.31−7.41 (m, 2 H), 7.12−7.20 (m, 2
H), 6.81−6.91 (m, 2 H), 5.26 (s, 2 H), 4.89 (s, 2 H), 3.95 (s, 2 H),
2.29 (s, 3 H). HRMS: calcd for C22H19FN4O3 + H+, 407.151 40; found
(ESI, [M + H]+ obsd), 407.1514.
2-(5-Fluoro-2-methyl-3-((6-oxo-1-(4-(trifluoromethyl)-
benzyl)-1,6-dihydropyridazin-3-yl)methyl)-1H-indol-1-yl)acetic
Acid (41). General procedure C and Scheme 5c were followed using
I-14-2 (prepared using general procedure D). Solid (overall yield,
30%). 1H NMR (400 MHz, DMSO-d6) δ 2.31 (s, 3 H), 3.95 (s, 2 H),
4.94 (s, 2 H), 5.32 (s, 2 H), 6.82−6.91 (m, 2 H), 7.11 (dd, J = 9.9, 2.5
Hz, 1 H), 7.19 (d, J = 9.6 Hz, 1 H), 7.35 (dd, J = 8.8, 4.3 Hz, 1 H),
7.49 (d, J = 8.1 Hz, 2 H), 7.69 (d, J = 8.1 Hz, 2 H). HRMS: calcd for
C24H19F4N3O3 + H+, 473.136 25 found (ESI-FTMS, [M + H]1+),
473.136 25.
2-(5-Fluoro-3-((1-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypro-
pan-2-yl)benzyl)-6-oxo-1,6-dihydropyridazin-3-yl)methyl)-2-
methyl-1H-indol-1-yl)acetic Acid (42). Scheme 5a was used. Solid
1
(overall yield, 35%). H NMR (400 MHz, MeOD) δ ppm 2.35 (s, 3
H), 4.06 (s, 2 H), 4.84 (s, 2 H), 5.39 (s, 2 H), 6.83−6.89 (m, 1 H),
6.86 (d, J = 9.60 Hz, 1 H), 7.14 (dd, J = 9.85, 2.53 Hz, 1 H), 7.23 (dd,
J = 8.84, 4.29 Hz, 1 H), 7.27 (d, J = 9.60 Hz, 1 H), 7.48 (d, J = 8.59
Hz, 2 H), 7.71 (d, J = 8.34 Hz, 2 H).
2-(3-((1-Benzyl-6-oxo-1,6-dihydropyridazin-3-yl)methyl)-2-
methyl-1H-indol-1-yl)acetic Acid (43). General procedure C and
Scheme 5c were followed using I-14-3 (prepared using general
procedure D). Solid (overall yield, 50%). 1H NMR (400 MHz,
DMSO-d6) δ 2.32 (s, 3 H), 3.97 (s, 2 H), 4.93 (s, 2 H), 5.24 (s, 2 H),
6.83 (d, J = 9.6 Hz, 1 H), 6.87−6.95 (m, 1 H), 6.99−7.07 (m, 1 H),
7.12 (d, J = 9.3 Hz, 1 H), 7.24−7.42 (m, 7 H). HRMS: calcd for
C23H21N3O3 + H+, 387.158 29; found (ESI-FTMS, [M + H]1+
387.158 09.
2-(3-((1-(2,4-Difluorobenzyl)-6-oxo-1,6-dihydropyridazin-3-
yl)methyl)-5,7-difluoro-2-methyl-1H-indol-1-yl)acetic Acid
(44). Scheme 5a was followed starting with 5,7-difluoro-2-methyl-
1H-indole, which was prepared from 2,4-difluoroaniline according to
Scheme 2.14,15 Solid (overall yield, 48%). 1H NMR (400 MHz,
MeOD) δ ppm 2.22 (s, 3 H), 3.86 (s, 2 H), 4.84 (s, 2 H), 5.24 (s, 2
H), 6.49−6.56 (m, 1 H), 6.70−6.86 (m, 3 H), 6.74 (d, J = 9.60 Hz, 1
H), 7.11 (d, J = 9.60 Hz, 1 H), 7.18−7.26 (m, 1H).
Methyl 2-(3-((3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)-
methyl)-5-chloro-2-methyl-1H-indol-1-yl)acetate (I-24). To a
100 mL round-bottom flask under a nitrogen atmosphere was added I-
23 (0.128 g, 0.31 mmol, 1.0 equiv), methyl bromoacetate (0.11 mL,
1.24 mmol, 4.0 equiv), potassium carbonate (0.256 g, 1.86 mmol, 6.0
equiv), and 50 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
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 to give a white solid
(0.081 g, 54%). 1H NMR (400 MHz, CDCl3) δ 0.88 (s, 1 H), 2.25 (s,
3 H), 3.70 (s, 3 H), 4.31 (s, 2 H), 4.74 (s, 2 H), 5.37 (s, 2 H), 6.97−
7.13 (m, 2 H), 7.21−7.34 (m, 4 H), 7.44 (dd, J = 8.1, 1.5 Hz, 2 H),
7.55 (d, J = 1.5 Hz, 1 H), 7.62−7.70 (m, 2 H), 7.73−7.81 (m, 1 H),
8.34−8.48 (m, 1 H).
2-(3-((3-Benzyl-4-oxo-3,4-dihydrophthalazin-1-yl)methyl)-5-
chloro-2-methyl-1H-indol-1-yl)acetic Acid (27) (Scheme 7).
Methyl 2-(5-Chloro-2-methyl-1H-indol-3-yl)acetate (I-19). To a
250 mL round-bottom flask under an atmosphere of nitrogen was
added 5-chloro-2-methylindole (5.0 g, 30.2 mmol, 1.0 equiv) and 100
mL of THF. The resulting solution was cooled to −78 °C in a dry ice/
acetone bath, and n-butyllithium (21 mL of 1.51 M solution, 31.72
mmol, 1.05 equiv) was added dropwise over 30 min. This was allowed
to stir at −78 °C for 30 min, at which point zinc chloride (4.12 g, 30.2
mmol, 1.0 equiv) was added as a solution in 5 mL of THF dropwise.
The resulting mixture was allowed to warm to room temperature.
Methyl bromoacetate (2.78 mL, 30.2 mmol, 1.0 equiv) was then added
and the mixture allowed to stir for 24 h. The reaction mixture was then
poured into 500 mL of saturated aqueous ammonium chloride and
extracted with three 100 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 the crude material
which was purified by silica gel chromatography to give the desired
product as a yellow oil (4.53 g, 63%). 1H NMR (400 MHz, CDCl3) δ
2.35 (s, 3 H), 3.64 (s, 2 H), 3.68 (s, 3 H), 6.99−7.07 (m, 1 H), 7.09−
7.14 (m, 1 H), 7.46 (d, J = 2.0 Hz, 1 H), 7.95 (br s, 1 H).
Compound 27. To a 100 mL round-bottom flask was added I-24
(0.155 g, 0.32 mmol, 1.0 equiv) and 20 mL of THF. To this was added
a solution of lithium hydroxide (0.038 g, 1.60 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
2-(5-Chloro-2-methyl-1H-indol-3-yl)acetic Acid (I-20). To a
250 mL round-bottom flask was added I-19 (4.53 g, 19.07 mmol, 1.0
equiv) and 50 mL of THF. To this was added a solution of lithium
hydroxide (2.28 g, 95.37 mmol, 5.0 equiv) in 25 mL of water. To the
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
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Journal of Medicinal Chemistry
Article
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 a white
were treated (q.d.) either intraperitoneally with dexamethasone (1
mg/kg) or orally with compound 2 (20 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 and preparation of lung tissue for
histopathology were performed as previously described.32 Results
represent the mean SEM. Statistical significance for all results was
determined using the Mann−Whitney U test. Results were considered
significant for P < 0.05.
1
powder (0.083 g, 55%). H NMR (400 MHz, DMSO-d6) δ 2.32 (s, 3
H), 4.39 (s, 2 H), 4.93 (s, 2 H), 5.30 (s, 2 H), 7.01 (dd, J = 8.7, 2.1 Hz,
1 H), 7.20−7.42 (m, 6 H), 7.52 (d, J = 1.8 Hz, 1 H), 7.83 (m, J = 7.5,
7.5, 7.5, 7.5, 1.6 Hz, 2 H), 7.96 (dd, J = 6.9, 1.1 Hz, 1 H), 8.28 (dd, J =
7.8, 1.5 Hz, 1 H), 13.09 (br s, 1 H). HRMS: calcd for C27H22ClN3O3 +
H+, 471.134 97 found (ESI-FTMS, [M + H]1+ 471.134 87.
hCRTH2 FRET Assay. The cAMP TR-FRET assay was performed
by incubating a dilution series of compound concentrations with
30 000 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 μM 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 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 the manufacturer’s directions. Data were 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. 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.
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 after challenge. The
following day (24 h) ear swelling was measured again with the
micrometer and the swelling was expressed as postchallenge −
prechallenge thickness (Δ) (Student’s unpaired t test used for
statistical analysis).
AUTHOR INFORMATION
Corresponding Author
*Phone: (617) 665-5626. E-mail: neelu.kaila@pfizer.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the DAC group for analytical data. We also thank
Professor William Abraham from the Mount Sinai Medical
Center at University of Miami, FL, for conducting the sheep
studies to explore efficacy of compounds in asthma.
ABBREVIATIONS USED
■
hCRTH2, human chemoattractant receptor homologous
molecule expressed on Th2 cells; AHR, airway hyper-
responsiveness; LAR, late airway response; EAR, early airway
response; FRET, fluorescence resonance energy transfer; BOP,
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate; Papp, apparent permeability coefficient; pH,
potential hydrogen; PGD2, prostaglandin D2; nM, nanomolar;
HDM, house dust mite; BAL, bronchoalveolar lavage; MgSO4,
magnesium sulfate; HCl, hydrochloric acid; SEM, standard
error of the mean; MED, minimal efficacious dose
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