Identification of 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1 H-pyrido[4,3-b ]indol-5(2 H)-yl)acetic acid (setipiprant/ACT-129968), a potent, selective, and orally bioavailable chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) antagonist

Article abstract of DOI:10.1021/jm400122f

Herein we describe the discovery of the novel CRTh2 antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 28 (setipiprant/ACT-129968), a clinical development candidate for the treatment of asthma and seasonal allergic rhinitis. A lead optimization program was started based on the discovery of the recently disclosed CRTh2 antagonist 2-(2-benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 5. An already favorable and druglike profile could be assessed for lead compound 5. Therefore, the lead optimization program mainly focused on the improvement in potency and oral bioavailability. Data of newly synthesized analogs were collected from in vitro pharmacological, physicochemical, in vitro ADME, and in vivo pharmacokinetic studies in the rat and the dog. The data were then analyzed using a traffic light selection tool as a visualization device in order to evaluate and prioritize candidates displaying a balanced overall profile. This data-driven process and the excellent results of the PK study in the rat (F = 44%) and the dog (F = 55%) facilitated the identification of 28 as a potent (IC₅₀ = 6 nM), selective, and orally available CRTh2 antagonist.

Full text of DOI:10.1021/jm400122f

Article
pubs.acs.org/jmc
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 Expressed on Th2
Cells (CRTH2) Antagonist
Heinz Fretz,* Anja Valdenaire, Julien Pothier, Kurt Hilpert, Carmela Gnerre, Oliver Peter, Xavier Leroy,
and Markus A. Riederer
Drug Discovery Unit, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
S
* Supporting Information
ABSTRACT: Herein we describe the discovery of the novel
CRTh2 antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-
1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 28 (setipiprant/
ACT-129968), a clinical development candidate for the
treatment of asthma and seasonal allergic rhinitis. A lead
optimization program was started based on the discovery of
the recently disclosed CRTh2 antagonist 2-(2-benzoyl-3,4-
dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid 5. An
already favorable and druglike profile could be assessed for
lead compound 5. Therefore, the lead optimization program
mainly focused on the improvement in potency and oral bioavailability. Data of newly synthesized analogs were collected from in
vitro pharmacological, physicochemical, in vitro ADME, and in vivo pharmacokinetic studies in the rat and the dog. The data
were then analyzed using a traffic light selection tool as a visualization device in order to evaluate and prioritize candidates
displaying a balanced overall profile. This data-driven process and the excellent results of the PK study in the rat (F = 44%) and
the dog (F = 55%) facilitated the identification of 28 as a potent (IC50 = 6 nM), selective, and orally available CRTh2 antagonist.
degranulation, and expression of adhesion molecules in
eosinophils and basophils.8,10,11 In addition, upon activation
of CRTh2 by PGD2, Th2 cells can secrete Th2-specific
cytokines, such as interleukin (IL) 4, IL-5, and IL-13,10,12
responsible for enhancing eosinophilia, tissue remodeling, and
airway hypersensitivity.13,14
Mice deficient in CRTh2 have been described to be resistant
to experimentally induced allergic asthma, suggesting that
CRTh2 might play a central role in the pathogenesis of allergic
diseases.15 Therefore, low molecular weight antagonists of
CRTh2 were expected to counteract pathophysiological effects
of PGD2 and thus to display controlling effects on allergic
inflammation.16
The first non-prostanoid CRTh2 modulators have already
been described a decade ago. For example, 2 (indomethacin), a
nonsteroidal anti-inflammatory drug (NSAID) acting as a
nonselective inhibitor of COX-1 and COX-2 enzymes that
participate in prostaglandin synthesis from arachidonic acid,17
was discovered to act as a partial agonist on CRTh2,18 whereas
3 (ramatroban, BAY u3405), a human thromboxane A2
(TXA2) receptor (hTP2) antagonist with clinical efficacy in
INTRODUCTION
■
Allergic disorders, such as asthma, allergic rhinitis, and atopic
dermatitis, represent an increasing global health problem,1 and
despite clear advances in therapy, the disease of many patients
is not adequately controlled. There is a clear medical need for
additional, efficacious, safe, and orally active drugs for allergic
disorders.2,3
Prostaglandin D2 (PGD2) 1 (Chart 1) is a lipid mediator
synthesized from arachidonic acid via the cyclooxygenase
(COX) and prostaglandin D2 synthase (PGDS) pathway.
PGD2 is produced in the brain where it is involved in regulating
central nervous functions such as sleep and pain perception. In
the periphery, mast cells are the initial source of PGD2. These
tissue-resident cells are presenting allergen-specific IgE-anti-
bodies on their surface. Upon encounter with allergens,
clustering of IgE antibodies induces an intracellular signaling
cascade, leading to the generation of PGD2.4,5 In patients
suffering from allergic asthma, PGD2 and its metabolites are
increased in the lung, plasma, and urine.6,7
Chemoattractant receptor-homologous molecule expressed
on T helper 2 cells (CRTh2) is a G-protein-coupled receptor
(GPCR) for PGD2.8 The expression of CRTh2 is limited to
eosinophils, basophils, Th2 cells, and innate lymphoid cells.8,9
Activation of CRTh2 induces a marked increase in mobility,
Received: January 25, 2013
© XXXX American Chemical Society
A
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Chart 1. Structure of Endogenous CRTh2 Agonist PGD2 (1), Partial Agonist Indomethacin (2), Antagonist Ramatroban (3),
Primary Screening Hit 4, Lead Compound 5, and Clinical Development Candidate Setipiprant/ACT-129968 (28)
asthma and allergic rhinitis,19−21 was the first small molecule
CRTh2 antagonist disclosed, displaying inhibitory activity of in
vitro PGD2-induced eosinophil migration.22 Meanwhile the
discovery of numerous CRTh2 antagonists have been described
and are currently in early proof-of-concept clinical studies for
treating the signs and symptoms of both asthma and allergic
rhinitis.23
liberation (Ca2+ flux, FLIPR) or cyclic (c) AMP were used to
give evidence that the compounds are truly antagonizing
hCRTh2 mediated downstream signaling. The results from
both assays are reported as IC50 values. Cross-selectivity with
other prostanoid receptors was addressed by counterscreening
the compounds against the human PGD2 receptor hDP1,
against the human prostaglandin E2 (PGE2) receptor subtypes
hEP1, hEP2, hEP3, and hEP4, and against the human
thromboxane A2 receptor hTP2. Cell lines stably expressing
recombinant human receptors were used: Chinese hamster
ovary (CHO) and HEK-293 cells for hEP1 and hEP3 receptors,
respectively, membranes from HEK-293 cells containing hEP2
or hEP4 receptors, and HEK-293 cells stably expressing human
hTP2 receptors. Reported are IC50 values defined as half-
maximal inhibition of receptor stimulation using an agonist
concentration previously determined to trigger 80% of maximal
response. Selectivity of the antagonists toward the hDP1
receptor was determined by using a competitive receptor
binding assay with CHO cells stably expressing high levels of
recombinant hDP1 receptor, employing [3H]PGD2 as ligand.25
Reported are the respective IC50 values and the selectivity
factor (f Sel) between hDP1 and hCRTh2 which is expressed as
the IC50(hDP1)/IC50(hCRTh2) ratio. Furthermore, the
potential of 2-(3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)-
acetic acid analogues to inhibit COX enzymes was addressed
because of the structural similarity with NSAID 2, a
nonselective inhibitor of COX enzymes.17 COX inhibition
was assessed using a commercially available ovine COX-1 assay
kit from Cayman Chemicals (catalog no. 760111). The
compounds were tested in duplicate at 10 μM final
concentration according to the protocol provided by the kit
manufacturer.
Herein, the lead optimization of a novel chemotype series
providing potent, selective, and orally available CRTh2
antagonists is described. Initially identified as a singleton out
of 80 000 compounds from a GPCR focused library, high-
throughput screening hit 4 was confirmed as a true hit
belonging to a cluster of close analogues that displayed a clear
structure−activity relationship (SAR).24 Attempts to eliminate
potentially unwanted structural features led to the successful
design of novel CRTh2 antagonist scaffolds. From this work, 2-
(2-benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic
acid 5 evolved as a novel druglike lead compound devoid of any
structural alerts.24 The goal of the subsequent lead optimization
program was mainly focused on the improvement in potency
and oral bioavailability. In vitro pharmacological and phys-
icochemical and in vitro absorption, distribution, and
metabolism (ADME) properties of the novel CRTh2
antagonists as well as in vivo pharmacokinetic studies in the
rat and the dog will be presented. Furthermore, the data driven
process will be discussed which led to the decision to select 2-
(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-
5(2H)-yl)acetic acid 28 (setipiprant/ACT-129968), as a clinical
development candidate for the treatment of asthma and
seasonal allergic rhinitis.
RESULTS AND DISCUSSION
■
Potent antagonists with binding IC50 < 100 nM were further
verified in a competitive radioligand displacement assay in the
presence of 0.5% human serum albumin (HSA) in order to
identify those antagonists that retained potency under
conditions where binding to plasma proteins occurs. The
positives were subsequently tested in the eosinophil shape
change (hESC) assay.26,27 In brief, human eosinophils were
isolated from whole blood of healthy volunteers and were
subsequently stimulated with 15 nM PGD2 in the presence of
Biological Assays. Following our screening cascade, a
competitive radioligand displacement assay was used as a
biological in vitro test system to establish the structure−activity
relationship (SAR) for all synthesized potential human (h)
CRTh2 modulators of this novel 2-(3,4-dihydro-1H-pyrido[4,3-
b]indol-5(2H)-yl)acetic acid series. The receptor binding assay
was performed with human embryonic kidney (HEK-293) cells
stably expressing hCRTh2 using [3H]PGD2 (2.5 nM, 170 Ci/
mmol) as radioligand. Quantification of intracellular Ca2+
B
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Scheme 1. General Synthetic Scheme for the Preparation of 2-(3,4-Dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic Acid
a
Derivatives 5 and 11−36
a
Reagents: (a) Boc2O, DIPEA or Et3N, DCM or THF, room temperature, 53−100%; (b) BrCH2CO2Et, Cs2CO3, DMF, 80 °C or acetone, room
temperature, 41−88% yield; (c) 4 N HCl in 1,4-dioxane, DCM, room temperature, 16−99% yield; (d) R-COCl, DIPEA, DCM, 0 °C to room
temperature, 28−98% yield; (e) 0.2 N aqueous NaOH, THF, room temperature, 18−93% yield.
50% human plasma at 37 °C for 5 min. Stimulation was
stopped by adding fixation solution, and increase in light
scattering reflecting changes in cell shape was measured by flow
cytometry. A dose dependent inhibition of this shape change
could be demonstrated with potent antagonists by preincubat-
ing the cells with increasing concentrations of the respective
antagonist for 10 min prior to stimulation with PGD2.
Table 1. Initial SAR Study Exploring R1 at the Indole
Moiety
a
Chemistry. About 200 novel 2-(3,4-dihydro-1H-pyrido[4,3-
b]indol-5(2H)-yl)acetic acid analogues were synthesized in four
and five steps as depicted in Scheme 1,28 starting with 2,3,4,5-
tetrahydro-1H-pyrido[4,3-b]indole analogues 6a−l, all available
from commercial sources or synthetically accessible following
well established procedures. Classical N-Boc-protection was
followed by an alkylation step of 7a−l with ethyl bromoacetate
in the presence of potassium carbonate in DMF at 80 °C or in
acetone at room temperature. Removal of the Boc group in
8a−l using 4 N HCl in 1,4-dioxane gave the corresponding
hydrochloride salts 9a−l, which were subsequently reacted with
the respective acid chloride R-COCl in DCM at 0 °C in the
presence of DIPEA as a base to provide amides 10a−l.
Saponification of the ethyl ester with 0.2 M aqueous NaOH
solution in THF and preparative reverse-phase LC−MS
purification provided 2-(3,4-dihydro-1H-pyrido[4,3-b]indol-
5(2H)-yl)acetic acid derivatives 5 and 11−36 with >95%
purity in 18−93% yield.
SAR Studies. An initial SAR was established with 2-(3,4-
dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid analogues
11−20 (Table 1). The focus of this set of compounds was
directed toward the tolerability of a substituent R1 attached to
the indole unit. As illustrated with 12, 15, 16, and 18,
compounds were obtained that were by a factor of 2−3 slightly
more potent in the radioligand binding assay than lead
compound 5, displaying IC50 values of 5, 11, 7, and 9 nM if
R1 at C(8) represented chlorine, methyl, fluorine, and
trifluoromethyl, respectively. Methoxy or isopropyl at C(8)
seemed to be less favorable (IC50 of 62 and 42 nM) as
illustrated with 19 and 20 (Table 1). Generally, up to 10-fold
less potent compounds were obtained if R1 represented
b
compd
R1
hCRTh2 IC50 [nM]
5
H
17
820
5
11
12
13
14
15
16
17
18
19
20
6-Cl
8-Cl
6-Me
7-Me
8-Me
8-F
107
1590
11
7
6,8-F2
8-CF3
8-MeO
8-i-Pr
35
9
62
42
a
Listed are the IC50 values of the hCRTh2 receptor radioligand
binding in buffer. The data represent the means from at least four
b
independent experiments.
chlorine or methyl connected to C(6) or if R1 was methyl-
attached to C(7), as exemplified with 11, 13, or 14,
respectively. Combining two fluorine atoms in the same
molecule, one connected to the more favorable C(8) position
and a second attached to the suboptimal C(6) position,
resulted in a more than 5-fold drop in binding affinity as
demonstrated with 17. The results of the SAR study exploring
R1 at the indole moiety were in accordance with the data
disclosed for the (E)-2-cyano-3-(1H-indol-3-yl)acrylamide hit
series.24
In another subset of analogues, preferred R1 residues were
combined with various R groups. One-third of those
compounds displayed a potent interaction with hCRTh2,
inhibiting [3H]PGD2 binding in a competitive manner with
C
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Table 2. SAR Study Exploring R and R1, with IC50 Binding Data for hCRTh2 of Selected Compounds in Comparison with Lead
a
Compound 5, Measured in the Presence and Absence of Human Serum Albumin (HSA) in the Assay Buffer
a
IC50 values are given for the effects in the cell based Ca2+ flux assay, the cAMP homogeneous time resolved fluorescence (HTRF) assay, and the
human eosinophil shape change assay. Data are given to demonstrate selectivity against prostanoid receptors hDP1, hEP1‑4, and hTP2. The IC50
b
values represent the mean from at least three independent experiments if not stated otherwise. Human serum albumin shift factor f HSA
IC50(HSA)/IC50(buffer). Selectivity factor f Sel = IC50(hDP1)/IC50(hCRTh2).
=
c
D
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Table 3. Physicochemical Properties and Ligand Efficiency of Selected Antagonists in Comparison with Lead Compound 5
solubility (μg/mL)
a
b
compd
MW (Da)
LE
cLogP
log D7.4
water (pH)
buffer, pH 4
buffer, pH 7
5
334.37
358.39
368.82
386.81
384.43
402.42
416.45
405.43
438.53
0.43
0.46
0.42
0.42
0.38
0.37
0.37
0.38
0.35
2.09
2.0
2.7
2.8
3.3
3.3
3.1
2.0
4.4
−1.2
0.2
64 (4.3)
50 (3.9)
110 (3.8)
25 (4.9)
335 (nd)
50 (4.3)
28 (5.7)
45 (3.8)
2 (4.3)
515
80
810
930
815
1000
830
880
560
800
30
22
23
24
26
28
32
33
35
−0.5
−0.3
0.2
180
140
90
0.1
90
0.2
21
−0.3
1.8
115
1
a
b
Ligand efficiency (LE): relative free binding energy in kcal/mol per non-H atom, calculated from IC50 values. pH of the final aqueous solution.
IC50 ≤ 15 nM. A representative selection is listed in Table 2 for
a continuing discussion of the SAR. From this list it became
evident that hydrophobic R groups were crucial for potent
hCRTh2 binding, for example, monocyclic five- and six-
membered aromatic rings as represented by thiophene and
phenyl, directly attached to the carbonyl group, as well as fused
aromatic ring systems as illustrated with naphthalene or the
nitrogen-containing nonbasic indole, connected either directly
or via a methylene or an ethylene bridge to the carbonyl group.
The most potent antagonist, 35, was obtained if R represented
a biphenyl moiety. Compared to 5, a significant drop in
potency was observed if R represents an alkyl group, for
example, methyl (IC50 = 1.6 μM) or pentyl (IC50 = 0.2 μM).24
Furthermore, a 5-fold drop in potency was noticed for the
respective cyclohexyl analogue 21 (IC50 = 70 nM),
consolidating the importance of an aromatic ring system at
this site of the molecule. In Table 1, the previously discussed
beneficial contributions of fluorine, chlorine, methyl, or
trifluoromethyl at C(8) could be strengthened with additional
examples listed in Table 2. Also the less favorable effect of C(6)
substitution was further established with 25, 31, and 36. These
examples displayed an approximately 20-, 12-, and 6-fold drop
in potency, respectively, if compared with their relatives 24, 28,
and 35 that were not substituted at C(6).
Acidic compounds tend to bind to basic serum albumin.
Therefore, not surprisingly the compounds discussed herein
displayed high plasma protein binding in an in vitro plasma
protein binding assay (PPB > 97%, data not shown). Since
targeted hCRTh2 is located on circulating cells, the impact of
plasma protein binding on the potency of the hCRTh2
antagonists was assessed by adding 0.5% human serum albumin
(HSA) to the binding assay buffer. Generally, a less than 10-
fold HSA shift factor f HSA, defined as the ratio IC50(HSA)/
IC50(buffer), was noticed. With the exceptions of 30 and 31, a
substantially higher albumin shift up to a factor fHSA of 57 was
observed in the cases where R was a naphthyl or a
naphthylmethyl moiety. No obvious correlation between f HSA
and physicochemical parameters of the antagonists (Table 3)
could be established. However, a comparable shift effect was
observed for the inhibition of the PGD2 induced morphological
shape change of human eosinophils measured in the presence
of human plasma.
effects with the IC50 values given in Table 2 for both cell based
functional assays, the Ca2+ flux and the cAMP assay. Moreover,
species selectivity was addressed in a concomitant study by
testing the compounds in a FLIPR assay against mouse, rat,
guinea pig, and dog CRTh2 using cells expressing the
respective recombinant species CRTh2. Antagonistic effects
with comparable potencies were found as for hCRTh2 except
for 24, 26, and 28. Unexpectedly, these three examples were
identified as partial agonists on the mouse and the guinea pig
receptor (data not shown), limiting the choice of potential in
vivo animal models for efficacy studies.
In addition, all antagonists inhibited the PGD2 induced shape
change of human eosinophils in the hESC assay. Compounds
22−24 and 33 were discovered to antagonize hESC most
effectively with IC50 < 100 nM. In fact, these antagonists belong
to the group that exhibited only a minor serum albumin shift
f HSA in the binding assay.
Selectivity against other prostanoid receptors was considered
important in order to avoid unwanted adverse effects from off-
target effects. All compounds were tested against the human
PGD2 receptor hDP1, the human PGE2 receptors hEP1, hEP2,
hEP3, and hEP4, and the human TXA2 receptor hTP2. Many of
the antagonists were highly selective with a selectivity factor f Sel
> 1000 in favor of hCRTh2 (Table 2). However, a few
antagonists were identified that manifested considerable
affinities in the low micromolar to submicromolar range for
the hDP1 receptor. Those compounds could be classified
mainly into two groups. For example, molecules where R1 is
representing a chlorine atom connected to C(6), as exemplified
with 25, 31, and 36, could be assigned to one group. In these
cases, a selectivity factor f Sel in the range between 4 and 20 was
obtained. This narrow selectivity window originates from an up
to 20-fold loss in potency for hCRTh2 on one hand and from a
considerable gain in affinity for the hDP1 receptor on the other.
The second group contained molecules with R representing the
large lipophilic naphthyl, as illustrated with 26−30, or biphenyl,
exemplified by 35. These compounds were identified as being
highly potent on hCRTh2 but were also found to inhibit the
hDP1 receptor in the low micromolar to submicromolar range,
resulting in a selectivity factor in the range 80 < f Sel < 1000. A
selectivity factor of f Sel > 200 in favor of hCRTh2 was applied
as a selection criterion in order to further profile a compound.
Some minor cross-selectivity was observed in the single digit
micromolar range with PGE2 receptor hEP2 measured in the β-
arrestin assay (IC50 > 10 μM in the binding assay); however,
the selectivity factors f Sel were all >400 in favor of the CRTh2
antagonists (Table 2). The compounds were found to be highly
selective against the other EP receptors (hEP1, IC50 > 25 μM
Since agonistic activity on the CRTh2 receptor was reported
for structurally related indolylacetic acid 2,18 2-(3,4-dihydro-
1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid derivatives 5 and
11−36 were consequently tested for their agonistic potential. It
could be demonstrated that all compounds were solely
antagonists on hCRTh2, fully antagonizing the PGD2 induced
E
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(Ca2+); hEP3, IC50 > 25 μM (Ca2+); hEP4, IC50 > 10 μM (β-
arrestin and binding). In contrast to structurally related 3, no
inhibition of hTP2 receptor was observed for this compound
series (IC50 > 25 μM (Ca2+)).
The inhibitory potential of 2-(3,4-dihydro-1H-pyrido[4,3-
b]indol-5(2H)-yl)acetic acid analogues 5 and 11−36 for
enzymes that are involved in the prostaglandin synthesis, for
example, the COX enzymes, was addressed because of the
structural similarity with NSAID 2, a nonselective COX
inhibitor.17 All hCRTh2 antagonists reported herein reduced
COX-1 enzyme activity by less than 30% at 10 μM compound
concentration (no detailed data shown); hence, all IC50 values
were determined well above 10 μM.
Physicochemical and in Vitro ADME Properties.
Physicochemical properties of the antagonists selected for
further profiling are given in Table 3. The molecular weights
were all below 450 Da, in an optimal range between 358 and
438 Da as targeted for small druglike molecules. Furthermore,
the attractive ligand efficiencies (LE) with values between 0.35
and 0.46 placed the compounds in the envisaged range of that
for oral drug candidates.29 The cLogP values were calculated
between 2.0 and 4.4 as a measure of molecular hydrophobicity,
and these values were well below 5, in accordance with
Lipinski’s rule of five.30 Generally, the low to medium
molecular weights (MWs) ranging from 358 to 416 Da,
cLogP values between 2.0 and 3.3, and log D7.4 values ranging
from −0.3 to +0.2 provided compounds that were highly
soluble in buffered aqueous solution at pH 7 with solubility
values between 560 and 1000 μg/mL. Still an acceptable
solubility was obtained in buffer at pH 4 (21−180 μg/mL) or
even in pure water (25−335 μg/mL). Unsurprisingly, the most
hydrophobic compound 35 listed in Table 3 displayed the
highest cLogP value of 4.4 and the highest log D7.4 value of 1.8
in this series and consequently was found to be insoluble in
buffer at pH 4 and water (1 and 2 μg/mL, respectively). Only
poor solubility was measured in buffer at pH 7 (30 μg/mL).
The drug−drug interaction potential for the selected
antagonists in Table 4 was assessed in vitro by measuring the
inhibition of the cytochrome P450 isoforms 2C9, 2D6, and
3A4. Since all IC50 values were well above 10 μM, a low drug−
drug interaction potential was considered for those compounds.
Chemical stability in various media was evaluated by
incubating the compounds in rat and human plasma for up
to 4 h, in simulated gastric fluid (SGF) for 1 h, and in simulated
intestinal fluid (SIF) for 4 h.31 Recovery of unchanged parent
compound was determined by LC−MS. The compounds were
stable under the described conditions. The elimination half-life
(T1/2) was found to be >4 h after exposure in rat as well as in
human plasma, and T1/2 > 1 h in SGF, and T1/2 > 4 h in SIF
(see Supporting Information).
Metabolic stability was assessed by incubating the com-
pounds with human and rat liver microsomes as well as with
plated rat hepatocytes (Table 4). With the exception of 35 all
compounds displayed a very low intrinsic clearance (Clint) with
values below 10 μL min−1 (mg protein)−1 after incubation with
human liver microsomes. An increased clearance (Clint = 85 μL
min−1 (mg protein)−1) was observed for 35. Only 22, 24, and
28 provided similarly low Clint values in rat liver microsomes,
whereas 23, 26, 32, 33, and 35 were found to be more prone to
oxidative metabolism, providing Clint values in the range
between 23 and 63 μL min−1 (mg protein−1). A low clearance
was observed in rat hepatocytes with all Clint values not
exceeding 5.3 μL min−1 (106 cells)−1.
In Vivo Pharmacokinetic Studies. Pharmacokinetic
studies with a representative set of potent and selective
CRTh2 antagonists (Table 5) were carried out in the Wistar rat
Table 5. Pharmacokinetic Profile of Selected Potent
Antagonists, Determined in Male Wistar Rats (Three
Animals for Each Experiment) after Intravenous (iv) and
a
Oral (po) Administrations as Solutions
compd AUC0−last (ng h−1 mL−1
)
CL (mL min−1 kg−1
)
T1/2 (h) F (%)
5
6300
244
4.6
36
2.3
0.6
2.3
5.8
6
14
5
22
24
26
28
33
35
6450
57300
58500
62
5.9
1.1
1.3
11
23
36
44
2
1.2
0.5
171
35
4
a
Vss = 0.4−0.8 L/kg for all compounds tested. iv: 2 mg/kg (28, 35), 1
mg/kg (5, 22, 24, 26), 0.2 mg/kg (33). po: 10 mg/kg (5, 22, 24, 26,
28, 35), 2 mg/kg (33).
at intravenous doses of 0.2, 1, and 2 mg/kg and at oral doses of
2 and 10 mg/kg. Among the six antagonists presented, 28 and
its des-fluoro analogue 26 displayed the most favorable
pharmacokinetic properties with an excellent exposure
(AUC0−last) of 58 500 and 57 300 ng h−1 mL−1, low plasmatic
clearance of 1.3 and 1.1 mL min−1 kg−1, and an oral
bioavailability of 44% and 36%, respectively. In comparison, a
5-fold higher plasma clearance was observed for 24. Indeed,
despite low plasmatic clearance of 5.9 mL min−1 kg−1, only a
moderate exposure of 6450 ng h−1 mL−1 was determined,
leading to an oral bioavailability of 23%. With 33, the exposure
dropped to 62 ng h−1 mL−1, giving rise to a bioavailability of
only 2%. The moderate plasmatic clearance of 11 mL min−1
kg−1 was not considered the only parameter attributed to the
low bioavailability of 33. Other additional effects like
incomplete absorption or active secretion into the gut lumen
could also be reasons for the low bioavailability. Compounds 22
and 35 displayed the highest clearance within this series, with
values of 35 and 36 mL min−1 kg−1. Consequently, their
exposures were well below 500 ng h−1 mL−1 and their
respective oral bioavailability did not exceed the 5% threshold.
For all the antagonists tested, a low volume of distribution Vss
Table 4. In Vitro ADME Properties of Selected Potent
Antagonists in Comparison with Lead Compound 5
Clint
(μL min−1
(mg protein)−1
)
a
CYP2C9,
Clint (μL min−1
compd
IC50 (μM)
HLM
RLM
(106 cells)−1), RHepa
5
15
43
5
<4
5
<4
8
0.9
2.1
1.4
1.9
1.5
3.1
nd
22
23
24
26
28
32
33
35
>50
>50
40
24
<4
36
5
<4
<4
5
>50
16
6
63
23
31
>50
14
<4
85
nd
5.3
a
CYP inhibition: IC50(2D6,3A4) > 50 μM.
F
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typical for acidic drugs (e.g., warfarin, aspirin) was found within
a range of 0.4−0.8 L/kg.
Pharmacokinetic studies with 24 and 28 were performed in
male Beagle dogs. Both compounds were administered orally at
a dose of 10 mg/kg and intravenously at a dose of 1 mg/kg as
solutions containing 10% propylene glycol (PG). The acquired
data are presented in Table 6. The exposure after oral
including their respective threshold ranges; the TLs of the
remaining parameters were not registered in Table 7 because
they all appeared in the green range for the best candidates.
This assessment revealed that lead structure 5 already displayed
a favorable and overall druglike profile. No red flags appeared,
and only 6 out of the 33 parameters really offered potential
room for improvement (Table 7). Applying the threshold
ranges with the respective color coding uncovered not more
than three close analogues (24, 26, and 28) with more green
TLs than 5. Ultimately, the envisaged optimization effort
concluded with the three potential candidates 24, 26, and 28.
For example, four parameters of 26 were clearly improved, as
shown with four yellow TL changing to green. However, at the
same time three other parameters changed for the worse; for
example, the TL for f HSA changed from green to yellow, the TL
f Sel from green to red, and TL hESC from yellow to red. More
importance was attached to the worsening of the last two
parameters, especially the shift toward much lower potency
with IC50 > 1 μM in the relevant functional hESC assay, than to
the positive almost 10-fold improved exposure, and thus, 26
was not considered a probable candidate. Actually, antagonist
24 was identified as the most balanced candidate with the most
green, the least yellow, and with no red TLs. Example 28 scored
second with one yellow TL more for the serum albumin shift
factor f HSA. A closer look at the TL alert for hESC of 28
revealed a 4-fold drop in potency if compared with 24.
However, by comparison of the plasma exposure of 28 (green
TL) with 24 (yellow TL), it became obvious that 28
outperformed 24 by a factor of 10 in this respect, thus
compensating the aforementioned 4-fold potency drop by more
than a factor of 2. Well knowing that the shift to lower potency
in plasma and consequently in whole blood could compromise
in vivo efficacy, 28 was nevertheless selected for further
development mainly because of its excellent oral uptake and the
high exposure that could be reached. It was believed that
therapeutically relevant drug levels still could be obtained,
especially at higher dosage. The full pharmacokinetic and
pharmacodynamic profile of 28 will be presented in a
forthcoming publication.
Table 6. Pharmacokinetic Profile of 24 and 28 Determined
in Male Beagle Dogs (Three Animals for Each Experiment)
after Intravenous and Oral Administrations at a Dose of 1
and 10 mg/kg, Respectively
compd
AUC0−last (ng h−1 mL−1
)
CLiv (mL min−1 kg−1
)
F (%)
24
28
9620
11
64
55
91100
1.3
administration of 24 and 28 was higher than in the rat with
values of 9620 and 91 100 ng h−1 mL−1, respectively. Both
compounds showed a similar bioavailability with 55% for 28
and 64% for 24 despite a 10-fold difference in plasmatic
clearance after intravenous administration. Indeed, while 28
was found to be a low clearance compound with a value of 1.3
mL min−1 kg−1, the clearance for 24 was elevated to 11 mL
min−1 kg−1.
Discussion. The optimization program of the 2-(3,4-
dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid lead series
with the main focus on the improvement in potency and oral
bioavailability offered numerous hCRTh2 antagonists with
interesting characteristics. A visualization device was considered
useful in order to simplify a data-driven analysis of the collected
data and to evaluate and prioritize candidates showing a
balanced profile with a great promise for further development.
Therefore, a traffic light (TL) selection tool,32 basically a
conditional formatting with the color coding red, yellow, and
green, was applied (Table 7). Threshold ranges were defined
Table 7. Applying Traffic Lights (TL) as a Tool for the Data-
a
Driven Selection of the Clinical Candidate
CONCLUSIONS
■
In summary, an optimization process with the main focus to
improve potency and oral bioavailability of a novel hCRTh2
antagonist series was presented starting with the recently
disclosed 2-(2-benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-
5(2H)-yl)acetic acid 5 as lead compound. Data were collected
on more than 30 parameters for a set of about 200 newly
synthesized analogues. A traffic light analysis was conducted to
visualize and to compare the data and to evaluate and prioritize
candidates displaying a balanced profile. This data-driven
process and the excellent results of the PK study in the rat
(F = 44%) and the dog (F = 55%) facilitated the identification
of 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]-
indol-5(2H)-yl)acetic acid 28 (setipiprant/ACT-129968) as a
potent, selective, and orally available hCRTh2 antagonist.
Unfortunately, it was not feasible to rely on the efficacy in
animal asthma models as a selection criterion for the
development candidate because the most balanced hCRTh2
antagonists 24, 26, and 28 turned out to act as partial agonists
on the mouse or guinea pig receptor. This fact limited the
choice of animal asthma models. For that reason and because of
the complex challenges concerning the in vivo pharmacological
profiling of 28 in animal models as well as the cell based in vitro
a
Color coded thresholds (green, go; yellow, alert; red, no go) are
given for selected properties of antagonist 24, 26, and 28 in
comparison with lead compound 5.
and applied for the more than 30 parameters that were
presented throughout Tables 2−6 and discussed in the
corresponding paragraphs. A red TL was used to visualize
values in a less favored range as a “no go”. A yellow TL was set
as an alert for moderate data, whereas a green TL was
employed to highlight a “go” for excellent parameter data. The
threshold ranges were applied on the full data set of eligible
antagonists. However, only the eight parameters that differed
most for 5, 24, 26, and 28 were listed in Table 7 as TLs,
G
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source temperature, 150 °C; desolvatation temperature, 400 °C;
desolvatation gas flow, 400 L/h; cone gas flow, 10 L/h; extraction
cone, 4 RF; lens, 0.1 V; sampling cone, 30; capillary, 1.5 kV; high
resolution mode; gain, 1.0; MS function, 0.2 s per scan; 120−1000
amu in full scan, centroid mode; lock spray, leucine enkephalin 2 ng/
mL (556.2771 Da); scan time, 0.2 s with interval of 10 s and average of
5 scans; DAD, Acquity UPLC PDA detector; column, Waters Acquity
UPLC BEH C18, 1.7 μm, 2.1 mm × 50 mm, thermostated in the
Acquity UPLC column manager at 60 °C; eluent A, water containing
0.05% formic acid; eluent B, acetonitrile containing 0.05% formic acid;
gradient, 2−98% B over 3.0 min; flow rate, 0.6 mL/min; detection, UV
214 nm and MS. Melting points were measured on a Sanyo
Gallenkamp digital apparatus and are uncorrected.
studies with cells originating from various species, no further
particulars on pharmacological data were discussed herein.
These data will be presented and discussed in a dedicated
publication (paper in preparation). Since the treatment of
patients has been the ultimate goal, selection priority was based
on the activity of the antagonists on relevant CRTh2 expressing
human cells and oral bioavailability. Ultimately, 28 was selected
for further studies and proposed as a clinical development
candidate for the treatment of asthma and seasonal allergic
rhinitis. At the time of selection, 28 was among the first
hCRTh2 antagonists available, and therefore, we decided to
commence clinical profiling in order to obtain proof of concept
for CRTh2 inhibition in humans, to evaluate safety aspects, and
to gain further experience with such a novel treatment
modality, however, being well aware of the fact that the
observed blood plasma shift to lower potency, especially in the
more important cellular test systems, could compromise the
outcome of clinical efficacy. Therapeutically relevant drug levels
were expected to be reached, especially at higher dosage,
because of the excellent oral uptake of 28 and hence its high
systemic exposure. Moreover, in the course of our subsequent
discovery and development efforts more emphasis was put on
the plasma protein effect in order to identify antagonists with
highly potent inhibitory activity on CRTh2 expressing human
cells and exhibiting no or only a minor shift to lower potency in
the presence of human blood plasma and human whole blood.
The clinical program and the generated novel understanding
concerning CRTh2 antagonism in human volunteers and
patients will be discussed in future reports.
All compounds tested for biological activity are reported as having
at least 95% purity as judged by HPLC method LC(1).
General Procedure for the Synthesis of 7a−l (Scheme 1,
Stage a). To suspensions of the respective 2,3,4,5-tetrahydro-1H-
pyrido[4,3-b]indole hydrochloride salts 6a−l (18.4 mmol) in DCM
(100 mL) were added DIPEA (22.1 mmol, 2.85 g, 3.78 mL) and di-
tert-butyl dicarbonate (19.3 mmol, 4.22 g). The resulting white
solutions were allowed to stir at ambient temperature for 1 h.
Saturated aqueous NH4Cl solution was added, and the resulting
aqueous phases were extracted twice with DCM. The combined
organic phases were washed with water and brine and then dried over
MgSO4. The solvent was evaporated in vacuo, and the resulting solids
were dried under high vacuum and used as such in the next step.
tert-Butyl 3,4-Dihydro-1H-pyrido[4,3-b]indole-2(5H)-carbox-
ylate (7a, R1 = H).33 Boc protection of 6a provided 7a as a white
solid in 83% yield. LC(1)/ESI-MS tR = 1.00 min; m/z [M + H+] =
273.07. 1H NMR (CDCl3) δ: 7.83 (s), 7.45 (d, J = 7.3 Hz, 1 H), 7.31
(m, 1 H), 7.13 (m, 2 H), 4.64 (s, 2 H), 3.82 (bs, 2 H), 2.83 (bs, 2 H),
1.50 (s, 9 H). HRMS (ESI): m/z calcd for C16H21N2O2 [M + H+]
273.1603, found 273.1598.
tert-Butyl 8-Fluoro-3,4-dihydro-1H-pyrido[4,3-b]indole-
2(5H)-carboxylate (7b, R1 = 8-F).34,35 Boc protection of 6b
provided 7b as a brown solid in 96% yield. LC(1)/ESI-MS tR = 0.91
min; m/z [M + H+] = 291.20. 1H NMR (DMSO-d6) δ: 10.99 (s, 1 H),
7.28 (dd, J1 = 8.8 Hz, JH−F = 4.5 Hz, 1 H), 7.18 (dd, JH−F = 9.9 Hz, J2 =
2.4 Hz, 1 H), 6.86 (ddd, JH−F = 9.9 Hz, J1 = 8.8 Hz, J2 = 2.4 Hz, 1 H),
4.50 (s, 2 H), 3.70 (t, J = 5.7 Hz, 2 H), 2.78 (t, J = 5.7 Hz, 2 H), 1.45
(s, 9 H). HRMS (ESI): m/z calcd for C16H20N2O2F [M + H+]
291.1509, found 291.1515.
General Procedure for the Synthesis of 8a−l (Scheme 1,
Stage b). To solutions of the respective tert-butyl 3,4-dihydro-1H-
pyrido[4,3-b]indole-2(5H)-carboxylate derivatives 7a−l (1.17 mmol)
and ethyl bromoacetate (1.4 mmol, 239 mg, 0.159 mL) in DMF (3
mL) was added Cs2CO3 (2.34 mmol, 761 mg). The resulting reaction
mixtures were allowed to stir at 80 °C for 1 h. After the mixtures were
cooled, 10 mL of water and 10 mL of brine were added and the
resulting aqueous phases were extracted three times with DCM. The
combined organic phases were washed with water and brine and dried
over MgSO4. Evaporation of the solvent in vacuo yielded brown pastes
from which the pure products 8a−l were isolated by flash
chromatography on silica gel using EtOAc/n-heptane as eluent or by
precipitation from cyclohexane.
For the preparation of 8b, 8d, and 8i, acetone was used as solvent
instead of DMF and the reaction was performed at room temperature.
tert-Butyl 5-(2-Ethoxy-2-oxoethyl)-3,4-dihydro-1H-pyrido-
[4,3-b]indole-2(5H)-carboxylate (8a, R1 = H).36 Alkylation of 7a
provided 8a as a yellow oil in 58% yield. LC(1)/ESI-MS tR = 0.97 min;
m/z [M + H+] = 359.29. 1H NMR (CD3OD) δ: 7.42 (d, J = 7.7 Hz, 1
H), 7.26 (m, 1 H), 7.15 (m, 1 H), 7.07 (m, 1 H), 4.89 (s, 2 H), 4.62
(s, 2 H), 4.19 (q, J = 7.3 Hz), 3.83 (t, J = 5.6 Hz, 2 H), 2.76 (t, J = 5.6
Hz, 2 H), 1.52 (m, 9 H), 1.26 (t, J = 7.1 Hz, 3 H). 13C NMR
(CD3OD) δ: 169.4, 155.6 (bs), 137.1, 133.6 (bs), 125.5, 121.1, 119.2,
117.0, 108.5, 106.9 (bs), 80.1, 61.2, 43.8, 41.6−40.5 (m), 27.4, 21.7
(bs), 13.1. HRMS (ESI): m/z calcd for C20H27N2O4 [M + H+]
359.1971, found 359.1977.
EXPERIMENTAL SECTION
■
Reagents and solvents were used as purchased without any further
purification. Reactions were performed under inert Ar or N2
atmosphere unless stated otherwise. NMR data were recorded at
room temperature on a Bruker Avance II 400 MHz spectrometer
equipped with a BBO 5 mm probe head unless stated otherwise.
Chemical shifts (δ) are reported in parts per million (ppm) relative to
proton resonances resulting from incomplete deuteration of the NMR
solvent, e.g., for dimethylsulfoxide δ(H) 2.49 ppm, for chloroform
δ(H) 7.24 ppm. The abbreviations s, d, t, q, m, and b refer to singlet,
doublet, triplet, quartet, multiplet, and broad, respectively.
The following instruments and conditions were used. For analytical
LC(1)/ESI-MS: Dionex HPG-3200RS pump system; MS, Thermo
MSQ Plus MS detector; (DAD)/ (ELSD), DAD-3000RS detector
equipped with Sedere Sedex 85 ELS detector; column, Zorbax SB-Aq,
3.5 μm, 4.6 mm × 50 mm, 40 °C; eluent A, water containing 0.04%
TFA; eluent B, acetonitrile; gradient, 5−95% B over 1 min; flow rate,
4.5 mL/min. For analytical LC(2)/ESI-MS: Dionex HPG-3200RS
pump system; MS, Thermo MSQ Plus MS detector; DAD/ELSD,
DAD-3000RS detector equipped with Sedere Sedex 85 ELS detector;
column, Atlantis T3, 5 μm, 4.6 mm × 30 mm, 40 °C; eluent A, water
containing 0.04% TFA; eluent B, acetonitrile; gradient, 5−95% B over
1 min; flow rate, 4.5 mL/min. For analytical LC(3)/ESI-MS: Waters
Acquity binary pump system; MS, Waters Aquity SQD; DAD/ELSD,
Waters Acquity PDA and ELS detector; column, UPLC BEH C18 1.7
μm, 2.1 mm × 50 mm; eluent A, water containing 0.05% formic acid;
eluent B, acetonitrile containing 0.05% formic acid; gradient, 2−95% B
over 1.4 min and then 95−98% B over 0.4 min; flow rate, 1.2 mL/min.
For preparative HPLC: Gilson 333/334 binary high pressure gradient
pump system equipped with Sedere Sedex V.F.S variable flow splitter,
Dionex P680 isocratic make-up pump; DAD, Dionex UVD340U DAD
detector; MS, Finnigan AQA MS detector; fraction collection, Gilson
215 autosampler and fraction collector; column, Atlantis Prep C18 (30
mm × 75 mm OBD); eluent A, water containing 0.05% formic acid;
eluent B, acetonitrile; flow rate, 75 mL/min. For HR-LC−MS: Waters
Acquity binary pump system, solvent manager; MS, SYNAPT G2MS;
tert-Butyl 5-(2-Ethoxy-2-oxoethyl)-8-fluoro-3,4-dihydro-1H-
pyrido[4,3-b]indole-2(5H)-carboxylate (8b, R1 = 8-F).37 Alkyla-
H
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Journal of Medicinal Chemistry
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tion of 7b provided 8b after precipitation from cyclohexane as a white
solid in 70% yield. LC(1)/ESI-MS tR = 0.97 min; m/z [M + H+] =
377.20. 1H NMR (CDCl3) δ: 7.13 (m, 2 H), 6.94 (td, JH−F = 9.1 Hz, J1
= 9.1 Hz, J2 = 2.3 Hz, 1 H), 4.73 (s, 2 H), 4.62 (bs, 2 H), 4.23 (q, J =
7.1 Hz, 2 H), 3.87 (bs, 2 H), 2.77 (bs, 2 H), 1.53 (s, 9 H), 1.29 (t, J =
7.1 Hz, 3 H). 13C NMR (CDCl3): δ 168.4, 158.0 (d, JC−F = 235 Hz),
155.1 (bs), 135.5 (m), 133.4, 125.9 (m), 109.5 (d, JC−F = 26 Hz),
109.2 (d, JC−F = 10 Hz), 108.1 (m), 103.3 (d, JC−F = 24 Hz), 80.0,
61.8, 44.8, 41.1−40.4 (m), 28.5, 22.5 (bs), 14.2. HRMS (ESI): m/z
calcd for C20H26N2O4F [M + H+] 377.1876, found 377.1871.
General Procedure for the Synthesis of 9a−l (Scheme 1,
Stage c). To solutions of the respective tert-butyl 5-(2-ethoxy-2-
oxoethyl)-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate de-
rivatives 8a−l (2 mmol) in DCM (5 mL) were added dropwise a
solution of 4 N HCl in 1,4-dioxane (5 mL) at ambient temperature.
The resulting mixtures were allowed to stir at ambient temperature for
3 h. Then the solvents were removed under reduced pressure and the
crude solids were dried under high vacuum and used without further
purification in the next step.
eluent, as a white solid in 67% yield. LC(1)/ESI-MS tR = 1.04 min; m/
1
z [M + H+] = 467.29. H NMR (DMSO-d6), 50:50 mixture of two
rotamers, δ: 7.64 (m, 2.5 H), 7.56 (m, 4 H), 7.30 (d, J = 7.5 Hz, 3 H),
7.00−7.25 (m, 2.5 H), 4.98 (bs, 1 H), 4.77 (m, 3 H), 4.22 (m, 3 H),
3.86 (m, 1 H), 2.89 (m, 2 H), 2.71 (q, J = 7.4 Hz, 2 H), 1.28 (m, 6 H).
Ethyl 2-(2-(1-Naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido-
[4,3-b]indol-5(2H)-yl)acetate (10b1). Acylation of 9b with 1-
naphthoyl chloride provided 10b1 after precipitation from n-heptane
as a brown solid in 98% yield. LC(1)/ESI-MS tR = 0.95 min; m/z [M
+ H+] = 431.18. 1H NMR (DMSO-d6), 55:45 mixture of two
rotamers, δ: 7.88−7.98 (m, 2.55 H), 7.85 (d, J = 8.3 Hz, 0.45 H),
7.44−7.59 (m, 4 H), 7.25 (dd, JH−F = 9.3 Hz, J1 = 2.4 Hz, 0.55 H),
7.17 (dd, J1 = 8.9 Hz, JH−F = 4.2 Hz, 0.55 H), 7.12 (dd, J1 = 8.8 Hz,
JH−F = 4.1 Hz, 0.45 H), 7.00 (ddd, JH−F = 9.1 Hz, J1 = 8.9 Hz, J2 = 2.4
Hz, 0.55 H), 6.89 (ddd, JH−F = 9.0 Hz, J1 = 8.8 Hz, J2 = 2.5 Hz, 0.45
H), 6.74 (dd, JH−F = 9.3 Hz, J2 = 2.5 Hz, 0.45 H), 5.14 (m, 1.1 H), 4.78
(s, 0.9 H), 4.70 (s, 1.1 H), 4.30−4.48 (m, 1.8 H), 4.26 (q, J = 7.3 Hz,
0.9 H), 4.21 (q, J = 7.0 Hz, 1.1 H), 3.64 (t, J = 5.7 Hz, 1.1 H), 3.01 (m,
0.9 H), 2.65 (m, 1.1 H), 1.32 (t, J = 7.1 Hz, 1.35 H), 1.27 (m, 1.65 H).
Ethyl 2-(8-Fluoro-2-(thiophene-2-carbonyl)-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetate (10b3). Acylation of 9b with
thiophene-2-carbonyl chloride provided 10b3 after precipitation from
acetonitrile/diethylether as a white solid in 69% yield. LC(1)/ESI-MS
tR = 1.09 min; m/z [M + H+] = 386.96. 1H NMR (DMSO-d6) δ: 7.81
(dd, J1 = 5.0 Hz, J2 = 1.0 Hz, 1 H), 7.56 (m, 1 H), 7.43 (dd, J1 = 8.9
Hz, J2 = 4.4 Hz, 1 H), 7.32 (m, 1 H), 7.19 (dd, J1 = 5.0 Hz, J2 = 3.7 Hz,
1 H), 6.95 (ddd, J1 = 9.3 Hz, J2 = 9.3 Hz, J3 = 2.5 Hz, 1 H), 5.08 (s, 2
H), 4.85 (m, 2 H), 3.99 (t, J = 5.7 Hz, 2 H), 2.90 (m, 2 H), 1.22 (t, J =
7.1 Hz, 3 H).
Ethyl 2-(3,4-Dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetate
Hydrochloride (9a). Deprotection of 8a provided 9a as a white solid
in 88% yield. LC(1)/ESI-MS tR = 0.59 min; m/z [M + H+] = 259.28.
1H NMR (CD3OD) δ: 7.50 (d, J = 7.8 Hz, 1 H), 7.36 (m, 1 H), 7.23
(t, J = 7.7 Hz, 1 H), 7.14 (t, J = 7.6 Hz, 1 H), 5.01 (s, 2 H), 4.47 (s, 2
H), 4.23 (q, J = 7.1 Hz, 2 H), 3.66 (m, 2 H), 3.13 (t, J = 6.1 Hz, 2 H),
1.29 (t, J = 7.1 Hz, 4 H).
Ethyl 2-(8-Fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-
yl)acetate Hydrochloride (9b). Deprotection of 8b provided 9b
as a white solid in 96% yield. LC(1)/ESI-MS tR = 0.63 min; m/z [M +
1
H+] = 277.22. H NMR (DMSO-d6) δ: 9.52 (m, 2 H), 7.46 (dd, J1 =
Ethyl 2-(2-(3-Chlorobenzoyl)-8-fluoro-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetate (10b4). Acylation of 9b with
3-chlorobenzoyl chloride provided 10b4 after precipitation from
acetonitrile/diisopropylether as a white solid in 70% yield. LC(1)/
9.2 Hz, JH−F = 4.4 Hz, 1 H), 7.34 (dd, JH−F = 9.7 Hz, J1 = 2.5 Hz, 1 H),
7.00 (td, JH−F = 9.3 Hz, J1 = 9.2 Hz, J2 = 2.6 Hz, 1 H), 5.12 (s, 2 H),
4.30 (bs, 2 H), 4.16 (q, J = 7.1 Hz, 2 H), 3.48 (s, 2 H), 2.99 (t, J = 5.9
Hz, 2 H), 1.23 (t, J = 7.1 Hz, 3 H).
1
ESI-MS tR = 1.04 min; m/z [M + H+] = 415.11. H NMR (DMSO-
d6), 53:47 mixture of two rotamers, δ: 7.40−7.47 (m, 1 H), 7.28−7.40
(m, 3 H), 7.08−7.20 (m, 1.53 H), 6.86−6.99 (m, 1.47 H), 5.06 (m,
0.53 H), 4.92 (m, 0.53 H), 4.74 (s, 0.94 H), 4.69 (m, 1.06 H), 4.49 (m,
0.47 H), 4.32−4.46 (m, 1 H), 4.22 (m, 2 H), 4.03 (ddd, J1 = 12.7 Hz,
J2 = 7.0 Hz, J3 = 5.4 Hz, 0.47 H), 3.66 (m, 1 H), 2.76−2.99 (m, 1.47
H), 2.70 (m, 0.53 H), 1.28 (t, J = 7.3 Hz, 1.41 Hz), 1.25 (t, J = 7.0 Hz,
1.59 Hz).
General Procedure for the Synthesis of 10a1 to 10l2 (Scheme
1, Stage d). To cooled suspensions of the respective ethyl 2-(3,4-
dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetate hydrochloride salts
9a−l (7 mmol) in DCM (20 mL) was added DIPEA at 0 °C (21
mmol), followed by the corresponding acyl chlorides (7.7 mmol). The
resulting brown suspensions were allowed to stir at room temperature
for 1 h. DCM was added, and the resulting organic phases were first
washed with saturated aqueous NaHCO3 solution, then with water and
brine. The combined organic phases were dried over MgSO4 and
yielded brown solids after evaporation of the solvent in vacuo. The
crude products were purified by flash chromatography on silica gel,
using EtOAc/n-heptane as eluent. The various purification methods
are mentioned within the corresponding sections.
Ethyl 2-(2-(2-(1H-Indol-3-yl)acetyl)-8-fluoro-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetate (10b6). Acylation of 9b with
2-(1H-indol-3-yl)acetyl chloride provided, after column chromatog-
raphy on silica gel using EtOAc/n-heptane as eluent, 10b6 as a
colorless resin in 81% yield. LC(1)/ESI-MS tR = 0.90 min; m/z [M +
1
H+] = 434.03. H NMR (CDCl3), 55:45 mixture of two rotamers, δ:
8.33 (m, 0.45 H), 8.12 (m, 0.55 H), 7.68 (m, 1 H), 7.37 (d, J = 7.9 Hz,
0.55 H), 7.05−7.28 (m, 4.45 H), 6.87−7.04 (m, 2 H), 4.84 (s, 0.9 H),
4.71 (s, 1.1 H), 4.68 (s, 1.1 H), 4.63 (s, 0.9 H), 4.21 (m, 2 H), 4.07 (t,
J = 5.5 Hz, 1.1 H), 3.98 (s, 2 H), 3.83 (t, J = 5.3 Hz, 0.9 H), 2.79 (m,
1.1 H), 2.52 (s, 0.9 H), 1.27 (m, 3 H).
General Procedure for the Synthesis of 5 and 11−36
(Scheme 1, Stage e). To solutions of the respective esters 10a−l
(11 mmol) in THF (100 mL) was added 0.2 M aqueous NaOH
solution (58 mL, 11.7 mmol). The resulting mixtures were allowed to
stir at ambient temperature for 30 min. The reaction mixtures were
extracted twice with diethyl ether. The pH of the aqueous phase was
set to pH 2 by slow addition of concentrated aqueous HCl solution.
The resulting acidic aqueous phases were extracted three times with
DCM. The DCM phases were combined and dried over Na2SO4. The
volatiles were removed under reduced pressure, and the obtained
solids were recrystallized from acetonitrile or purified by preparative
HPLC.
Ethyl 2-(2-Benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-
yl)acetate (10a1). Acylation of 9a with benzoyl chloride provided
10a1 after preparative HPLC purification as a beige solid in 70% yield.
1
LC(1)/ESI-MS tR = 0.89 min; m/z [M + H+] = 363.11. H NMR
(CDCl3) δ: 7.40−7.61 (m, 5 H), 7.02−7.32 (m, 3 H), 5.01 (bs, 1 H),
4.78 (s, 2 H), 4.70 (bs, 1 H), 4.24 (q, J = 7.1 Hz, 3 H), 4.22 (bs, 1H),
3.81 (bs, 1 H), 2.79−3.00 (m, 2 H), 1.30 (t, J = 7.1 Hz, 3 H).
Ethyl 2-(2-(1-Naphthoyl)-3,4-dihydro-1H-pyrido[4,3-b]indol-
5(2H)-yl)acetate (10a3). Acylation of 9a with 1-naphthoyl chloride
provided crude 10a3 as yellow solid in 97% yield. LC(1)/ESI-MS tR =
1
1.14 min; m/z [M + H+] = 413.16. H NMR (DMSO-d6), 60:40
mixture of two rotamers, δ: 8.02 (m, 2 H), 7.76 (m, 1 H), 7.64−7.40
(m, 5.4 H), 7.37 (d, J = 8.2 Hz, 0.4 H), 7.16 (m, 0.6 H), 7.10 (m, 0.6
H), 7.03 (m, 0.6 H), 6.84 (t, J = 7.4 Hz, 0.4 H), 5.10 (s, 0.8 H), 4.99
(m, 2.2 H), 4.36 (m, 1 H), 4.19 (m, J = 7.1 Hz, 0.8 H), 4.14 (q, J = 7.1
Hz, 1.2 H), 4.08 (m, 0.8 H), 3.51 (t, J = 5.6 Hz, 1 H), 2.97 (m, 1 H),
2.68 (m, 1 H), 2.55 (m, 1 H), 1.25 (t, J = 7.1 Hz, 1.2 H), 1.19 (t, J =
7.1 Hz, 1.8 H). The compound was used without further purification
in the next step.
Ethyl 2-(2-(4′-Ethyl-[1,1′-biphenyl]-4-carbonyl)-3,4-dihydro-
1H-pyrido[4,3-b]indol-5(2H)-yl)acetate (10a4). Acylation of 9a
with 4′-ethyl-[1,1′-biphenyl]-4-carbonyl chloride provided 10a4 after
column chromatography on silica gel, using EtOAc/n-heptane as
2-(2-Benzoyl-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)-
acetic Acid (5). Saponification of 10a1 provided pure 5 after
recrystallization from acetonitrile as an orange solid in 73% yield, mp
225.8−226.9 °C (dec). LC(1)/ESI-MS tR = 0.77 min; m/z [M + H+]
1
= 335.06. H NMR (DMSO-d6), 60:40 mixture of two rotamers, δ:
13.04 (bs, 1 H), 7.44−7.57 (m, 5.6 H), 7.40 (m, 1 H), 7.27 (m, 0.4 H),
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7.00−7.18 (m, 1.6 H), 6.97 (bs, 0.4 H), 4.94 (s, 2 H), 4.82 (bs, 1.2 H),
4.61 (bs, 0.8 H), 4.03 (bs, 0.8 H), 3.64 (bs, 1.2 H), 2.81 (s, 2 H). 13C
NMR (DMSO-d6) δ: 170.9, 170.3 (bs), 137.2 (bs), 136.8 (bs), 134.2
(s), 130.1 (bs), 129.0, 127.5 (bs), 127.1 (bs), 121.5, 119.6, 118.0 (bs),
109.9, 106.4, 45.3 (bs), 45.1 (bs), 44.5, 22.9 (bs), 22.0 (bs). HRMS
(ESI): m/z calcd for C20H19N2O3 [M + H+] 335.1395, found
335.1396.
44.73, 44.70, 44.5, 44.4, 39.5, 39.3, 23.1, 22.3. HRMS (ESI): m/z calcd
for C24H20N2O3F [M + H+] 403.1458, found 403.1458.
2-(2-(2-(1H-Indol-3-yl)acetyl)-8-fluoro-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetic Acid (33). Saponification of
10b6 provided pure 33 after preparative HPLC as a beige foam in 88%
1
yield. LC(1)/ESI-MS tR = 0.79 min; m/z [M + H+] = 406.11. H
NMR (DMSO-d6), 60:40 mixture of two rotamers, δ: 12.75 (m, 1 H),
10.92 (s, 0.6 H), 10.86 (s, 0.4 H), 7.61 (m, 1 H), 7.32−7.44 (m, 2 H),
7.24−7.32 (m, 2 H), 7.07 (m, 1 H), 6.95 (m, 2 H), 4.92 (s, 0.8 H),
4.90 (s, 1.2 H), 4.80 (s, 0.8 H), 4.65 (s, 1.2 H), 3.88 (m, 4 H), 2.69 (d,
J = 5.1 Hz, 2 H). 13C NMR (DMSO-d6) δ: 170.8, 170.4, 170.3, 157.5
(d, JC−F = 232 Hz), 157.5 (d, JC−F = 231 Hz), 137.0, 136.6, 136.5,
136.4, 133.9, 133.8, 127.8, 127.7, 125.7 (d, JC−F = 10 Hz), 125.4 (d,
JC−F = 10 Hz), 124.0, 123.9, 121.5, 121.4, 119.3, 119.2, 118.9, 118.8,
111.8, 111.7, 110.8, 110.7, 109.0 (d, JC−F = 26 Hz), 108.7, 108.6, 107.1
(d, JC−F = 4 Hz), 107.0 (d, JC−F = 4 Hz), 103.2 (d, JC−F = 24 Hz),
103.1 (d, JC−F = 24 Hz), 44.7, 43.4, 43.0, 39.1, 31.6, 31.5, 23.0, 22.1.
HRMS (ESI): m/z calcd for C23H21N3O3F [M + H+] 406.1567, found
406.1564.
2-(8-Fluoro-2-(thiophene-2-carbonyl)-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetic Acid (22). Saponification of
10b3 provided pure 22 after recrystallization from acetonitrile as a
white solid in 82% yield, mp 239.4−241.9 °C (dec). LC(1)/ESI-MS tR
1
= 0.78 min; m/z [M + H+] = 358.94. H NMR (DMSO-d6) δ: 13.09
(m, 1 H), 7.81 (dd, J1 = 5.0 Hz, J2 = 0.7 Hz, 1 H), 7.57 (d, J = 0.3 Hz,
1 H), 7.43 (dd, J1 = 8.9 Hz, J2 = 4.4 Hz, 1 H), 7.31 (d, J = 8.8 Hz, 1
H), 7.19 (dd, J1 = 4.9 Hz, J2 = 3.8 Hz, 1 H), 6.95 (ddd, J1 = 9.3 Hz, J2 =
9.3 Hz, J2 = 2.4 Hz, 1 H), 4.97 (s, 2 H), 4.85 (bs, 2 H), 4.00 (t, J = 5.6
Hz, 2 H), 2.87 (s, 2 H). 13C NMR (DMSO-d6) δ: 170.8, 163.5, 157.6
(d, JC−F = 232 Hz), 138.0, 136.6, 133.9, 130.3 (bs), 129.6 (bs), 127.8,
125.4 (d, JC−F = 10 Hz), 110.9 (d, JC−F = 10 Hz), 109.2 (d, JC−F = 26
Hz), 106.7 (d, JC−F = 4 Hz), 103.2 (d, JC−F = 24 Hz), 44.7, 22.7 (bs).
HRMS (ESI): m/z calcd for C18H16N2O3FS [M + H+] 359.0865,
found 359.0863.
2-(2-(4′-Ethyl-[1,1′-biphenyl]-4-carbonyl)-3,4-dihydro-1H-
pyrido[4,3-b]indol-5(2H)-yl)acetic Acid (35). Saponification of
10a4 provided pure 35 after precipitation from acetonitrile as a beige
foam in 79% yield. LC(1)/ESI-MS tR = 0.94 min; m/z [M + H+] =
439.00. 1H NMR (DMSO-d6), 60:40 mixture of two rotamers, δ: 13.06
(m, 1 H), 7.76 (d, J = 7.9 Hz, 2 H), 7.64 (d, J = 8.1 Hz, 2 H), 7.55 (m,
2.5 H), 7.21−7.48 (m, 3.5 H), 6.83−7.20 (m, 2 H), 4.95 (bs, 2 H),
4.80 (bs, 1.2 H), 4.73 (bs, 0.8 H), 4.05 (bs, 0.8 H), 3.75 (bs, 1.2 H),
2.85 (bs, 2 H), 2.67 (q, J = 7.5 Hz, 2 H), 1.23 (t, J = 7.6 Hz, 3 H). 13C
NMR (DMSO-d6) δ: 170.9, 170.2 (bs), 144.1, 141.8, 137.2 (m), 135.4
(m), 134.3 (m), 129.0, 128.3−127.9 (m), 127.2, 127.0 (bs), 125.4
(bs), 121.5 (bs), 119.6 (bs), 117.7−118.0 (m), 109.9, 106.4 (bs), 45.2
(m, 2 C atoms), 44.6, 28.3, 23.0 (bs), 22.0 (bs), 16.0. HRMS (ESI):
m/z calcd for C28H27N2O3 [M + H+] 439.2021, found 439.2021.
Solubility Measurements. For each compound 40 μL of 10 mM
DMSO stock solution was dispensed into a Millipore MSHVN4510
Multiscreen 96-well filter plate for solubility by means of a BIOMEK
NXP (Beckman Coulter) laboratory automated workstation. The
solvents were evaporated under reduced pressure in a Hettich AG
Combidancer. Each well was filled with 200 μL of water, 60 mM
citrate buffer, pH4, or 70 mM phosphate buffer, pH7. The mixture was
stirred on a Eppendorf Thermomixer 5355 comfort at 300 rpm at 25
°C for 24 h. The resulting biphasic system was filtered under reduced
pressure using a Millipore MAVM0960R vacuum manifold. The
filtrates were collected into a 96-well Thermo Scientific AB0600PCR
plate. Solubility was determined by measuring the concentration of the
solutions using a high pressure mixing Shimadzu modular HPLC
system with a Phenomenex column Synergi Polar-RP 4 μm, 80 Å, 2.0
cm × 50 mm warmed at 50 °C: eluent A, water containing 0.05%
formic acid; eluent B, acetonitrile containing 0.05% formic acid;
gradient, 5−100% A nonlinear gradient over 2.4 min; flow rate of 1.5
mL/min; injection volume, 5 μL. UV detection occurred at 250 nm.
Concentrations were determined by comparison of the peak area with
the calibration curve.
2-(2-(3-Chlorobenzoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-
b]indol-5(2H)-yl)acetic Acid (24). Saponification of 10b4 provided
pure 24 after recrystallization from acetonitrile as a white solid in 89%
yield, mp 251.8−256.2 °C (dec). LC(1)/ESI-MS tR = 0.83 min; m/z
1
[M + H+] = 386.87. H NMR (DMSO-d6), 60:40 mixture of two
rotamers, δ: 7.57 (m, 1 H), 7.50 (m, 2 H), 7.41 (d, J = 7.5 Hz, 1 H),
7.25−7.38 (m, 1.6 H), 7.03−7.17 (m, 0.4 H), 6.79−6.95 (m, 1 H),
4.78 (s, 1.2 H), 4.63 (s, 2 H), 4.56 (s, 0.8 H), 3.99 (s, 0.8 H), 3.64 (s,
1.2 H), 2.81 (m, 2 H). 13C NMR (DMSO-d6) δ: 170.8, 168.7 (bs),
157.6 (d, JC−F = 232 Hz), 138.9 (bs), 138.6 (m), 136.9 (bs), 136.3
(bs), 133.9, 133.8, 131.0, 130.3−129.9 (m), 127.3 (bs), 127.0 (bs),
126.1 (bs), 125.7−125.2 (m), 110.9 (d, JC−F = 10 Hz), 109.2 (d, JC−F
=
26 Hz), 106.5 (bs), 103.2 (d, JC−F = 24 Hz), 45.1 (bs), 44.9 (bs), 44.7,
23.0 (bs), 22.6 (bs). HRMS (ESI): m/z calcd for C20H17N2O3ClF [M
+ H+] 387.0911, found 387.0910.
2-(2-(1-Naphthoyl)-3,4-dihydro-1H-pyrido[4,3-b]indol-
5(2H)-yl)acetic Acid (26). Saponification of 10a3 provided pure 26
after recrystallization from acetonitrile as a light yellow solid in 73%
yield, mp 218.4−219.4 °C (dec). LC(1)/ESI-MS tR = 0.82 min; m/z
1
[M + H+] = 385.03. H NMR (DMSO-d6), 65:35 mixture of two
rotamers, δ: 13.03 (m, 1 H), 8.02 (m, 2 H), 7.75 (m, 1 H), 7.39−7.63
(m, 5.35 H), 7.36 (d, J = 8.4 Hz, 0.35 H), 7.15 (t, J = 7.2 Hz, 0.65 H),
7.09 (m, 0.65 H), 7.03 (m, 0.65 H), 6.83 (t, J = 7.5 Hz, 0.35 H), 5.06
(d, J = 15.5 Hz, 0.65 H), 4.99 (m, 1.35 H), 4.90 (m, 1.35 H), 4.42 (m,
0.35 H), 4.31 (m, 0.65 H), 4.08 (m, 0.35 H), 3.51 (t, J = 5.4 Hz, 1.35
H), 2.96 (m, 0.70 H), 2.68 (m, 0.65 H), 2.56 (m, 0.65 H). 13C NMR
(DMSO-d6) δ: 170.9, 169.2, 137.3, 137.1, 135.2, 135.0, 134.1, 133.5,
133.5, 129.6, 129.5, 129.4, 129.3, 128.9, 128.9, 127.5, 127.0, 126.9,
126.0, 125.9, 125.5, 125.1, 125.0, 124.9, 124.1, 123.9, 121.6, 121.4,
119.7, 119.5, 118.0, 117.6, 109.9, 109.8, 106.5, 106.4, 44.6 (bs), 44.5,
39.3, 23.0, 22.2. HRMS (ESI): m/z calcd for C24H21N2O3 [M + H+]
385.1552, found 385.1551.
2-(2-(1-Naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]-
indol-5(2H)-yl)acetic Acid (28). Saponification of 10b1 provided
pure 28 after recrystallization from acetonitrile as a beige solid in 93%
yield, mp 224.0 °C. LC(1)/ESI-MS tR = 0.83 min; m/z [M + H+] =
403.09. 1H NMR (DMSO-d6), 65:35 mixture of two rotamers, δ: 8.02
(m, 2 H), 7.76 (d, J = 7.8 Hz, 0.65 H), 7.72 (m, 0.35 H), 7.49−7.64
(m, 3.35 H), 7.35−7.49 (m, 2.35 H), 6.98 (ddd, JH−F = 9.3 Hz, J1 = 9.3
Hz, J2 = 2.4 Hz, 0.65 H), 6.88 (m, 0.65 H), 4.85−5.14 (m, 3.3 H), 4.42
(m, 0.35 H), 4.32 (m, 0.7 H), 4.06 (m, 0.35 H), 3.50 (t, J = 5.5 Hz, 1.3
H), 2.95 (m, 0.70 H), 2.68 (m, 0.65 H), 2.58 (m, 0.65 H). 13C NMR
hCRTh2 Radioligand Binding Assay. The CRTh2 binding assay
was established based on previous reports.38−40 Membranes of
HEK293 cells with recombinant expression of the human CRTH2
receptor were generated. Cells were detached from culture plates in 5
mL of buffer A (5 mM Tris, 1 mM MgCl2, 0.1 mM PMSF, 0.1 mM
phenanthroline, pH 7.4) per plate using a rubber policeman,
transferred into centrifugation tubes, and frozen at −80 °C. After
thawing, the cells were fragmented by homogenization with a Polytron
cell homogenizer for 30 s. The membrane fragments were collected by
centrifugation at 30000g for 20 min and resuspended in buffer B (75
mM Tris, 25 mM MgCl2, 250 mM saccharose, pH 7.4). Aliquots were
stored at −20 °C.
(DMSO-d6) δ: 170.7, 169.2, 157.7 (d, JC−F = 232 Hz), 157.4 (d, JC−F
=
233 Hz), 137.1, 136.2, 135.1, 134.9, 134.0, 133.8, 133.5, 129.6, 129.5,
129.4, 129.3, 128.9, 128.8, 127.5, 127.4, 127.0, 126.9, 126.0, 125.9,
125.7 (d, JC−F = 10 Hz), 125.2, 125.1, 125.0, 124.1, 123.9, 110.9 (d,
JC−F = 10 Hz), 110.8 (m), 109.3 (d, JC−F = 26 Hz), 109.1 (d, JC−F = 26
Hz), 106.7 (m), 103.3 (d, JC−F = 23 Hz), 103.0 (d, JC−F = 23 Hz),
The binding assay was performed in a final assay volume of 250 μL.
In each well, an amount of 75 μL of buffer C (50 mM Tris, pH 7.4,
100 mM NaCl, 1 mM EDTA, 0.1% protease free BSA, 0.01% NaN3)
was mixed with 50 μL of [3H]PGD2 (2.5 nM, 220 000 dpm/well,
Amersham Biosciences, TRK734) and 25 μL of a solution of test
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Journal of Medicinal Chemistry
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compound in buffer C containing 10% DMSO. The binding assay was
started by adding 100 μL of a membrane suspension in buffer C,
reaching a final concentration of 80 μg of membrane protein per well.
Nonspecific binding was determined in the presence of 10 μM PGD2.
This binding assay mix was incubated at room temperature for 90 min
and then filtered through a GF/C filter 96-well plate which was
presoaked in 0.5% PEI for 3 h. The filter wells were washed three
times with ice cold binding buffer C. Then Microscint-40 (Packard, 40
μL per well) was added and the receptor bound radioactivity was
quantified by scintillation counting in a “TopCount” benchtop
microplate scintillation counter (Packard).
protocol (Axis-Shield). In brief, anticoagulated whole blood was
layered onto a Polymorphprep gradient (density, 1.113 g/mL) and
centrifuged at 500g for 30 min. The polymorphonuclear fraction was
harvested and depleted for erythrocytes by hypotonic saline lysis.
The polymorphonuclears (PMNs) were resuspended in assay buffer
composed of PBS with Ca2+/Mg2+ supplemented with 0.1% BSA, 10
mM Hepes, and 10 mM glucose, pH 7.4, with 106 cells/mL and
stained with CD49d-APC (allophycocyanin conjugated mouse anti-
human VLA-4) for 1 h at room temperature. Next, 45 μL of human
plasma was mixed with 10 μL of assay buffer and 30 μL of PMNs (1.3
× 107 cells/mL) were mixed. The PMNs were then activated by
addition of 15 nM PGD2 and incubated for 5 min at 37 °C. The
activation was stopped by addition of 100 μL of ice cold 1%
paraformaldehyde. The samples were analyzed with a FACSAria flow
cytometer (BD Biosciences). Eosinophils were identified based on the
fluorescence signal (CD49d-APC (Allophycocyanin (APC)) and side
scatter (SSC). The data recorded in our experiments were gated to
detect the eosinophil cell population only. Eosinophil activation was
quantified based on increase in forward scatter (FSC). Activation was
expressed as the percent of cells with a forward scatter larger than that
of nonactivated eosinophils.
hCRTh2 Intracellular Calcium Liberation (FLIPR) Assay.
Human embryonic kidney cells (HEK-293, DSMZ) stably expressing
the human CRTh2 receptor under the control of the CMV promoter
were grown to near confluency in DMEM medium (1 g/L glucose,
Gibco) supplemented with 10% fetal calf serum (Pan) containing
penicillin and streptomycin (100 units/mL each, Gibco) under
standard mammalian cell culture conditions at 37 °C in a humidified
atmosphere of 5% CO2. Cells were detached from culture dishes with a
cell dissociation buffer (0.02% EDTA in PBS, Gibco) for 1 min,
washed off with DMEM without phenol red (Gibco), and collected by
centrifugation with 200g at room temperature for 5 min. The cells
were incubated with 4 μM Fluo-4 AM ester (Teflabs) in DMEM
(without phenol red) in 20 mM HEPES, pH 7.2 (Gibco), at 37 °C in a
humidified atmosphere of 5% CO2 for 40 min. Then they were washed
with and resuspended in assay buffer (HBSS with 20 mM HEPES, pH
7.2). Resuspended cells, 100 000 in 60 μL per well, were seeded onto a
384-well clear-bottom black assay plate (Greiner), sedimented by
centrifugation, and incubated at room temperature for 15 min. Stock
solutions of test compounds were made up at a concentration of 10
mM in DMSO and serially diluted in assay buffer to concentrations
required for dose−response curves. PGD2 (Biomol, Plymouth
Meeting, PA) is used as an agonist. A FLIPRII instrument (Molecular
Devices) was operated following the manufacturer’s instructions. Test
compound (10 μL) was added to each well and incubated for 5 min.
Cells were activated by a final concentration of 50 nM PGD2 (Biomol,
Plymouth Meeting, PA) dissolved in the assay buffer supplemented
with 0.05% bovine serum albumin (fatty acid content of <0.02%,
Sigma). Fluorescence emission was recorded during test compound
and PGD2 addition, and emission peak values above base level after
PGD2 addition were exported. Values were normalized to high-level
control (no antagonist added) after subtraction of baseline value
control (no PGD2 added). The IC50 values were calculated by four-
parameter dose−response curve fitting (XLfit, ISBS).
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures and analytical data (Tables
S1−S5) of synthesized compounds 11−21, 23, 25, 27, 29−32,
34, 36 and the respective precursors; method for the
determination of the log D values; experimental details for
the biological assays used to assess the receptor selectivity, for
the cytochrome P450 enzyme inhibition assays, and for the
metabolic stability studies; and the detailed description of the
pharmacokinetic studies in the rat and the dog. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +41-61-565-6208. E-mail: heinz.fretz@actelion.com.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Intracellular cAMP Assay. Cells were cultured in 96-well plates to
reach about 80% confluency (10 000 cells seeded 3 days before
experiment) using the same growth conditions as described above.
The culture medium was removed and the stimulation started by
addition of 100 μL/well DMEM containing 50 μM IBMX (3-isobutyl-
1-methylxanthine), various concentrations of test compound, 100 nM
PGD2, and 10 μM forskolin. After a 20 min incubation time at 37 °C
intracellular cAMP concentrations were quantified using the Tropix
cAMP-screen chemiluminescent ELISA system (Applied Biosystems)
following the instructions provided in the kit. Briefly, cells were
solubilized by addition of 100 μL of lysis buffer (provided in assay kit)
and incubated for 30 min at 37 °C. Then an amount of 60 μL per well
of the cAMP−alkaline phosphatase conjugate, diluted 1:100, followed
by 60 μL of anti-cAMP antibody was added. After 1 h of incubation,
the solution was removed and washed six times with washing buffer.
Then 100 μL/well CSPD/Sapphire-II RTU substrate enhancer
solution was added and incubated for 30 min. The signal was
quantified in the luminometer PHERAstar (BMG Labtech). Absolute
cAMP concentrations were then determined using a standard cAMP
curve that was generated in parallel.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank S. Koch, D. Lotz, A. Jonuzi, P. Risch, B.
Butscha, J. Giller, B. Lack, M. Baumann, and S. Brand for
technical support; B. Capeleto for physicochemical and
Francois Le Goff for stability measurements in different
solution media; R. Bravo for formulation work; S. Delahaye
and H. Kletzl for providing DMPK data; M. Holdener for
supportive discussions; and J. Williams for proofreading the
manuscript.
ABBREVIATIONS USED
■
APC, allophycocyanin; Boc2O, di-tert-butyl dicarbonate; bs,
broad singlet; CHO, chinese hamster ovary; Cl, clearance; Clint,
intrinsic clearance; CRTh2, chemoattractant receptor-homolo-
gous molecule expressed on Th2 cells; Da, dalton; DAD, diode-
array detector; DIPEA, diisopropylethylamine; DMEM,
Dulbecco’s modified Eagle medium; ELSD, evaporative light
scattering detector; EtOAc, ethyl acetate; FCS, fetal calf serum;
Shape Change of Human Eosinophils. After healthy volunteers
had given informed consent, blood samples were drawn by
venipuncture according to the protocol approved by the ethics
committee of Basel (Switzerland). Polymorphonuclear leukocytes
(containing eosinophils, basophils, and neutrophils) were isolated
using the Polymorphprep method according to the manufacturer’s
f
HSA, shift factor in human serum albumin; FLIPR, fluorometric
imaging plate reader; FSC, forward scatter; f Sel, selectivity
K
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factor; HBSS, Hank’s balanced salt solution; hDP1, human
prostaglandin D2 receptor subtype 1; hEP1, human prostaglan-
din E2 receptor subtype 1; hEP2, human prostaglandin E2
receptor subtype 2; hEP3, human prostaglandin E2 receptor
subtype 3; hEP4, human prostaglandin E2 receptor subtype 4;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
hESC, human eosinophil shape change assay; HLM, human
liver microsomes; HSA, human serum albumin; hTP, human
thromboxane A2 receptor; HTRF, homogeneous time-resolved
fluorescence; IBMX, 3-isobutyl-1-methylxanthine; IL, interleu-
kin; LE, ligand efficiency; log D7.4, measured log D at pH 7.4;
mmol, millimole; PDA, photodiode array; PEI, polyethylenei-
mine; PG, propylene glycol; PGD2, prostaglandin D2; PGDS,
prostaglandin synthase; PMSF, phenylmethanesulfonyl fluo-
ride; PMN, polymorphonuclear; RHepa, rat hepatocyte; RLM,
rat liver microsome; SGF, simulated gastric fluid; SIF, simulated
intestinal fluid; SSC, side scatter; Th2, T-helper 2; TL, traffic
light; TXA2, thromboxane A2; Vss, volume of distribution at
steady state
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