Discovery of a new class of potent, selective, and orally bioavailable CRTH2 (DP2) receptor antagonists for the treatment of allergic inflammatory diseases

Article abstract of DOI:10.1021/jm701383e

A novel chemical class of potent chemoattractant receptor-homologous expressed on Th2 lymphocytes (CRTH2 or DP2) antagonists is reported. An initial and moderately potent spiro-indolinone compound (5) was found during a high-throughput screening campaign.

Full text of DOI:10.1021/jm701383e

J. Med. Chem. 2008, 51, 2227–2243
2227
Discovery of a New Class of Potent, Selective, and Orally Bioavailable CRTH2 (DP2) Receptor
Antagonists for the Treatment of Allergic Inflammatory Diseases#
Stefano Crosignani,† Patrick Page,† Marc Missotten,† Véronique Colovray,† Christophe Cleva,† Jean-François Arrighi,†
John Atherall,§ Jackie Macritchie,§ Thierry Martin,† Yves Humbert,† Marilène Gaudet,† Doris Pupowicz,† Maurizio Maio,†
Pierre-André Pittet,† Lucia Golzio,‡ Claudio Giachetti,‡ Cynthia Rocha,‡ Gérald Bernardinelli,4 Yaroslav Filinchuk,
Alexander Scheer,† Matthias K. Schwarz,† and André Chollet*,†
Merck Serono International S.A., 9 chemin des Mines, 1202 GeneVa, Switzerland, Istituto di Ricerche Biomediche A. Marxer, Merck Serono,
I-10010 Colleretto Giacosa, Italy, BioFocus DPI, Saffron Walden, Essex CB10 1XL, U.K., Laboratory of X-Ray Crystallography, UniVersity of
GeneVa, 1211 GeneVa 4, Switzerland, and Swiss-Norwegian Beam Line, ESRF, F-38043 Grenoble, France
ReceiVed NoVember 5, 2007
A novel chemical class of potent chemoattractant receptor-homologous expressed on Th2 lymphocytes
(CRTH2 or DP2) antagonists is reported. An initial and moderately potent spiro-indolinone compound (5)
was found during a high-throughput screening campaign. Structure-activity relationship (SAR) investigation
around the carboxylic acid group revealed that changes in this part of the molecule could lead to a reversal
of functional activity, yielding weakly potent agonists. SAR investigation of the succinimide functional
group led to the discovery of several single-digit nanomolar antagonists. The potency of these compounds
was confirmed in a human eosinophil chemotaxis assay. Moreover, compounds (R)-58 and (R)-71 were
shown to possess pharmacokinetic properties suitable for development as an orally bioavailable drug.
Introduction
are associated with a higher degree of bronchial hyper-
responsiveness and the occurrence of fatal asthma.8
Prostaglandin D2 (PGD2, 1, Figure 1) is the major prostanoid
species produced by mast cells in response to stimulation by
allergens and plays a key role in inflammatory processes. PGD2
exerts its effect through two high-affinity G-protein-coupled
receptors: the classical DP receptor and the recently discovered
chemoattractant receptor-homologous expressed on Th2 lym-
phocytes (CRTH2 also known as DP2).a In humans, CRTH2 is
predominantly expressed by Th2 cells, eosinophils, and baso-
phils all known to play a key role in allergic diseases.1
Activation of the Gi-coupled CRTH2 by prostaglandin D2
(PGD2) or the selective agonist DK-PGD2 (2) (Figure 1)
stimulates chemotaxis of human Th2 cells, eosinophils, and
basophils, in vitro and in vivo,2,3 suggesting that the CRTH2
receptor may directly mediate the recruitment of inflammatory
cells in allergic diseases such as asthma, allergic rhinitis, and
atopic dermatitis. Severity of atopic dermatitis has been cor-
related with increased numbers of circulating Th2 cells.4 Atopic
dermatitis subjects or those with sensitivity to pollen or to dust-
mite antigens have significantly increased levels of CD4+ T
cells expressing CRTH2 receptors.2,5,6 CRTH2 is expressed by
Ag-specific Th2 cells in allergic individuals, supporting the
notion that CRTH2 receptor is important in the recruitment of
Th2 in allergic diseases in humans.7 Finally, sequence variants
of the CRTH2 receptor that confer increased mRNA stability
Further data in animal models likewise point to a potential
role for CRTH2 modulation in allergic disorders. CRTH2
receptor activation induces the release of eosinophils from
guinea-pig bone marrow.9 In mouse models of allergic asthma
and atopic dermatitis, CRTH2 receptor activation promotes
eosinophilia and exacerbates pathology.10 Intratracheal admin-
istration of PGD2 in rats, pretreated with systemic IL-5 injection
or not, induces eosinophil trafficking into the airways. This effect
is mimicked by selective CRTH2 receptor agonists but not by
a DP (DP1) selective agonist.3,11 Inhibition by selective small
molecules of the CRTH2 receptor but not of the DP or TP
receptor abolishes inflammatory responses in a mouse model
of contact hypersensitivity.12 Quite surprisingly, mice lacking
the functional gene that encodes CRTH2 show enhanced
eosinophil recruitment and increased IL-5 production by sple-
nocytes, suggesting that CRTH2 limits rather than enhances
inflammation.13 Recently, it has been postulated that DP and
CRTH2 might have opposite effects in complex inflammatory
processes.14
PGD2 is enzymatically and nonenzymatically metabolized
into many different products, several of which are biologically
active.14,15 The thromboxane A2 antagonist ramatroban (3)
(Figure 1), a drug marketed for allergic rhinitis, was shown to
be also a potent CRTH2 antagonist. In addition, the finding that
indomethacin (4) (Figure 1) was able to activate CRTH2, among
other pharmacological actions, was the starting point for the
discovery of other indole acetic acid derivatives and further
spurred much effort in the pharmaceutical industry aimed at
discovering selective agents to modulate its action.16 The
availability of highly selective ligands will provide new insights
on the role of CRTH2 in inflammatory processes.
#
Dedicated to the memory of Dr. Serge Halazy.
* To whom correspondence should be addressed. Phone: +41224149826.
Fax: +41224149565. E-mail: andre.chollet@merckserono.net.
†
Merck Serono International S.A.
BioFocus DPI.
Merck Serono.
University of Geneva.
§
‡
4
ESRF.
a Abbreviations: ADME, absorption-distribution-metabolism-excretion;
CRTH2, chemoattractant receptor-homologous expressed on Th2 lympho-
cytes, also known as DP2; DMF, dimethylformamide; DMSO, dimethyl
sulfoxide; NOE, nuclear Overhauser effect; Papp, permeability coefficient;
PGD2, prostaglandin D2; SAR, structure-activity relationship.
We set out to discover and develop a new class of potent,
selective, and orally active DP2 antagonists as potential new
10.1021/jm701383e CCC: $40.75
2008 American Chemical Society
Published on Web 03/05/2008

2228 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
Table 1. Spiro-indolinone Compounds from High-Throughput Screening
Campaign
Figure 1. Structures of PGD2 (1), selective CRTH2 agonist DK-PGD2
(2), ramatroban (3), indomethacin (4), and a new spiro-indolinone
compound (5) identified as a primary screening hit.
agents for the pharmacological management of allergic and
inflammatory diseases.
Results and Discussion
Primary Screening Results. A high-throughput screening
campaign based on a radioligand binding assay was performed
on 19 200 compounds selected for their potential to interact with
G-protein-coupled receptors. Several spiro-indolinones were
found as positive and confirmed in a dose-response IC50
determination experiment. In particular, it appeared that a
carboxylic acid moiety was present in the most potent com-
pounds (Table 1). The most potent compound was 5 with Ki
values of 196 and 150 nM for human and mouse CRTH2,
respectively, in a competitive 3H-PGD2 binding assay. The very
preliminary structure-activity relationship (SAR) that could be
obtained from this limited set of compounds also suggested that
the presence of a lipophilic substituent on the succinimide
nitrogen could have a beneficial effect, as compared to that of
a simple methyl group or nonaromatic substituent (e.g., 10, Ki
) 3829 nM).
Compound 5 was further evaluated in a receptor functional
assay and assessed for early absorption-distribution-
metabolism-excretion (ADME ) properties (Table 2). Testing
of the compound in a GTPγS binding assay showed antagonist
activity (IC50 ) 418 nM) and even inverse agonism (-11%
stimulation at 20 µM). The in vitro early ADME profile of 5
showed a remarkable stability toward oxidative metabolism in
both rat and human liver microsomes but a poor permeability
in the Caco-2 cell assay, as expected for a carboxylic acid. No
inhibition effect on five major cytochrome P450 isoforms could
be observed at 10 µM. The compound was shown to be more
than 100-fold selective against other prostanoid receptors and
also on a panel of 50 receptors and ion-channel pharmacological
targets.
a Competitive displacement radioligand binding assay on human CRTH2
(see Experimental Section).
Table 2. Pharmacological and Early in Vitro ADME Profile of Hit 5
Ki bindinga (hCRTH2)
Ki bindingb (mCRTH2)
IC50 GTPγSc (hCRTH2)
Cl (h-LM)d
196 ( 124 nM
150 nM
418 ( 95 nM
0 µL/min/mg protein
9 µL/min/mg protein
>10 µM
Cl (r-LM)d
IC50 (1A2)e
IC50 (2C19)e
>10 µM
IC50 (2C9)e
>10 µM
IC50 (2D6)e
>10 µM
IC50 (3A4)e
>10 µM
Caco-2 (Papp)f
3.0 × 10-6 cm/s
a Competitive displacement radioligand binding assay on human CRTH2.
b Competitive displacement radioligand binding assay on murine CRTH2.
c GTPγS radioligand binding assay. d Microsome stability experiment. h-LM,
human liver microsomes; r-LM, rat liver microsomes. e Inhibition of
cytochrome P450 isoforms. f Caco-2 cell permeability assay (see Experi-
mental Section). Papp, permeability coefficient.
with respect to the substituents on the succinimide and indoli-
none nitrogen atoms, as well as modifications of the succinimide
moiety.
On the basis of these data, 5 was considered as a suitable
starting point for lead optimization. In the following sections,
we describe the SAR around the core structure, in particular
Chemistry. Hit 5 was readily prepared as depicted in Scheme
1. By using the commercially available chloroisatin 19 as the
starting point, derivative 20 was obtained by Knoevenagel

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2229
Scheme 1a
a Reagents: (a) Methyl cyanoacetate, piperidine. MeOH, reflux, 90%. (b) KCN, MeOH, H2O, rt, 86%. (c) HCl, MeOH, rt to reflux. (d) AcOH, reflux,
66%. (e) KOtBu, DMF, then benzyl bromide, rt, 73%. (f) NaH, THF, tert-butyl bromoacetate, rt, 91%. (g) TFA, DCM, 0 °C to rt, 57%.
Scheme 2a
a Reagents: (a) KOtBu, DMF, then aryl bromide, r.t. (b) NaH, THF, then tert-butyl bromoacetate, r.t. or tert-butyl bromopropionate, reflux. (c) TFA,
DCM, 0 °C to r.t.
Scheme 3a
Scheme 4
a Reagents: (a) NaH (1.1 equiv), DMF, then bromoacetonitrile (0.95
equiv). (b) NaN3 (1.5 equiv), NH4Cl (1.5 equiv), DMF, 125 °C.
introduced first, and then, the final alkylation with tert-butyl
bromoacetate be carried out on all compounds. A more
streamlined approach would be to reverse the order of reactions
and alkylate on the indolinone nitrogen before the diversification
step on the succinimide (Scheme 4).
Changing the conditions for the alkylation of the spiro-
indolinone-succinimide system, it was possible to switch its
regioselectivity (Scheme 5); whereas the use of 1 equiv of
potassium tert-butoxide led to alkylation on the succinimide
nitrogen, the use of 2 equiv of sodium hydride gave rise
exclusively to the product of alkylation on the indolinone
nitrogen. In order to verify the identity of the products, nuclear
Overhauser effect (NOE) experiments were performed. The
structure of 33 was confirmed by the distinct NOE on Hd
upon irradiation of Hc, whereas no such effect was seen for
34 (Figure 2).
reaction. Subsequent reaction with potassium cyanide, followed
by cyclization under acidic conditions, generated the desired
spirocycle 22. Alkylation with benzyl bromide predominantly
occurred on the succinimide ring, and purification by column
chromatography gave 23. The final alkylation was carried out
with tert-butyl bromoacetate, and deprotection yielded the
desired product 5.
By using the common intermediate 22 as the starting point,
the same synthetic strategy was applied to investigate the SAR
of the southern part of the molecule (Scheme 2). The reaction
using tert-butyl-3-bromopropionate required stronger conditions
than those for the corresponding bromoacetic ester, but the
reaction was nonetheless successful. For the synthesis of
tetrazole derivative 29, a two-step protocol was used (Scheme
3): intermediate 23 was alkylated with bromoacetonitrile, and
the resulting nitrile was converted into the desired tetrazole by
reaction with sodium azide.
Over 40 compounds were prepared rapidly by using the
strategy depicted in Scheme 4. Because these molecules have
a chiral center, they were first prepared and tested as racemates.
The preparation of pure enantiomers was carried out for
derivatives that displayed good potency in vitro and a good
pharmacological profile. Possible strategies for the enantiose-
In order to explore more efficiently the northern part of the
molecule, a modification of the synthesis was sought; the
original route would necessitate that the main diversity be

2230 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
Scheme 5a
two nitrogen atoms were then introduced by using the same
strategy employed for the other pyrrolidinone derivatives
(Scheme 8).
Biological Activity. At first, the SAR around of the southern
part of the molecule, including the key carboxylic acid feature,
was explored (Table 3). The importance of the carboxylic acid
was confirmed by the fact that the corresponding ethyl ester
derivative 51 proved to be very weakly active.
The length of the spacer connecting the carboxylic acid group
to the indolinone core was found to be of crucial importance;
whereas a two-carbon chain led to almost complete loss of
potency (data not shown), a three-carbon chain (as was seen
with the first set of commercial compounds) is allowed, although
with about 10-fold loss of potency in the binding assay (e.g.,
52, Ki ) 1759 nM and 58, Ki ) 37 nM). Remarkably, these
compounds exhibited a profound difference compared to the
hit compound 5 when tested in the GTPγS assay; whereas 5
behaved as an inverse agonist, all the compounds bearing a
three-carbon chain exhibited full agonist behavior, with poten-
cies similar to those obtained in the binding assay. This behavior
can be rationalized by comparing the 3D structure of one- and
three-carbon chain compounds with known antagonists and
agonists. As shown in Figure 3, one-carbon chain compounds
give a very good overlay with ramatroban, whereas three-carbon
chain compounds give better results when overlaid with a
CRTH2 agonist.
a Reagents: (a) KOtBu, DMF, then tert-butyl bromoacetate. (b) NaH (2.1
equiv), DMF, then tert-butyl bromoacetate (0.95 equiv).
Figure 2. Elucidation of the regiochemistry of the alkylation by NOE.
lective syntheses were envisaged, but all entailed a large number
of steps. As, at the time, the configuration of the active
enantiomer was not known, it was decided to perform a chiral
resolution in order to obtain both enantiomers for evaluation of
the biological activity. Several attempts to perform the chiral
resolution by crystallization of the carboxylic acids by using a
variety of chiral bases failed to yield enantiomerically pure
compounds (data not shown). However, several compounds
could be separated by using preparative HPLC packed with
a chiral stationary phase (see Experimental Section). The
absolute configuration of the compounds was determined for
three representative compounds ((R)-5, (R)-58, and (S)-90) by
single X-ray diffraction crystallography.
Synthesis of the spiro-4-pyrrolidinone compounds proved to
be less straightforward. The synthesis of the spiro-4-pyrrolidi-
nones 41 is outlined in Scheme 6. Intermediate 20 could be
reacted with nitromethane in the presence of catalytic piperidine
to give the 1,4-addition product 35, which could be readily
decarboxylated by ester hydrolysis followed by heating in
dimethylformamide (DMF). Reaction with excess acetamide
catalyzed by palladium dichloride converted the nitrile to the
primary amide group.17 Reduction of the nitro group proved
the most troublesome step in the sequence; among the many
reducing agents that were tested, only zinc in hot acetic acid
gave a clean reduction. Under the conditions used, spontaneous
cyclization occurred to directly give the key spirocyclic
intermediate 38. Sequential alkylation of the two amide moieties,
first with tert-butyl bromoacetic acid, then with the appropriate
benzyl bromide, followed by deprotection of the tert-butyl ester
group with TFA, yielded the final products 41.
Finally, a derivative bearing a tetrazole (29), a well-known
carboxylic acid bioisostere, was tested and found to be markedly
less potent than 5.
After it was determined that the optimal substituent for the
southern part of the molecule is a methylenecarboxylic acid,
the SAR on the benzylic substituent was explored (Table 4).
Initially, the spacer between the succinimide nitrogen and the
aromatic group was investigated. A compound with a two-
carbon chain (55) was found to be almost equipotent with 5,
whereas the vinylogous compound 56 showed a 4-fold reduction
in potency. Introduction of a branching on the spacer (57) led
to a 2-fold reduction in potency, as well as the creation of a
further stereocenter in the molecule.
Substitution on the phenyl ring gave rise to compounds that
were generally more potent than the unsubstituted 5. Only the
introduction of a methylsulfonyl group in the para position (65)
led to a substantial loss of potency (Ki ) 1506 nM). It is
noteworthy that bulky groups (-OPh, 64, Ki ) 143 nM) were
still accepted and showed a small increase in potency compared
to 5.
In general, halogens proved to be preferred substituents, and
incorporation at the ortho and meta positions are particularly
favored, for example, o-fluoro derivative 58 (Ki ) 37 nM) and
m-chloro derivative 60 (Ki ) 68 nM), compared with the
m-methoxy derivative 63 (Ki ) 169 nM). The effect of these
substituents was furthermore found to be additive, so that the
simultaneous presence of two halogens in ortho and meta
positions led to some of the most potent compounds in this
subseries: 71 (Ki ) 10 nM), 66 (Ki ) 25 nM), and 68 (Ki ) 37
nM).
The high activity demonstrated by 76 (Ki ) 38 nM), in which
the substitution of the phenyl ring of 5 with a thiazole led to a
5-fold improvement in potency, led us to explore the effect of
heterocycles in this position (Table 5). Modification of the
2-methyl substituent on the thiazole moiety with substituted
phenyl groups gave rise to two other interesting compounds
with comparable activities (77, Ki ) 24 nM and 78, Ki ) 55
nM). Replacement of the thiazole with oxazole, oxadiazole, or
For the synthesis of the isomeric structure 50, the route
starting from the described intermediate 4318 was attempted first
(Scheme 7). However, upon deprotection of the ester group,
spontaneous decarboxylation was found to occur even under
mild conditions. The same behavior was observed after basic
hydrolysis of the ethyl ester derivative. However, construction
of the spiro-cyclic system could be easily performed in a single
step from 45, prepared as described by the procedure of Simig,18
by simple reaction of its sodium salt with 2-bromoethylisocy-
anate (Scheme 8). Subsequent hydrogenation on platinum
dioxide gave the key intermediate 47. The substituents on the

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2231
Scheme 6a
a Reagents: (a) Piperidine, nitromethane, r.t., 74%. (b) KOH, MeOH, r.t. (c) HCl (aq), r.t. (d) DMF, 160 °C, quant (3 steps). (e) PdCl2, acetamide,
THF/H2O. (f) Zn, AcOH, r.t. to reflux, 54%. (g) KOtBu, THF, then tert-butyl bromoacetate, 0 °C to r.t., 52%. (h) NaH, THF, then ArCH2Br, r.t., 52%. (i)
TFA, DCM, 0 °C to r.t., 95%.
Scheme 7a
of the most active racemic compounds (Table 7). Rewardingly,
we found that for each couple, one of the two enantiomers
showed a much greater affinity for the CRTH2 receptor than
the other, with a 16- to 40-fold difference between them. X-ray
analysis of three active enantiomers indicated that the most
active enantiomers had the same configuration (Figure 4). All
the other absolute structures were assigned by analogy.
The selectivity for three of these highly active, enantiomeri-
cally pure compounds was investigated. For a panel of over 50
receptors and enzymes, a more than 100-fold difference in
a Reagents: (a) Benzylchloroformate, TEA, THF, r.t. (b) DMAP, DMF,
r.t. (c) HCl, H2O, r.t. (d) H2, Pd/C, EtOAc.
activity was found for (R)-58, (R)-60, and (R)-71. In particular,
these compounds were tested against five other prostaglandin
furan groups led to a decrease in potency. However, the
receptors (DP, EP2, EP3, EP4, and FP) and in each case were
isoxazole group was found to be a very good replacement. The
found to have an IC50 > 30 µM.
first derivative prepared (85, Ki ) 51 nM) already showed a
The activity of a number of compounds was also tested in a
GTPγS binding assay (Table 8). All compounds having a
methylenecarboxylic acid substituent on the indolinone nitrogen
proved to be antagonists, with activities comparable with those
measured in the binding experiment. Some of the most promis-
ing compounds were also tested for affinity on the mouse
CRTH2 receptor (Table 9). This is of particular importance,
because in the field of GPCRs, many examples of receptors
showing significant interspecies differences are known, so that
a molecule behaving as an effective antagonist on the human
receptor could prove to be inactive on the receptors expressed
by other species. This, of course, has a high relevance for the
feasibility of pharmacological studies in animal models and for
the choice of the species for those studies. In our case, the five
compounds tested showed a very good correlation between the
results obtained on both human and murine CRTH2, indicating
that mouse models could be used for in vivo pharmacological
evaluation of the compounds.
potency comparable to that of the thiazole derivative. More
importantly, substitution of one of the two methyl groups with
a phenyl group led to the discovery of two very active
compounds (86, Ki ) 7.6 nM and 87, Ki ) 5.3 nM).
Interestingly, these two regioisomers showed practically the
same potency.
A more limited effort was undertaken for replacement with
six-membered heterocycles. Although a pyridine derivative
proved to be very potent (88, Ki ) 12 nM), its instability toward
oxidative metabolism (see Table 11) discouraged us from further
investigations on this type of structures.
The possibility to replace the succinimide moiety was also
explored (Table 6). Removal of either of the carbonyl groups
gave either of the regioisomeric pyrrolidinones, which showed
a very different behavior. Compounds 89-91 retained most of
the potency of the corresponding succinimides; 91 (Ki ) 3.8
nM) was equipotent to its counterpart 87. In contrast, 92 was
found to be very poorly active, clearly suggesting that the
carbonyl in the 5-position on the succinimide ring is important
to the binding.
CRTH2 antagonists were then tested for their ability to inhibit
DK-PGD2-induced eosinophil chemotactic activity (Table 10).
The effectiveness correlated with potency at CRTH2, thus
providing a key indication on the mechanism of action of
CRTH2 antagonists in relevant human cells. In addition, CRTH2
Having found a number of active structures, we then moved
on to find out whether only one of the two enantiomers was
responsible for the activity. The comparison was made for six

2232 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
Scheme 8a
a Reagents: (a) Sodium hydride, DMF, then bromoethylisocyanate, 77%. (b) H2, PtO2, DMF, r.t. 95%. (c) KOtBu, THF, then tert-butyl bromoacetate, 0
°C to r.t., 30%. (d) NaH, THF, then ArCH2Br, r.t. (e) TFA, DCM, 0 °C to r.t.
Table 3. SAR around the Southern Part
tested (10 µM). There was one exception; only (R)-71 showed
some inhibition on one isoform with an estimated IC50 ≈ 5.3
µM on CYP 2C19. Overall, the series demonstrated a low
potential for P450-mediated drug-drug interaction.
In contrast, more problems were encountered with regard to
the in vitro permeability of these compounds. This was not
entirely surprising, considering both the structures of the
compounds, which have a carboxylic acid, the negative effect
on permeability of which is well-known, and the data obtained
for the hit compound 5. In most cases, the Papp observed in
the Caco-2 experiments varied between 0.8 × 10-6 and 2.4 ×
10-6 cm·s-1 (low permeability range in our assay). The notable
exception was (R)-71, for which the increase of lipophilicity
on the phenyl substituent led to a substantial improvement on
the in vitro permeability (Papp 26.4 × 10-6 cm·s-1, high
permeability range).
On the basis of the data gathered, four compounds were
selected for PK studies in rat. The results are summarized in
Table 12. Both succinimide compounds (R)-58 and (R)-71
showed moderate and very similar clearance and oral bioavail-
ability values. Although the clearance was in line with the values
predicted by the results of the in vitro rat liver microsome
a Competitive displacement radioligand binding assay on human CRTH2.
experiment, the similar oral bioavailability of both compounds
was unexpected when incorporating the results of the Caco-2
b GTPγS radioligand binding assay.
experiments. Because of the high permeability ranking of (R)-
71 (with equivalent clearance), we expected this compound to
have an oral bioavailability higher than that of (R)-58. (R)-71
displayed a much higher volume of distribution than that of
(R)-58 (Vss ) 7 L/kg for (R)-71 vs Vss ) 0.9 L/kg for (R)-
58), indicating that whereas the latter compound appears to
remain in the central plasma compartment (Vss ≈ total body
water), the former appears to be distributing into some tissues,
possibly because of its higher lipophilicity. This observation
was confirmed in the PK studies in dog (Vss ) 2 L/kg for (R)-
58 vs Vss ) 5 L/kg for (R)-71). For both compounds, oral
bioavailability was moderate to high in this species; however,
in contrast to rats, (R)-58 showed a lower clearance (approxi-
matively 20% of liver blood flow) and higher oral bioavailability
than (R)-71 in dogs. The reason for this was not investigated.
As for the pyrrolidinone compounds, both molecules tested
gave much poorer results in vivo in rat than what could be
expected on the basis of the data obtained in in vitro metabolic
tests, both compounds not being metabolized at all in the
presence of rat liver microsomes. For 91, the very high clearance
observed after intravenous dosing (Cl ≈ liver blood flow, QH)
antagonists did not inhibit eotaxin-induced eosinophil chemo-
tactic activity (data not shown). Finally, no obvious cytotoxic
effect could be observed with the concentrations of chemoat-
tractants or the various doses of CRTH2 antagonists by using
the classical MTS assay (data not shown).
In order to select the best compounds for PK studies, the
most active compounds were subjected to in vitro profiling, in
particular with respect to oxidative metabolism in human and
rat liver microsomes and a Caco-2 cellular permeation model.
The results of the microsome stability and Caco-2 permeability
screens are summarized in Table 11.
Most compounds were moderately to highly resistant to
oxidative metabolism, demonstrating low intrinsic clearance in
both species studied. Notable exceptions were 63 and 88, which
exhibited high intrinsic clearance (>60 µL/min/mg protein) in
the rat liver microsomes; 88 was also the only compound to
have high values for the corresponding human microsome
experiment. This might be due to oxidative demethylation of
the arylmethoxy groups present in these compounds. The
molecules did not inhibit five human P450 isoforms (CYP 1A2,
2C19, 2C9, 2D6, and 3A4) up to the maximum concentration

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2233
Figure 3. (a) Overlay of 5 (R enantiomer) with ramatroban. (b) Overlay of the three-carbon chain analogue of 5 (R enantiomer) with the CRTH2
agonist L-888,607.19
Table 4. SAR around the Northern Part (1)
albeit acceptable and similar to that of the succinimide compounds
(low to medium, Cl ≈ 20% QH). For this molecule, the very low
oral bioavailability could probably be better explained by poor and
prolonged absorption, especially considering that a concentration
plateau was observed between 0.25 and 2 h after oral dosing, as
well as by the fact that t1/2 per oral route was found to be
significantly longer than t1/2 per IV (7.9 vs 0.9 h), indicating that
it was not the elimination phase that was observed but rather a
mixture of absorption-elimination.
The relatively poor correlation of the in vitro-in vivo
behavior we observed with the compounds in this chemical
series highlights the importance of conducting in vivo PK studies
early in the lead optimization stage. With this knowledge, we
could customize the in vitro ADME screening cascade for better
predictive ability before launching into the labor-intensive full
lead optimization effort and further in vivo testing.
Conclusion
We have identified a novel family of CRTH2 antagonists.
The initial hit 5, having a Ki of 196 nM, was optimized, and
molecules with Ki in the low single-digit nanomolar range were
discovered. The potency of these compounds was confirmed in
a human eosinophil chemotaxis experiment. The compounds
were characterized in terms of pharmaco-kinetic properties both
in vitro and in vivo, and two molecules ((R)-58 and (R)-71)
having suitable characteristics for further pharmacological
evaluation in vivo were identified.
Experimental Section
All chemicals were purchased from Fluka-Aldrich (Buchs,
Switzerland) unless otherwise stated. Melting points were measured
with a Büchi Melting Point B-545 apparatus and were uncorrected.
NMR spectra were recorded on a Bruker DPX-300 MHz spec-
trometer. Data were reported as follows: chemical shift in ppm by
using either residual dimethyl sulfoxide (DMSO, 2.49 ppm), CHCl3
(7.24 ppm), or MeOH (3.35 ppm) as internal standards on the scale,
multiplicity (s, singlet; br s, broad singlet; d, doublet; t, triplet; q,
quadruplet; and m, multiplet), coupling constants (J) in hertz, and
integration. MS data provided were obtained by using a Waters
ZMD mass spectrometer operated in positive or negative electro-
spray. Analytical HPLC method A: Xterra MSC8, 50 × 4.6 mm,
3.5 µm, 0.1% TFA in H2O (solvent A), 0.1% TFA in MeCN
(solvent B), linear gradient 5% to 100% B (8 min, flow rate 2 mL/
min), UV detection Max Plot 230-400 nm. Chiral HPLC method
a Competitive displacement radioligand binding assay on human CRTH2.
highlighted the possibility of an extra-hepatic route of clearance
and/or phase 2 metabolism, which was however unconfirmed.
For (S)-90, the clearance value was higher than the value that would
be predicted on the basis of the liver microsomes experiments alone,

2234 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Table 5. SAR around the Northern Part (2): Heteroaromatics
Crosignani et al.
a Competitive displacement radioligand binding assay on human CRTH2.
A: Chiralpak AD-H (Daicel) 250 × 4.6 mm column, isocratic
conditions ethanol:formic acid 100:0.1 (v/v) (30 min, flow rate 1
mL/min, column temperature 30 °C), UV detection 254 nm. Chiral
HPLC method B: Chiralcel OJ-H (Daicel) 250 × 4.6 mm column,
isocratic conditions methanol:formic acid 100:0.1 (v/v) (20 min,
flow rate 1 mL/min, column temperature 40 °C), UV detection 254
nm. Elemental analyses were performed on an Erba Science 11108
CHN analyzer.
for 7 h, and the resultant black solution was left to stand overnight.
The solvent was removed in Vacuo, water (500 mL) was added,
and the mixture was acidified by the addition of 2 M hydrochloric
acid. [Caution: Traces of cyanide may still be present!] The mixture
was extracted with dichloromethane (3 × 300 mL). The combined
extracts were washed with water (2 × 300 mL) and dried (MgSO4).
The solvent was removed in Vacuo to give the crude (5-chloro-3-
cyano-2-oxo-2,3-dihydro-1H-indol-3-yl)-cyanoacetic acid methyl
1
ester as a pale-brown solid (53.3 g, 86%). H NMR (300 MHz,
Methyl (5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)(cy-
ano)acetate (20). A stirred mixture of 5-chloro-1H-indole-2,3-dione
(90.8 g, 0.500 mol) and methyl cyanoacetate (44.1 mL, 0.500 mol)
in methanol (1000 mL) was treated with piperidine (2 mL) and
heated under reflux for 5 h before standing overnight at ambient
temperature. The crude (5-chloro-2-oxo-1,2-dihydro-indol-3-ylidene)-
cyanoacetic acid methyl ester was removed by filtration, washed
with cold methanol (2 × 150 mL), and dried in vacuo to give 117.6
g (90%) of a dark purple solid (6.5:1 mixture of geometric isomers)
DMSO-d6) δ 11.7 and 11.69 (1H, 2s), 7.70 and 7.76 (1H, 2m),
7.52 and 7.49 (1H, 2m), 7.04 and 7.02 (1H, 2m), 5.92 and 5.83
(1H, 2s), 3.74 (3H, s).
5-Chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione (22).
A stirred solution of the crude (5-chloro-3-cyano-2-oxo-2,3-dihydro-
1H-indol-3-y)-cyanoacetic acid methyl ester 21 (53.3 g, 0.180 mol)
in methanol (450 mL) was cooled in an ice-water bath and
saturated with hydrogen chloride gas, keeping the temperature below
20 °C. The resultant solution was left to stand at ambient
temperature overnight and then heated cautiously under reflux for
5 h to give a yellow suspension. The solvent was removed in Vacuo
to give a semi-solid residue that was mixed with glacial acetic acid
(375 mL) and heated under reflux, with stirring, for 16 h. After
cooling in an ice-water bath, the crude 5-chloro-1H-spiro[indole-
3,3′-pyrrolidine]-2,2′,5′-trione was removed by filtration, washed
with cold glacial acetic acid (100 mL), then with water (100 mL),
and then diethyl ether (100 mL), and dried in Vacuo to give an
off-white solid. A second crop was obtained by removing the
1
which was sufficiently pure by H NMR for subsequent reaction.
1H NMR (300 MHz, DMSO-d6) δ 11.22 and 11.11 (1H, 2br s),
8.27 and 7.78 (1H, 2br s), 7.55 and 7.42 (1H, 2m), 6.91 (1H, m),
3.95 and 3.88 (3H, 2s).
(5-Chloro-3-cyano-2-oxo-2,3-dihydro-1H-indol-3-yl)-cyanoace-
tic Acid Methyl Ester (21). A stirred suspension of crude (5-chloro-
2-oxo-1,2-dihydro-indol-3-ylidene)-cyanoacetic acid methyl ester
20 (56.0 g, 0.210 mol) in methanol (750 mL) was treated, in
portions, with potassium cyanide (13.8 g, 0.210 mol) and then with
water (45 mL). The mixture was stirred at ambient temperature

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2235
Table 6. Modifications of Succinimide Core
(1′-Benzyl-5-chloro-2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-
1-yl)-acetic Acid tert-Butyl Ester (24). A stirred suspension of
sodium hydride (60% dispersion in oil [116 mg, 2.90 mmol]) in
anhydrous tetrahydrofuran (30 mL) was treated with 1′-benzyl-5-
chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione 23 (900 mg,
2.64 mmol). After being stirred at ambient temperature for 30 min,
the clear solution was treated with tert-butyl bromoacetate (0.51
mL, 3.4 mmol). The resultant suspension was stirred for 3 h and
then left to stand overnight. Water (100 mL) was added, and the
mixture extracted with ethyl acetate (3 × 100 mL). The combined
extracts were washed with brine (100 mL) and dried (MgSO4), and
the solvent was removed in Vacuo to give the crude (1′-benzyl-5-
chloro-2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)-acetic acid
tert-butyl ester. This was purified by flash chromatography (silica)
eluting with petroleum ether (40-60) containing an increasing
amount (0 to 50%) of ethyl acetate to give the title compound as
1
a white solid (1.088 g, 91%). H NMR (300 MHz, DMSO-d6) δ
7.80 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.37-7.27 (5H, m), 7.13 (1H, d, J ) 8.5 Hz), 4.71 (1H, d, J )
15.5 Hz), 4.65 (1H, d, J ) 15.5 Hz), 4.53 (1H, d, J ) 18.0 Hz),
4.48 (1H, d, J ) 18.0 Hz), 3.41 (1H, d, J ) 18.0 Hz), 3.10 (1H, d,
J ) 18.0 Hz), 1.38 (9H, s). MS: 399.3 [M - H - tBu]+; 453.4 [M
- H]-.
(1′-Benzyl-5-chloro-2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-
1-yl)acetic Acid (5). A stirred solution of (1′-benzyl-5-chloro-
2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)-acetic acid tert-butyl
ester 24 (800 mg, 1.76 mmol) in anhydrous dichloromethane (20
mL) was cooled in an ice-water bath and treated with trifluoroacetic
acid (2.0 mL). After being stirred in the cold for 30 min, the mixture
was allowed to reach ambient temperature, and the reaction mixture
was stirred at ambient temperature for 48 h. The solvent was
removed in Vacuo, and toluene (50 mL) was added. The crude (1′-
benzyl-5-chloro-2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)ace-
tic acid was removed by filtration and washed with more toluene
(25 mL) and then with petroleum ether (25 mL). Purification by
flash chromatography (silica) eluting with ethyl acetate gave a white
solid (401 mg, 57%), mp 205-207 °C. 1H NMR (300 MHz,
DMSO-d6) δ 13.2 (1H, br s), 7.79 (1H, d, J ) 2.0 Hz), 7.46 (1H,
dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.38-7.26 (5H, m), 7.16 (1H, d, J )
8.5 Hz), 4.77 (1H, d, J ) 15.5 Hz), 4.65 (1H, d, J ) 15.5 Hz),
4.55 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0 Hz), 3.41 (1H, d,
J ) 18.0 Hz), 3.10 (1H, d, J ) 18.0 Hz). MS: 399.1 [M - H]+;
397.1 [M - H]-. Anal. (C20H15ClN2O5) C, H, N.
a Competitive displacement radioligand binding assay on human CRTH2.
Table 7. Activity of Pure Enantiomers
compound
Ki (nM)a
(R)-5
(S)-5
81 ( 41
2044 ( 16
23 ( 11
926 ( 400
23 ( 8
870 ( 129
72 ( 13
(R)-58
(S)-58
(R)-60
(S)-60
(R)-63
(S)-63
(S)-71
(R)-71
(S)-90
(R)-90
1172 ( 182
302 ( 56
5.3 ( 1.1
13 ( 2
683 ( 74
a Competitive displacement radioligand binding assay on human CRTH2.
1′-benzyl-5-chloro-1-(1H-tetrazol-5-ylmethyl)-2′H,5′H-spiro[in-
dole-3,3′-pyrrolidine]-2,2′,5′(1H)-trione (29). A stirred suspension
of sodium hydride (60% dispersion in oil [29 mg, 0.73 mmol]) in
anhydrous tetrahydrofuran (8.0 mL) was treated with 1′-benzyl-5-
chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione 23 (225 mg,
0.66 mmol). After being stirred at ambient temperature for 30 min,
the clear solution was treated with bromoacetonitrile (0.060 mL,
0.86 mmol). The resultant suspension was stirred overnight. Water
(50 mL) was added, and the mixture extracted with ethyl acetate
(3 × 50 mL). The combined extracts were washed with brine (50
mL) and dried (MgSO4), and the solvent was removed in Vacuo to
give the crude intermediate, which was purified by flash chroma-
tography (silica) eluting with petroleum ether (40-60) containing
an increasing amount (0 to 50%) of ethyl acetate. The purified
intermediate (115 mg, 0.303 mmol) in anhydrous DMF (2.3 mL)
was treated with sodium azide (29 mg, 0.45 mmol) and with
ammonium chloride (25 mg, 0.45 mmol). The reaction mixture was
heated at 125 °C for 2 h before cooling to room temperature. Water
was added, and the mixture was extracted with ethyl acetate. The
combined organic phases were washed with brine and dried
(MgSO4), and the solvent was removed in Vacuo to give the crude
1′-benzyl-5-chloro-1-(1H-tetrazol-5-ylmethyl)-2′H,5′H-spiro[indole-
3,3′-pyrrolidine]-2,2′,5′(1H)-trione, which was purified by recrys-
tallization from chloroform to give a white solid (71 mg, 25%).
1H NMR (300 MHz, DMSO-d6) δ 7.82 (1H, d, J ) 2.0 Hz), 7.47
(1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.38-7.26 (5H, m), 7.16 (1H,
d, J ) 8.5 Hz), 5.35 (1H, d, J ) 16.5 Hz), 5.30 (1H, d, J ) 16.5
Hz), 4.71 (1H, d, J ) 15.5 Hz), 4.66 (1H, d, J ) 15.5 Hz), 3.41
solvent in Vacuo from the combined acetic acid filtrates and
repeating the purification procedure to give a total of 30.5 g (66%).
1H NMR (300 MHz, DMSO-d6) δ 11.8 (1H, br s), 10.9 (1H, br s),
7.69 (1H, d, J ) 2.0 Hz), 7.32 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
6.89 (1H, d, J ) 8.5 Hz), 3.16 (1H, d, J ) 18.0 Hz), 2.95 (1H, d,
J ) 18.0 Hz). MS: 249.1 [M - H]-.
1′-Benzyl-5-chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-
trione (23). A stirred solution of 5-chloro-1H-spiro[indole-3,3′-
pyrrolidine]-2,2′,5′-trione 22 (800 mg, 3.19 mmol) in anhydrous
N,N-dimethylformamide (6 mL) was treated with potassium tert-
butoxide (377 mg, 3.19 mmol). After stirring at ambient temperature
for 40 min, benzyl bromide (0.38 mL, 3.19 mmol) was added. The
mixture was stirred for 2 h and left to stand overnight. The resultant
mixture was diluted with water (50 mL) and extracted with ethyl
acetate (3 × 50 mL). The combined extracts were dried (MgSO4),
and the solvent was removed in Vacuo to give the crude 1′-benzyl-
5-chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione, which was
purified by flash chromatography (silica) eluting with petroleum
ether (40-60) containing an increasing amount (0 to 75%) of ethyl
acetate. This gave 789 mg (73%) of the title compound as a yellow
1
solid. H NMR (300 MHz, DMSO-d6) δ 11.01 (1H, s), 7.68 (1H,
d, J ) 2.0 Hz), 7.37-7.25 (6H, m), 6.92 (1H, d, J ) 8.5 Hz), 4.68
(1H, d, J ) 15.5 Hz), 4.63 (1H, d, J ) 15.5 Hz), 3.31 (1H, d, J )
18.0 Hz), 3.14 (1H, d, J ) 18.0 Hz). MS: 341.3 [M + H]+; 339.2
[M - H]-.

2236 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
Figure 4. X-ray structure of (R)-58 (left) and (S)-90 (right).
Table 8. Activity in the GTPγS Assay
Table 11. In Vitro ADME Characterization of Selected Compounds
Cl (h-LM, µL/ Cl (r-LM, µL/ Caco-2
compound min/mg protein)a min/mg protein)a (Papp, 10-6 cm/s)b log D
compound
IC50 (nM)a
58
59
60
66
71
72
76
77
85
86
87
91
212 ( 26
182 ( 18
122 ( 28
296 ( 104
48 ( 0.1
82 ( 5
236 ( 8
48 ( 15
220 ( 23
21 ( 12
23 ( 10
19 ( 2
(R)-58
(R)-60
(R)-63
67
68
69
70
(R)-71
73
86
88
15
3
9
6
4
3
2
5
10
0
23
8
68
0
2.4
ND
0.8
ND
ND
ND
ND
26.4
ND
ND
0
-1
-0.4
ND
-0.9
-0.9
-0.7
-0.3
-0.5
-0.2
-0.6
ND
0
31
24
19
8
26
66
0
71
3
(R)-60
(R)-71
107 ( 6
66 ( 8
(S)-90
91
1.8
2.1
-0.4
0
0
-0.3
a GTPγS radioligand binding assay on human CRTH2.
a Microsome stability experiment. h-LM, human liver microsomes; r-LM,
rat liver microsomes. b Caco-2 cell permeability assay.
Table 9. Activity on Human and Murine CRTH2
mL, 19.0 mmol) in anhydrous N,N-dimethylformamide (46 mL).
The resultant dark yellow mixture was allowed to reach ambient
temperature and stirred for 4 h. The resultant mixture was diluted
with saturated aqueous ammonium chloride (500 mL) and extracted
with ethyl acetate (3 × 200 mL). The combined extracts were dried
(MgSO4), and the solvent was removed to give the crude (5-chloro-
2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)acetic acid tert-butyl
ester, which was purified by flash chromatography (silica) eluting
with petroleum ether (40-60) containing an increasing amount (0
to 80%) of ethyl acetate. This gave 4.76 g (95%) of a yellow solid.
1H NMR (300 MHz, DMSO-d6) δ 11.9 (1H, br s), 7.81 (1H, d, J
) 2.0 Hz), 7.44 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.11 (1H, d, J
) 8.5 Hz), 4.52 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0 Hz),
3.27 (1H, d, J ) 18.0 Hz), 2.93 (1H, d, J ) 18.0 Hz), 1.39 (9H, s).
MS: 309.2 [M - H - tBu]+; 363.3 [M - H]-.
compound
Ki (hCRTH2, nM)a
Ki (mCRTH2, nM)b
5
89
(R)-58
(R)-60
(R)-71
(S)-90
196 ( 124
702 ( 173
23 ( 11
23 ( 8
5.3 ( 1.1
13 ( 2
150
480
22
33
5
88
a Competitive displacement radioligand binding assay on human CRTH2.
b Competitive displacement radioligand binding assay on murine CRTH2.
Table 10. Inhibition of Human Eosinophil Chemotaxis Induced by
DK-PGD2 2
compound
IC50 (nM)
3
5
169 ( 79
156 ( 90
104 ( 72
40 ( 30
(1′-Benzyl-5-chloro-2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-
1-yl)acetic Acid Ethyl Ester (51). To a stirred suspension of
sodium hydride (60% dispersion in oil [39 mg, 0.97 mmol]) in
anhydrous tetrahydrofuran (10 mL) was added 1′-benzyl-5-chloro-
1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione 23 (300 mg, 0.88
mmol). After being stirred at ambient temperature for 30 min, the
clear solution was treated with ethyl bromoacetate (0.13 mL, 1.2
mmol). The resultant suspension was stirred for 3 h and then left
to stand overnight. Water (50 mL) was added, and the mixture was
extracted with ethyl acetate (3 × 50 mL). The combined extracts
were washed with brine (50 mL) and dried (MgSO4), and the solvent
was removed in Vacuo to give the crude (1′-benzyl-5-chloro-2,2′,5′-
trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)-acetic acid ethyl ester.
Purification by flash chromatography (silica) eluting with petroleum
ether containing an increasing amount (0 to 50%) of ethyl acetate,
followed by trituration with di-isopropyl ether gave the title
(R)-5
(R)-58
(R)-60
(R)-63
71
(R)-71
89
90
(S)-90
91
76 ( 39
18 ( 9
24 ( 11
7.3 ( 4.2
1350 ( 276
97 ( 33
62 ( 36
0.23 ( 0.16
(1H, d, J ) 18.0 Hz), 3.19 (1H, d, J ) 18.0 Hz). MS: 423.0 [M -
H]+; 421.0 [M - H]-.
(5-Chloro-2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1-yl)ace-
tic Acid tert-Butyl Ester (30). To a stirred suspension of sodium
hydride (60% dispersion in mineral oil, 1.68 g, 42.0 mmol) in
anhydrous N,N-dimethylformamide (140 mL) was added 22 (5.00
g, 20.0 mmol). After being stirred at ambient temperature for 40
min, the dark orange solution was cooled in an ice bath and treated
for 30 min with a solution of tert-butyl bromoacetate (3.24 g, 2.26
1
compound as a white solid (264 mg, 70%), mp 143-144 °C. H
NMR (300 MHz, DMSO-d6) δ 7.80 (1H, d, J ) 2.0 Hz), 7.47 (1H,
dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.37-7.26 (5H, m), 7.18 (1H, d, J )
8.5 Hz), 4.74-4.57 (4H, m), 4.13 (2H, q, J ) 7.0 Hz), 3.42 (1H,

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2237
Table 12. Pharmacokinetic Profile of Selected Compounds
dose
(mpk)
AUC0-last t
(h·ng/mL)
C0 or Cmax
(ng/mL)
species
Fz (%)
tmax (h)
t1/2 (h)
Cl (L/kg/h)
Vss (L/kg)
(R)-58
1990
297
2570
503
rat
rat
dog
dog
iv
os
iv
1
5
1
1
1250
2480
2020
2350
0
0.5
0
1
4.2
7
0.8
0.5
0.9
2
40%
os
120%
2
3
(R)-71
1700
163
1950
68.8
rat
rat
dog
dog
iv
os
iv
1
5
1
1
1180
1910
637
0.
1.5
0
13
5.4
3
0.7
2
7
5
32%
52%
os
339
2.67
n.e.
(S)-90
5060
55
rat
rat
iv
os
1
5
1390
319
0
1.5
0.9
8
0.7
4
0.3
0.4
5%
91
1970
6
rat
rat
iv
os
1
5
247
2.01
0
0.25
0.1
n.e.
0.20%
d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz), 1.18 (3H, t, J ) 7.0
Hz). MS: 427.2 [M - H]+; 425.0 [M - H]-. Anal. (C22H19ClN2O5)
C, H, N.
(1H, br s), 7.73 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J
) 2.0 Hz), 7.21 (3H, m), 6.90 (2H, d, J ) 8.5 Hz), 4.59 (2H, s),
3.74 (3H, s), 3.73 (2H, m), 3.32 (1H, d, J ) 18.0 Hz), 3.13 (1H,
d, J ) 18.0 Hz), 2.27 (2H, t, J ) 7.0 Hz), 1.79 (2H, m). MS: 457.1
[M - H]+; 455.0 [M - H]-.
General Procedure for the Synthesis of Spiro-indolinone
Derivatives-Method A. Stage 1: Reaction vessels were charged
with 5-chloro-1H-spiro[indole-3,3′-pyrrolidine]-2,2′,5′-trione 22
(500 mg, 1.90 mmol), anhydrous N,N-dimethylformamide (3 mL),
and potassium tert-butoxide (235 mg, 1.90 mmol). After the
mixtures was stirred at ambient temperature for 40 min, the
appropriate alkylating agent (1 eq) was added, and the mixtures
were stirred at ambient temperature for 18 h. Water (25 mL) was
added, and the mixtures were extracted with ethyl acetate (3 × 25
mL). The combined extracts were dried (MgSO4), and the solvent
was removed in Vacuo to give the crude products that were purified
by flash chromatography (silica) eluting with petroleum ether
containing an increasing amount of ethyl acetate. The products were
checked by 1H NMR, and if they were sufficiently pure, they were
taken on to the next stage.
Stage 2: Each tube was charged with the appropriate product
from Stage 1 (0.20 mmol) and dissolved in anhydrous N,N-
dimethylformamide (2 mL). Potassium tert-butoxide (25 mg, 0.22
mmol) was added. Then, tert-butyl 4-bromobutyrate (0.04 mL, 0.22
mmol) was added, and the reaction mixtures were heated at 50 °C
for 18 h. The reactions were worked up by adding water (about 5
mL) and extracting with ethyl acetate (3 × 5 mL). The extracts
were evaporated in tubes by using a Genevac HT4 for 2 h at 60
°C. They were then taken on to the next stage.
Stage 3: Each of the crude products from Stage 2 were treated
with a 10% (v/v) solution of trifluoroacetic acid in anhydrous
dichloromethane (3 mL) and stirred at ambient temperature for 72 h.
Toluene (1 mL) was added to each reaction mixture, the magnetic
stirrer beads were removed, and the solvents were removed by using
a Genevac HT4, first under vac-ramp conditions at 45 °C for 110
min and then at full vacuum at 50 °C for 45 min. N,N-
dimethylformamide (1 mL) was added to each tube to dissolve the
residues which were transferred to a microtiter plate for automated
reverse-phase preparative chromatography.
4-[5-Chloro-1′-(4-chlorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]butanoic Acid (54). Isolated as a white solid
1
in 62% yield. H NMR (300 MHz, DMSO-d6) δ 13.1 (1H, br s),
7.77 (1H, d, J ) 2.0 Hz), 7.48 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.42 (2H, d, J ) 8.5 Hz), 7.31 (2H, d, J ) 8.5 Hz), 7.23 (1H, d,
J ) 8.5 Hz), 4.66 (2H, s), 3.73 (2H, m), 3.34 (1H, d, J ) 18.0
Hz), 3.16 (1H, d, J ) 18.0 Hz), 2.26 (2H, t, J ) 7.0 Hz), 1.79 (2H,
m). MS: 461.0 [M - H]+; 459.1 [M - H]-.
General Procedure for the Synthesis of Spiro-indolinone
Derivatives-Method B. Stage 1: Each tube was charged with (5-
chloro-2,2′,5′-trioxo-spiro[indole-3,3′-pyrrolidin]-1-yl)acetic acid
tert-butyl ester 30 (73 mg, 0.20 mmol), potassium carbonate (83
mg, 0.60 mmol), and anhydrous N,N-dimethylformamide (2 mL).
The appropriate alkylating agent (0.24 mmol) was added, and the
reaction mixtures were stirred at 50 °C overnight. When the
alkylating agent was alpha-methyl benzyl bromide or analogues,
the reaction was carried out in a CEM Discover Microwave reactor
at 150 °C for 30 min. In the cases where the alkylating agent was
in the form of the hydrochloride salt, twice the normal amount of
potassium carbonate was used. Each of the reaction mixtures was
worked up by adding water (about 5 mL) and extracting with ethyl
acetate (3 × 5 mL). The extracts were evaporated in tubes by using
a Genevac HT4 for 2 h at 60 °C. They were then taken on to the
next stage.
Stage 2: Each of the crude products from Stage 1 were treated
with a 10% (v/v) solution of trifluoroacetic acid in anhydrous
dichloromethane (3 mL) and stirred at ambient temperature for 72 h.
Toluene (1 mL) was added to each reaction mixture, the magnetic
stirrer beads were removed, and the solvents were removed by using
a Genevac HT4, first under vac-ramp conditions at 45 °C for 110
min and then at full vacuum at 50 °C for 45 min. N,N-
dimethylformamide (1 mL) was added to each tube to dissolve the
residues which were transferred to a microtiter plate for automated
reverse-phase preparative chromatography.
Following this procedure, the following products could be
prepared:
4-[5-Chloro-1′-(2-fluorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]butanoic Acid (52). Isolated as a pale-yellow
solid in 54% yield. 1H NMR (300 MHz, DMSO-d6) δ 12.1 (1H, br
s), 7.76 (1H, d, J ) 2.0 Hz), 7.48 (1H, dd, J ) 8.5 Hz; J ) 2.0
Hz), 7.40-7.16 (5H, m), 4.74 (1H, d, J ) 15.5 Hz), 4.68 (1H, d,
J ) 15.5 Hz), 3.73 (2H, m), 3.35 (1H, d, J ) 18.0 Hz), 3.18 (1H,
d, J ) 18.0 Hz), 2.27 (2H, t, J ) 7.0 Hz), 1.79 (2H, m). MS: 445.0
[M - H]+; 443.1 [M - H]-.
Following this procedure, the following products could be
prepared:
[5-Chloro-2,2′,5′-trioxo-1′-(2-phenylethyl)spiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (55). Isolated as a brown solid
1
in 67% yield. H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s),
7.55 (1H, d, J ) 2.0 Hz), 7.44 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.33-7.20 (5H, m), 7.15 (1H, d, J ) 8.5 Hz), 4.54 (1H, d, J )
18.0 Hz), 4.48 (1H, d, J ) 18.0 Hz), 3.71 (2H, t, J ) 7.0 Hz), 3.28
(1H, d, J ) 18.0 Hz), 3.01 (1H, d, J ) 18.0 Hz), 2.84 (2H, m).
MS: 413.1 [M - H]+; 411.1 [M - H]-. Anal. (C21H17ClN2O5) C,
H, N.
4-[5-Chloro-1′-(4-methoxybenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]butanoic Acid (53). Isolated as a pale-
yellow solid in 61% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.1

2238 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
[5-Chloro-2,2′,5′-trioxo-1′-[(2E)-3-phenylprop-2-en-1-yl]spiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (56). Isolated as a beige
solid in 60% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br
s), 7.83 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0
Hz), 7.41 (2H, m), 7.34 (2H, t, J ) 7.5 Hz), 7.26 (1H, m), 7.17
(1H, d, J ) 8.5 Hz), 6.56 (1H, t, J ) 15.5 Hz), 6.23 (1H, dt, J )
15.5 Hz; J ) 5.5 Hz), 4.56 (1H, d, J ) 18.0 Hz), 4.50 (1H, d, J )
18.0 Hz), 4.26 (2H, m), 3.39 (1H, d, J ) 18.0 Hz), 3.10 (1H, d, J
) 18.0 Hz). MS: 425.0 [M - H]+; 423.1 [M - H]-. Anal.
(C22H17ClN2O5) C, H, N.
d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS(ESI+): 429.4
[M-H]+; 427.3 [M-H]-. Anal. (C21H17ClN2O6) C, H, N.
[5-Chloro-2,2′,5′-trioxo-1′-(3-phenoxybenzyl)spiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (64). Isolated as a black solid in
72% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.79
(1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.40
(2H, m), 7.30 (2H, m), 7.15 (2H, m), 7.03-6.95 (4H, m), 4.68
(1H, d, J ) 15.5 Hz), 4.63 (1H, d, J ) 15.5 Hz), 4.53 (1H, d, J )
17.5 Hz), 4.47 (1H, d, J ) 17.5 Hz), 3.40 (1H, d, J ) 18.0 Hz),
3.09 (1H, d, J ) 18.0 Hz). MS: 491.0 [M - H]+; 489.1 [M -
H]-. Anal. (C26H19ClN2O6) C, H, N.
[5-Chloro-1′-[4-(methylsulfonyl)benzyl]-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (65). Isolated as an off-
white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.3 (1H, br s),
7.91 (2H, d, J ) 8.0 Hz), 7.84 (1H, d, J ) 2.0 Hz), 7.56 (2H, d,
J ) 8.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.18 (2H, d,
J ) 8.5 Hz), 4.83 (1H, d, J ) 15.5 Hz), 4.77 (1H, d, J ) 15.5 Hz),
4.55 (1H, d, J ) 17.5 Hz), 4.48 (1H, d, J ) 17.5 Hz), 3.44 (1H, d,
J ) 18.0 Hz), 3.22 (3H, s), 3.13 (1H, d, J ) 18.0 Hz). MS: 494.0
[M - NH4]+; 475.0 [M - H]-.
[5-Chloro-2,2′,5′-trioxo-1′-(1-phenylethyl)spiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (57). Isolated as a orange solid
1
in 26% yield. H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s),
7.83 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.41 (2H, m), 7.34 (2H, t, J ) 7.5 Hz), 7.26 (1H, m), 7.17 (1H, d,
J ) 8.5 Hz), 6.56 (1H, t, J ) 15.5 Hz), 6.23 (1H, dt, J ) 15.5 Hz;
J ) 5.5 Hz), 4.56 (1H, d, J ) 18.0 Hz), 4.50 (1H, d, J ) 18.0 Hz),
4.26 (2H, m), 3.39 (1H, d, J ) 18.0 Hz), 3.10 (1H, d, J ) 18.0
Hz). MS: 413.1 [M - H]+; 411.1 [M - H]-. Anal. (C21H17ClN2O5)
H, N; C: calcd 61.10; found 60.36.
[5-Chloro-1′-(2-fluorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (58). Isolated as a beige solid in
72% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.79
(1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.40-7.15 (5H, m), 4.74 (1H, d, J ) 15.5 Hz), 4.69 (1H, d, J )
15.5 Hz), 4.54 (1H, d, J ) 18.0 Hz), 4.49 (1H, d, J ) 18.0 Hz),
3.41 (1H, d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS: 417.2
[M - H]+; 414.9 [M - H]-. Anal. (C20H14ClFN2O5) C, H, N.
[5-Chloro-1′-(4-fluorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (59). Isolated as a beige solid in
73% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.80
(1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.33
(2H, dd, J ) 8.5 Hz; J ) 5.5 Hz), 7.21-7.15 (3H, m), 4.69 (1H,
d, J ) 15.5 Hz), 4.64 (1H, d, J ) 15.5 Hz), 4.54 (1H, d, J ) 17.5
Hz), 4.48 (1H, d, J ) 17.5 Hz), 3.40 (1H, d, J ) 18.0 Hz), 3.08
(1H, d, J ) 18.0 Hz). MS: 417.3 [M - H]+; 415.2 [M - H]-.
[5-Chloro-1′-(3-chlorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (60). Isolated as a beige solid in
61% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.80
(1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.40-7.28 (3H, m), 7.24 (1H, d, J ) 6.5 Hz), 7.16 (1H, d, J ) 8.5
Hz), 4.70 (2H, s), 4.56 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0
Hz), 3.41 (1H, d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS:
433.4 [M - H]+; 431.4 [M - H]-.
[5-Chloro-1′-(2,3-difluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (66). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.79 (1H,
d, J ) 2.0 Hz), 7.45 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.44-7.34
(1H, m), 7.25-7.10 (3H, m), 4.79 (1H, d, J ) 15.5 Hz), 4.73 (1H,
d, J ) 15.5 Hz), 4.55 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0
Hz), 3.41 (1H, d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS:
435.0 [M - H]+; 433.1 [M - H]-. Anal. (C20H13ClF2N2O5) C,
H, N.
[5-Chloro-1′-(2,5-difluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (67). Isolated as an off-white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.81 (1H,
d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.34-7.08
(4H, m), 4.74 (1H, d, J ) 15.5 Hz), 4.69 (1H, d, J ) 15.5 Hz),
4.56 (1H, d, J ) 18.0 Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.43 (1H, d,
J ) 18.0 Hz), 3.13 (1H, d, J ) 18.0 Hz). MS: 435.1 [M - H]+;
433.1 [M - H]-.
[5-Chloro-1′-(3,5-difluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (68). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.85 (1H,
d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.22-7.14
(2H, m), 7.06-6.99 (2H, m), 4.73 (2H, s), 4.57 (1H, d, J ) 18.0
Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.44 (1H, d, J ) 18.0 Hz), 3.13
(1H, d, J ) 18.0 Hz). MS: 435.0 [M - H]+; 433.1 [M - H]-.
Anal. (C20H13ClF2N2O5) C, H, N.
[5-Chloro-1′-(3,4-difluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (69). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.83 (1H,
d, J ) 2.0 Hz), 7.49-7.30 (3H, m), 7.19-7.12 (2H, m), 4.69 (2H,
s), 4.56 (1H, d, J ) 18.0 Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.42 (1H,
d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS: 435.0 [M - H]+;
433.1 [M - H]-.
[5-Chloro-1′-(4-chlorobenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (61). Isolated as a white solid
1
in 58% yield. H NMR (300 MHz, DMSO-d6) δ 13.3 (1H, br s),
7.79 (1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
7.41 (2H, d, J ) 8.5 Hz), 7.31 (2H, d, J ) 8.5 Hz), 7.16 (1H, d,
J ) 8.5 Hz), 4.70 (1H, d, J ) 15.5 Hz), 4.65 (1H, d, J ) 15.5 Hz),
4.53 (1H, d, J ) 17.5 Hz), 4.47 (1H, d, J ) 17.5 Hz), 3.40 (1H, d,
J ) 18.0 Hz), 3.09 (1H, d, J ) 18.0 Hz). MS: 433.4 [M - H]+;
431.4 [M - H]-. Anal. (C20H14Cl2N2O5) C, H, N.
[5-Chloro-1′-(2-chloro-5-fluorobenzyl)-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (70). Isolated as a pale
[5-Chloro-1′-(2-methoxybenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (62). Isolated as an off-white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.3 (1H, br s), 7.78 (1H,
d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.28 (1H,
td, J ) 8.0 Hz; J ) 1.5 Hz), 7.17 (1H, d, J ) 8.5 Hz), 7.11
(1H, dd, J ) 7.5 Hz; J ) 1.5 Hz), 7.02 (1H, d, J ) 7.5 Hz), 6.90
(1H, td, J ) 7.5 Hz; J ) 1.0 Hz), 4.65 (1H, d, J ) 15.5 Hz), 4.58
(1H, d, J ) 15.5 Hz), 4.56 (1H, d, J ) 17.5 Hz), 4.50 (1H, d, J )
17.5 Hz), 3.81 (3H, s), 3.40 (1H, d, J ) 18.0 Hz), 3.12 (1H, d, J
) 18.0 Hz). MS: 429.1 [M - H]+; 427.1 [M - H]-. Anal.
(C21H17ClN2O6) C, H, N.
[5-Chloro-1′-(3-methoxybenzyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (63). Isolated as a beige solid in
75% yield. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.80
(1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.25
(1H, t, J ) 7.5 Hz), 7.17 (1H, d, J ) 8.5 Hz), 6.86-6.83 (3H, m),
4.69 (1H, d, J ) 15.5 Hz), 4.63 (1H, d, J ) 15.5 Hz), 4.56 (1H, d,
J ) 18.0 Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.74 (3H, s), 3.41 (1H,
1
yellow gum. H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s),
7.84 (1H, d, J ) 2.0 Hz), 7.58 (1H, dd, J ) 9.0 Hz; J ) 5.0 Hz),
7.48 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.24 (1H, td, J ) 9.0 Hz,
J ) 3.0 Hz), 7.18 (1H, d, J ) 8.5 Hz), 7.12 (1H, dd, J ) 9.0 Hz,
J ) 3.0 Hz), 4.75 (1H, d, J ) 16.5 Hz), 4.69 (1H, d, J ) 16.5 Hz),
4.57 (1H, d, J ) 18.0 Hz), 4.50 (1H, d, J ) 18.0 Hz), 3.47 (1H, d,
J ) 18.0 Hz), 3.19 (1H, d, J ) 18.0 Hz). MS: 451.0 [M - H]+;
449.0 [M - H]-.
[5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (71). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.78 (1H,
d, J ) 2.0 Hz), 7.48-7.40 (2H, m), 7.35-7.27 (2H, m), 7.16 (1H,
d, J ) 8.5 Hz), 4.73 (1H, d, J ) 15.5 Hz), 4.68 (1H, d, J ) 15.5
Hz), 4.55 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0 Hz), 3.41
(1H, d, J ) 18.0 Hz), 3.11 (1H, d, J ) 18.0 Hz). MS: 451.3 [M -
H]+; 449.3 [M - H]-. Anal. (C20H13Cl2FN2O5) H, N; C: calcd
53.24; found 52.55.

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2239
[5-Chloro-1′-(3,5-dichlorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (72). Isolated as a pale brown
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.3 (1H, br s), 7.84 (1H,
d, J ) 2.0 Hz), 7.56 (1H, d, J ) 2.0 Hz), 7.48 (1H, dd, J ) 8.5
Hz; J ) 2.0 Hz), 7.35 (2H, d, J ) 2.0 Hz), 7.17 (1H, d, J ) 8.5
Hz), 4.73 (2H, br s), 4.56 (1H, d, J ) 17.5 Hz), 4.50 (1H, d, J )
17.5 Hz), 3.43 (1H, d, J ) 18.0 Hz), 3.13 (1H, d, J ) 18.0 Hz).
MS: 467.0 [M - H]+; 465.1 [M - H]-.
[5-Chloro-1′-(3-chloro-5-fluorobenzyl)-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (73). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.84 (1H,
d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.38 (1H,
dt, J ) 9.0 Hz; J ) 2.0 Hz), 7.25 (1H, m), 7.18 (1H, d, J ) 8.5
Hz), 7.13 (1H, d, J ) 3.0 Hz), 4.73 (2H, s), 4.57 (1H, d, J ) 18.0
Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.43 (1H, d, J ) 18.0 Hz), 3.13
(1H, d, J ) 18.0 Hz). MS: 451.0 [M - H]+; 449.0 [M - H]-.
Anal. (C20H13Cl2FN2O5) C, H, N.
[5-Chloro-1′-(3-fluoro-4-methoxybenzyl)-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (74). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.80 (1H,
d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.18-7.05
(4H, m), 7.06-6.99 (2H, m), 4.65 (1H, d, J ) 15.5 Hz), 4.59 (1H,
d, J ) 15.5 Hz), 4.56 (1H, d, J ) 18.0 Hz), 4.47 (1H, d, J ) 18.0
Hz), 3.82 (3H, s), 3.40 (1H, d, J ) 18.0 Hz), 3.08 (1H, d, J ) 18.0
Hz). MS: 447.0 [M - H]+; 445.1 [M - H]-. Anal. (C21H16Cl-
FN2O6) C, H, N.
(1H, br s), 7.60 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5 Hz; J
) 2.0 Hz), 7.17 (1H, d, J ) 8.5 Hz), 4.83 (2H, s), 4.56 (1H, d, J
) 18.0 Hz), 4.48 (1H, d, J ) 18.0 Hz), 3.43 (1H, d, J ) 18.0 Hz),
3.21 (1H, d, J ) 18.0 Hz), 1.38 (9H, s). MS: 447.1 [M - H]+;
445.1 [M - H]-.
[5-Chloro-1′-(1,3-oxazol-2-ylmethyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (81). Isolated as a white
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 8.11 (1H,
d, J ) 1.0 Hz), 7.72 (1H, d, J ) 2.0 Hz), 7.46 (1H, dd, J ) 8.5
Hz; J ) 2.0 Hz), 7.19 (1H, d, J ) 1.0 Hz), 7.16 (1H, t, J ) 8.5
Hz), 4.82 (2H, s), 4.54 (1H, d, J ) 18.0 Hz), 4.48 (1H, d, J ) 18.0
Hz), 3.41 (1H, d, J ) 18.0 Hz), 3.16 (1H, d, J ) 18.0 Hz). MS:
390.1 [M - H]+; 388.1 [M - H]-.
[5-Chloro-1′-[(5-cyclopropyl-1,3,4-thiadiazol-2-yl)methyl]-
2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid
1
(82). Isolated as an off-white solid. H NMR (300 MHz, DMSO-
d6) δ 13.2 (1H, br s), 7.75 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J )
8.5 Hz; J ) 2.0 Hz), 7.16 (1H, d, J ) 8.5 Hz), 5.05 (2H, s), 4.54
(1H, d, J ) 18.0 Hz), 4.46 (1H, d, J ) 18.0 Hz), 3.41 (1H, d, J )
18.0 Hz), 3.12 (1H, d, J ) 18.0 Hz), 2.54 (1H, m), 1.20 (2H, m),
1.02 (2H, m). MS: 447.1 [M - H]+; 445.1 [M - H]-. Anal.
(C19H15ClN4O5S) H, N; C: calcd 55.61; found 54.84.
[5-Chloro-1′-(3-furylmethyl)-2,2′,5′-trioxospiro[indole-3,3′-
pyrrolidin]-1(2H)-yl]acetic Acid (83). Isolated as a white solid.
1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.77 (1H, d, J
) 2.0 Hz), 7.63 (1H, t, J ) 1.5 Hz), 7.61 (1H, m), 7.46 (1H, dd,
J ) 8.5 Hz; J ) 2.0 Hz), 7.17 (1H, d, J ) 8.5 Hz), 6.38 (1H, dd,
J ) 1.5, Hz, J ) 1.0 Hz), 4.51 (4H, m), 3.37 (1H, d, J ) 18.0 Hz),
3.06 (1H, d, J ) 18.0 Hz). MS: 389.0 [M - H]+; 387.3 [M -
H]-. Anal. (C18H13ClN2O6) H, N; C: calcd 55.61; found 54.84.
[5-Chloro-1′-(1H-indol-3-ylmethyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (84). Isolated as a brown
solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 11.06
(1H, s), 7.66 (1H, d, J ) 2.0 Hz), 7.59 (1H, d, J ) 9.0 Hz), 7.44
(1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.36 (1H, d, J ) 8.0 Hz), 7.28
(1H, d, J ) 2.5 Hz), 7.15 (1H, d, J ) 8.5 Hz), 7.10 (1H, m), 7.00
(1H, m), 4.82 (1H, d, J ) 15.5 Hz), 4.75 (1H, d, J ) 15.5 Hz),
4.52 (1H, d, J ) 18.0 Hz), 4.45 (1H, d, J ) 18.0 Hz), 3.35 (1H, d,
J ) 18.0 Hz), 3.03 (1H, d, J ) 18.0 Hz). MS: 438.2 [M - H]+;
436.1 [M - H]-.
[5-Chloro-1′-[(3,5-dimethylisoxazol-4-yl)methyl]-2,2′,5′-triox-
ospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (85). Isolated
as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H,
br s), 7.78 (1H, d, J ) 2.0 Hz), 7.45 (1H, dd, J ) 8.5 Hz; J ) 2.0
Hz), 7.15 (1H, d, J ) 8.5 Hz), 4.52 (1H, d, J ) 18.0 Hz), 4.48
(1H, d, J ) 15.5 Hz), 4.46 (1H, d, J ) 18.0 Hz), 4.38 (1H, d, J )
15.5 Hz), 3.34 (1H, d, J ) 18.0 Hz), 3.01 (1H, d, J ) 18.0 Hz),
2.38 (3H, s), 2.16 (3H, s). MS: 418.2 [M - H]+; 416.1 [M - H]-.
[5-Chloro-1′-[(5-methyl-3-phenylisoxazol-4-yl)methyl]-2,2′,5′-
trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (86). Iso-
lated as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H,
br s), 7.65-7.58 (3H, m), 7.51-7.43 (4H, m), 7.14 (1H, d, J )
8.5 Hz), 4.65 (1H, d, J ) 15.5 Hz), 4.52 (1H, d, J ) 18.0 Hz),
4.46 (1H, d, J ) 18.0 Hz), 4.45 (1H, d, J ) 15.5 Hz), 3.26 (1H, d,
J ) 18.0 Hz), 3.00 (1H, d, J ) 18.0 Hz), 2.45 (3H, s). MS: 480.2
[M - H]+; 478.2 [M - H]-.
[5-Chloro-1′-[(1-methyl-1H-1,2,3-benzotriazol-5-yl)methyl]-
2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid
1
(75). Isolated as an off-white solid. H NMR (300 MHz, DMSO-
d6) δ 13.2 (1H, br s), 7.91 (1H, br s), 7.87-7.82 (2H, m), 7.50-7.45
(2H, m), 7.17 (1H, d, J ) 8.5 Hz), 4.89 (1H, d, J ) 15.5 Hz), 4.83
(1H, d, J ) 15.5 Hz), 4.56 (1H, d, J ) 18.0 Hz), 4.48 (1H, d, J )
18.0 Hz), 4.30 (3H, s), 3.44 (1H, d, J ) 18.0 Hz), 3.11 (1H, d, J
) 18.0 Hz). MS: 454.1 [M - H]+; 452.1 [M - H]-.
[5-Chloro-1′-[(2-methyl-1,3-thiazol-4-yl)methyl]-2,2′,5′-triox-
ospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (76). Isolated
1
as a white solid in 60% yield. H NMR (300 MHz, DMSO-d6) δ
13.2 (1H, br s), 7.74 (1H, d, J ) 2.0 Hz), 7.47 (1H, dd, J ) 8.5
Hz; J ) 2.0 Hz), 7.25 (1H, s), 7.18 (1H, d, J ) 8.5 Hz), 4.72 (2H,
s), 4.56 (1H, d, J ) 18.0 Hz), 4.49 (1H, d, J ) 18.0 Hz), 3.41 (1H,
d, J ) 18.0 Hz), 3.14 (1H, d, J ) 18.0 Hz), 2.64 (3H, s). MS:
420.1 [M - H]+; 418.0 [M - H]-.
[5-Chloro-1′-{[2-(3-chlorophenyl)-1,3-thiazol-4-yl]meth-
yl}-2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid
1
(77). Isolated as an off-white solid. H NMR (300 MHz, DMSO-
d6) δ 13.2 (1H, br s), 7.96 (1H, m), 7.88 (1H, dt, J ) 6.5 Hz, J )
2.0 Hz), 7.74 (1H, d, J ) 2.0 Hz), 7.60-7.51 (3H, m), 7.46 (1H,
dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.18 (1H, d, J ) 8.5 Hz), 4.85 (2H,
s), 4.58 (1H, d, J ) 18.0 Hz), 4.50 (1H, d, J ) 18.0 Hz), 3.46 (1H,
d, J ) 18.0 Hz), 3.16 (1H, d, J ) 18.0 Hz). MS: 516.0 [M - H]+;
514.1 [M - H]-.
[5-Chloro-1′-{[2-(4-chlorophenyl)-1,3-thiazol-4-yl]meth-
yl}-2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid
1
(78). Isolated as an off-white solid. H NMR (300 MHz, DMSO-
d6) δ 13.2 (1H, br s), 7.94 (2H, m), 7.74 (1H, d, J ) 2.0 Hz),
7.60-7.55 (3H, m), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.18
(1H, d, J ) 8.5 Hz), 4.84 (2H, s), 4.58 (1H, d, J ) 18.0 Hz), 4.50
(1H, d, J ) 18.0 Hz), 3.46 (1H, d, J ) 18.0 Hz), 3.16 (1H, d, J )
18.0 Hz). MS: 516.1 [M - H]+; 514.1 [M - H]-.
[5-Chloro-1′-{[5-(4-methoxyphenyl)-1,2,4-oxadiazol-3-yl]me-
thyl}-2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]ace-
tic Acid (79). Isolated as a white solid. 1H NMR (300 MHz,
DMSO-d6) δ 13.2 (1H, br s), 8.05 (2H, m), 7.69 (1H, d, J ) 2.0
Hz), 7.47 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.20-7.16 (3H, m),
4.90 (2H, s), 4.58 (1H, d, J ) 18.0 Hz), 4.50 (1H, d, J ) 18.0 Hz),
3.88 (3H, s), 3.46 (1H, d, J ) 18.0 Hz), 3.22 (1H, d, J ) 18.0 Hz).
MS: 497.0 [M - H]+; 495.1 [M - H]-. Anal. (C23H17ClN4O7) H,
N; C: calcd 55.60; found 54.76.
[5-Chloro-1′-[(3-methyl-5-phenylisoxazol-4-yl)methyl]-2,2′,5′-
trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (87). Iso-
lated as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2
(1H, br s), 7.81 (2H, m), 7.68 (1H, d, J ) 2.0 Hz), 7.54 (3H, m),
7.45 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.14 (1H, d, J ) 8.5 Hz),
4.78 (1H, d, J ) 15.5 Hz), 4.59 (1H, d, J ) 18.0 Hz), 4.52 (1H, d,
J ) 18.0 Hz), 4.46 (1H, d, J ) 15.5 Hz), 3.33 (1H, d, J ) 18.0
Hz), 3.07 (1H, d, J ) 18.0 Hz), 2.25 (3H, s). MS: 480.2 [M -
H]+; 478.2 [M - H]-.
[5-Chloro-1′-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-
2,2′,5′-trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid
1
(88). Isolated as a white solid. H NMR (300 MHz, DMSO-d6) δ
[1′-[(5-tert-Butyl-1,2,4-oxadiazol-3-yl)methyl]-5-chloro-2,2′,5′-
trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (80). Iso-
lated as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.2
13.2 (1H, br s), 8.14 (1H, s), 7.69 (1H, d, J ) 2.0 Hz), 7.47 (1H,
dd, J ) 8.5 Hz; J ) 2.0 Hz), 7.17 (1H, d, J ) 8.5 Hz), 4.79 (1H,
d, J ) 15.5 Hz), 4.73 (1H, d, J ) 15.5 Hz), 4.58 (1H, d, J ) 18.0

2240 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
Hz), 4.52 (1H, d, J ) 18.0 Hz), 3.74 (3H, s), 3.40 (1H, d, J ) 18.0
Hz), 3.26 (1H, d, J ) 18.0 Hz), 2.23 (3H, s), 2.20 (3H, s). MS:
458.2 [M - H]+; 456.1 [M - H]-.
Separation of Enantiomers. [(3R)-1′-Benzyl-5-chloro-2,2′,5′-
trioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-5.
After purification by chiral preparative HPLC on a Chiralpak AD
column, eluting with ethanol containing 0.05% of formic acid, the
title compound was isolated as a white solid. Retention time (chiral
HPLC): 5.62 min.
[(3S)-1′-Benzyl-5-chloro-2,2′,5′-trioxospiro[indole-3,3′-pyrro-
lidin]-1(2H)-yl]acetic Acid (S)-5. After purification by chiral
preparative HPLC on a Chiralpak AD column, eluting with ethanol
containing 0.05% of formic acid, the title compound was isolated
as a white solid. Retention time (chiral HPLC): 8.30 min.
[(3R)-5-Chloro-1′-(2-fluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-58. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.05% of trifluoroacetic acid, the title compound
was isolated as a white solid. Retention time (chiral HPLC): 5.22
min.
[(3S)-5-Chloro-1′-(2-fluorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (S)-58. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.05% of trifluoroacetic acid, the title compound
was isolated as a white solid. Retention time (chiral HPLC): 6.76
min.
[(3R)-5-Chloro-1′-(3-chlorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-60. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.1% of formic acid, the title compound was
isolated as a white solid. Retention time (chiral HPLC): 7.23 min.
[(3S)-5-Chloro-1′-(3-chlorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (S)-60. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.1% of formic acid, the title compound was
isolated as a white solid. Retention time (chiral HPLC): 11.22 min.
[(3R)-5-Chloro-1′-(3-methoxybenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-63. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.2% of formic acid, the title compound was
isolated as a white solid. Retention time (chiral HPLC): 5.86 min.
[(3S)-5-Chloro-1′-(3-chlorobenzyl)-2,2′,5′-trioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (S)-63. After purification by
chiral preparative HPLC on a Chiralpak AD column, eluting with
ethanol containing 0.2% of formic acid, the title compound was
isolated as a white solid. Retention time (chiral HPLC): 9.61 min.
[(3R)-5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,2′,5′-trioxo-
spiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-71. After
purification by chiral preparative HPLC on a CHIRALCEL OJ
column, eluting with carbon dioxide/methanol/acetonitrile (60/32/
8), the title compound was isolated as a white solid. Retention time
(chiral HPLC): 12.56 min.
Methyl [5-chloro-3-(nitromethyl)-2-oxo-2,3-dihydro-1H-indol-
3-yl](cyano)acetate (35). A suspension of methyl (2Z)-(5-chloro-
2-oxo-1,2-dihydro-3H-indol-3-ylidene)(cyano)acetate 20 (10.0 g;
38.1 mmol), in nitromethane (61 mL; 1.1 mol) was treated with
piperidine (189 µl; 1.90 mmol). After being stirred for 3 h, the
mixture was diluted with ethyl acetate and filtered through silica.
Evaporation of the solvent gave methyl [5-chloro-3-(nitromethyl)-
2-oxo-2,3-dihydro-1H-indol-3-yl](cyano)acetate as an oil (9.10 g,
73.8%) as a mixture of diastereomers, which was used without
further purification for the subsequent step. MS: 324.2 [M - H]+;
322.2 [M - H]-.
[5-Chloro-3-(nitromethyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]ac-
etonitrile (36). To a solution of methyl [5-chloro-3-(nitromethyl)-
2-oxo-2,3-dihydro-1H-indol-3-yl](cyano)acetate 35 (5.00 g; 15.4
mmol) in MeOH (150 mL) was added a potassium hydroxide
solution in water (9.27 mL; 5.00 M; 46.3 mmol). After the mixture
was stirred for 2.5 h, the solvent was evaporated. The residue was
taken up with ethyl acetate, the organic phase was extracted with
1N HCl and dried (MgSO4), and the solvent was removed in Vacuo
to give the crude [5-chloro-3-(nitromethyl)-2-oxo-2,3-dihydro-1H-
indol-3-yl](cyano)acetic acid (4.89 g, quant.) as a mixture of
diastereomers, which was sufficiently pure for subsequent reaction.
A solution of [5-chloro-3-(nitromethyl)-2-oxo-2,3-dihydro-1H-
indol-3-yl](cyano)acetic acid (2.00 g; 6.46 mmol) in anhydrous
DMF (15 mL) was heated at 160 °C for 1 h. After evaporation of
the solvent, the residue was taken up in ethyl acetate, extracted
with brine, and dried (MgSO4), and the solvent was removed in
Vacuo to give the crude [5-chloro-3-(nitromethyl)-2-oxo-2,3-
1
dihydro-1H-indol-3-yl]acetonitrile (1.74 g, quant). H NMR (300
MHz, DMSO-d6) δ 11.1 (1H, br s), 7.63 (1H, d, J ) 2.0 Hz), 7.35
(1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 6.94 (1H, d, J ) 8.5 Hz), 5.31
(1H, d, J ) 14.5 Hz), 5.21 (1H, d, J ) 14.5 Hz), 3.23 (1H, d, J )
17.0 Hz), 3.10 (1H, d, J ) 17.0 Hz). MS: 264.1 [M - H]-.
2-[5-Chloro-3-(nitromethyl)-2-oxo-2,3-dihydro-1H-indol-3-
yl]acetamide (37). A solution of [5-chloro-3-(nitromethyl)-2-oxo-
2,3-dihydro-1H-indol-3-yl]acetonitrile 36 (775 mg; 2.92 mmol) in
THF (6 mL) and water (2 mL) was treated with acetamide (775
mg; 13.1 mmol) and palladium(II) chloride (51.7 mg; 0.29 mmol)
and stirred overnight. Ethyl acetate was added, and the organic
phase was washed with brine, dried (MgSO4), and concentrated in
Vacuo to give the title compound (740 mg, 89%) that was used as
1
such for the next step. H NMR (300 MHz, DMSO-d6) δ 10.69
(1H, s), 7.52 (1H, d, J ) 2.0 Hz), 7.33 (1H, br s), 7.24 (1H, dd, J
) 8.5 Hz; J ) 2.0 Hz), 6.88 (1H, br s), 6.82 (1H, d, J ) 8.5 Hz),
5.34 (1H, d, J ) 14.0 Hz), 5.06 (1H, d, J ) 14.0 Hz), 2.69 (1H, d,
J ) 15.5 Hz), 2.51 (1H, partially covered by DMSO peak). MS:
284.1 [M - H]+; 282.1 [M - H]-.
5-Chloro-5′H-spiro[indole-3,3′-pyrrolidine]-2,5′(1H)-di-
one (38). To an ice-cold suspension of 2-[5-chloro-3-(nitromethyl)-
2-oxo-2,3-dihydro-1H-indol-3-yl]acetamide 37 (1.10 g; 3.88 mmol)
in AcOH (25 mL), zinc (2.5 g; 39 mmol) was added in portions
over 5 min. After the mixture was stirred for 5 min, the ice bath
was removed, and the reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was heated to reflux for 2 h.
The solvent was removed in Vacuo, and the residue was purified
by flash chromatography (silica) by using DCM containing increas-
ing amounts of MeOH (3% to 20%) to give the title compound
(420 mg, 46%) which was used as such for the next step (HPLC
[(3S)-5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,2′,5′-trioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (S)-71. After purifica-
tion by chiral preparative HPLC on a CHIRALCEL OJ column,
eluting with carbon dioxide/methanol/acetonitrile (60/32/8), the title
compound was isolated as a white solid. Retention time (chiral
HPLC): 5.60 min.
1
Method A, purity 77%). H NMR (300 MHz, DMSO-d6) δ 10.6
[(3S)-5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,5′-dioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (S)-90. After purifica-
tion by chiral preparative HPLC on a Chiralpak AD column, eluting
with ethanol containing 0.05% of trifluoroacetic acid, the title
compound was isolated as a white solid. Retention time (chiral
HPLC): 4.38 min.
[(3R)-5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,5′-dioxospiro[in-
dole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (R)-90. After purifica-
tion by chiral preparative HPLC on a Chiralpak AD column, eluting
with ethanol containing 0.05% of trifluoroacetic acid, the title
compound was isolated as a white solid. Retention time (chiral
HPLC): 4.90 min.
(1H, br s), 7.96 (1H, br s), 7.45 (1H, d, J ) 2.0 Hz), 7.25 (1H, dd,
J ) 8.5 Hz; J ) 2.0 Hz), 6.85 (1H, d, J ) 8.5 Hz), 3.46 (1H, d, J
) 10.0 Hz), 3.39 (1H, d, J ) 10.0 Hz), 2.50 (1H, covered by DMSO
peak), 2.44 (1H, d, J ) 16.5 Hz). MS: 237.1 [M - H]+; 235.1 [M
- H]-.
tert-Butyl (5-chloro-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-
1(2H)-yl)acetate (39). To a solution of 5-chloro-5′H-spiro[indole-
3,3′-pyrrolidine]-2,5′(1H)-dione 38 (230 mg; 0.97 mmol) in dry
DMF (10 mL) was added potassium tert-butoxide (109 mg; 0.97
mmol). The reaction mixture was stirred at room temperature for
30 min and then cooled to 0 °C, and tert-butyl bromoacetate (190
mg; 0.97 mmol) was added, dissolved in 3 mL of dry DMF. The

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2241
reaction mixture was stirred for 1 h at room temperature. The
solvent was evaporated in Vacuo, the residue was redissolved in
ethyl acetate, washed with water, and dried (MgSO4), and the
solvent was removed in Vacuo. The residue was purified by flash
chromatography (silica) by using cyclohexane containing increasing
amounts of ethyl acetate (50% to 100%) to give the title compound
(341 mg, 52.4%). 1H NMR (300 MHz, DMSO-d6) δ 8.02 (1H, br
s), 7.58 (1H, d, J ) 2.0 Hz), 7.36 (1H, dd, J ) 8.5 Hz; J ) 2.0
Hz), 7.04 (1H, d, J ) 8.5 Hz), 4.45 (2H, s), 3.48 (1H, d, J ) 10.0
Hz), 3.43 (1H, d, J ) 10.0 Hz), 2.62 (1H, d, J ) 16.5 Hz), 2.40
(1H, d, J ) 16.5 Hz), 1.38 (9H, s). MS: 351.1 [M - H]+; 349.2
[M - H]-.
m), 7.43-7.33 (2H, m), 7.27 (1H, t, J ) 9.0 Hz), 7.06 (1H, d, J )
8.5 Hz), 4.57 (1H, d, J ) 15.0 Hz), 4.46 (1H, d, J ) 15.0 Hz),
4.45 (2H, s), 3.57 (1H, d, J ) 10.0 Hz), 3.51 (1H, d, J ) 10.0 Hz),
2.84 (1H, d, J ) 16.5 Hz), 2.61 (1H, d, J ) 16.5 Hz). MS: 437.1
[M - H]+; 435.1 [M - H]-.
tert-Butyl [5-chloro-1′-[(3-methyl-5-phenylisoxazol-4-yl)methyl]-
2,5′-dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetate (40c). Fol-
lowing the same protocol used for the preparation of 40a, starting
from tert-butyl [5-chloro-1′-(2-fluorobenzyl)-2,5′-dioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetate 39 and 4-(bromomethyl)-3-methyl-
5-phenylisoxazole, the title compound was isolated, after purification
by chromatography (silica) eluting with cyclohexane containing
increasing amounts of ethyl acetate (10 to 50%), as a white solid
in 53% yield. 1H NMR (300 MHz, CDCl3) δ 7.72-7.69 (2H, m),
7.56-7.47 (3H, m), 7.22 (1H, dd, J ) 8.5 Hz, J ) 2.0 Hz), 6.87
(1H, d, J ) 2.0 Hz), 6.61 (1H, d, J ) 8.5 Hz), 4.83 (1H, d, J )
15.5 Hz), 4.53 (1H, d, J ) 15.5 Hz), 4.30 (2H, s), 3.55 (1H, d, J
) 10.0 Hz), 3.18 (1H, d, J ) 10.0 Hz), 2.99 (1H, d, J ) 17.0 Hz),
2.57 (1H, d, J ) 17.0 Hz), 2.40 (3H, s), 1.41 (9H, s). MS: 522.4
[M - H]+; 520.1 [M - H]-.
[5-Chloro-1′-[(3-methyl-5-phenylisoxazol-4-yl)methyl]-2,5′-di-
oxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (91). A
cooled (0 °C) solution of tert-butyl [5-chloro-1′-[(3-methyl-5-
phenylisoxazol-4-yl)methyl]-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-
1(2H)-yl]acetate 40c (794 mg; 1.52 mmol) in DCM (28 mL) was
treated with trifluoroacetic acid (12 mL). The solution was stirred
at room temperature for 6 h. The solution was diluted with DCM
and washed with 0.1 N HCl, and the organic layer was dried over
MgSO4, filtered, and evaporated to give a white residue, which was
crystallized from ethanol, to give the title compound as a white
solid (500 mg, 71%). 1H NMR (300 MHz, DMSO-d6) δ 13.1 (1H,
br s), 7.75-7.72 (2H, m), 7.55-7.52 (3H, m), 7.42 (1H, d, J )
2.0 Hz), 7.31 (1H, dd, J ) 8.5 Hz, J ) 2.0 Hz), 7.03 (1H, d, J )
8.5 Hz), 4.69 (1H, d, J ) 15.0 Hz), 4.39 (2H, s), 4.36 (1H, d, J )
15.0 Hz), 3.48 (1H, d, J ) 9.5 Hz), 3.31 (1H, d, J ) 9.5 Hz), 2.80
(1H, d, J ) 16.5 Hz), 2.56 (1H, d, J ) 16.5 Hz), 2.30 (3H, s). MS:
466.3 [M - H]+; 464.5 [M - H]-.
tert-Butyl [1′-benzyl-5-chloro-2,5′-dioxospiro[indole-3,3′-pyr-
rolidin]-1(2H)-yl]acetate (40a). To a solution of tert-butyl (5-
chloro-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl)acetate 39
(175 mg; 0.50 mmol) in dry THF (5 mL) was added sodium hydride
(24 mg; 0.60 mmol). The reaction mixture was stirred for 15 min,
and benzyl bromide (71 µl; 0.60 mmol) was added. After the
mixture was stirred for 10 min, the solvent was evaporated, and
the residue was redissolved in ethyl acetate, washed with water
and brine, dried (MgSO4), and removed in Vacuo to give a residue
which was purified by chromatography (silica) eluting with cyclo-
hexane containing increasing amounts of ethyl acetate (20 to 50%)
1
to give the title compound (220 mg, 52%). H NMR (300 MHz,
CDCl3) δ 7.37-7.26 (5H, m), 7.21 (1H, d, J ) 8.5 Hz), 7.09 (1H,
br s), 6.59 (1H, d, J ) 8.5 Hz), 4.70 (1H, d, J ) 14.5 Hz), 4.42
(1H, d, J ) 14.5 Hz), 4.35 (1H, d, J ) 17.5 Hz), 4.25 (1H, d, J )
17.5 Hz), 3.71 (1H, d, J ) 10.0 Hz), 3.27 (1H, d, J ) 10.0 Hz),
3.08 (1H, d, J ) 17.0 Hz), 2.58 (1H, d, J ) 17.0 Hz), 1.40 (9H, s).
MS: 441.4 [M - H]+; 439.2 [M - H]-.
[1′-Benzyl-5-chloro-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-
1(2H)-yl]acetatic Acid (89). A cooled (0 °C) solution of tert-butyl
(1′-benzyl-5-chloro-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-
yl)acetate 40a (110.6 mg; 0.25 mmol) in DCM (8 mL) was treated
with trifluoroacetic acid (2.0 mL). The solution was stirred at room
temperature for 3.5 h. The solvents were evaporated in Vacuo, and
the residue was taken up with toluene and evaporated in Vacuo.
After the mixture was dried under high vacuum for 12 h, the title
compound was obtained as a white solid (91.9 mg, 95%). 1H NMR
(300 MHz, DMSO-d6) δ 13.1 (1H, br s), 7.47 (1H, d, J ) 2.0 Hz),
7.38-7.25 (6H, m), 7.05 (1H, d, J ) 8.5 Hz), 4.56(1H, d, J )
15.0 Hz), 4.43 (2H, s), 4.40 (1H, d, J ) 15.0 Hz), 3.49 (1H, d, J
) 10.0 Hz), 3.45 (1H, d, J ) 10.0 Hz), 2.81 (1H, d, J ) 16.5 Hz),
2.63 (1H, d, J ) 16.5 Hz). MS: 385.3 [M - H]+; 383.2 [M -
H]-.
Benzyl 5-chloro-2,2′-dioxospiro[indole-3,3′-pyrrolidine]-1(2H)-
carboxylate (46). A solution of benzyl 5-chloro-2-oxoindoline-1-
carboxylate 45 (2.00 g; 6.63 mmol) in dry DMF (30 mL) was
treated with sodium hydride (265 mg; 6.63 mmol). After 10 min,
2-bromoethyl isocyanate (600 µL; 6.63 mmol) was added. After
another 10 min, ethyl acetate was added and washed with water.
The organic phase was dried (MgSO4), and the solvent was removed
in Vacuo to give a solid residue that was purified by recrystallization
(ethyl acetate) to give the title compound as a white solid (1.895
tert-Butyl [5-chloro-1′-(5-chloro-2-fluorobenzyl)-2,5′-dioxo-
spiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetate (40b). Following
the same protocol used for the preparation of 40a, starting from
tert-butyl (5-chloro-2,5′-dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-
yl)acetate 39 and 5-chloro-2-fluorobenzyl bromide, the title com-
pound was isolated, after purification by chromatography (silica)
eluting with cyclohexane containing increasing amounts of ethyl
1
g, 77%). H NMR (300 MHz, DMSO-d6) δ 9.45 (1H, br s), 7.74
(1H, d, J ) 8.5 Hz), 7.54 (2H, m), 7.42-7.33 (3H, m), 7.27 (1H,
d, J ) 2.0 Hz), 6.98 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz), 5.40 (2H,
s), 4.76 (2H, t, J ) 8.5 Hz), 3.80 (2H, t, J ) 8.5 Hz). MS: 371.1
[M - H]+; 369.1 [M - H]-.
5-Chloro-2′H-spiro[indole-3,3′-pyrrolidine]-2,2′(1H)-di-
one (47). A solution of benzyl 5-chloro-2,2′-dioxospiro[indole-
3,3′-pyrrolidine]-1(2H)-carboxylate 46 (1.000 g; 2.70 mmol) in
DMF (50 mL) was treated with platinum dioxide (200 mg; 0.88
mmol). The mixture was placed in a hydrogenation vessel under
20 atm of hydrogen. After the mixture was stirred overnight, a
further quantity of platinum dioxide (2.50 g) was added, and
hydrogenation continued at 20 atm H2 overnight. The reaction
mixture was filtered on a paper filter, and the solvent was removed
in Vacuo to give the title compound as a gray solid (610 mg, 96%).
1H NMR (300 MHz, DMSO-d6) δ 10.15 (1H, br s), 9.0 (1H, br s),
7.12 (1H, d, J ) 2.0 Hz), 6.82 (1H, dd, J ) 8.5 Hz; J ) 2.0 Hz),
6.73 (1H, d, J ) 8.5 Hz), 4.70 (2H, t, J ) 8.5 Hz), 3.76 (2H, t, J
) 8.5 Hz). MS: 237.0 [M - H]+; 235.0 [M - H]-.
tert-Butyl (5-chloro-2,2′-dioxospiro[indole-3,3′-pyrrolidin]-
1(2H)-yl)acetate (48). A solution of 5-chloro-5′H-spiro[indole-3,3′-
pyrrolidine]-2,5′(1H)-dione 47 (600 mg; 2.16 mmol) in 30 mL of
dry DMF was treated with potassium tert-butoxide (241 mg; 2.16
mmol; 1.00 eq.). After 10 min, the reaction mixture was treated
with a solution of tert-butyl bromoacetate (420 mg; 2.16 mmol) in
1
acetate (5 to 40%), as a white solid in 73% yield. H NMR (300
MHz, CDCl3) δ 7.27 (1H, dd, J ) 6.5 Hz, J ) 2.0 Hz), 7.14–7.09
(2H, m), 6.99 (1H, d, J ) 2.0 Hz), 6.87 (1H, t, J ) 9.0 Hz), 6.49
(1H, d, J ) 8.5 Hz), 4.55 (1H, d, J ) 15.0 Hz), 4.36 (1H, d, J )
15.0 Hz), 4.23 (1H, d, J ) 16.0 Hz), 4.14 (1H, d, J ) 16.0 Hz),
3.62 (1H, d, J ) 10.0 Hz), 3.21 (1H, d, J ) 10.0 Hz), 2.92 (1H, d,
J ) 17.0 Hz), 2.45 (1H, d, J ) 17.0 Hz), 1.28 (9H, s). MS: 493.4
[M - H]+; 491.0 [M - H]-.
[5-Chloro-1′-(5-chloro-2-fluorobenzyl)-2,5′-dioxospiro[indole-
3,3′-pyrrolidin]-1(2H)-yl]acetatic Acid (90). A cooled (0 °C)
solution of tert-butyl [5-chloro-1′-(5-chloro-2-fluorobenzyl)-2,5′-
dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetate 40b (2.045 g;
4.15 mmol) in DCM (28 mL) was treated with trifluoroacetic acid
(12 mL). The solution was stirred at room temperature for 5 h.
The solution was diluted with DCM and washed with 0.1 N HCl,
and the organic layer was dried over MgSO4, filtered, and
evaporated to give a white residue, which was crystallized from
ethanol, to give the title compound as a white solid (1.580 g, 87%).
1H NMR (300 MHz, DMSO-d6) δ 13.2 (1H, br s), 7.54-7.52 (2H,

2242 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Crosignani et al.
6 mL of dry DMF. After 4 h, the solvent was evaporated, and the
residue was dissolved in ethyl acetate. After addition of water, a
solid precipitated and was filtered to give the title compound as a
gray solid (225 mg, 30%). 1H NMR (300 MHz, DMSO-d6) δ 10.07
(1H, br s), 7.30 (1H, d, J ) 2.0 Hz), 6.84 (1H, dd, J ) 8.5 Hz; J
) 2.0 Hz), 6.69 (1H, d, J ) 8.5 Hz), 5.10 (2H, s), 4.73 (2H, t, J
) 8.5 Hz), 3.84 (2H, t, J ) 8.5 Hz), 1.38 (9H, s). MS: 351.2 [M
- H]+; 348.9 [M - H]-.
Radioligand Binding Assay. A scintillation proximity assay was
used for radioligand competition and saturation binding assays. For
each assay point, 2 to 5 µg of human or mouse CRTH2 cell
membranes were incubated in a final volume of 100 µL in 96 well
plates (Corning, NY) for 90 min and shaken at room temperature
in the presence of 100 µg of wheat germ agglutinin-coated
scintillation proximity assay beads (WGA-SPA, RPNQ0001, GE
Healthcare), 1.5 nM [3H]PGD2 (Amersham, 156 Cie/mmol) in
binding buffer (10 mM HEPES/KOH pH 7.4, 10 mM MnCl2, with
protease inhibitor cocktail tablets). Nonspecific binding was
determined in the presence of 1 µM PGD2 (Cayman, Ann Arbor,
MI). Assay was performed in the presence of 1% dimethylsulfoxide
(Me2SO). Binding activity was determined by using a 1450
Microbeta scintillation counter (Wallac, UK). Ki values were
calculated by using the Cheng-Prusoff equation (Cheng and
Prusoff, 1973) and represent the average of at least three independent-
dose response experiments.
tert-Butyl [5-chloro-1′-[(5-methyl-3-phenylisoxazol-4-yl)methyl]-
2,2′-dioxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetate (49). A
suspension of tert-butyl (5-chloro-2,2′-dioxospiro[indole-3,3′-pyr-
rolidin]-1(2H)-yl)acetate 48 (100 mg; 0.29 mmol) in dry DMF (5
mL) was treated with sodium hydride (14.8 mg; 0.37 mmol). After
the mixture was stirred for 20 min, 4-(bromomethyl)-5-methyl-3-
phenylisoxazole (86 mg; 0.34 mmol) was added. After the mixture
was stirred for 1 h, the solvent was evaporated to give a residue,
which was dissolved in ethyl acetate. The organic phase was washed
with brine, dried (MgSO4), and removed in Vacuo to give a solid
residue, which was purified by chromatography (silica), eluting with
cyclohexane containing increasing amounts of ethyl acetate (30 to
50%) to give the title compound as a white solid (86.0 mg, 58%
yield). 1H NMR (300 MHz, DMSO-d6) δ 7.62–7.59 (2H, m),
7.51-7.47 (3H, m), 7.32 (1H, d, J ) 2.0 Hz), 6.72 (1H, dd, J )
8.5 Hz; J ) 2.0 Hz), 6.28 (1H, d, J ) 8.5 Hz), 5.05 (2H, s), 4.85
(2H, s), 4.75 (2H, t, J ) 8.5 Hz), 3.86 (2H, t, J ) 8.5 Hz), 2.18
(3H, s), 1.38 (9H, s). MS: 522.4 [M - H]+; 520.1 [M - H]-.
[5-Chloro-1′-[(5-methyl-3-phenylisoxazol-4-yl)methyl]-2,2′-di-
oxospiro[indole-3,3′-pyrrolidin]-1(2H)-yl]acetic Acid (92). A
cooled (0 °C) solution of tert-butyl [5-chloro-1′-[(5-methyl-3-
phenylisoxazol-4-yl)methyl]-2,2′-dioxospiro[indole-3,3′-pyrrolidin]-
1(2H)-yl]acetate 49 (80 mg; 0.15 mmol) in DCM (8 mL) was treated
with trifluoroacetic acid (2.0 mL). The solution was stirred at room
temperature for 5 h. The solvents were evaporated in Vacuo, and
the residue was taken up with toluene and evaporated in Vacuo.
After the mixture was dried under high vacuum for 5 h, the title
compound was obtained as an off-white solid (65.0 mg, 91%). 1H
NMR (300 MHz, DMSO-d6) δ 12.9 (1H, br s), 7.60-7.57 (2H,
m), 7.50-7.47 (3H, m), 7.33 (1H, d, J ) 2.0 Hz), 6.73 (1H, dd, J
) 8.5 Hz, J ) 2.0 Hz), 6.26 (1H, d, J ) 8.5 Hz), 5.13 (2H, s),
4.87 (2H, s), 4.75 (2H, t, J ) 8.5 Hz), 3.89 (2H, t, J ) 8.5 Hz),
2.18 (3H, s). MS: 466.3 [M - H]+; 464.4 [M - H]-.
Biological Assays. Construction of pCEP4-hCRTH2 Mam-
malian Expression Vector. Mouse CRTH2 cDNA was amplified
by polymerase chain reaction by using a mouse cDNA library and
cloned into the pCEP4 vector (Invitrogen Life technology, Carlsbad,
CA).
Stable Transfected Cell Lines. Human chinese hamster ovary
cell line expressing hCRTH2 was purchased at Euroscreen (Bel-
gium) and cultured in HAM’s F12 containing 10% fetal calf serum
(Cancerra, Australia), 50 µg/mL streptomycin, 50 U/mL penicillin
(Invitrogen), and 400 µg/mL Geneticin (G418) (Calbiochem, San
Diego, CA), according to the manufacturer.
Human embryonic kidney (HEK) 293(EBNA) cells (Invitrogen
Life Technologies, catalog no. R620-07) were cultured in Dulbec-
co’s modified Eagle’s F-12 medium (DMEM-F-12, Invitrogen Life
Technologies) containing 10% heat inactivated fetal calf serum, 2
mM glutamine, 50 units/mL penicillin, 50 µg/mL streptomycin, and
250 µg/mL G418 (for EBNA selection). By using the GeneJammer
transfection Reagent (Stratagene, La Jolla, CA), HEK 293 (EBNA)
cells were transfected with the mouse CRTH2 pCEP4 vector
(containing the gene for the hygromycin resistance). Cells were
maintained in culture for 48 h post-transfection and then grown in
the presence of 300 µg/mL of hygromycin B (Calbiochem) for 4
weeks to select for resistant cells expressing the mCRTH2.
Membrane Preparation. Cells expressing the human and mouse
CRTH2 were disrupted by nitrogen cavitation (Parr Instruments,
Moline, IL) at 4 °C, 800 psi for 30 min. Membranes were prepared
by differential centrifugation (1000g for 10 min and then 100 000g
for 60 min). Membrane pellets were resuspended in 50 mM Tris
HCl pH 7.4, 1 mM EDTA, and 250 mM sucrose and stored at
-80 °C.
[35S]GTPγS Binding Assay. The [35S]GTPγS binding assay was
performed at 30 °C with gentle agitation in 96-well scintillating
white polystyrene plates (Perkin-Elmer, Waltham, MA) in a final
volume of 200 µL containing 2% of Me2SO.
Briefly, 10 µg of membrane expressing human CRTH2 were
incubated in 20 mM HEPES/KOH pH 7.4, 10 mM MgCl2, 10 µg/
mL Saponin, 3µM GDP, and 150 mM NaCl for 10 min, with
various concentration of PGD2. Nonspecific binding was determined
in the presence of 10 µM GTPγS. Antagonist activity of compounds
was measured in the presence of 80 nM PGD2. A total of 0.15 nM
[35S]GTPγS (reference) was subsequently added to each sample,
and after incubation of 30min, reactions were stopped by centrifu-
gation at 700g for 10 min. Supernatant was removed, and
[35S]GTPγS binding was determined by using a 1450 Microbeta
scintillation counter. Data were analyzed using Prism (GraphPad
Software Inc., San Diego, CA).
Human Eosinophil Chemotactic Activity. Eosinophils were
purified from buffy coats of normal donors obtained from a regional
hospital according to institutional guidelines of the ethical com-
mittee. Purity of the eosinophils was assessed by determining
surface antigen expression or appropriate isotypic controls by using
flow cytometric analysis. The purity of eosinophil preparations was
>95% (CD16-, CD11b+, VLA-4+, CCR3+, and CRTH2+) with
>98% viability. Eosinophil chemotaxis assay was performed in
Boyden chambers with a 5 µm pore size polycarbonate filter. The
plates were incubated at 37 °C, 5% CO2, for 90 min. Migrated
cells were transferred to 96-well plates via centrifugation by using
a funnel adaptator and quantified by measuring fluorescence with
a fluorescence counter. DK-PGD2, a metabolite of PGD2 that is
selective for CRTH2 (100 nM), and CCL11/eotaxin (10 nM) were
used as chemoattractants. Eosinophils were incubated with varying
concentrations of CRTH2 antagonists for 30 min at 37 °C, 5% CO2,
before being applied to the chemotaxis chamber; migration toward
DK-PGD2 or eotaxin was then observed.
Oxidative Metabolism. Rat and human microsomes were used
to screen the metabolic instability resulting from phase I oxidation.
Microsomes (final concentration 0.5 mg/mL), 0.1 M phosphate
buffer pH 7.4, and test compound (final concentration 3 µM, 1%
DMSO) were added to the assay plate and preincubated at 37 °C.
NADPH solution (final incubation concentration of 1 mM) was
added to initiate the reaction. The reaction was stopped by the
addition of 100 µL of cold acetonitrile at the appropriate time points
(time 0, 15, 30, and 45 min). The samples were centrifuged at 5000
rpm for 5 min at 4 °C to precipitate the protein. Samples were
analyzed by LC/MS-MS. The percentage of compound disappear-
ance, the in vitro intrinsic clearance, was calculated, and the
predicted in vivo clearance was determined.20
Cytochrome P450 Inhibition. Five human recombinant cyto-
chrome P450 isoenzymes (BD Gentest, Woburn, MA) were tested
(CYP 1A2, CYP 2C9, CYP 2C19, CYP 2D6, and CYP 3A4). Each
cytochrome P450 isoenzyme converts a specific fluorogenic sub-
strate to a fluorescent product by the enzymatic activity. Test
compound (up to final incubation concentration of 10 µM) in 1%
DMSO was added to the incubation mixture. The fluorescence

CRTH2 (DP2) Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2243
airways inflammation is mediated by CRTH2 receptor. J. Pharmacol.
Exp. Ther. 2005, 312, 954–960.
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intensity after a defined reaction time serves as the readout
(Multilabel Counter VICTOR2, EG&G WALLAC, Turku, Finland).
For each compound, the inhibition percentage at each concentration
and the IC50 were estimated.
Caco-2 Permeability. Caco-2 cells were obtained from American
type culture collection (Rockville, MD). The cells were seeded onto
a 24-well polycarbonate filter membrane (Transwell inserts, surface
area: 0.33 cm2, Corning) at a cell density of 1 × 105 cell/cm2 and
grown in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum, 1% nonessential amino acids, and 1%
L-glutamine. Permeability studies were performed with the mono-
layers cultured for 21 days. Prior to all experiments, the cell
monolayer integrity was evaluated by trans epithelial electrical
resistance; values greater than 800 ohm ·cm2 were used. The
permeability studies were initiated by adding an appropriate volume
of Hank’s balanced salt solution buffer containing 10 µM test
compound to either the apical (for apical to basolateral transport;
A to B) or basolateral (for basolateral to apical transport; B to A)
side of the monolayer. The monolayers were then placed in an
incubator at 37 °C. At the end of the incubation time (2 h), samples
were taken from both the apical and basolateral compartments. The
concentrations of test compound were analyzed by LC/MS-MS.
The permeability coefficient (Papp) was calculated according to
the following equation: Papp ) (dA/dt)(1/SC0), where dA/dt is the
flux of the test compound across the monolayer (nmol/s), S is the
surface area of the cell monolayer (cm2), and C0 is the initial
concentration (µM) in the donor compartment.
In vivo Pharmacokinetic Studies. Rat. The test compound was
administered orally by gavage or as an intravenous bolus dose to
three male Sprague–Dawley or Janvier rats per dose route. Serial
blood samples were collected via an indwelling carotid catheter
up to 24 h post dose.
Dog. The test compound was administered orally by gavage and
as an intravenous bolus dose to three male beagle dogs in a cross-
over study design. Each dog received 1 mg/kg of compound
formulated as a solution. Serial blood samples were collected up
to 48 h post dose. For both species, plasma samples (pooled by
time point for rat, individual for dog) were analyzed by LC-MS/
MS. Pharmacokinetic parameters were determined by using non-
compartmental analysis.
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are associated with asthma and differentially influence mRNA stability.
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Acknowledgment. Y.F. is grateful to SNBL for the provision
of the in-house beam time.
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Supporting Information Available: Analytical data related to
the chemical purity of compounds 5, 29, and 51–92. Full structural
details for the crystal structures of (R)-5, (R)-58, and (S)-90 in the
CIF format. This material is available free of charge via the Internet
at http://pubs.acs.org.
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