Identification of an indole series of prostaglandin D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.02.062

A novel indole series of PGD₂ receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with K

Full text of DOI:10.1016/j.bmcl.2006.02.062

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3043–3048
Identification of an indole series of prostaglandin D2
receptor antagonists
Claudio F. Sturino,a,* Nicolas Lachance,a Michael Boyd,a Carl Berthelette,a Marc Labelle,a
Lianhai Li,a Bruno Roy,a John Scheigetz,a Nancy Tsou,c Christine Brideau,b
Elizabeth Cauchon,b Marie-Claude Carriere,b Danielle Denis,b Gillian Greig,b
Stacia Kargman,b Sonia Lamontagne,b Marie-Claude Mathieu,b Nicole Sawyer,b
Deborah Slipetz,b Gary O’Neill,b Zhaoyin Wang,a Robert Zamboni,a
Kathleen M. Mettersb and Robert N. Younga
aDepartment of Medicinal Chemistry, Merck Frosst Canada & Co., 16711 Trans Canada Hwy. Kirkland, Que., Canada H9H 3L1
bDepartment of Biochemistry, Merck Frosst Canada & Co., 16711 Trans Canada Hwy. Kirkland, Que., Canada H9H 3L1
cDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 12 January 2006; revised 20 February 2006; accepted 20 February 2006
Available online 10 March 2006
Abstract—A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the iden-
tification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6
and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced
cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure–activity relationships of
this indole series of DP receptor antagonists will also be discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
Seasonal allergic rhinitis (SAR) is a multisymptom dis-
ease characterized by nasal congestion, itching, and rhi-
norrhea. The nasal congestion in SAR results from
tissue edema and vasodilatation in the nasal mucosa.
A number of treatments are currently available for
SAR but they are either ineffective at treating all the
symptoms of this disease or suffer from unwanted side-
effects. For example, antihistamines are effective at con-
trolling the rhinorrhea associated with SAR but are not
effective at controlling the congestive response. The
symptoms of SAR are induced by a number of media-
tors such as histamine, cysteinyl leukotrienes, and
PGD2. It has been demonstrated in humans that intra-
nasal instillation of prostaglandin D2 results in upper
airway obstruction with 10-fold greater potency than
histamine.1 PGD2 is the major cyclooxygenase derived
metabolite produced by degranulation of mast cells2,3
and is present in the nasal washings of atopic individuals
following allergen challenge. The potential role of PGD2
in allergic rhinitis prompted us to initiate a program
aimed at identifying PGD2 receptor4 (DP receptor)
antagonists to block the inflammatory effects of
PGD2.5–9 To this end, we wish to disclose herein the
identification of an indole series of potent and selective
PGD2 receptor antagonists.
The search for PGD2 receptor antagonists was initiated
through the screening of the Merck sample collection.
From this effort, a number of hits were identified. One
of these hits was the difluoroindole 1 (Fig. 1). This com-
pound displayed good binding affinity10 (11 nM) for the
DP receptor although this compound was actually more
potent on the thromboxane A2 (TP) receptor (1.7 nM).
Compound 1 displayed moderate to good selectivity
versus the remaining six prostanoid receptors (Fig. 1).
At this point, medicinal chemistry was initiated in an ef-
fort to improve the DP potency and selectivity of this in-
dole series. At the outset, with the structure–activity
information available to us from the screening effort,
Keywords: Prostaglandin D2; PGD2; DP receptor antagonist; Indole.
*
Corresponding author. Tel.: +1 514 428 2630; fax: +1 514 428
4900; e-mail: claudio_sturino@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.062

3044
C. F. Sturino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3043–3048
responding methylsulfoxide analog, it was significantly
less selective versus the TP receptor.
F
O
N
At this point sulfoxide 10, which is a mixture of four
stereoisomers, was resolved into its individual isomers.
The DP and TP binding affinities of the two more potent
isomers (16 and 17) are presented in Figure 2. These two
isomers were found to have R configuration at the acetic
acid side chain as determined by X-ray crystallography
of sulfoxide 17 (Fig. 3). The corresponding S stereoiso-
mers were 50- to 100-fold less active on the DP receptor.
The stereochemistry at sulfur has a minimal effect on the
activity or selectivity of these antagonists as illustrated
in Figure 2.
OH
F
Cl
1
Ki (nM)
EP1
EP2
EP3
EP4
DP
11 5.1
TP
FP
IP
1.7 1.6 >7000 >4600 >300 >2900 >20000 >2000
Figure 1. Prostanoid binding profile of lead difluoroindole 1.
we decided to focus our initial attention on modifying
the benzoid substituents of the indole core. Table 1 sum-
marizes the results from modification of the indole 7-
position. The unsubstituted indole 2 possess good DP
activity (Ki = 36 nM) but is 15-fold more potent on the
TP receptor (Ki = 2.4 nM). Introduction of an isopropyl
group (3) not only increased the DP activity but also
inverted the selectivity in favour of the DP receptor.
Concurrent with the SAR studies at the 7-position of the
indole, other benzoid positions were explored. The effect
of modifying the right-hand ring was also investigated.
Figure 4 below highlights two initial analogs that were
prepared in this regard. Both of these methylsulfone
analogs were found to be modestly selective for the
DP receptor, though less potent than the corresponding
7-substituted sulfoxide analog 17. Since both 18 and 19
displayed similar binding profiles, it was decided to
focus the initial structure–activity studies on the cyclo-
pentyl series (19). Table 2 highlights some of these re-
A bromine or aldehyde substituent as in 4 or 5 leads to
potent, non-selective, antagonists. The dimethylamide
analog 7 is somewhat less active on DP but displays
good selectivity versus TP (100-fold). The corresponding
phenyl amide 8 is less potent on DP and is also non-se-
lective. Interestingly, while the methyl sulfide analog 9
leads to a potent, non-selective antagonist, the corre-
sponding sulfoxide 10 is significantly more selective
versus the TP receptor (60-fold). Other sulfoxide ana-
logs (11–14) did not result in improved DP activity or
selectivity relative to the methylsulfoxide. While the sul-
fone analog 15 exhibited similar DP potency as the cor-
sults. Replacement of the methylsulfone with
a
sulfonamide group resulted in somewhat diminished
activity. Interestingly, introduction of a bromine atom
at the 7-position, as present in antagonist 21, resulted
in an approximate 270-fold gain in potency. Initial
OH
O
OH
O
N
N
S
S
O
O
:
:
Cl
Cl
Table 1. DP and TP binding affinities of 7-substituted indoles
17
16
DP Ki = 1.7 0.7 nM
TP Ki = 130 48 nM
DP Ki = 1.8 0.7 nM
TP Ki = 47 8.4 nM
O
OH
N
Figure 2. Prostanoid binding profile of sulfoxides 16 and 17.
R
Cl
Compound
R
Kia (nM)
TP
2.4 ( 1.2)
DP
36 ( 11)
2
3
H
i-Pr
1.7 ( 0.6)
2.6 ( 0.9)
6.3 ( 4.8)
16 ( 5.3)
28 ( 2.9)b
110 ( 74)
1.0 ( 0.0)
2.8 ( 0.4)
4.0 ( 0.4)
23 ( 12)
9.1 ( 1.7)
2.2 ( 0.8)
6.6 ( 5.8)
7.8 ( 3.3)
3700 ( 1000)
88 ( 48)
4
Br
CHO
5
6
CH2OH
CONMe2
CONHPh
SMe
7
8
9
2.6 ( 1.2)
172 ( 24)
340 ( 170)
1800 ( 1200)
96 ( 49)
10
11
12
13
14
15
SOMe
SOEt
SO-i-Pr
SOPh
SOBn
SO2Me
29 ( 7.8)
3.9 ( 1.5)
1.9 ( 0.2)
140 ( 45)
5.1 ( 1.7)
a Values are means of at least three experiments unless otherwise
stated.
b n = 2.
Figure 3. ORTEP representation of 17. Non-hydrogen atoms are
represented by ellipsoids corresponding to 20% probability. Hydrogen
atoms have been drawn at arbitrary size.

C. F. Sturino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3043–3048
3045
O
O
O O
S
in an approximate 1000-fold loss of DP activity. The
meta-chloro analog 31 or the meta-trifluoromethoxy ana-
log 32 provided equipotent antagonists relative to the
chloro analog 22. Interestingly, several heterocycles were
found to be good 4-chlorobenzyl replacements leading to
antagonists such as the 2,3-dichloro-5-thieno[3,2-b]pyri-
dine 33 and the chloroquinoline 34.
O
S
O
OH
N
N
OH
Cl
Cl
18
19
DP Ki = 80 6.6 nM
TP Ki = 990 310 nM
DP Ki = 300 95 nM
TP Ki = 2100 750 nM
While a number of benzyl replacements were identified,
none proved to further enhance the TP selectivity rela-
tive to the corresponding 4-chlorobenzyl analog 22.
Thus, from the above studies, antagonists 27 and 28
were found to display the optimal in vitro profiles. As
before, since antagonists 27 and 28 are mixtures of iso-
mers, it was deemed advantageous at this point to syn-
thesize and evaluate the prostanoid binding profile of
the individual isomers. Table 4 summarizes the prosta-
noid binding profile of the most active isomers, namely
35 and 36.11 Both 35 and 36 are potent DP receptor
antagonists with high levels of selectivity versus the
remaining seven prostanoid receptors as measured by
the receptor binding affinities. In addition, antagonists
35 and 36 display excellent potencies in a DP functional
assay where they inhibit the PGD2 induced production
of cAMP in platelet rich plasma12 (PRP) with IC50 val-
ues of 7.9 and 8.6 nM, respectively. Also, the TP selec-
tivities of 35 and 36 were maintained in a TP
functional assay13 which measures a compound’s ability
to inhibit the U46619-induced platelet aggregation in
platelet rich plasma (see Table 5).
Figure 4. Prostanoid binding profile of sulfones 18 and 19.
pharmacokinetic studies in rats with sulfonamide 21 re-
vealed that this compound was extensively demethylat-
ed. Re-introduction of the more metabolically stable
methylsulfone group, as in 22, improved both the DP
potency and TP selectivity. Compound 22 is thus a po-
tent (Ki = 1.7 nM) DP antagonist with a high level of
TP selectivity (120-fold). At this point, having achieved
desirable level of DP potency we turned our attention on
exploring additional bromine replacements with the aim
of maintaining DP activity while improving the TP
selectivity. To this end, replacement of the bromine with
a vinyl, cyclopropyl, cyano or thiophene unit provided
potent compounds though with comparable levels of
selectivity. Gratifyingly, the level of selectivity could be
further improved with the introduction of a methyl ke-
tone (27) or a secondary alcohol (28) substituent at the
7-position. These data illustrate how, within this indole
series, both the DP activity and TP selectivity can be
effectively modulated with the proper choice of
substituents.
The preparation of the tetrahydrocarbazole compounds
2–12 presented in Table 1 involved a Fischer-indole reac-
tion as the key step in their synthesis (Scheme 1).14,15
Thus, arylhydrazines (37) are treated with ethyl 2-oxocyc-
lohexanecarboxylate (38) in acetic acid to furnish the
corresponding indoles (39) in moderate to good yields.
Standard benzylation and ester hydrolysis provided the
target compounds (41).
We next examined potential replacements for the para-
chloro benzylic group. Table 3 presents some of the
analogs prepared in this series. Replacement of the chlo-
rine atom with the larger tert-butyl substituent, as in 29,
resulted in a modest loss of activity. Introduction of a
methylsulfonyl unit to generate the bis-sulfone 30 resulted
Table 2. Modification of the indole 5- and 7-positions
O
1
R
OH
N
2
R
Cl
Compound
R1
R2
Kia (nM)
DP
300 ( 95)
TP
2100 ( 750)
19
20
21
22
23
24
25
26
27
28
SO2Me
SO2NMe2
SO2NMe2
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
H
H
1100 ( 850)
4.1 ( 0.7)b
1.7 ( 0.2)
3.6 ( 2.0)
2.8 ( 1.4)
9.7 ( 0.8)
4.5 ( 0.7)
9.5 ( 3.0)
8.5 ( 2.7)
2300 ( 1300)
100 ( 9.1)
200 ( 40)
Br
Br
CH@CH2
Cy
CN
520 ( 210)
330 ( 210)
1100 ( 240)
730 ( 300)
2-Thiophene
COMe
CH(OH)Me
3600 ( 2300)
16,000 ( 16,000)
a Values are means of at least three experiments unless otherwise stated.
b n = 2.

3046
C. F. Sturino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3043–3048
Table 3. Modification of the indole benzylic substituents
O
O
O
O
O
O
O
O
S
O
S
S
OH
O
OH
OH
N
N
31
N
N
Br
Br
Br
S
tBu
O
Cl
29
30
O
O
O O
S
O
O
O
O
S
O
S
OH
OH
OH
N
N
Br
Br
Br
N
N
S
OCF
3
Cl
Cl
Cl
32
33
34
Compound
Kia (nM)
DP
TP
820 ( 120)
29
30
31
32
33
34
7.5 ( 1.0)
1900 ( 720)
3.5 ( 0.6)
4.1 ( 1.1)
4.4 ( 2.3)
1.0 ( 0.4)
2700 ( 1100)
760 ( 390)
260 ( 130)
220 ( 73)
180 ( 43)
a Values are means of at least three experiments.
Table 4. Prostanoid receptor binding profiles of 35 and 36
O
S
O
O O
S
O
O
OH
OH
N
N
O
HO
Cl
Cl
35
36
Compound
Kia (nM)
EP3
DP
TP
EPI
EP2
EP4
FP
IP
35
36
2.6 0.7
1.8 0.7
1200 470
7100 820
>20,000
>20,000
230
390
>20,000
>20,000
>20,000
>2900
>20,000
>20,000
>20,000
>20,000
a Values are means of at least three experiments.
Table 5. DP and TP receptor functional activity of indoles 35 and 36
NHNH2
O
OEt
a
IC50 (nM)
a
c
b
O
Compound
+
R
O
DP WPb
DP PRPc
TP PRPd
N
H
OEt
R
37
38
39
35
36
2.0 ( 0.7)
2.0 ( 1.4)
7.9 ( 2.8)
8.6 ( 4.3)
8400 ( 4700)
43,000 ( 16,000)
O
O
a Values are means of at least three experiments.
R
R
b DP WP assay, inhibition of the accumulation of cAMP in washed
platelets challenged with PGD2.
N
N
OEt
Cl
OH
Cl
c DP PRP assay, inhibition of the accumulation of cAMP in platelet
rich plasma challenged with PGD2.
40
41
d TP PRP assay, the inhibition of U46619-induced platelet aggregation
in platelet rich plasma.
Scheme 1. Synthesis of tetrahydrocarbazole series. Reagents and
condition: (a) AcOH; (b) NaH, DMF, 0 °C, 4-ClC6H4CH2Br; (c)
NaOH, H2O, MeOH, THF.
In the case of the tetrahydrocyclopenta[b]indole series, a
direct Fischer-indole approach proved unsuccessful,
giving rise to low yields and mixtures of products. To
overcome this shortcoming, the indole core was con-
structed via a palladium mediated reaction16 between
an ortho-iodoaniline and a cyclopentanone as illustrated

C. F. Sturino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3043–3048
3047
O
O
O
O
O
O
O
S
S
I
S
a
b
OEt
NH2
N
H
NH2
42
43
44
O
O
O O
S
O
O
S
d
c
OEt
OEt
Cl
N
H
N
Br
Br
46
45
O
O
O
O
O
e
S
O
f
S
OEt
Cl
OH
Cl
N
N
O
O
47
27
Scheme 2. Synthesis of tetrahydrocyclopenta[b]indole series. Reagents and conditions: (a) I2, EtOH, AgSO4; (b) ethyl 2-oxocyclopentane-
carboxylate, Pd(OAc)2 DMF; (c) pyrHBr–Br2, pyr, Zn, AcOH; (d) NaH, DMF, 0 °C; 4-ClC6H4CH2Br; (e) 1—1-ethoxyvinylstannane, Pd2(dba)3,
Ph3As, DMF, 100 °C; 2—aq HCl; (f) NaOH, H2O, MeOH, THF.
5. Giles, H.; Leff, P.; Bolofo, M. L.; Kelly, M. G.; Robert-
son, A. D. Br. J. Pharmacol. 1989, 96, 291.
by the synthesis of 27 shown in Scheme 2. Thus, after
iodination of aniline 42, condensation with ethyl 2-oxo-
cyclopentane-carboxylate followed by treatment with
Pd(OAc)2 in DMF yielded the desired indole 44 in good
yields. Selective bromination at the 7-position was
achieved through the actions of pyridinium tribromide
to provide 45.17 Following standard benzylation, a Stille
coupling of 46 with 1-(ethoxyvinyl) tributylstannane fol-
lowed by acidic work-up generated ketone 47.18 Ester
hydrolysis thus delivered the DP antagonist 27.
6. Torisu, K.; Kobayashi, K.; Iwahashi, M.; Egashira, H.;
Nakai, Y.; Okada, Y.; Nanbu, F.; Ohuchida, S.; Nakai,
H.; Toda, M. Eur. J. Med. Chem. 2005, 40, 505.
7. Mitsumori, S.; Tsuri, T.; Honma, T.; Hiramatsu, Y.;
Okada, T.; Hashizume, H.; Inagaki, M.; Arimura, A.;
Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M. J. Med.
Chem. 2003, 46, 2436.
8. Mitsumori, S.; Tsuri, T.; Honma, T.; Hiramatsu, Y.;
Okada, T.; Hashizume, H.; Kida, S.; Inagaki, M.; Arim-
ura, A.; Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M.
J. Med. Chem. 2003, 46, 2446.
9. Tsuri, T.; Honma, T.; Hiramatsu, Y.; Okada, T.; Hash-
izume, H.; Mitsumori, S.; Inagaki, M.; Arimura, A.;
Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M. J. Med.
Chem. 1997, 40, 3504.
10. Abramovitz, M.; Adam, M.; Boie, Y.; Carriere, M.-C.;
Denis, D.; Godbout, C.; Lamontagne, S.; Rochette, C.;
Sawyer, N.; Tremblay, N. M.; Belley, M.; Gallant, M.;
Dufresne, C.; Gareau, Y.; Ruel, R.; Juteau, H.; Labelle,
M.; Ouimet, N.; Metters, K. M. Biochim. Biophys. Acta
2000, 1483, 285.
In summary, we have presented a number of potent and
selective DP receptor antagonists. In particular, indoles
35 and 36 were shown to have the optimal potency and
selectivity. These compounds demonstrate high levels of
DP activity in the DP PRP functional assay and are
amongst the most potent DP antagonist reported to
date. In addition, the structure–activity profile highlight-
ed herein illustrate how, within this indole class of com-
pounds, one can successfully modulate both the DP
potency and TP selectivity of these antagonists.
11. The absolute stereochemistry of the acetic acid side chain
present in 35 and 36 was established by X-ray analysis of
the following acid which was obtained from ester hydro-
lysis and resolution of 45:
Acknowledgment
The authors thank Mr. Oliver Bouchard for his help in
exploring with some of the chemistry described in
Schemes 1 and 2.
O O
S
O
OH
N
H
Br
48
References and notes
Crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 297169(17) and CCDC 297363
(48). Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK [e-mail: deposit@ccdc.cam.ac.uk].
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12. The DP functional assays were performed on blood
collected from normal human volunteers. Platelet-rich
plasma (PRP) was prepared by centrifugation at 150g for
15 min. A portion of the PRP fraction was used to prepare
the washed platelet fraction by isolating the platelets by
4. Boie, Y.; Sawyer, N.; Slipetz, D. M.; Metters, K. M.;
Abramovitz, M. J. Biol. Chem. 1995, 270, 18910.

3048
C. F. Sturino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3043–3048
centrifugation (10 min at 800g) and resuspension of the
cuvette at 37 °C for 3 min. Twenty-five microliters of the
test compound diluted in buffer [0.9% (w/v) or 0.15 M
NaCl containing 0.25% (w/v) BSA] or vehicle (buffer), as
control was added to 250 lL stirring platelets for a 10 min
preincubation period. Twenty microliters of agonist
(1.36 lM U46619, final concentration) was then added
to the stirring PRP mixture to induce aggregation and the
incubation continued for five additional minutes. Maxi-
mum aggregation was determined at approximately 5 min.
The maximum extent of aggregation achieved in the
presence of U46619 was set to 100%.
platelet cell pellet in buffer (25 mM HEPES, pH 7.4, HBSS
without Ca2+, Mg2+). The WP and PRP assays were
conducted as follows: isobutylmethylxanthine (IBMX;
2 mM final concentration) was added to prevent degrada-
tion of cAMP. Samples (100 lL) of either human WP or
PRP were then preincubated (10 min at 37 °C) with
increasing concentrations of test compound in DMSO.
Samples were then challenged with PGD2 (300 nM final)
added in DMSO and incubated for an additional 2 min at
37 °C. The reaction was then terminated by the addition of
200 lL ethanol to disrupt the cells and extract the cAMP.
The samples were mixed thoroughly and centrifuged at
1400g for 10 min at 4 °C. Supernatant aliquots (100 lL)
were removed and the ethanol removed by evaporation.
cAMP was measured by [125I]cAMP scintillation proxim-
ity assay (SPA) (RPA556, Amersham).
14. All new compounds provided satisfactory spectral data
along with mass spectral and/or elemental analysis.
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13. TP functional assay was performed as follows: Test
compounds were assessed for their ability to inhibit
platelet aggregation in human PRP using aggregometry.
Two hundred and fifty microliters of PRP was stirred in a
18. Gan, T.; Van Ornum, S. G.; Cook, J. M. Tetrahedron Lett.
1997, 38, 8453.