Discovery of AMG 853, a CRTH2 and DP dual antagonist

Article abstract of DOI:10.1021/ml1002234

Prostaglandin D₂ (PGD₂) plays a key role in mediating allergic reactions seen in asthma, allergic rhinitis, and atopic dermatitis. PGD₂ exerts its activity through two G protein-coupled receptors (GPCRs), prostanoid D rece

Full text of DOI:10.1021/ml1002234

LETTER
pubs.acs.org/acsmedchemlett
Discovery of AMG 853, a CRTH2 and DP Dual Antagonist
Jiwen Liu, An-Rong Li, Yingcai Wang, Mike G. Johnson, Yongli Su, Wang Shen, Xuemei Wang, Sarah Lively,
Matthew Brown, SuJen Lai, Felix Gonzalez Lopez De Turiso, Qingge Xu, Bettina Van Lengerich,
Mike Schmitt, Zice Fu, Ying Sun, Shanna Lawlis, Lisa Seitz, Jay Danao, Jill Wait, Qiuping Ye, Hua Lucy Tang,
Mark Grillo, Tassie L. Collins, Timothy J. Sullivan, and Julio C. Medina*
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
S Supporting Information
b
ABSTRACT: Prostaglandin D2 (PGD2) plays a key role in mediating allergic reactions
seen in asthma, allergic rhinitis, and atopic dermatitis. PGD2 exerts its activity through
two G protein-coupled receptors (GPCRs), prostanoid D receptor (DP or DP1), and
chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or
DP2). We report the optimization of a series of phenylacetic acid derivatives in an effort
to improve the dual activity of AMG 009 against DP and CRTH2. These efforts led to the
discovery of AMG 853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl
sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), which is being evaluated
in human clinical trials for asthma.
KEYWORDS: AMG 853, CRTH2 receptor, DP receptor, prostaglandin D2, antagonist, asthma
rostaglandin D2 (PGD2) is produced by mast cells in high
concentrations during allergic responses and plays a key
P
role in mediating allergic reactions seen in asthma, allergic
rhinitis, atopic dermatitis, and allergic conjunctivitis.1,2 PGD2
exerts its activity through two G protein-coupled receptors
(GPCRs), DP (prostanoid D receptor, DP1) and CRTH2
(chemoattractant receptor-homologous molecule expressed
on Th2 cells, DP2). These two GPCRs act in concert to
promote a number of biological effects associated with the
development and maintenance of the allergic responses.
Numerous studies using DP and CRTH2 antagonists, com-
bined with genetic analysis, support the view that these
receptors play a pivotal role in mediating allergic diseases.3-6
Therefore, there has been significant interest in the develop-
ment of selective DP and CRTH2 antagonists.7-23 We
have pursued the thesis that blockade of both receptors may
prove more beneficial in alleviating allergic diseases triggered
by PGD2 than inhibiting either one separately; therefore, we
have been interested in identifying potent CRTH2/DP dual
inhibitors. We previously reported on the discovery of AMG
009 (1; Figure 1) as a potent CRTH2 and DP dual
antagonist.23 In this article, we report our continued optimi-
zation efforts, which led to the discovery of AMG 853 (2),
which possesses increased DP and CRTH2 potency as
compared to 1.
Figure 1. AMG 009 and AMG 853.
The anilines were converted to the benzene sulfonamides by
treating with sulfonyl chlorides, and then, hydrolysis of the
methyl esters using sodium hydroxide afforded the final com-
pounds (1-25).
Compound 1 had high affinity toward the CRTH2 receptor,
even in the presence of human plasma. It inhibited the binding of
3H-PGD2 to the CRTH2 receptors on HEK-293 cells with an
IC50 of 0.021 μM in the presence of human plasma. It also
3
inhibited the binding of H-PGD2 to the DP receptors with a
moderate IC50 of 0.28 μM in the presence of plasma. Therefore,
we set out to find compounds with improved activity on DP
while maintaining the excellent activity on CRTH2 displayed by
1. In addition, in vitro metabolism studies with 1 identified the
methoxy, amide, and sulfonamide phenyl groups as potential
metabolic sites. Thus, we focused our efforts on modifying these
regions.
Compound 1 was one of the most potent dual CRTH2 and
DP inhibitors identified from this series when it was selected
for preclinical studies. Potency comparisons were mainly
based on IC50 values in the presence of 50% human plasma,
Compounds 1-25 were synthesized according to the chem-
istry depicted in Scheme 1. 4-Chloro-3-nitrobenzoyl chloride
was reacted with amines to form the corresponding amides.
Displacement of the chlorine adjacent to the nitro group with
4-hydroxyphenylacetic acids gave the bis-aryl ethers in good
yields. Reduction of the nitro group followed by protection
of the carboxylic acids as methyl esters provided the aniline
methyl esters. The final two steps were carried out in one pot.
Received: September 23, 2010
Accepted: February 22, 2011
Published: March 02, 2011
r
2011 American Chemical Society
326
dx.doi.org/10.1021/ml1002234 ACS Med. Chem. Lett. 2011, 2, 326–330
|

ACS Medicinal Chemistry Letters
LETTER
Scheme 1a
Table 2. Phenylacetic Acid Modifications
IC50a in buffer/plasma (μM)
compd
R1
R2
CRTH2
DP
1
OMe
Me
Cl
H
H
H
H
H
F
0.003/0.021
0.003/0.026
0.006/0.090
0.007/0.11
0.018/0.21
0.004/0.032
0.012/0.28
0.006/0.23
0.008/1.33
0.030/0.44
0.31/ND
10
11
12
13
14
a Reagents and conditions: (a) Amines, triethylamine, THF, 0 °C, 12 h,
∼90%. (b) Phenylacetic acids, cesium carbonate, DMSO, 70 °C, 9 h,
∼85%. (c) H2, Pd/C, NaOH, water, room temperature, 3 h, 100%; or
SnCl2, EtOAc, 60 °C, 4 h, 80%. (d) H2SO4, MeOH, 60 °C, 5 h, 100%.
(e) Sulfonyl chlorides, 2,6-lutidine, THF, 60 °C, 12 h; then water,
NaOH, room temperature, 2 h, ∼70% two steps in one pot.
F
CN
F
0.007/0.60
a Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed
on HEK 293 cells. Assay run in buffer containing 0.5% bovine serum
albumin or in 50% human plasma. See ref 24 for assay protocol. Values
are means of three experiments, and the standard deviation is (30%.
Table 1. Sulfonamide Modifications
Table 3. Combination of Amide and Phenylacetic Acid
Modifications
IC50a in buffer/plasma (μM)
compd
R
CRTH2
DP
1
3
4
5
6
7
8
9
2,4-Cl
0.003/0.021
0.005/0.023
0.008/0.069
0.68/4.38
0.012/0.28
0.021/0.12
0.050/3.64
0.93/>10
0.52/2.34
>10/ND
IC50a in buffer/plasma (μM)
3,4-Cl
2,5-Cl
compd
R1
R2
CRTH2
DP
3,5-Cl
1
OMe
OMe
Cl
n-Bu
c-Pr
0.003/0.021
0.004/0.028
0.006/0.090
0.007/0.025
0.012/0.28
0.041/1.03
0.008/1.33
0.014/0.22
2,3-Cl
0.032/0.30
0.012/1.18
0.004/0.047
0.004/0.10
15
11
16
2,6-Cl
n-Bu
2-Cl-4-CF3
0.021/0.19
Cl
c-Pr
4-Cl
3
0.013/ND
a Displacement of 3H-PGD2 from the CRTH2 or DP receptor ex-
pressed on HEK 293 cells. Assay run in buffer containing 0.5% bovine
serum albumin or in 50% human plasma. See ref 24 for assay protocol.
Values are means of three experiments, and the standard deviation is
(30%.
a Displacement of H-lPGD2 from the CRTH2 receptor expressed on
HEK 293 cells. Assay run in buffer containing 0.5% bovine serum
albumin. See ref 24 for assay protocol. Values are means of three
experiments, and the standard deviation is (30%.
which correlated well with the functional activity in human
whole blood. During earlier optimization efforts, it was found
that single changes to the structure of 1 failed to yield significant
improvements in potency. For example, a variety of chloro-sub-
stituted benzene sulfonamides were studied (Table 1). Two
compounds (3 and 8) had similar potency as compared to 1; all
other compounds suffered a loss in potency.
Substitution of the phenyl ring of the phenylacetic acid moiety
was also investigated (Table 2). A few substituents (methyl in 10
and difluoro in 14) provided similar CRTH2 and DP potencies
as the methoxy group in 1. However, other substitutions, such as
mono halogen (11 and 12) and cyano (13), afforded weaker
potencies, especially in the presence of human plasma.
Furthermore, a previous study around the amide demon-
strated that the n-butyl amide as in 1 was one of the most
potent amides.23 A structure-activity relationship study also
showed that the bis-aryl ether linker was preferred. It could be
replaced by a methylene linker, and the potencies were
maintained. All other linker modifications, such as sulfide,
sulfoxide, sulfone, and amine linkers, afforded compounds
exhibiting weaker potencies (data not shown).
When more than one change was made to the structure of
1, and interesting combination effects were seen. For exam-
ple, substitution of the n-butyl amide by a cyclopropyl amide
in combination with replacement of the methoxy moiety by
327
dx.doi.org/10.1021/ml1002234 |ACS Med. Chem. Lett. 2011, 2, 326–330

ACS Medicinal Chemistry Letters
LETTER
Table 4. Combination of Sulfonamide and Amide
Table 5. Potent CRTH2 and DP Dual Antagonists
Modifications
IC50a in buffer/plasma (μM)
compd
R1
R2
CRTH2
DP
17
18
19
20
c-Bu
t-Bu
c-Bu
t-Bu
4-Cl
0.006/0.012
0.009/0.015
0.005/0.019
0.005/0.012
0.016/0.085
0.046/0.45
0.009/0.21
0.015/0.23
4-Cl
2,4-Cl
2,4-Cl
a Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed
on HEK 293 cells. Assay run in buffer containing 0.5% bovine serum
albumin or in 50% human plasma. See ref 24 for assay protocol. Values
are means of three experiments, and the standard deviation is (30%.
chlorine afforded a compound (16) with similar potency to 1
in the displacement assays (Table 3). On the other hand,
substitution of the n-butyl amide moiety of 1 by a cyclopropyl
amide group provided a compound (15) with decreased DP
potency (Table 3). Likewise, replacement of the methoxy
group in the phenylacetic acid moiety of 1 by a chlorine atom
also resulted in a compound (11) that was less potent than 1
in the presence of plasma. This suggested that the combina-
tion of modifications at different parts of the molecule
needed to be taken into consideration when optimizing this
series for potency in the presence of plasma.
CRTH2 IC50a (μM)
DP IC50a (μM)
compd
in buffer
in plasma
in buffer
in plasma
1
0.003
0.004
0.013
0.025
0.005
0.009
0.003
0.021
0.013
0.041
0.053
0.008
0.019
0.008
0.012
0.013
0.009
0.020
0.010
0.014
0.004
0.28
21
22
23
24
25
2
0.045
0.022
0.042
0.019
0.038
0.035
Table 4 further illustrates the effect that combination of
different groups had on this series. The t-butyl amide func-
tional group in combination with the 4-chlorobenzenesulfo-
namide moiety afforded a molecule (18) with decreased
activity on the DP receptor, as compared to the analogous
cyclo-butyl amide derivative (17). However, compound 20,
which contained a t-butyl amide group in combination with
the 2,4-dichlorobenzenesulfonamide moiety, had similar affi-
nity for the DP receptor as the corresponding cyclo-butyl
amide (19) in the presence of plasma.
On the basis of the observations exemplified in Tables 3 and 4,
we explored multiple combinations of preferred groups in the
phenylacetic acid, amide, and sulfonamide regions. The combi-
nation produced dual CRTH2 and DP inhibitors with signifi-
cantly improved DP potency as compared to 1. Table 5 shows a
few examples of the potent dual antagonists. Compound 2 was
among the potent dual antagonists discovered.
Key compounds were also evaluated in CRTH2 and DP
human whole blood functional assays. Consistent with the
displacement assay data, 2 was also significantly more potent
than 1 in the human whole blood functional assays. Compound 2
inhibited the PGD2-induced down-modulation of CRTH2 on
CD16 negative granulocytes (eosinophils) in human whole blood
with a Kb of 0.2 nM, while 1 had a Kb of 1 nM.25 Compound 2 also
inhibited PGD2-induced cAMP response in platelets in 80%
human whole blood with a Kb of 4.7 nM, which was significantly
improved, as compared to the Kb of 148 nM of 1.26 In addition, 2
demonstrated similar antagonist activity in an aequorin assay
a Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed
on HEK 293 cells. Assay run in buffer containing 0.5% bovine serum
albumin or in 50% human plasma. See ref 24 for assay protocol. Values
are means of three experiments, and the standard deviation is (30%.
using CRTH2-transfected HEK 293 cells and an eosinophil shape
change assay, as compared to the CRTH2 receptor down-
modulation human whole blood assay.25
The significant improvement of DP potency of 2 over 1 was
also demonstrated in vivo in a guinea pig model of PGD2-
induced airway constriction (Figure 2).27 In this model, airway
resistance (Penh, enhanced pause) was measured in response
to PGD2 challenge. The efficacy seen in this model was most
likely due to the effect on the DP receptors, because CRTH2
selective antagonist had no effect in the model.23 The in vitro
guinea pig DP potency was evaluated in guinea pig whole
blood cAMP assay. Compound 2 had a Kb of 5 nM, while the
Kb of 1 was 82 nM.
The pharmacokinetic properties of 2 were evaluated in
several species (Table 6) and were similar to those of 1.23
Compound 2 had low to moderate clearance across species,
excellent oral absorption, and low potential of inducing drug-
drug interactions as indicated by CYP inhibition and induc-
tion tests.
In summary, further optimization of a series of phenylacetic
acid derivatives led to the discovery of 2 (AMG 853), which
displayed significantly improved potency, as compared to 1
(AMG 009), in CRTH2 and DP displacement and functional
328
dx.doi.org/10.1021/ml1002234 |ACS Med. Chem. Lett. 2011, 2, 326–330

ACS Medicinal Chemistry Letters
LETTER
Figure 2. Evaluation of 2 and 1 in the guinea pig model of PGD2-induced airway constriction. Compounds 2 and 1 were dosed sc 4 h prior to aerosolized
PGD2 exposure (0.6 mg/mL); n = 4 animals per group. bPlasma exposure at 4 h after dosing; *p < 0.02, †p < 0.05, and n.s., not significant relative to
vehicle treatment.
Nagai, H.; Narumiya, S. Prostaglandin D2 as a Mediator of Allergic
Asthma. Science 2000, 287, 2013–2017.
(7) Norman, P. DP2 receptor antagonists in development. Expert
Opin. Invest. Drugs 2010, 19, 947–961.
(8) Medina, J. C.; Liu, J. PGD2 Antagonists. Annu. Rep. Med. Chem.
2006, 41, 221–235.
Table 6. Pharmacokinetic Properties of 2
oral
bioavailabilityb
(%)
clearance
(L/h/kg)a
MRT
(h)
Vdss
species
(L/kg)
rat
0.19
0.9
3.9
3.0
1.0
0.71
2.72
0.97
87
100
79
(9) Pettipher, R.; Hansel, T. T. Antagonists of the prostaglandin D2
receptor CRTH2. Drug News Perspect. 2008, 21, 317–322.
dog
cynoc
(10) Philip, G.; van Adelsberg, J.; Loeys, T.; Liu, N.; Wong, P.; Lai, E.;
Balachandra, S.; Reiss, T. F. Clinical studies of the DP1 antagonist laropiprant
in asthma and allergic rhinitis. J. Allergy Clin. Pharmacol. 2009, 124, 942–948.
(11) Sturino, C. F.; O'Neill, G.; Lachance, N.; Boyd, M.; Berthelette,
C.; Labelle, M.; Li, L.; Roy, B.; Scheigetz, J.; Tsou, N.; Aubin, Y.;
Bateman, K. P.; Chauret, N.; Day, S. H.; Lꢀevesque, J.; Seto, C.; Silva,
J. H.; Trimble, L. A.; Carriere, M.; Denis, D.; Greig, G.; Kargman, S.;
Lamontagne, S.; Mathieu, M.; Sawyer, N.; Slipetz, D.; Abraham, W. M.;
Jones, T.; McAuliffe, M.; Piechuta, H.; Griffith, D. A. N.; Wang, Z.;
Zamboni, R.; Young, R. N.; Metters, K. M. Discovery of a Potent and
Selective Prostaglandin D2 Receptor Antagonist, [(3R)-4-(4-Chloro-
benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta-
[b]indol-3-yl]-acetic Acid (MK-0524). J. Med. Chem. 2007, 50, 794–806.
(12) Beaulieu, C.; Guay, D.; Wang, Z.; Leblanc, Y.; Roy, P.; Dufresne,
C.; Zamboni, R.; Berthelette, C.; Day, S.; Tsou, N.; Denis, D.; Greig, G.;
Mathieu, M. C.; O'Neill, G. Identification of prostaglandin D2 receptor
antagonists based on a tetrahydropyridoindole scaffold. Bioorg. Med.
Chem. Lett. 2008, 18, 2696–2700.
(13) Arimura, A. Discovery of a First-in-Class Drug, a Prostaglandin
D2 Antagonist, for the Treatment of Allergic Diseases. In Molecular
Imaging for Integrated Medical Therapy and Drug Development; Tamaki,
N., Kuge, Y., Eds.; Springer: New York, 2010; Part IV, pp 281-287.
(14) Mitsumori, S.; Tsuri, T.; Honma, T.; Hiramatsu, Y.; Okada, T.;
Hashizume, H.; Kida, S.; Inagaki, M.; Arimura, A.; Yasui, K.; Asanuma,
F.; Kishino, J.; Ohtani, M. Synthesis and Biological Activity of Various
Derivatives of a Novel Class of Potent, Selective, and Orally Active
Prostaglandin D2 Receptor Antagonists. 2. 6,6-Dimethylbicyclo-
[3.1.1]heptane Derivatives. J. Med. Chem. 2003, 46, 2446–2456.
(15) Oxagen Ltd. Novel CRTH2 Antagonist Shows Potential in Mod-
erate Persistent Asthma: Presented at 19th Annual Congress of the European
Respiratory Society (ERS), September 18, 2009; http://www.docguide.com/
news/content.nsf/news/852576140048867A85257635005BC1C3.
(16) Tumey, L. N.; Robarge, M. J.; Gleason, E.; Song, J.; Murphy, S. M.;
Ekema, G.; Doucette, C.; Hanniford, D.; Palmer, M.; Pawlowski, G.; Danzig,
J.; Loftus, M.; Hunady, K.; Sherf, B.; Mays, R. W.; Stricker-Krongrad, A.;
Brunden, K. R.; Bennani, Y. L.; Harrington, J. J. 3-Indolyl sultams as selective
CRTh2 antagonists. Bioorg. Med. Chem. Lett. 2010, 20, 3287–3290.
(17) Grimstrup, M.; Receveur, J. M.; Rist, O.; Frimurer, T. M.;
Nielsen, P. A.; Mathiesen, J. M.; H€ogberg, T. Exploration of SAR features
0.96
a iv dose at 0.5 mg/kg. b Oral dose at 2 mg/kg. c Cynomolgus monkey.
assays, including assays performed in human whole blood, and
in vivo. Compound 2 was selected for evaluation in human
clinical trials for asthma.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed synthetic experimen-
b
tal procedures and characterization for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 650-244-2425. E-mail: medinaj@amgen.com.
’ REFERENCES
(1) Mitsumori, S. Recent Progress in Work on PGD2 Antagonists for
Drugs Targeting Allergic Diseases. Curr. Pharm. Des. 2004, 10, 3533–3538.
(2) Nagai, H. Prostaglandin as a Target Molecule for Pharmacother-
apy of Allergic Inflammatory Diseases. Allergol. Int. 2008, 57, 187–196.
(3) Schuligoi, R.; Sturm, E.; Luschnig, P.; Konya, V.; Philipose, S.;
Sedej, M.; Waldhoer, M.; Peskar, B. A.; Heinemann, A. CRTH2 and
D-Type Prostanoid Receptor Antagonists as Novel Therapeutic Agents
for Inflammatory Diseases. Pharmacology 2010, 85, 372–382.
(4) Pettipher, R. The roles of the prostaglandin D2 receptors DP1
and CRTH2 in promoting allergic responses. Br. J. Pharmacol. 2008,
153, s191–s199.
(5) Pettipher, R.; Hansel, T. T.; Armer, R. Antagonism of the
prostaglandin D2 receptors DP1 and CRTH2 as an approach to treat
allergic diseases. Nat. Rev. Drug Discovery 2007, 6, 313–325.
(6) Matsuoka, T.; Hirata, M.; Tanaka, H.; Takahashi, Y.; Murata, T.;
Kabashima, K.;Sugimoto, Y.; Kobayashi, T.;Ushikubi, F.; Aze, Y.; Eguchi,
N.; Urade, Y.; Yoshida, N.; Kimura, K.; Mizoguchi, A.; Honda, Y.;
329
dx.doi.org/10.1021/ml1002234 |ACS Med. Chem. Lett. 2011, 2, 326–330

ACS Medicinal Chemistry Letters
LETTER
by modifications of thiazoleacetic acids as CRTH2 antagonists. Bioorg.
Med. Chem. Lett. 2010, 20, 1638–1641.
(18) Liu, J.; Wang, Y.; Sun, Y.; Marshall, D.; Miao, S.; Tonn, G.;
Anders, P.; Tocker, J.; Tang, H. L.; Medina, J. Tetrahydroquinoline
derivatives as CRTH2 antagonists. Bioorg. Med. Chem. Lett. 2009,
19, 6840–6844.
(19) Stearns, B. A.; Baccei, C.; Bain, G.; Broadhead, A.; Clark, R. C.;
Coate, H.; Evans, J. F.; Fagan, P.; Hutchinson, J. H.; King, C.; Lee, C.;
Lorrain, D. S.; Prasit, P.; Prodanovich, P.; Santini, A.; Scott, J. M.; Stock,
N. S.; Truong, Y. P. Novel tricyclic antagonists of the prostaglandin D2
receptor DP2 with efficacy in a murine model of allergic rhinitis. Bioorg.
Med. Chem. Lett. 2009, 19, 4647–4651.
(20) Ulven, T.; Kostenis, E. Minor Structural Modifications Convert
the Dual TP/CRTH2 Antagonist Ramatroban into a Highly Selective
and Potent CRTH2 Antagonist. J. Med. Chem. 2005, 48, 897–900.
(21) Crosignani, S.; Page, P.; Missotten, M.; Colovray, V.; Cleva, C.;
Arrighi, J. F.; Atherall, J.; Macritchie, J.; Martin, T.; Humbert, Y.; Gaudet,
M.; Pupowicz, D.; Maio, M.; Pittet, P. A.; Golzio, L.; Giachetti, C.;
Rocha, C.; Bernardinelli, G.; Filinchuk, Y.; Scheer, A.; Schwarz, M. K.;
Chollet, A. Discovery of a New Class of Potent, Selective, and Orally
Bioavailable CRTH2 (DP2) Receptor Antagonists for the Treatment of
Allergic Inflammatory. Diseases. J. Med. Chem. 2008, 51, 2227–2243.
(22) Armer, R. E.; Ashton, M. R.; Boyd, E. A.; Brennan, C. J.;
Brookfield, F. A.; Gazi, L.; Gyles, S. L.; Hay, P. A.; Hunter, M. G.;
Middlemiss, D.; Whittaker, M.; Xue, L.; Pettipher, R. Indole-3-acetic
Acid Antagonists of the Prostaglandin D2 Receptor CRTH2. J. Med.
Chem. 2005, 48, 6174–6177.
(23) Liu, J.; Fu, Z.; Wang, Y.; Schmitt, M.; Huang, A.; Marshall, D.;
Tonn, G.; Seitz, L.; Sullivan, T.; Tang, H. L.; Collins, T.; Medina, J.
Discovery and optimization of CRTH2 and DP dual antagonists. Bioorg.
Med. Chem. Lett. 2009, 19, 6419–6423.
(24) The CRTH2 or DP radioligand binding assay was performed
on HEK-293 cells stably expressing human CRTH2 or DP. To measure
binding, [3H]-PGD2 was incubated together with 293 (hCRTH2 or
hDP) cells in the presence of increasing concentrations of compounds.
After teh cells were washed, the amount of [3H]-PGD2 that remained
bound to the cells was measured by scintillation counting, and the
concentration of compounds required to achieve a 50% inhibition of
[3H]-PGD2 binding (the IC50) was determined. The binding buffer
contains either 0.5% BSA (buffer binding) or 50% human plasma
(plasma binding).
(25) Human whole blood was drawn into acid-citrate-dextrose
(ACD) anticoagulated tubes, treated with compounds or DMSO, and
then stimulated with a dose response of PGD2. Fluorochrome con-
jugated antibodies were used to label CRTH2 positive granulocytes, and
CRTH2 receptor internalization was monitored by flow cytometry. The
Kb was determined using the Schild equation.
(26) Human whole blood was drawn into ACD vacutainer tubes,
treated with compounds or DMSO, and then stimulated with a dose
response of PGD2. Cells were lysed, and cAMP was measured using a
competitive ELISA. Comparison of the dose response to PGD2 in
samples containing DMSO only and samples containing compounds
was used in determining Kb using the Schild equation.
(27) Hamelman, E.; Schwarze, J.; Takeda, K.; Oshiba, A.; Larsen,
G. L.; Irvin, G.; Gelfand, G. W. Am. J. Respir. Crit. Care Med. 1997,
156, 766–775.
330
dx.doi.org/10.1021/ml1002234 |ACS Med. Chem. Lett. 2011, 2, 326–330