Optimization of phenylacetic acid derivatives for balanced CRTH2 and DP dual antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.12.107

Our first generation CRTH2 and DP dual antagonists, represented by AMG 009, are more potent toward the CRTH2 receptor than to the DP receptor. Here we report our efforts in the discovery of CRTH2 and DP dual antagonists with more balanced potencies to both receptors, such as compound 15.

Full text of DOI:10.1016/j.bmcl.2011.12.107

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1686–1689
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of phenylacetic acid derivatives for balanced CRTH2 and DP
dual antagonists
⇑
Jiwen (Jim) Liu , Yingcai Wang, Michael G. Johnson, An-Rong Li, Wang Shen, Xuemei Wang, Yongli Su,
Matthew Brown, Bettina Van Lengerich, Erika Rickel, Tod Martin, Alison Budelsky, Lisa Seitz, Jay Danao,
H. Lucy Tang, Tassie Collins, Julio C. Medina
Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Our first generation CRTH2 and DP dual antagonists, represented by AMG 009, are more potent toward
the CRTH2 receptor than to the DP receptor. Here we report our efforts in the discovery of CRTH2 and
DP dual antagonists with more balanced potencies to both receptors, such as compound 15.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 December 2011
Revised 20 December 2011
Accepted 21 December 2011
Available online 30 December 2011
Keywords:
CRTH2
DP
PGD2
GPCR
Dual Antagonist
SAR
Asthma
CRTH2 (chemoattractant receptor-homologous molecule ex-
pressed on Th2 cells, also known as DP2) and DP (prostanoid D
receptor, also known as DP1) are two G-protein coupled receptors
activated by prostaglandin D2 (PGD2). Numerous studies using DP
and CRTH2 antagonists, combined with genetic analysis, support
the view that these receptors play a pivotal role in mediating aller-
gic diseases.1–3 Therefore, there have been a lot of research and
development activities for DP and CRTH2 antagonists, especially
for CRTH2 antagonists.4–19 We believe that blockade of both recep-
tors may prove more beneficial in alleviating allergic diseases trig-
gered by PGD2 than inhibiting either one; therefore we have been
interested in identifying potent CRTH2/DP dual inhibitors, such as
AMG 009 and AMG 853 (Fig. 1).20,21
AMG 009, our first-generation dual antagonist, is more potent
against CRTH2 than DP. It displaced 3H-PGD2 from the CRTH2
receptors expressed on HEK 293 cells with IC50 of 3 nM in buffer
and 21 nM in the presence of 50% plasma. It displaced 3H-PGD2
from the DP receptors expressed on HEK 293 cells with IC50 of
12 nM in buffer and 280 nM in the presence of 50% plasma. AMG
853, our second-generation dual antagonist, is a more balanced
dual antagonist.21 Here we report our efforts in identifying other
more potent and balanced dual antagonists.
Cl
Cl
O
O
S
O
NH
O
NH
S
Cl
Cl
F
OMe
O
O
O
O
H
N
H
N
OH
OH
O
O
AMG 853
AMG 009
Figure 1.
Most of the phenylacetic acid derivatives in this Letter (1–8)
were prepared using the same route reported in our early commu-
nication.20 Sulfoxide 9 and sulfone 10 were obtained from the oxi-
dation of sulfide 8 using 3-chloroperoxybenzoic acid. Compounds
in Table 3 were synthesized through derivatizing the benzoic acid
intermediate, a product of step (a) in Scheme 1. Synthesis of com-
pound 15 is illustrated in Scheme 1 as an example. The benzoic
acid intermediate was converted into the acid chloride, which
was treated with 1-pentyne to afford the acetylenic ketone (step
b, ii).22 The acetylenic ketone was converted into the pyrazole
using hydrazine (step c).23 Reduction of the nitro group followed
by treatment with sulfonyl chloride in pyridine afforded com-
pound 15 after the final ester hydrolysis.
⇑
Corresponding author.
E-mail address: jiwenl@amgen.com (J. Liu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.107

J. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1686–1689
1687
Table 1
Table 3
Cl
O
Cl
O
O
NH
O
NH
Cl
S
Cl
S
OMe
O
O
R
O
n-BuHN
R
OH
DP IC50a in buffer (M)
O
a
Compd
R
CRTH2 IC50 in buffer
(M)
a
a
Compd
R
CRTH2 IC50 in buffer
(M)
DP IC50 in
buffer (M)
n-BuHN
AMG
009
0.003
0.006
0.77
0.012
0.015
0.023
0.010
0.010
0.004
OMe
O
O
O
O
AMG
009
O
O
O
0.003
0.012
N
Et
11
12
13
14
15
OH
OH
OH
N
HN
N
N
Et
1
2
0.31
0.48
0.23
O
N
N
N
n-Pr
0.004
0.002
0.002
0.010
N
N
HN
N
H
N
n-Pr
O
O
O
O
HN
3
0.003
0.083
n-Pr
COOH
COOH
COOH
COOH
HN
Displacement of 3H-PGD2 from the CRTH2 or DP receptors expressed on HEK
293 cells. Assay run in buffer containing 0.5% BSA. See Ref. 25 for assay protocol.
Values are means of three experiments, standard deviation is 30%.
a
4
5
6
0.002
0.58
0.029
0.15
N
O
sulfonamide
linker
O
Cl
O
O
NH
0.005
0.005
Cl
S
OMe
O
O
H
N
Displacement of 3H-PGD2 from the CRTH2 or DP receptors expressed on HEK
293 cells. Assay run in buffer containing 0.5% BSA. See Ref. 25 for assay protocol.
Values are means of three experiments, standard deviation is 30%.
a
OH
O
phenylacetic acid
amide
Figure 2. Areas of optimization.
Table 2
Cl
O
moiety have been reported, such as bioisoteric replacements of the
carboxylic acid, and the substitutions on the phenyl ring and at the
a
O
NH
Cl
S
R
-position of the phenylacetic acid.20,24 Table 1 shows another
X
O
modification in the area of the phenylacetic acid, the heterocyclic
replacements of the phenyl ring. Most of the heterocyclic replace-
ments resulted in a reduction in potency, especially for DP. The
benzofuran derivative (6) was an exception. It maintained the
CRTH2 potency and slightly increased the DP potency in buffer.
However, the improved DP binding potency in buffer was not
maintained in the presence of plasma, as compared to that of
AMG 009 (data not shown).
n-BuHN
OH
O
a
a
Compd
X
R
CRTH2 IC50 in buffer (M) DP IC50 in buffer (M)
AMG 009
7
8
9
O
CH2
S
S(O)
SO2
OMe 0.003
OMe 0.002
0.012
0.006
0.63
>10
H
H
H
0.021
0.66
0.40
10
>10
The results of bisaryl ether linker study are shown in Table 2. It
was found that the methylene linker (7) moderately improved the
binding to the DP receptor. The increased binding potency was also
diminished in the presence of plasma, possibly due to higher plas-
ma protein binding. Other linkers, such as the sulfide (8), sulfoxide
(9) and sulfone (10), all decreased the potencies, especially the DP
potency.
In general, optimization in the areas of phenylacetic acid, sul-
fonamide and ether linker was not fruitful in identifying balanced
dual antagonists. Optimization in the area of the amide proved to
be key for improving DP activity (Table 3). Replacing the amide
functional group with a heterocycle proved to be a successful strat-
egy. The heterocycles with NH were good replacements for the n-
butyl amide, such as those in compound 11, and 13–15. Compound
15 is a more balanced dual antagonist; its DP potency was signifi-
a
Displacement of 3H-PGD2 from the CRTH2 or DP receptors expressed on HEK
293 cells. Assay run in buffer containing 0.5% BSA. See Ref. 25 for assay protocol.
Values are means of three experiments, standard deviation is 30%.
Optimization of this series of phenylacetic acid derivatives is
mainly divided into four areas: phenylacetic acid, bisaryl ether lin-
ker, sulfonamide and amide (Fig. 2). A portion of the structure–
activity relationship (SAR) has been reported.20,21,24 Here we dis-
cuss the SAR that led to the identification of more balanced dual
antagonists and present a more complete picture of the SAR of this
chemical series.
The sulfonamide was important for the DP activity: no potent
dual antagonists were identified through the modification of this
functional group.24 Multiple modifications of the phenylacetic acid

1688
J. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1686–1689
NO2
O
NO2
O
O
a
O
b
O
O
HO
O
d
O
HO
OMe
OMe
OH
O
O
Cl
O O
S
NO2
O
NH
O
O
c
O
O
Cl
O
OMe
OH
N
N
H
N
N
H
15
Scheme 1. reagents and conditions: (a) (i) TMSCHN2, MeOH/benzene, 23 °C, 1 h; (ii) 4-chloro-3-nitrobenzoic acid, Cs2CO3, DMSO, 60 °C, 18 h; 90%, two steps; (b) (i) oxalyl
chloride, DMF, DCM, 23 °C, 17 h; (ii) Pd(PPh3)2Cl2, CuI, triethylamine, 1-pentyne, toluene, 23 °C, 16 h; 67%, two steps; (c) hydrazine, MeOH, 70 °C, 17 h, 76%; (d) (i) H2, Pd/C,
EtOH/EtOAc, 23 °C, 4 h; (ii) 2,4-dichlorobenzenesulfonyl chloride, pyridine, 23 °C, 60 h; (iii) LiOH, THF/H2O, 23 °C, 2 h, 58%, three steps.
8. Sturino, C. F.; O’Neill, G.; Lachance, N.; Boyd, M.; Berthelette, C.; Labelle, M.; Li,
Table 4
L.; Roy, B.; Scheigetz, J.; Tsou, N.; Aubin, Y.; Bateman, K. P.; Chauret, N.; Day, S.
H.; Lévesque, J.; Seto, C.; Silva, J. H.; Trimble, L. A.; Carriere, M.; Denis, D.; Greig,
a
a
a
a
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. J. Med. Chem. 2007, 50, 794.
9. 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.
Bioorg. Med. Chem. Lett. 2008, 18, 2696.
Compd
CRTH2 IC50 in CRTH2 IC50 in DP IC50 in
buffer (M)
DP IC50 in
plasma (M)
buffer (M)
plasma (M)
AMG 009
14
15
0.003
0.002
0.002
0.021
0.020
0.008
0.012
0.010
0.004
0.28
0.11
0.046
10. Arimura, A. In Molecular Imaging for Integrated Medical Therapy and Drug
Development; Tamaki, N., Kuge, Y., Eds.; Springer, 2010; pp 281–287. Part IV.
11. 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.
J. Med. Chem. 2003, 46, 2446.
a
Displacement of 3H-PGD2 from the CRTH2 or DP receptor expressed on HEK 293
cells. Assay run in buffer containing 0.5% BSA or in 50% human plasma. See Ref. 25
for assay protocol. Values are means of three experiments, standard deviation is
30%.
12. Oxagen Ltd Novel CRTH2 Antagonist Shows Potential in Moderate 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.
13. 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. Bioorg. Med. Chem. Lett. 2010, 20, 3287.
14. Grimstrup, M.; Receveur, J. M.; Rist, O.; Frimurer, T. M.; Nielsen, P. A.;
Mathiesen, J. M.; Högberg, T. Bioorg. Med. Chem. Lett. 2010, 20, 1638.
15. Liu, J.; Wang, Y.; Sun, Y.; Marshall, D.; Miao, S.; Tonn, G.; Anders, P.; Tocker, J.;
Tang, H. L.; Medina, J. Bioorg. Med. Chem. Lett. 2009, 19, 6840.
cantly improved compared to that of AMG 009 (Table 4). The oxa-
diazole (12), which has no NH, had significantly weaker CRTH2
potency.
Compounds 14 and 15 were also evaluated for their CRTH2 and
DP functional activities, and compound 15 was also more potent in
inhibiting DP functional activity than AMG 009. Compounds 14
and 15 inhibited human eosinophil shape change mediated by
PGD2 through the CRTH2 receptors with Kb of 0.18 nM and
0.084 nM, respectively.26 AMG 009 had a Kb of 0.050 nM in the
same test. They also inhibited PGD2 induced cAMP response med-
iated by DP in platelets in 80% human whole blood with Kb of
50 nM and 5 nM, respectively, while AMG 009 had a Kb of 42 nM.27
Since CRTH2 and DP share little sequence homology, it is not
surprising to see a divergent SAR for the two receptors in this
chemical series. This phenylacetic acid series is versatile. It satisfies
the demand of the two receptors unrelated in GPCR family and in
sequence. We recently reported that selective antagonists for
CRTH2 and DP were generated through modifications in the areas
of sulfonamide and phenylacetic acid.24 We also reported that
more balanced dual antagonists, such as AMG 853, were discov-
ered through combining good modifications in the areas of amide,
phenylacetic acid and phenyl substituents of the phenyl sulfon-
amide.21 Here we demonstrate that heterocyclic replacement of
the amide of AMG 009 can also yield more balanced dual antago-
nist, such as 15. Its DP potency was significantly improved and
its CRTH2 potency was maintained compared to AMG 009.
16. 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. Bioorg. Med.
Chem. Lett. 2009, 19, 4647.
17. Ulven, T.; Kostenis, E. J. Med. Chem. 2005, 48, 897.
18. 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. J. Med. Chem. 2008, 51, 2227.
19. 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. J. Med. Chem. 2005, 48, 6174.
20. 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. Bioorg. Med. Chem. Lett. 2009, 19,
6419.
21. Liu, J.; Li, A.; Wang, Y.; Johnson, M. G.; Su, Y.; Shen, W.; Wang, X.; Lively, S.;
Brown, M.; Lai, S. J.; Lopez De Turiso, F. G.; Xu, Q.; Van Lengerich, B.; Schmitt,
M.; Fu, Z.; Sun, Y.; Lawlis, S.; Seitz, L.; Danao, J.; Wait, J.; Ye, Q.; Tang, L.; Grillo,
M.; Collins, T. L.; Sullivan, T. J.; Medina, J. ACS Med. Chem. Lett. 2011, 2, 326.
22. Collett, M. J.; Jones, D. W.; Renyard, S. J. J. Chem. Soc., Perkin Trans. 1 1986, 1471.
23. García, H.; Iborra, S.; Miranda, M. A.; Morera, I. M.; Primo, J. Heterocycles 1991,
32, 1745.
24. Wang, Y.; Fu, Z.; Schmitt, M.; Wang, X.; Shen, W.; Rickel, E.; Martin, T.;
Budelsky, A.; Marshall, D.; Collins, T.; Tang, L.; Medina, J; Liu, J. Bioorg. Med.
Chem. Lett. 2011, 21, doi:10.1016/j.bmcl.2011.10.123.
25. The CRTH2 or DP radioligand binding assay was performed on HEK 293 cells
stably expressing human CRTH2 or DP, respectively. To measure binding, [3H]-
PGD2 was incubated together with HEK 293(hCRTH2) cells in the presence of
increasing concentrations of compounds. After washing, 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).
26. Human erythrocytes and granulocytes were enriched from normal donor
peripheral blood by Isolymph (Gallard-Schlesinger Industries, Plainview, NY)
gradient centrifugation. The erythrocytes were removed using ACK lysing
buffer (Gibco, Carlsbad, CA). The mixed granulocyte population was pre-
incubated with vehicle (0.05% DMSO) or antagonists for 10 min at room
References and notes
1. Schuligoi, R.; Sturm, E.; Luschnig, P.; Konya, V.; Philipose, S.; Sedej, M.;
Waldhoer, M.; Peskar, B. A.; Heinemann, A. Pharmacology 2010, 85, 372.
2. Pettipher, R. Br. J. Pharm. 2008, 153, s191.
3. Pettipher, R.; Hansel, T. T.; Armer, R. Nat. Rev. Drug Discov. 2007, 6, 313.
4. Norman, P. Exp. Opin. Invest. Drugs 2010, 19, 947.
5. Medina, J. C.; Liu, J. Annu. Rep. Med. Chem. 2006, 41, 221.
6. Pettipher, R.; Hansel, T. T. Drug News Perspect. 2008, 21, 317.
7. Philip, G.; van Adelsberg, J.; Loeys, T.; Liu, N.; Wong, P.; Lai, E.; Balachandra, S.;
Reiss, T. F. J. Allerg. Clin. Pharmacol. 2009, 124, 942.

J. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1686–1689
1689
temperature prior to stimulation with PGD2 (600–0.003 nM at 1:3 dilution)
(Cayman Chemical Co, Ann Arbor, MI) for 10 min at 37 °C. The cells were fixed
using 1% final paraformaldehyde (Alpha Aesar, Ward Hill, MA) and were
analyzed on a FACS caliber (BD Biosciences, San Jose, CA) flow cytometer.
Leukocytes were gated on using forward/side scatter parameters. The FL2
positive cells (eosinophils) were then gated and their geometric mean of the
forward scatter was calculated. The geometric means were graphed using
GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA) and IC50 values were
calculated. The Kb values were calculated from their IC50 values using the
equation A/(RÀ1) where A is the concentration of the inhibitor used. The value
R = X/Y where X is the IC50 value of PGD2 in the presence of the inhibitor and Y
is the IC50 value of PGD2 alone.
27. Whole blood was collected from healthy human volunteers into ACD
Vacutainer tubes (VWR). IBMX (3-Isobutyl-1-methylxanthine, Sigma) was
added to whole blood at a final concentration of 1 mM. 200
treated blood was incubated in the presence of a small molecule DP antagonist
in a 96 well plate at 37 °C for 30 min. 20 L of the antagonist incubated blood
lL/well of IBMX
l
was then aliquoted into a 96 well plate containing PGD2 (Cayman Chemicals)
at various concentrations. The plate was incubated at 37 °C for 60 min. After
the PGD2 stimulation, the blood was lysed with 55 lL/well of Tropix Lysis
buffer (ABI) and subsequently assayed for cAMP levels using the cAMP-Screen
DirectÒ Immunoassay System (ABI). The PGD2 stimulated cAMP curves were fit
Genedata Screener Condoseo (Genedata AG) using a Hill fit model. A Schild plot
is the constructed and Kb is calculated.