Minor structural modifications convert the dual TP/CRTH2 antagonist ramatroban into a highly selective and potent CRTH2 antagonist

Article abstract of DOI:10.1021/jm049036i

Ramatroban, a thromboxane A₂ receptor (TP) antagonist with clinical efficacy in asthma and allergic rhinitis, was recently shown to also antagonize the prostaglandin D₂ receptor CRTH2. Here we report that minor structural changes to

Full text of DOI:10.1021/jm049036i

Supporting Information
Minor Structural Modifications Convert the Dual TP/CRTH2 Antagonist
Ramatroban into a Highly Selective and Potent CRTH2 Antagonist
Trond Ulven* and Evi Kostenis
7TM Pharma A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark.
Table of Contents:
Synthetic procedures and characterization of compounds
Biological assays
Generation/origin of the cDNA Constructs
Cell Culture and Transfection
Binding experiments.
BRET assay
Inositol phosphate accumulation assays
cAMP accumulation assays
Materials
Calculations and data analysis
S1

Synthetic procedures and characterization of compounds
General Comments. Melting points were determined on an Electrothermal 9200
1
13
melting point apparatus. H and C NMR spectra were recorded on a Bruker AMX 300
spectrometer, operating at 300.13 and 75.47 MHz respectively. Spectra were calibrated
relative to tetramethylsilane internal standard or residual solvent peak. Infrared spectra
were recorded from films on a NaCl plate or from KBr disks on a Perkin Elmer Spectrum
One FT-IR spectrophotometer. High resolution mass spectra (HRMS) were obtained on a
JEOL-SX 102 in FAB+ mode with NBA matrix. Analytical HPLC was performed on an
Agilent 1100-series instrument equipped with UV-detector and a VL mass detector
operating under electrospray ionisation conditions in positive (ESI+) or negative (ESI-)
mode. Conditions: column: XTerra MS C18; flow: 1.0 mL/min; gradient: 0-5 min: 15-
100% acetonitrile in water, 5-7½ min: 100% acetonitrile, modifier: 5 mM ammonium
formate. Elemental analyses were performed by Johannes Theiner at Mikroanalytisches
Laboratorium, University of Vienna, Austria, and results are within 0.4% of calculated
values, unless otherwise stated. All reactions were conducted under an atmosphere of
argon, unless otherwise stated. Starting materials and reagents were purchased from
Aldrich. Dry solvents were purchased from Fluka and used as obtained.
4-Fluoro-N-methyl-N-(2,3,4,9-tetrahydro-1H-carbazol-3-yl)benzenesulfonamide
(4). A solution of 1,2,4,9-tetrahydrocarbazole-3-one1 (800 mg, 4.32 mmol) in dry MeOH
(80 mL) was added methylamine (2M in THF, 15 mL, 30 mmol), acetic acid (1.72 mL,
30 mmol) and dry MgSO4 (5 g). The reaction mixture was stirred at room temperature for
15 min before NaBH3CN (534 mg, 8.5 mmol) was added, and stirring was continued for
12 h. The reaction was added EtOAc, solids were filtered off and washed with EtOAc.
The organic phase was washed with 1 M Na2CO3 (2x) and brine (1x), dried (MgSO4) and
concentrated to give methyl-(2,3,4,9-tetrahydro-1H-carbazol-3-yl)-amine as a yellow oil
(750 mg), which was used directly in the next step.
The methyl-(2,3,4,9-tetrahydro-1H-carbazol-3-yl)-amine (3.8 g, 19 mmol) in CH2Cl2
(100 mL) was added Et3N (4.2 mL, 30 mmol) and fluorobenzenesufonyl chloride (5.8 g,
30 mmol), and the reaction was stirred at room temperature. After 12 h the reaction
S2

mixture was diluted with CH2Cl2 and the organic phase was washed with 3% HCl (2x), 1
M NaOH (2x) and brine (1x), dried (MgSO4) and concentrated. The residue was purified
by flash chromatography (SiO2, EtOAc:heptane, 1:2), and the product was recrystallized
o
1
from MeOH to give 4.6 g (68%) of 4 as yellow crystals: mp: 211-212 C; H NMR
(CDCl3): δ 1.83 (m, 1H), 2.02 (ddd, J = 17.7, 11.9, 5.8 Hz, 1H), 2.63-2.80 (m, 2H), 2.81-
3.02 (m, 2H), 2.92 (s, 3H), 4.33 (m, 1H), 7.09 (m, 1H), 7.16 (m, 1H), 7.20-7.31 (m, 3H),
7.34 (d, J = 4.1 Hz, 1H), 7.76 (br s, 1H), 7.90 (m, 2H); 13C NMR (CDCl3): δ 23.1, 24.5,
27.6, 29.2, 54.6, 108.4, 110.8, 116.6 (d, 2JCF = 22 Hz), 117.8, 119.7, 121.9, 127.6, 129.9
(d, 3JCF = 9 Hz),132.4, 136.5, 165.4 (d, 1JCF = 253 Hz); IR νmax 3400, 1592, 1492, 1333,
1236, 1166, 1154, 1088, 959, 840, 741 cm-1; MS ESI+ m/z 358.9 [M + H]+.
3-{3-[(4-Fluorobenzenesulfonyl)methylamino]-1,2,3,4-tetrahydrocarbazol-9-yl}-
propionic acid (5). The indole 4 (339 mg, 0.95 mmol) in dry DMF (18 mL) was added
NaH (60% in mineral oil, 44 mg, 1.1 mmol) and the reaction mixture was stirred under
argon at room temperature. After 60 min ethyl 3-bromopropionate (0.25 mL, 2.0 mmol)
was added, and stirring at room temperature was continued. After 12 h the reaction
mixture was added EtOAc. The organic phase was washed with water (3x) and brine
(1x), dried (MgSO4) and concentrated. The crude product was purified by flash
chromatography (SiO2, EtOAc:heptane, 1:2) to give 365 mg (87%) white foam,
consisting of a 10:7 mixture of the alkylated product and the starting material (4). The
mixture was inseparable on TLC, and was used directly in the next step: TLC
(EtOAc:heptane, 1:2): Rf 0.20.
The product mixture containing the ethyl ester in THF (15 mL) was added LiOH·H2O
(65 mg, 1.6 mmol) in water (6 mL). After 12 h the reaction was acidified with 3% HCl
and extracted with CH2Cl2 (2x). The extract was washed with brine (1x), dried (MgSO4)
and concentrated. The residue was purified by flash chromatography (SiO2,
EtOAc:heptane, 1:1, then [MeOH w/10%AcOH]:EtOAc, 1:9) to give 186 mg (47% over
2 steps) of 5 as a white solid: mp 148-151 oC; TLC (SiO2, [MeOH w/10%AcOH]:EtOAc,
1
1:9) Rf 0.5; H NMR (CDCl3): δ 1.83-2.01 (m, 2H), 2.60-2.75 (m, 2H), 2.79 (t, J = 7.1
Hz, 2H), 2.80-3.00 (m, 2H), 2.89 (s, 3H), 4.25-4.44 (m, 1H), 4.34 (td, J = 7.1, 3.4 Hz,
13
2H), 7.04-7.11 (m, 1H), 7.14-7.29 (m, 4H), 7.33 (d, J = 7.5 Hz, 1H), 7.88 (m, 2H); C
S3

NMR (CDCl3): δ 22.0, 24.5, 27.6, 29.2, 34.5, 38.6, 54.4, 108.1, 109.0, 116.6 (d, 2JCF = 22
Hz),118.1, 119.7, 121.7, 127.3, 129.9 (d, 3JCF = 9 Hz), 133.4, 136.5, 165.2 (d, 1JCF = 253
Hz), 176.2; IR νmax 1712, 1592, 1493, 1467, 1337, 1167, 958, 840, 737 cm-1; HRMS
calcd for C22H23FN2O4S [M]+: 430.1363, found 430.1353; LC/MS ESI- Rt 3.23 min, m/z
428.8 [M
-
H]-, purity: >95% by UV-trace, >95 by MS-trace. Anal.
(C22H23FN2O4S·¼H2O) C, H, N, S.
Ethyl (3-oxo-1,2,3,4-tetrahydrocarbazol-9-yl)-acetate glycol ketal (6).The indol 2
(4.53 g, 19.8 mmol) in dry DMF (225 ml) under argon at room temperature was added
NaH (60% in mineral oil, 948 mg, 23.7 mmol) in one portion. After 60 min ethyl
bromoacetate (4.4 mL, 40 mmol) was added and stirring was continued. After 12 h the
reaction mixture was added EtOAc and the organic phase was washed with water (3x)
and brine (1x), dried (MgSO4) and concentrated. The crude product was recrystallized
from EtOAc:heptane (1:2), and the crystals were washed with EtOAc:heptane (1:5) to
give 4.71 g white powder. The residue was concentrated and purified by flash
chromatography (SiO2, EtOAc:hepante, 1:2), and the product was recrystallized from
Et2O to provide another 0.77 g white powder, in total 5.48 g (88%) of 6 as white fine
o
1
crystals: mp 112-113 C; TLC (SiO2, EtOAc:heptane, 1:2) Rf 0.2; H NMR (CDCl3): δ
1.26 (t, J = 7.0 Hz, 3H), 2.11 (t, J = 6.4 Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H), 2.99 (s, 2H),
4.06 (m, 4H), 4.20 (q, J = 7.1 Hz, 2H), 4.72 (s, 2H), 7.05-7.19 (m, 3H), 7.42 (d, J = 7.9
Hz, 1H); 13C NMR (CDCl3): δ 14.4, 20.6, 31.9, 32.1, 45.1, 61.8, 64.9, 108.5, 108.6,
109.2, 118.1, 119.6, 121.6, 127.6, 133.6, 137.7, 169.0; IR νmax 1751, 1468, 1198, 1115,
1055, 1017, 740 cm-1; MS ESI+ m/z 315.9 [M + H]+.
Ethyl (3-oxo-1,2,3,4-tetrahydrocarbazol-9-yl)-acetate (7). Ketal 6 (1.43 g, 4.54
mmol) in THF (40 mL) was added 15% aq. HCl (10 mL), and the homogenous reaction
mixture was stirred at room temperature under inert atmosphere. After 16 h the reaction
was added water and extracted with CH2Cl2 (3x). The extract was washed with brine
(1x), dried (MgSO4) and concentrated to give 1.32 g red oil consisting of a mixture of the
title compound and the corresponding carboxylic acid. The crude product was dissolved
in dry CH2Cl2 (40 mL) and added EDC (600 mg, 4 mmol). The mixture was stirred at
S4

room temperature under argon for 15 min, and dry EtOH (10 mL) was added. Stirring
was continued for 3 h. The reaction was concentrated, added CH2Cl2, washed with 3%
HCl, and brine, dried (MgSO4) and concentrated. The residue was subject to flash
chromatography (SiO2, EtOAc:heptane, 1:2) to provide 1.0 g (83%) of compound 7 as
o
1
white fine crystals: mp 92-94 C; TLC (SiO2, EtOAc:heptane, 1:2): Rf 0.2; H NMR
(CDCl3): δ 1.27 (t, J = 7.2 Hz, 3H), 2.83 (t, J = 7.0 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H), 3.64
(s, 2H), 4.22 (q, J = 7.2 Hz; 2H), 4.78 (s, 2H), 7.11-7.18 (m, 1H), 7.20-7.24 (m, 2H), 7.44
(d, J = 7.7 Hz, 1H); 13C NMR (CDCl3): δ 14.4, 21.7, 36.6, 38.6, 45.0, 62.0, 107.7, 108.9,
118.9, 120.2, 122.4, 126.9, 133.9, 137.7, 168.7, 209.3; IR νmax 1748, 1714, 1467, 1200,
1179, 741 cm-1; MS ESI+ m/z 272.0 [M + H]+.
Ethyl [3-(4-fluorobenzenesulfonylamino)-1,2,3,4-tetrahydrocarbazol-9-yl]acetate
(10). Ketone 7 (136 mg, 0.5 mmol) in EtOH (3 mL) was added ammonium formate (385
mg, 5 mmol) and sodium cyanoborohydride (31 mg, 0.5 mmol). The reaction was stirred
under argon at room temperature. After 24 h the reaction mixture was concentrated and
the residue was partitioned between CH2Cl2 and 1 M Na2CO3. The organic phase was
dried (MgSO4) and concentrated to give amine 8 as a green oil, which was used in the
next step without purification: LC/MS: Rt 2.59 min, m/z 272.9 [M + H]+.
The crude amine 8 in dry CH2Cl2 (8 mL) was added triethylamine (0.08 mL, 0.6 mmol)
and 4-fluorophenylsulfonyl chloride (117 mg, 0.6 mmol). The reaction was stirred under
argon at room temperature. After 16 h the reaction mixture was added CH2Cl2 and
washed with 3% HCl (2x), 1 M Na2CO3 (1x) and brine (1x), dried (MgSO4) and
concentrated. The residue was purified by flash chromatography (SiO2, EtOAc:heptane,
1:2) to give 56 mg (26% yield over 2 steps) 10 as a white amorphous solid: TLC(SiO2,
EtOAc:heptane, 1:2): Rf 0.15; 1H NMR (CDCl3): δ 1.27 (t, J = 7.0 Hz, 3H), 1.98-2.07 (m,
2H), 2.52 (dd, J = 15.6, 6.0 Hz, 1H), 2.68-2.78 (m, 2H), 2.89 (dd, J = 15.5, 4.7 Hz, 1H),
3.82 (m, 1H), 4.20 (q, J = 7.0 Hz, 2H), 4.70 (s, 2H), 4.86 (d, J = 8.1 Hz, 1H), 7.04-7.11
(m, 1H), 7.11-7.22 (m, 4H), 7.30 (d, J = 7.0 Hz, 1H), 7.89 (m, 2H); 13C NMR (CDCl3): δ
2
14.4, 19.2, 28.6, 29.3, 44.8, 49.4, 61.9, 106.9, 108.7, 116.6 (d, JCF = 22 Hz), 118.1,
119.9, 122.0, 127.4, 129.9 (d, 3JCF = 9 Hz), 134.0, 137.3, 137.4, 165.2 (d, 1JCF = 253 Hz),
S5

168.9; IR νmax 3286, 2931, 1740, 1592, 1468, 1166, 1153, 839, 740 cm-1; MS ESI+ m/z
430.8 [M + H]+.
[3-(4-Fluorobenzenesulfonylamino)-1,2,3,4-tetrahydrocarbazol-9-yl]acetic
acid
(12). The ester 10 (15 mg, 0.034 mmol) in THF (1 mL) was added LiOH·H2O (5 mg, 0.1
mmol) in water (0.6 mL). The homogenous reaction mixture was stirred at room
temperature. After 1 h the reaction was added 3% HCl until pH <1 and the mixture was
extracted with CH2Cl2 (2x). The extract was washed with brine (1x), dried (MgSO4) and
concentrated to give 13 mg (95%) of acid 12 as a white foam: TLC(SiO2, [MeOH w/5%
AcOH]:EtOAc, 1:9) Rf ~0.25; 1H NMR (CDCl3): δ 1.95-2.06 (m, 2H), 2.50 (dd, J = 15.6,
6.0 Hz, 1H), 2.59-2.78 (m, 2H), 2.85 (dd, J = 15.6, 4.9 Hz, 1H), 3.76-3.86 (m, 1H), 4.69
(s, 2H), 5.00 (d, J = 7.7 Hz, 1H), 7.04-7.21 (m, 5H), 7.29 (d, J = 7.5 Hz, 1H), 7.81-7.90
(m, 2H); 13C NMR (CDCl3): δ 19.1, 28.6, 29.1, 44.2, 49.4, 107.2, 108.7, 116.6 (d, 2JCF
=
3
22 Hz), 118.2, 120.1, 122.1, 129.9 (d, JCF = 9 Hz), 134.0, 137.2, 173.2; IR νmax3273,
2928, 1732, 1592, 1494, 1468, 1166, 1153, 910, 839, 736 cm-1. HRMS calcd for
C20H19FN2O4S [M]+: 402.1050, found 402.1042. LC/MS ESI- Rt 2.56 min, m/z 400.8 [M
- H]-, purity: >97% by UV-trace, >97 by MS-trace.
Ethyl
{3-[(4-fluorobenzenesulfonyl)methylamino]-1,2,3,4-tetrahydrocarbazol-9-
yl}-acetate (11). The carbazolone 7 (900 mg, 3.3 mmol) in dry EtOH (60 mL) was added
acetic acid (1.32 mL, 23 mmol), methyl amine (33 wt% in EtOH, 2.86 mL, 23 mmol) and
3Å molecular sieves (7 g). The reaction mixture was stirred for 15 min before sodium
cyanoborohydride (600 mg, 7 mmol) was added. Stirring was continued over night. The
reaction was then added EtOAc, solids were filtered off and washed with EtOAc. The
organic phase was washed with 1 M Na2CO3 (2x) and brine (1x), dried (MgSO4) and
concentrated to give 0.9 g of methylamine 9 as a green solid foam, which was used
directly in the next step.
The crude methylamine 9 (0.9 g) in CH2Cl2 (20 mL) was added Et3N (0.81 mL, 5.8
mmol) and fluorobenzenesufonyl chloride (1.23 g, 5.8 mmol), and the reaction mixture
was stirred at room temperature. After 12 h the reaction mixture was diluted with CH2Cl2
(250 mL), washed with 3% HCl (2x), 1 M NaOH (2x) and brine (1x), dried (MgSO4) and
S6

concentrated. The residue was purified by flash chromatography (SiO2, EtOAc:heptane,
1:2) to give 0.92 g (54%) of 11 as a white foam: TLC (SiO2, EtOAc:heptane, 1:2) Rf 0.2;
1H NMR (CDCl3): δ 1.26 (t, J = 7.2 Hz, 3H), 1.82-2.06 (m, 2H), 2.61-2.91 (m, 4H), 2.90
(s, 3H), 4.20 (q, J = 7.1 Hz, 2H), 2.26-2-39 (m, 1H), 4.69 (s, 2H), 7.03-7.12 (m, 1H),
13
7.12-7.27 (m, 4H), 7.33 (d, J = 7.7 Hz, 1H), 7.84-7.92 (m, 2H); C NMR (CDCl3): δ
14.4, 21.8, 24.4, 27.5, 29.2, 44.8, 54.3, 61.9, 108.4, 108.7, 116.6 (d, 2JCF = 22 Hz), 118.1,
119.9, 121.9, 127.3, 129.9, 133.8, 136.48, 136.52, 137.3, 165.2 (d, 1JCF = 253 Hz), 166.9,
168.8; IR νmax 1749, 1591, 1493, 1467, 1338, 1195, 1167, 1088, 958, 840, 740 cm-1; MS
ESI+ m/z 444.6 [M + H]+.
{3-[(4-Fluorobenzenesulfonyl)methylamino]-1,2,3,4-tetrahydrocarbazol-9-
yl}acetic acid (13). The ester 11 (0.56 g, 1.26 mmol) in THF (20 mL) was added
LiOH·H2O (105 mg, 2.5 mmol) in water (8 mL) and the reaction was stirred at room
temperature. After 12 h the reaction was added 3% HCl until pH <1 and the mixture was
extracted with CH2Cl2 (2x). The extract was washed with brine (1x), dried (MgSO4) and
concentrated to give 0.49 g (94%) orange solid foam. The crude product was subject to
flash chromatography (SiO2, EtOAc:Hep, 1:1, then [MeOH w/10%AcOH]:EtOAc, 1:9).
The residue was dissolved in CH2Cl2 and washed with 3% HCl (1x) and brine (1x), dried
(MgSO4) and concentrated to give 0.30 g (57%) solid as orange foam. The product was
recrystallized from CH2Cl2 to give 178 mg (34%) of 13 as white crystals: mp 165-173 oC;
1
TLC (SiO2, [MeOH w/5% AcOH]:EtOAc, 1:9) Rf 0.40; H NMR (DMSO-d6): δ 1.48-
1.58 (m, 1H), 1.79-1.98 (m, 1H), 2.45-2.59 (m, 1H), 2.60-2.85 (m, 3H), 2.83 (s, 3H), 4.12
(m, 1H), 4.84 (s, 2H), 6.93-7.09 (m, 2H), 7.26-7.33 (m, 2H), 7.42-7.52 (m, 2H), 7.89-
13
8.00 (m, 2H); C NMR (DMSO-d6): δ 21.0, 24.3, 26.0, 28.9, 44.0, 54.1, 106.8, 109.1,
116.6 (d, 2JCF = 23 Hz), 117.3, 118.9, 120.8, 126.5, 129.9 (d, 3JCF = 10 Hz), 134.4, 135.9,
1
137.0, 164.4 (d, JCF = 249 Hz), 170.4; IR νmax 1726, 1591, 1493, 1466, 1334, 1166,
1155, 958, 839, 741 cm-1; HRMS calcd for C21H21FN2O4S [M]+: 416.1206, found
416.1208. LC/MS ESI- Rt 4.40 min, m/z 415.0 [M - H], purity: >99% by UV-trace, >99%
by MS-trace. Anal. (C21H21FN2O4S) C, H, N, S.
S7

Purity of target compounds
Elemental analysis
Cmpd
Formula
Calc
Found
C
H
N
S
C
H
N
S
5
13
C22H23FN2O4S·¼H2O 60.60 5.35 6.42 7.37
C21H21FN2O4S 60.56 5.08 6.73 7.70
60.74 5.45 6.43 7.24
60.23 5.08 6.74 7.61
LC/MS purity
Compound Rt (min)
m/z
Purity by UV-trace Purity by MS-trace
5
12
13
3.23
2.56
4.40
428.8 [M – H]
400.8 [M – H]
415.0 [M – H]
>95%
>97%
>99%
>95%
>97%
>99%
S8

Biological assays
Generation/origin of the cDNA Constructs. The coding sequence of human CRTH2
(genbank accession no NM_004778) was amplified by PCR from a human hippocampus cDNA
library and inserted into the pcDNA3.1(+) expression vector (invitrogen) via 5’ HindIII and 3’
EcoRI. To generate a CRTH2-Renilla luciferase (CRTH2-Rluc) fusion protein, the CRTH2
coding sequence without a STOP codon and Rluc were amplified, fused in frame by PCR and
subcloned into the pcDNA3.1(+)Zeo expression vector. The thromboxane A2 (TXA2) receptor
(gen bank accession no BC074749, version beta) has been cloned from a leukocyte cDNA library
and inserted via 5’ HindIII and 3’ BamHI into pcDNA3.1(+). A TXA2-receptor-Rluc fusion
protein was generated by PCR and inserted via 5’ HindIII and 3’ XbaI into pcDNA3.1(+). The
prostaglandin D2 DP receptor (gen bank accession no NM_000953) was cloned from a bone
marrow cDNA library and inserted via 5’ HindIII and 3’ BamHI into pcDNA3.1(+). Human β-
arrestin2 (β-arr2) N-terminally tagged with GFP2 (GFP2-β-arr2) and Renilla luciferase were
purchased from BioSignal Packard Inc, Montreal, Canada). The sequence identity of the
constructs was verified by restriction endonuclease digests and sequencing in both directions on
an ABI Prism (Applied Biosystems, Foster City, CA).
Cell Culture and Transfection. COS-7 cells were grown in Dulbecco's modified Eagle's
medium (DMEM) 1885 supplemented with 10% fetal bovine serum, 100 units/ml penicillin,
1000 µg/ml streptomycin, and kept at 37°C in a 10% CO2 atmosphere. HEK293 cells were
maintained in Minimum Essential medium (MEM) supplemented with 10% (v/v) heat inactivated
fetal calf serum (HIFCS), 2mM GlutamaxTM-I, 1% non essential amino acids (NEAA), 1%
sodium pyruvate and 10 µg/ml gentamicin. For transient transfections, cells were allowed to
express the exogeneously added DNA for 48 hours before performing the binding or functional
experiments. Transient transfections on COS7 cells were performed essentially as described by
Heydorn et al.2 using a calcium phosphate-DNA co-precipitation method with the addition of
chloroquine. For functional inositolphosphate assays of CRTH2, COS7 cells were transiently
cotransfected with the receptor cDNA and a promiscuous Gα protein3 facilitating
inositolphosphate production by the otherwise Gi/o-selective receptor. Inositolphosphate assays
of the Gq/11-coupled TXA2 receptor were performed in the absence of any cotransfected Gα
protein as was the case for cAMP assays performed in DP receptor expressing cells. To perform
the binding and functional Bioluminescence Resonance Energy Transfer (BRET) assays, a
HEK293 cell clone stably expressing βarr2-GFP2 and CRTH2-Rluc was generated. BRET assays
S9

on the TXA2 receptor were performed upon transient transfection of the TXA2-Rluc fusion
protein into a HEK293 cell clone stably expressing βarr2-GFP2.
Binding experiments. – whole cell binding - 24h after transfection COS-7 cells were seeded
into 96well plates at a density of 30.000 cells/well. Competition binding experiments on whole
cells were then performed about 18-24 h later using the appropriate radioligands (see below) in a
binding buffer consisting of HBSS (GIBCO) and 10 mM HEPES (pH 7.5). The following
radioligands were used: (i) 1.2 nM [3H]PGD2 (NEN, 172 Ci/mmol) for CRTH2 expressing cells,
(ii) 0,5 nM [3H]PGD2 for DP receptor expressing cells, and (iii) 5 nM [3H]SQ29548 (Perkin
Elmer, 48,2 Ci/mmol) for TXA2 receptor expressing cells. Competing ligands were diluted in
DMSO which was kept constant at 1% (v/v) of the final incubation volume. Total and nonspecific
binding were determined in the absence and presence of 10 µM PGD2 for CRTH2 and DP
expressing cells, and 50 µM U46619, respectively, for TXA2 expressing cells. Binding reactions
were routinely conducted for 3 h at 4°C and terminated by 2 washes (100 µl each) with ice cold
binding buffer. Radioactivity was determined by liquid scintillation counting in a TOPCOUNT
(Packard) following over night incubation in Microscint 20. Stable HEK293 cells expressing
CRTH2-Rluc were seeded at a density of 30.000 cells/well 18-24 h prior to the binding assay
which was performed essentially as described for COS7 cells above. Fusion of Renilla luciferase
to the C-terminus of CRTH2 resulted in a fusion protein that displayed virtually indistinguishable
pharmacology from CRTH2wt. Determinations were made in duplicates.
BRET assay. Functional BRET assays were performed on HEK293 cells stably expressing
human CRTH2-Rluc and GFP2-β-arr2 or transiently expressing the TXA2-Rluc fusion protein
together with GFP2-β-arr2. Prior to their use in the BRET assay cells were detached and re-
suspended in D-PBS with 1000 mg/l L-Glucose at a density of 2x106 cells/ml. DeepBlueC™ was
diluted to 50 µM in D-PBS with 1000 mg/l L-Glucose (light sensitive). 100 µl of cells were
transferred to wells in a 96-well microplate (white OptiPlate) and placed in the Mithras LB 940
instrument (BERTHOLD TECHNOLOGIES, Bad Wildbad, Germany). 12 µl/well agonist were
then injected by injector 1 and 10 µl DeepBlueC™/well were injected simultaneously by injector
2. 5 seconds after the injections the light output from the well was measured sequentially at 400
nm and 515 nm, and the BRET signal was determined by calculating the ratio of the fluorescence
emitted by GFP2-β-arr2 (515 nM) over the light emitted by the receptor-Rluc (400 nM).
Antagonists were added before placing the microplates into the Mithras LB 940 and allowed to
preincubate for 15 minutes prior to the addition of agonist and DeepBlueC™. Compounds were
dissolved in DMSO and the final DMSO concentration was kept constant at 1% in the assay.
S10

Inositol phosphate accumulation assays. 24 h after transfection cells were seeded in 96-well
tissue culture plates and loaded with 5 µCi myo-[2-3H]-Inositol (16 Ci/mmol, TRK911,
Amersham Biosciences). The next day cells were then washed twice in HBSS buffer (including
CaCl2 and MgCl2, GIBCO cat. 14025-050) and stimulated with the respective agonists in HBSS
buffer supplemented with 5 mM LiCl for 45 minutes at 37°C. The reactions were terminated by
aspiration and addition of 10 mM ice-cold formic acid, and incubated for 30 minutes on ice. The
lysate was applied to AG 1-X8 anion-exchange resin (Bio-Rad, Hercules, CA) and washed twice
with buffer containing 60 mM sodium formate and 5 mM borax. The [3H]-inositol-phosphate
fraction was then eluted by adding 1 M ammonium formate and 100 mM formic acid solution and
counted after addition of HiSafe3 scintillation fluid (PerkinElmer, Boston, MA).
cAMP accumulation assays. COS7 cells transiently expressing the DP receptor were
metabolically labeled with 2 µCi of [3H]adenine (Amersham, TRK311) in 6-well plates for 18-24
hours at 37°C. They were then washed twice with PBS and stimulated with increasing
concentrations of PGD2 in HEPES-buffered saline supplemented with
1
mM
isobutylmethylxanthine (IBMX) for 30 minutes at 37°C. The reaction was stopped by adding 5%
(w/v) ice-cold trichloroacetic acid supplemented with 0.1 mM cAMP and 0.1 mM ATP.
[3H]cAMP was separated from the remaining nucleotides using anion exchange chromatography
as described4 and radioactivity was counted after addition of HiSafe3 scintillation fluid
(PerkinElmer, Boston, MA).
Materials. White 96well Optiplates and DeepBlueC™ were obtained from Packard
BioScience, Montreal, Canada. Tissue culture media and reagents were purchased from the
GIBCO invitrogen corporation (Breda, Netherlands). PGD2 was from Cayman and [3H]PGD2
from NEN. All other laboratory reagents were from Sigma (St. Louis, MO), unless explicitly
specified.
Calculations and data analysis - IC50 and EC50 values were determined by nonlinear
regression using the Prism 3.0 software (GraphPad Software, San Diego). Values of the
dissociation and inhibition constants (Kd and Ki) were estimated from competition binding
experiments using the equations Kd = IC50 L and Ki = IC50/(1 + L/Kd), where L is the
concentration of radioactive ligand and Kd is its dissociation constant. Antagonistic potencies in
functional assays are given as IC50 values that were obtained by competing with increasing
concentrations of inhibitor compound for an agonist concentration required to elicit about 75-80%
of the maximal agonist efficacy. IC50 values generated by this procedure are very closely
matching the true affinity of antagonists determined by the analysis according to Arunlakshana &
Schild.5
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References:
1. Urrutia, A.; Rodríguez, J. G. Synthesis of Aspidospermidine Alkaloids from 1,2,3,4-
tetrahydrocarbazole: Total Stereoselective Synthesis of (±)-18-Noraspidospermidine.
Tetrahedron 1999, 55, 11095-11108.
2. Heydorn, A.; Ward, R. J.; Jorgensen, R.; Rosenkilde, M. M.; Frimurer, T. M.; Milligan, G.,
Kostenis, E. Identification of a novel site within G protein alpha subunits important for
specificity of receptor-G protein interaction. Mol. Pharmacol. 2004, 66, 250-259.
3. Kostenis, E. Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled
receptors? Trends Pharmacol. Sci. 2001, 22, 560-564.
4. Holst, B.; Hastrup, H.; Raffetseder, U.; Martini, L.; Schwartz, T. W. Two active molecular
phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis. J.
Biol. Chem. 2001 276, 19793-19799.
5. Arunlakshana, O.; Schild, H. O. Some quantitative uses of drug antagonists. Br J. Pharmacol.
1959 14, 48-58.
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