Chemistry & Biology, Volume 18

Supplemental Information

Chemogenomic Discovery of Allosteric Antagonists

at the GPRC6A Receptor

David E. Gloriam, Petrine Wellendorph, Lars D. Johansen, Alex Rojas Bie Thomsen,

Karina Phonekeo, Daniel Sejer Pedersen, and Hans Bräuner-Osborne

Inventory of Supplemental Information Relates to Figure 1 and Table 1, Table S2: Effects of L-Orn and the 2-phenyl indoles 1 and 3 on wildtype and mutated mouse GPRC6A receptors This table displays mutagenesis data that validates the most crucial ligand-receptor interaction described in Figure 1 and Table 1; a hydrogen bond between the indole nitrogen to a backbone carbonyl in TMH5. Relates to Figure 3, Figure S1: Inhibitory effect of Cpd 3 at mGPRC6A expressed in Xenopus laevis oocytes This Figure confirms the inhibitory activity of the compound 3, seen in Figure 3, in a Xenopus laevis expression system that is independent on co-expression of chimeric G proteins and does not risk effects from L-amino acids in the media/buffers Relates to Figure 4, Figure S2: Schild plot of Cpd 1 at mGPRC6A This concentration-response curve demonstrates a non-competitive mode of action. This supports the chemogenomic hypothesis of a binding site located in the transmembrane region i.e. distinct from the orthosteric site in the amino terminus. Relates to Table 2, Table S1: Overview of evaluated 2-phenyl-indole compounds Table S1 describes the potencies (IC50 values) of all tested 2-phenyl-indoles, whereas Table 2 shows the potencies (and solubility) of the lead compounds (active and selective). Relates to Figure 2, Supplemental Experimental Procedures This is a detailed description of the synthesis of the compounds in Figure 2.

Table of Contents Supplemental Data ............................................................................................................................................. 3 

Table S1: Overview of evaluated 2-phenyl-indole compounds, related to Table 2 ............................. 3 

Figure S1: Inhibitory effect of Cpd 3 at mGPRC6A expressed in Xenopus laevis oocytes, related to

Figure 3 ............................................................................................................................................................ 6 

Figure S2: Schild plot of Cpd 1 at mGPRC6A, Related to Figure 4 ...................................................... 7 

Table S2: Effects of L-Orn and the 2-phenyl indoles 1 and 3 on wildtype and mutated mouse

GPRC6A receptors, Related to Figure 1 and Table 1 .............................................................................. 8 

Supplemental Experimental Procedures, Relates to Figure 2 ...................................................................... 9 

General experimental .................................................................................................................................... 9 

Synthesis of Ligand 1 .................................................................................................................................. 10 

Synthesis of ligand 2 .................................................................................................................................... 13 

Synthesis of the N-methylated analogue 3 ............................................................................................... 16 

Materials ........................................................................................................................................................ 19 

Supplemental References ................................................................................................................................ 19 

Supplemental Data 

Table S1: Overview of evaluated 2­phenyl­indole compounds, related to Table 2 Chemical structures and pharmacological activities of the 25 2-phenyl-indole compounds

evaluated at mGPRC6A in the IP turnover assay. Inhibitory activities are reported as mean

IC50 values of at least 3 independent experiments (n). Concentration-inhibition curves for

compounds that displayed less than 90% inhibition at the maximal tested concentration

were fitted using the value of the negative control. For compounds that displayed less than

50% inhibition at the maximal tested concentration, approximate IC50 values are given.

Off-targets from the selectivity screenings (see Table 2) are indicated as “+receptor name

activity” in the IC50 column.

Cmpd Molecular structure IC50 pIC50 SEM (µM) n

1

27 4.58 0.09

+ m1 activity

9

2

18 4.74 0.08 4

3

18 4.75 0.26

+ m1, m3 & mGlu5 activity

5

4

>100 3

5

>100 3

6

+ 5-HT2C activity 3

7

+ 5-HT2C activity 3

8

+ CaR activity 3

9

+ 5-HT2C activity 3

10

>50 3

11

>100 3

12

>50 3

13

>50 3

14

>100 3

15

>100 3

16

+ 5-HT2C activity 3

17

>100 3

18

>100 3

19

>100 3

20

>100 3

21

>100 3

22

>100 3

23

+ CaR activity 3

24

>100 3

25

>100 3

Figure S1: Inhibitory effect of Cpd 3 at mGPRC6A expressed in Xenopus  laevis oocytes, related to Figure 3 

Figure S1: Representative traces obtained from two-electrode voltage clamp recordings on Xenopus oocytes injected

with cRNA encoding mGPRC6A. L-Ornithine (300 µM) (duration indicated by grey filled bars) activated a maximal

inward current clamped at -60 mV. L-Ornithine (100 µM) (duration indicated by a closed bar) activated a

submaximal inward current. After a 3 min preincubation, Cpd 3 (100 µM, duration indicated by open bar),

suppressed the 100 µM submaximal L-ornithine-mediated inward current. After thorough washing, L-ornithine (300

µM) was able to reactivate the receptor. The recordings were reproduced in two independent experiments.

Figure S2: Schild plot of Cpd 1 at mGPRC6A, Related to Figure 4 

Figure S2: Concentration-response curves of L-Orn in the presence of various concentrations of Cpd 1 at

mGPRC6A, demonstrating a non-competitive mode of action. Results are expressed as counts per minute (CPM) and

are means ± SD of triplicate determinations of a single representative experiment. Two additional experiments gave

similar results.

 

Table S2: Effects of  L­Orn and  the 2­phenyl  indoles 1 and 3 on wildtype and mutated mouse GPRC6A receptors, Related to Figure 1 and Table 1 

The IP turnover assay (described under Methods) was used to determine the EC50 values for L-ornithine at wildtype

and mutated mGPRC6As. Inhibition curves for Cpd 1 and Cpd 3 were generated in the presence of L-ornithine (500

µM). Effective and inhibitory activities are reported as mean EC50 and IC50 values, respectively, of 3 independent

experiments (n). Mutated receptors I759R and I759W were insensitive to Cpds 1 and 3, in concentrations below 200

µM or 300 µM, respectively.

EC50 pEC50 SEM (µM) IC50 pIC50 SEM (µM) n

L-Ornithine Cpd 1 Cpd 3

wildtype 41 4.38 0.10 28 4.55 0.02 11 4.98 0.09 3 I759R 52 4.29 0.13 >200 >300 3 I759W 47 4.33 0.04 >200 >300 3

 

 

Supplemental Experimental Procedures, Relates to Figure 2 

General experimental For reactions conducted under anhydrous conditions, glassware was dried overnight in an oven at

150 C and was allowed to cool in a dessicator over anhydrous KOH. Anhydrous reactions were

carried out under nitrogen. Reagents/solvents for anhydrous reactions were dried as follows: THF

was distilled from sodium wire with benzophenone as indicator. Dichloromethane, toluene,

pyridine and N,N-dimethylformamide were dried and stored over 4 Å molecular sieves. Sulfate

buffer was prepared by dissolving 1.5 mol of Na2SO4 in 0.5 mol H2SO4 and adding water to give

a total volume of 2000 cm3.

Thin layer chromatography (TLC) was carried out on commercially available pre-coated

aluminium sheets (Merck 60F254). The quoted Rf values are rounded to the nearest 0.05. 1H- and 13C-NMR was run on a Varian Mercury 300 MHz and a Varian Gemini 300 MHz Fourier

transform spectrometers, respectively, using an internal deuterium lock. Solvents were used as

internal standard when assigning NMR spectra (Gottlieb et al., 1997). J values are given in Hz

and rounded to the nearest 0.5 Hz.

Dry column vacuum chromatography (DCVC) was carried out according to the published

procedure(Pedersen and Rosenbohm, 2001). HPLC-MS was run on an Agilent 1100 LC-MS

system on a C18 analytical reverse phase Ascentis Express RP-amide column (5 cm × 2.1mm,

2.7 m) using the following gradient: 0-1 min 100% solvent A, 1-6 min 100% solvent A to 100%

solvent B, 6-10 min 100% solvent B (solvent A: 0.1% formic acid, 5% acetonitrile, 94.9% water

(v/v/v); solvent B: 0.05% formic acid, 5% water, 94.95% acetonitrile) with a flow rate of 0.5

ml/min.

High resolution mass spectra were recorded on a Micromass Q-TOF 1.5, UB137.

Synthesis of Ligand 1 

3­(2'­Benzyloxy­carboxyeth­1'­yl)­2­phenyl­1H­indole (5) 

2-Phenyl indole (1.0 g, 5.2 mmol) was dissolved in anhydrous dichloromethane (40 ml) and

cooled with a water/ice bath. Benzyloxyacetyl chloride (0.82 ml, 5.2.mmol) was added followed

by titanium tetrachloride (0.63 ml, 5.7 mmol). After 20 minutes saturated aqueous ammonium

chloride (50 ml) was added and the dichloromethane was removed in vacuo. The residue was

transferred to a separatory funnel with ethyl acetate (100 ml) and saturated aqueous ammonium

chloride (50 ml). The organic phase was washed with a half saturated aqueous sodium, potassium

tartrate (2 × 100 ml), saturated aqueous NaHCO3 (100 ml), brine (50 ml), dried (Na2SO4), filtered

and concentrated in vacuo to give a purple, amorphous solid. Purification by DCVC [id. 4 cm; 20

ml fractions; 2 × heptanes; 0-100% EtOAc in n-heptane (v/v) – 5% increments] gave ketone 5

(0.62 g, 35%) as a grey amorphous solid.

m/z (+ESI) found: MNa+, 364.1313. (C23H19NO2Na requires M, 364.1313); Rf = 0.30 (30%

EtOAc in n-heptane, v/v); HPLC RT (min)= 6.4; 1H NMR (300 MHz; CDCl3) 8.48 (1H, br s,

NH), 8.33-8.30 (1H, m, indole-H4), 7.50-7.20 (13H, m, Ar), 7.47 (2H, s, CH2Ph), 4.18 (2H, s,

CH2C=O); 13C NMR (300 MHz; CDCl3) 193.8, 144.4, 137.8, 135.7, 132.9, 130.1, 129.7, 129.1,

128.6, 128.4, 128.0, 127.6, 124.1, 123.1, 122.6, 113.5, 111.5, 74.4, 73.5.

NH

PhNH

Ph

OOH

5 6

OOBn

3­(2'­Hydroxy­carboxyeth­1'­yl)­2­phenyl­1H­indole (6) 

Benzylether 5 (0.26 g, 0.76 mmol) was dissolved in freshly distilled THF (7 ml) and acetic acid

(10 ml) was added. The flask was flushed with nitrogen and palladium on charcoal (10 wt%, 60

mg) was added. The flask was flushed with hydrogen and fitted with a hydrogen balloon. The

reaction mixture was stirred vigorously for 2 days, filtered through a pad of Celite, diluted with

toluene and concentrated in vacuo to give a yellow solid. Purification by DCVC [id. 4 cm; 20 ml

fractions; 2 × heptanes; 0-100% EtOAc in n-heptane (v/v) – 5% increments] gave alcohol 6 (0.10

g, 53%) as a pale yellow foam.

m/z (+ESI) found: MNa+, 274.0836. (C16H13NO2Na requires M, 274.0844); Rf = 0.30 (40%

EtOAc in n-heptane, v/v); 1H NMR (300 MHz; CDCl3) 8.56 (1H, br s, NH), 8.34-8.31 (1H, m,

indole-H4), 7.57-7.53 (4H, m, Ar), 7.44-7.33 (4H, m, Ar), 4.28 (2H, s, CH2); 13C NMR (300

MHz; CDCl3) 194.1, 145.6, 135.5, 132.1, 130.1, 129.3, 128.8, 126.8, 123.9, 123.1, 122.0,

111.5, 66.9.

2­Oxo­2­(2'­phenyl­1H­indol­3'­yl)ethyl­3­aminopyrazine­2­carboxylate (1) 

Alcohol 6 (85 mg, 0.34 mmol) was dissolved in anhydrous DMF (7 ml) and 3-amino-2-

pyrazinecarboxylic acid (57 mg, 0.41 mmol), N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide

hydrochloride (79 mg, 0.41 mmol) and DMAP (50 mg, 0.41 mmol) was added. The reaction

mixture was stirred at ambient temperature overnight and transferred to a separatory funnel with

ethyl acetate (50 ml), aqueous sulfate buffer (25 ml), and brine (25 ml), and extracted with ethyl

acetate (2 × 25 ml). The combined organic phases were washed with saturated aqueous NaHCO3

(50 ml), filtered on Whatman 1PS filter paper and concentrated in vacuo to give a yellow

amorphous solid. Purification by DCVC [id. 4 cm; 20 ml fractions; 2 × heptanes; 50-100%

EtOAc in n-heptane (v/v) – 5% increments; 6 × EtOAc] gave carboxylic ester 1 (62 mg, 49%) as

a clear, colourless film.

m/z (+ESI) found: MH+, 373.1301. (C21H17N4O3 requires M, 373.1301); Rf = 0.30 (80% EtOAc in

n-heptane, v/v); HPLC RT (min)= 5.21; 1H NMR (300 MHz; Acetone-d6) 11.20 (1H, br s,

indole-NH), 8.32-8.27 (1H, m, indole-H4), 8.21 (1H, d, J = 2 Hz, pyrazine-H), 7.92-7.91 (1H, m,

Ar), 7.81-7.76 (2H, m, Ar), 7.61-7.59 (2H, m, Ar), 7.53-7.50 (1H, m, Ar), 7.32-7.23 (2H, m, Ar),

6.96 (2H, br s, NH2), 5.00 (2H, s, CH2); 13C NMR (300 MHz; DMSO-d6) 187.5, 165.0, 155.7,

147.8, 145.2, 135.5, 132.4, 132.2, 129.6, 128.5, 126.6, 123.1, 122.9, 122.1, 121.3, 111.8, 110.9,

67.9.

Synthesis of ligand 2 

2­(Methylamino)­1­morpholinoethanone hydrochloride (8) 

N-tert-Butoxycarbonyl sarcosine (2.0 g, 10.6 mmol) was dissolved in anhydrous dichloromethane

(40 ml) and morpholine (1.10 ml, 12.7 mmol), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide

hydrochloride (2.43 g, 12.7 mmol), diisopropylamine (7.39 ml, 42.4 mmol), and DMAP (1.68 g,

13.8 mmol) was added to give a yellow solution that was stirred at ambient temperature

overnight. The reaction mixture was diluted with ethyl acetate (20 ml) and the dichloromethane

was removed in vacuo. The residue was transferred to a separatory funnel with ethyl acetate (50

ml) and washed with aqueous sulfate buffer (25 ml), saturated aqueous NaHCO3 (50 ml), brine

(25 ml), dried (Na2SO4), filtered and concentrated in vacuo to give crude amide 7 (2.6 g) as a

yellow oil.

The crude product was dissolved in dioxane (25 ml) and 4M aqueous HCl (25 ml) was added.

After stirring at ambient temperature for 4 hours toluene was added and the mixture was

concentrated in vacuo to give yellow needles. The crude product was dissolved in refluxing

absolute ethanol (20 ml) and ether (20 ml) was added. The mixture was allowed to cool slowly to

give a white precipitate. The slurry was cooled with a water/ice bath and the solid was collected

by filtration and washed with ether (2 x 20 ml) to give amine 8 (1.10 g, 52% over two steps) as a

white amorphous solid after drying in a vacuum dessicator over KOH.

m/z (+ESI) found: MNa+, 181.0962. (C7H14N2O2Na requires M, 181.0953); 1H NMR (300 MHz;

DMSO-d6) 9.01 (2H, br s, NH2), 4.02 (2H, s, CH2C=O), 3.61-3.55 (4H, m, 2×CH2O), 3.49-3.46

(2H, m, CH2N), 3.37-3.33 (2H, m, CH2N), 2.53 (3H, s, CH3); 13C NMR (300 MHz; DMSO-d6)

163.7, 65.8 (×2), 48.1, 44.6, 41.7, 32.8.

3­(2'­Chloro­carboxyeth­1'­yl)­2­phenyl­1H­indole (9) 

According to the method of Roy et al. (Roy et al., 2006), 2-phenyl indole (48.8 g, 0.25 mol) was

suspended in anhydrous toluene (500 ml) and pyridine (20.5 ml) was added. The mixture was

heated to 60 oC under N2, and chloroacetyl chloride (19.9 ml, 0.25 mol) was added dropwise over

2 hours to give a green slurry. After completed addition stirring was continued for 30 minutes at

which time the heating was stopped. MeOH (100 ml) was added slowly to give a clear solution.

Upon addition of water (500 ml) a green precipitate was formed that was collected by filtration.

The solid was washed with water (2 × 250 ml) and dried in a vacuum dessicator over KOH. The

crude product (53 g) was recrystallised from refluxing absolute ethanol (450 ml) to give green

crystals that were collected by filtration, and washed with cold absolute ethanol (4 × 50 ml). The

product was dried in a vacuum dessicator over potassium hydroxide to give ketone 9 (39.53 g,

59%) as green needles.

m/z (+ESI) found: MH+, 270.0681. (C16H13ClNO requires M, 270.0686); Rf = 0.50 (50% EtOAc

in n-heptane, v/v); HPLC RT (min)= 6.1; 1H NMR (300 MHz; DMSO-d6) 12.31 (1H, br s, NH),

8.17-8.12 (1H, m, indole-H4), 7.67-7.63 (2H, m, Ar), 7.59-7.55 (3H, m, Ar), 7.47-7.42 (1H, m,

Ar), 7.29-7.20 (2H, m, Ar), 4.33 (2H, s, CH2); 13C NMR (300 MHz; DMSO-d6) 186.6, 145.3,

135.5, 132.0, 129.7, 128.5, 126.8, 123.2, 122.1, 121.3, 111.8, 111.5, 48.2.

2­[N­methyl(2'­morpholino­2'­oxoethyl)amino]­1­(2''­phenyl­1H­indol­3''­yl)­

ethanone (3) 

A 10 ml microwave vial was charged with ketone 9 (0.62 g, 2.3 mmol), ammonium salt 7 (0.45 g,

2.3 mmol), NaHCO3 (0.48 g, 5.8 mmol) and NaI (69 mg, 0.5 mmol) and suspended in anhydrous

acetone (10 ml). The vial was sealed and heated at 60 oC for 2 hr. The slurry was filtered and the

solids washed with acetone. The combined organic phases were concentrated in vacuo to give a

yellow foam that was suspended in ethyl acetate (15 ml) and iso-propanol (7 ml) was added to

dissolve almost most of the material. The precipitate was removed by filtration of the hot solution

and the solvents were removed in vacuo to give amine 3 (0.85 g, 94%) as a tan amorphous solid.

m/z (+ESI) found: MNa+, 414.1780. (C23H25N3O3Na requires M, 414.1794); 1H NMR (300 MHz;

DMSO-d6) 12.08 (1H, br s, NH), 8.10-8.06 (1H, m, indole-H4), 7.63-7.51 (5H, m, Ar), 7.43-

7.40 (1H, m, Ar), 7.25-7.16 (2H, m, Ar), 3.46-3.33 (10H, m, 2×CH2O, 2×CH2N, CH2C=OAr),

3.14 (2H, s, CH2C=ON), 2.12 (3H, s, CH3); 13C NMR (300 MHz; DMSO-d6) 194.2, 167.7,

144.1, 135.3, 132.6, 129.7, 129.2, 128.3, 126.8, 122.7, 121.6, 121.2, 112.8, 111.6, 66.1, 66.2,

64.7, 58.9, 45.4, 42.3, 41.5.

Synthesis of the N­methylated analogue 3 

3­(2'­Benzyloxy­carboxyeth­1'­yl)­1­methyl­2­phenyl­1H­indole (10) 

By the same method described above for the synthesis of compound 5, 1-methyl-2-phenyl indole

(1.1 g, 5.2 mmol) gave the desired ketone 10 (0.90 g, 49%) as a yellow gum.

m/z (+ESI) found: MH+, 356.1657. (C24H22NO2 requires M, 356.1651); Rf = 0.25 (30% EtOAc in

n-heptane, v/v); HPLC RT (min)= 6.5; 1H NMR (300 MHz; CDCl3) 8.52-8.48 (1H, m, indole-

H4), 7.55-7.45 (3H, m, Ar), 7.35-7.32 (5H, m, Ar), 7.28-7.25 (3H, m, Ar), 7.21-7.18 (2H, m, Ar),

4.44 (2H, s, CH2Ph), 3.91 (2H, s, CH2C=O), 3.49 (3H, s, CH3); 13C NMR (300 MHz; CDCl3)

192.7, 146.0, 138.0, 137.1, 132.2, 130.3 (×2), 129.4, 128.6, 128.5, 128.0, 127.0, 123.9, 123.3,

123.1, 113.9, 110.0, 74.2, 73.5, 31.4.

3­(2'­Hydroxy­carboxyeth­1'­yl)­1­methyl­2­phenyl­1H­indole (10) 

By the same method described above for the synthesis of compound 6, benzyl ether 9 (0.72 g, 2.0

mmol) gave the desired alcohol 11 (0.34 g, 63%) as a yellow amorphous solid.

m/z (+ESI) found: MH+, 266.1181. (C17H16NO2 requires M, 266.1181); Rf = 0.40 (40% EtOAc in

n-heptane, v/v); HPLC RT (min)= 4.7; 1H NMR (300 MHz; CDCl3) 8.50-8.43 (1H, m, indole-

H4), 7.61-7.54 (3H, m, Ar), 7.44-7.37 (5H, m, Ar), 3.99 (2H, s, CH2), 3.53 (3H, s, CH3); 13C

NMR (300 MHz; CDCl3) 193.5, 147.0, 136.8, 131.3, 130.2, 129.6, 129.1, 126.2, 123.7, 123.2,

122.3, 111.8, 109.9, 66.7, 31.0.

2­Oxo­2­(1'­methyl­2'­phenyl­1H­indol­3'­yl)ethyl­3­aminopyrazine­2­carboxylate 

(4) 

By the same method described above for the synthesis of compound 1, alcohol 11 (82 mg, 0.31

mmol) gave carboxylic ester 4 (62 mg, 50%) as a white amorphous solid.

m/z (+ESI) found: MNa+, 409.1277. (C22H18N4O3Na requires M, 409.1354); Rf = 0.30 (80%

EtOAc in n-heptane, v/v); HPLC RT (min)= 4.8; 1H NMR (300 MHz; Acetone-d6) 8.39-8.36

(1H, m, indole-NH), 8.18 (1H, d, J = 2 Hz, pyrazine-H), 7.89 (1H, d, J = 2 Hz, pyrazine-H), 7.71-

7.66 (4H, m, Ar), 7.54 (1H, d, J = 7.5 Hz, Ar), 7.37-7.26 (2H, m, Ar), 6.93 (2H, br s, NH2), 4.69

(2H, s, CH2), 3.60 (3H, s, CH3); 13C NMR (300 MHz; DMSO-d6) 186.8, 164.8, 155.7, 147.7,

146.5, 136.4, 132.4, 131.0, 130.1, 130.0, 129.0, 125.8, 123.3, 122.8, 122.7, 121.4, 111.6, 110.8,

67.5, 31.0.

Materials GlutaMAX-I DMEM, dialyzed fetal bovine serum, penicillin, streptomycin, HBSS and BSA were all

from Invitrogen (Paisley, UK). Inositol-free DMEM was homemade from compounds purchased

from Sigma-Aldrich (St Louis, MO). Initial pharmacological screening with commercially available 2-

phenyl-indole compounds were from Enamine (Kiev, Ukraine). All additional buffer reagents and

tested compounds were from Sigma-Aldrich.

Supplemental References Gottlieb, H.E., Kotlyar, V., and Nudelman, A. (1997). NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities. J Org Chem 62, 7512-7515. Pedersen, D.S., and Rosenbohm, C. (2001). Dry Column Vacuum Chromatography. Synthesis 16, 2431-2434. Roy, S., Haque, S., and Gribble, G.W. (2006). Synthesis of Novel Oxazolyl-indoles Synthesis 23, 3948-3954.