CRYSTALLINE FORM OF (R)-2-(TERT-BUTYLAMINO)-1-(5-FLUOROPYRIDIN-3-YL)-ETHAN-1-OL HEMI-TARTRATE SALT FOR THE TREATMENT OF HYPERGLYCEMIA AND DIABETES 2

Abstract

The present invention discloses a crystalline form of the (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethanol (formula (I)) hemi-(2R,3R)-tartrate salt characterised by a melting point of 211 C. and the XRPD pattern of FIG. 1. The present invention also relates to said hemi-tartrate salt for use in the treatment of hyperglycaemia and type 2 diabetes through activation of the beta2-adrenergic receptor. Importantly, such salts are thought to have a beneficial side-effect profile as they do not exert their effect through significant cAMP release.

Claims

1. A hemi-tartrate salt of a compound of formula I: ##STR00011##

2. The salt according to claim 1, wherein the tartrate comprises (2R,3R)-tartrate.

3. The salt according to claim 2, wherein the tartrate essentially consists of (2R, 3R)-tartrate.

4. The salt according to any one of the previous claims, wherein the salt has a purity of greater than about 90%, preferably greater than about 95%, more preferably greater than about 99%.

5. The salt according to any one of the previous claims, wherein the salt has a melting point of from about 209 to about 213 C. at atmospheric pressure, such as from about 210 to about 212 C., e.g. about 211 C.

6. The salt according to any one of the preceding claims for use in medicine.

7. A pharmaceutical composition comprising the salt as defined in any of the preceding claims, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier.

8. The pharmaceutical composition according to claim 7, wherein the compound of formula I has an enantiomeric excess of at least 90%, preferably at least 95%, even more preferably at least 98%, most preferably at least 99%.

9. A salt as defined any one of claims 1 to 5 for use in the treatment of hyperglycaemia or a disorder characterized by hyperglycaemia.

10. The use of a salt as defined in one of claims 1 to 5 for the manufacture of a medicament for the treatment of hyperglycaemia or a disorder characterized by hyperglycaemia.

11. A method of treating hyperglycaemia or a disorder characterized by hyperglycaemia comprising administering to a patient in need thereof a therapeutically effective amount of a salt as defined in any one of claims 1 to 5.

12. A pharmaceutical composition as defined in any one of claims 7 to 8 for use in the treatment of hyperglycaemia or a disorder characterized by hyperglycaemia.

13. The salt or composition for use, method or use according to any one of claims 9 to 12, wherein the hyperglycaemia or disorder characterised by hyperglycaemia is, or is characterised by, the patient displaying severe insulin resistance.

14. The salt or composition for use, method or use according to any one of claims 9 to 12, wherein the disorder characterised by hyperglycaemia is selected from the group consisting of Type 2 diabetes, Rabson-Mendenhall syndrome, Donohue's syndrome (leprechaunism), Type A and Type B syndromes of insulin resistance, the HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndromes, pseudoacromegaly, and lipodystrophy.

15. A combination product comprising: (a) a salt as defined in any one of claims 1 to 5; and (b) one or more other therapeutic agent that is useful in the treatment of hyperglycaemia or a disorder characterised by hyperglycaemia, wherein each of components (a) and (b) is formulated in admixture, optionally with one or more a pharmaceutically-acceptable adjuvant, diluent or carrier.

16. A kit-of-parts comprising: (a) a pharmaceutical composition as defined in any one of claims 7 to 8, and (b) one or more other therapeutic agent that is useful in the treatment of hyperglycaemia or a disorder characterised by hyperglycaemia, optionally in admixture with one or more pharmaceutically-acceptable adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

17. A process for the preparation of the salt as defined in any one of claims 1 to 5, comprising the step of reacting a compound of formula (I): ##STR00012## with tartaric acid or a solution of tartaric acid, such as wherein the solvent of the solution of tartaric acid is ethanol or an ethanol-water mixture.

Description

FIGURES

[0159] FIG. 1 shows an XRPD analysis of the hemitartrate salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol.

EXAMPLES

[0160] The present invention is illustrated by way of the following examples.

[0161] Chemicals and reagents were obtained from commercial suppliers and were used as received unless otherwise stated. All reactions involving moisture sensitive reagents were performed in oven or flame dried glassware under a positive pressure of nitrogen or argon.

Abbreviations

[0162] Abbreviations as used herein will be known to those skilled in the art. In particular, the following abbreviations may be used herein. [0163] aq aqueous [0164] Et2O diethylether [0165] EtOAc ethyl acetate [0166] EtOH ethanol [0167] iPrOAc isopropyl acetate [0168] iPrOH isopropanol [0169] MeCN acetonitrile [0170] rt room temperature [0171] THF tetrahydrofuran

Example Compounds and Salts

[0172] In the event that there is a discrepancy between nomenclature and the structure of compounds as depicted graphically, it is the latter that presides (unless contradicted by any experimental details that may be given and/or unless it is clear from the context).

Compound Example: (R)-2-(tert-Butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol

##STR00007## [0173] (a) 2-Chloro-1-(5-fluoropyridin-3-yl)ethan-1-one

##STR00008##

[0174] Isopropylmagnesium chloride (2 M in THF, 10.47 mL, 20.94 mmol) was added to a solution of LiCl (887.69 mg, 20.94 mmol) in THF (8 mL) at rt. After 15 min at rt, 3-bromo-5-fluoropyridine (3.35 g, 19.04 mmol) in THF (30 mL) was added dropwise at 0 C. The mixture was stirred at rt for 2 h and cooled in an ice-bath. A solution of 2-chloro-N-methoxy-N-methylacetamide (2.62 g, 19.04 mmol) in THF (30 mL) was added dropwise, and the mixture was stirred at rt for 2 h. NH.sub.4Cl (aq, 10%) was added and the mixture was extracted with Et.sub.2O. The combined extracts were washed with brine, dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified by chromatography to give the sub-title compound (1.52 g, 20.94 mmol, 46%).

(b) (R)-2-Chloro-1-(5-fluoropyridin-3-yl)ethan-1-one

##STR00009##

[0175] iPrOAc (250 mL, 10 vol), pentamethylcyclopentadienylrhodium(III) chloride dimer (74 mg, 0.001 eq), (1S,2S)-(+)-N-p-tosyl-1,2-diphenylethylenediamine (87 mg, 0.002 eq) and NEt.sub.3 (66 L, 0.004 eq) were stirred at rt for 1h20, before 2-chloro-1-(5-fluoropyridin-3-yl)ethan-1-one hydrochloride (25 g, 1.00 eq) was added. The mixture was cooled to 7 C., NEt.sub.3 (83 mL, 5.00 eq) was added and then HCOOH (9 mL, 2.00 eq) was added. The mixture was stirred at rt for 1 h, before being diluted with 0.5 M aq HCl, extracted with iPrOAc, washed with H.sub.2O and concentrated. The residue was diluted with iPrOH, cooled to 0 C. and then 5.56 M HCl in iPrOH (22 mL, 1.00 eq) was added. The solids were collected to give the sub-title compound (18.08 g, 72%, ee=98.7%).

(c) (R)-2-(tert-Butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol

##STR00010##

[0176] tert-Butylamine (11.37 mL, 108.21 mmol) followed by NaOH (476.08 mg, 11.90 mmol) were added to a mixture of 2-chloro-1-(5-fluoropyridin-3-yl)ethan-1-one (1.90 g, 10.82 mmol) and iPrOH (1.66 mL, 21.64 mmol) at rt. The mixture was heated at 75 C. for 4 h, allowed to cool, diluted with EtOAc, washed with H.sub.2O and brine, dried (Na.sub.2SO.sub.4) and concentrated. The residue was dissolved in hot EtOAc and allowed to cool. Pentane was added and the mixture kept at 20 C. overnight. The solids were collected and purified by chromatography to give the title compound (1.43 g, 6.74 mmol, 62%, ee=98%). .sup.1H NMR (400 MHz, CDCl.sub.3): 8.43-8.27 (m, 2H), 7.57-7.42 (m, 1H), 4.62 (dd, J=8.8, 3.7 Hz, 1H), 2.94 (dd, J=12.1, 3.8 Hz, 1H), 2.53 (dd, J=12.1, 8.8 Hz, 1H), 1.10 (s, 9H).

[0177] In the above-described experiments, the title compound was isolated as the free base by evaporation to dryness. However, this isolation method typically conducted at laboratory scale. Therefore, different solvents and solvent mixtures were tested to crystallize the free base. Surprisingly, all initial attempts resulted in either formation of crystals with relatively low yield (below 50%) or oil separation. It was therefore decided to investigate salt formation.

Salt Example 1: Hemi-Tartrate

[0178] A solution of L-(+)-tartaric acid (6.21 g, 0.5 eq) in EtOH (175 mL) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (17.57 g) in EtOH (525 mL, vol) and H.sub.2O (7 mL) at rt. The mixture was refluxed until all precipitate was dissolved, then cooled. The resultant slurry was stirred at rt overnight and then at 0 to 5 C. for 2 h. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (19.9 g, 83%, 100.0% purity by HPLC, 99.8% ee by HPLC).

[0179] Formation of the hemitartrate was confirmed by 1H-NMR spectrum, which indicated an amine:acid ratio of 2:1.

[0180] Characterization of solid-state properties of the title salt using XRPD, DSC and DVS techniques indicated that material is crystalline with a high melting point at 211 C. The salt was not hygroscopic and was physically stable under the tested conditions (2 days at 40 C. and 75% RH).

[0181] It was observed during the experiment that filtration of the hemitartrate salt was very good, and so further experiments were carried out to optimise the conditions for the formation of the hemitartrate salt at increased scale. The results are shown in the table below.

TABLE-US-00002 Used Purity, Scale, base:acid Yield, HPLC Batch g Conditions ratio, eq % (area-%) Comments 1a 1.2 EtOH 1:1.sup. 65 99.9 Thick slurry was (30 vol) formed. Mixture was rt .fwdarw. +5 C. diluted with additional 5 vol of EtOH to improve mixing and then filtered. 1b 2.0 EtOH/H.sub.2O 1:0.6 71 99.9 Thick slurry was (0.6 vol-%; formed. Water 0.6 42 vol) vol % was added to rt .fwdarw. reflux .fwdarw. improve mixing. RT .fwdarw. +5 C. 1c 2.0 EtOH/H.sub.2O 1:0.6 76 99.8 Full dissolution by (1 vol-%; heating was achieved 30.fwdarw. 40 vol) only after addition of rt .fwdarw. reflux .fwdarw. extra 10 vol of EtOH. rt .fwdarw. +5 C. 1d 2.0 EtOH/H.sub.2O 1:0.5 84 99.9 Full dissolution at (1 vol; reflux temperature 40 vol) was observed. Good 40 C. .fwdarw. mixing and filtration. reflux .fwdarw. rt .fwdarw. +5 C. 1e 5.0 EtOH/H.sub.2O 1:0.5 83 99.9 Full dissolution at (1 vol-%; reflux temperature. 40 vol) Crystallization started rt .fwdarw. reflux at 57 C. .fwdarw. +5 C. 1f 20.0 EtOH/H.sub.2O 1:0.5 83 100.0 Upscale experiment. (1 vol-%; Good mixing and 40 vol) filterability. rt .fwdarw. reflux .fwdarw. +5 C.

[0182] Overall, the hemitartrate salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol was obtained in high yield, high purity and with an enantiomeric excess over 99% for (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol. In particular, use of the free base of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol having a purity of only 95.6% (by HPLC) was used to prepare the hemitartrate salt having a purity in excess of 99.8% (by HPLC), indicating a significant purification effect upon crystallization.

[0183] Furthermore, the synthesis could be easily upscaled and, unlike many of the other salt forms of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol, had a low hygroscopicity and did not have any problems with slow filtration, which was thought to be caused by small crystal formation.

[0184] A sample of the hemitartrate salt was analysed by X-ray powder diffraction and found to have a diffraction pattern generating peaks as shown in FIG. 1. The data for the relevant peaks are summarised in the table below.

TABLE-US-00003 # 2 [] d [] Intensity 1 5.74 15.38 7 2 11.35 7.79 100 3 14.04 6.30 9 4 14.64 6.04 11 5 17.24 5.14 58 6 18.19 4.87 44 7 18.91 4.69 40 8 20.04 4.43 9 9 21.21 4.19 21 10 22.01 4.03 10 11 22.80 3.90 38 12 24.96 3.56 10 13 25.65 3.47 14 14 26.53 3.36 3 15 27.66 3.22 6 16 28.12 3.17 19 17 30.09 2.97 10 18 31.41 2.85 4 19 32.05 2.79 3 20 32.83 2.73 10

Comparative Salt Example 2: Dihydrochloride

[0185] Aqueous hydrochloric acid (37%, 1.1 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (300 mg, 1.0 eq) in iPrOH (10 vol) at 40 C. The resultant slurry was stirred at rt overnight. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (47%).

[0186] The synthesis was repeated using different solvents and acids, the results of which are shown in the table below.

TABLE-US-00004 Scale Solvent Yield, Purity, Batch (mg) (10 vol) Acid (%) (area-% HPLC) 2a 500 iPrOH 5.6M HCl 60 98.2 in iPrOH 2b 500 iPrOH:iPrOAc 5.6M HCl 84 94.9 (1:1) in iPrOH

[0187] The DSC analysis of the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol showed a broad endothermic event at 150 C. with mass loss due to possible dehydration or desolvation and a second endothermic event at 220 C. Based on observations and information collected during synthesis of the dihydrochloride salt, it is supposed that the product has high tendency to form hydrate or solvate during a salt preparation. Since good yield and purity of the dihydrochloride salt was not achieved, it was decided to discontinue work on this salt form.

Comparative Salt Example 3: Edisylate

[0188] 1,2-Ethanedisulfonic acid (1.05 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (500 mg, 1.0 eq) in iPrOH (20 vol) at rt. The resultant slurry was stirred at rt overnight. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol.

[0189] The salt was found to be hygroscopic under the tested conditions (2 days at 40 C. and 75% RH).

[0190] It was observed during the experiment that filtration of the edisylate salt was very slow due to the formation of very fine solids.

[0191] The synthesis was repeated using different solvents and acids, the results of which are shown in the table below.

TABLE-US-00005 Scale Batch (mg) Conditions Comment 3a 500 iPrOH, Thick slurry was formed. Filtration 20 vol, rt was very slow. 3b 400 2-vol % H.sub.2O Slurry was formed. Moderate in THF, filtration speed. 15 vol, rt 3c 200 EtOH, Very fine suspension was formed. The 15 vol, rt fine solids went through the glass filter frit 3 during the filtration. 3d 200 EtOH/H.sub.2O Very fine suspension was formed. The (2 vol-%), fine solids went through the glass 15 vol, rt filter frit 3 during the filtration. 3e 200 i-PrOH/H.sub.2O Very fine suspension was formed. The (2 vol-%), fine solids went through the glass 15 vol, rt filter frit 3 during the filtration. 3f 200 THF/H.sub.2O Slurry was formed. Filtration was (1 vol-%), fast. Low purity (94.7% by HPLC). 15 vol, rt

[0192] Further development of the edisylate salt for production was not carried out due to the low purification achieved from salt formation and due to the limited commercial availability of pharmaceutical grade 1,2-ethanedisulfonic acid in bulk amounts.

Comparative Salt Example 4: Maleate

[0193] Maleic acid (1.1 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (50 mg, 1.0 eq) in iPrOH (10 vol) at 40 C. The resultant slurry was stirred at rt overnight. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol.

[0194] The salt was slightly hygroscopic under the tested conditions (2 days at 40 C. and 75% RH), with a mass loss of between 0.9 wt. % and 1.4 wt. % due to water seen between 25 and 155 C. upon subsequent TGMS analysis.

[0195] It was observed during the experiment that filtration of the maleate salt was very slow due to the formation of very fine solids.

[0196] The title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol was used in recrystallisation attempts to find conditions which facilitate formation of larger particles and improved filtration. Conditions identified in screening experiments were further applied for salt formation experiments on 50 mg scale, the results of which are shown in the table below.

TABLE-US-00006 Comparative Example Solvent/Volumes Observations 4a THF/1.5-vol % H.sub.2O (30 vol) Sticky solids formed. 4b iPrOH (30 vol) Slow filtration. 4c iPrOH/5-vol % H.sub.2O (10 vol) Slow filtration. 4d iPrOH/i-PrOAc (3:1, 40 vol) Slow filtration. 4e iPrOH/5-vol % EtOH (20 vol) Slow filtration 4f THF/10-vol % EtOH (10 vol) Small amount precipitated. 4g MeCN (30 vol) Slow filtration. 4h MeCN/iPrOAc (3:1, 40 vol) Slow filtration. 4i MeCN/5-vol % H.sub.2O (10 vol) Precipitation was not started.

Comparative Salt Example 5: Citrate

[0197] An aqueous solution of citric acid (1 M, 1.1 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (40 mg, 1.0 eq) in THF/water (50:50 v:v) at rt. The resultant mixture was freeze-dried overnight. No crystallisation occurred.

[0198] Half of the mixture was dissolved in EtOAc; the other half of the mixture was dissolved in MeCN. The solutions were subjected to three temperature cycles between 5 and 50 C. of 12 h each, then left at rt for 3 days. No crystallisation occurred.

Comparative Salt Example 6: Succinate

[0199] Succinic acid (1.0 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (100 mg, 1.0 eq) in iPrOH (20 vol) at 40 C. The resultant mixture was stirred at rt overnight. No crystallisation occurred.

Comparative Salt Example 7: p-Toluenesulfonate

[0200] p-Toluenesulfonic acid (1.2 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (100 mg, 1.0 eq) in i-PrOH (20 vol) at 40 C. The resultant slurry was stirred at rt overnight. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (106%, 86.9% purity by HPLC).

[0201] It was observed during the experiment that filtration of the p-toluenesufonate salt was very slow due to the formation of very fine solids.

[0202] The salt was slightly hygroscopic under the tested conditions (2 days at 40 C. and 75% RH), with a mass loss of between 1.6 wt. % and 2.1 wt. % due to water seen between 25 and 160 C. upon subsequent TGMS analysis.

Comparative Salt Example 8: Fumarate

[0203] Fumaric acid (1.2 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (100 mg, 1.0 eq) in EtOH (10 vol) at 40 C. The resultant mixture was stirred at rt overnight. No crystallisation occurred.

[0204] The mixture was evaporate to dryness and then crystallised from iPrOH. The solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol.

Comparative Salt Example 9: Oxalate

[0205] An aqueous solution of oxalic acid (1 M, 1.1 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (40 mg, 1.0 eq) in THF/water (50:50 v:v) at rt. The resultant mixture was freeze-dried overnight.

[0206] Half of the mixture was dissolved in EtOAc; the other half of the mixture was dissolved in MeCN. The solutions were subjected to three temperature cycles between 5 and 50 C. of 12 h each, then left at rt for 3 days. In each case, the solids were collected to give the title salt of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (98.1% purity by HPLC.

[0207] Formation of the oxalate was confirmed by 1H-NMR spectrum, which indicated an amine:acid ratio of 1:1.

[0208] Characterization of solid-state properties of the title salt using XRPD, DSC and DVS techniques indicated that material is crystalline with a high melting point at 171 C. The salt was physically stable and was not hygroscopic under the tested conditions (2 days at 40 C. and 75% RH), showing no significant mass loss between 6 and 160 C. upon subsequent TGMS analysis.

[0209] Further scale-up of the title salt was not carried out due to concerns about potential nephrotoxicity in patients with diabetes.

Comparative Salt Example 10: Tartrate

[0210] An aqueous solution of L-(+)-tartaric acid (1 M, 1.1 eq) was added to a solution of (R)-2-(tert-butylamino)-1-(5-fluoropyridin-3-yl)ethan-1-ol (40 mg, 1.0 eq) in THF/water (50:50 v:v) at rt. The resultant mixture was freeze-dried overnight. No crystallisation occurred.

[0211] Half of the mixture was dissolved in EtOAc; the other half of the mixture was dissolved in MeCN. The solutions were subjected to three temperature cycles between 5 and 50 C. of 12 h each, then left at rt for 3 days. No crystallisation occurred.

BIOLOGICAL EXAMPLES

[0212] L6-myoblasts were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 g/L glucose supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 50 U/mL penicillin, 50 g/mL streptomycin and 10 mM HEPES. Cells were plated at 110.sup.5 cells per mL in 24-well plates. After reaching 90% confluence the cells were grown in medium containing 2% FBS for 7 days where upon cells differentiated into myotubes.

Biological Example 1: Glucose Uptake

[0213] Differentiated L6-myotubes were serum-starved overnight in medium containing 0.5% fatty-acid free BSA and stimulated with an agonist, with a final concentration of 110.sup.5 M. After 1 h 40 min the cells were washed with warm glucose free medium or PBS twice and another portion of agonist was added to the glucose free medium. After 20 min the cells were exposed to 50 nM .sup.3H-2-deoxyglucose for 10 min before washed with ice cold glucose free medium or PBS three times and lysed with 0.2 M NaOH, 400 L/well, for 1 h at 60 C. The cell lysate was mixed with 4 mL scintillation buffer (Emulsifier Safe, Perkin Elmer) and the radioactivity was detected in a -counter (Tri-Carb 4810TR, Perkin Elmer).

[0214] The compound of Compound Example 1 showed an activity of more than 75% of that of isoproterenol.

Biological Example 2: Measurement of Intracellular cAMP Levels

[0215] Differentiated cells were serum-starved overnight and stimulated with an agonist, final concentration 110.sup.5 M, for 15 min in stimulation buffer (HBSS supplemented with 1% BSA, 5 mM HEPES and 1 mM IBMX, pH 7.4). The medium was aspirated and 100 L of 95% EtOH was added to each well of the 24-well plate and cells were kept at 20 C. overnight. The EtOH was allowed to evaporate and 500 L of lysis buffer (1% BSA, 5 mM HEPES and 0.3% Tween-20, pH 7.4) was added to each well. The plate was kept at 80 C. for 30 min and then at 20 C. until the day of detection when the samples were thawed. Intracellular cAMP levels were detected using an alpha screen cAMP kit (6760635D from Perkin Elmer).

[0216] The compound of Compound Example 1 showed an activity of less than 50% of that of isoproterenol.