METHODS FOR TREATING NEUROLOGICAL SYMPTOMS ASSOCIATED WITH LYSOSOMAL STORAGE DISEASES

Abstract

Methods are provided for treating or preventing neurological symptoms and disorders which are associated with, e.g., lysosomal storage diseases. The methods include enhancing neuronal connectivity within the brain of a subject, increasing brain tissue volume, or preventing or delaying loss of brain tissue volume in a subject. Also provided are methods for monitoring the progression or regression of a neurological disorder, or assessing the onset of a neurological disorder, associated with a lysosomal storage disease, in which brain tissue volume of the subject is measured.

Claims

1. A method for enhancing neuronal connectivity within the brain of a subject, such as in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), ##STR00015## or a pharmaceutically acceptable salt or prodrug thereof, wherein: R.sup.1 is selected from hydrogen, halogen (e.g., fluorine), cyano, nitro, hydroxy, thio, amino, C.sub.1-6-alkyl (e.g., methyl or ethyl), C.sub.2-6-alkenyl, C.sub.2-6-alkynyl, C.sub.1-6-alkyloxy, C.sub.2-6-alkenyloxy, and C.sub.2-6-alkynyloxy, wherein said alkyl, alkenyl, alkynyl, alkyloxy, alkenyloxy, or alkynyloxy is optionally substituted with one or more (e.g., 1, 2, or 3) groups selected from halogen, cyano, nitro, hydroxy, thio, and amino; R.sup.2 and R.sup.3 are independently selected from C.sub.1-3-alkyl, optionally substituted by one or more (e.g., 1, 2, or 3) halogens, or R.sup.2 and R.sup.3 together form a cyclopropyl or cyclobutyl group, optionally substituted by one or more (e.g., 1 or 2) halogens; R.sup.4, R.sup.5, and R.sup.6 are each independently selected from hydrogen, halogen, nitro, hydroxy, thio, amino, C.sub.1-6-alkyl, and C.sub.1-6-alkyloxy, wherein said alkyl or alkyloxy is optionally substituted by one or more (e.g., 1, 2 or 3) groups selected from halogen, hydroxy, cyano, and C.sub.1-6-alkyloxy; and A is a 5- or 6-membered aryl or heteroaryl group (e.g., phenyl or thiazolyl), optionally substituted with 1, 2, or 3 groups independently selected from halogen, hydroxy, thio, amino, nitro, C.sub.1-6-alkoxy, and C.sub.1-6-alkyl.

2. The method of claim 1, wherein said compound is selected from: quinuclidin-3-yl (2-(4′-fluoro-[1,1′-biphenyl]-3-yl)propan-2-yl)carbamate; (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate; and (S)-quinuclidin-3-yl (2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate; and the pharmaceutically acceptable salts and prodrugs thereof.

3. The method of claim 1, wherein said compound is quinuclidin-3-yl (2-(4′-fluoro-[1,1′-biphenyl]-3-yl)propan-2-yl)carbamate.

4. The method of claim 1, wherein said compound is (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate.

5. The method of claim 1, wherein said compound is (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate in malate salt form.

6. The method of claim 1, wherein the subject has Gaucher disease Type 3.

7. The method of claim 1, wherein said compound, or pharmaceutically acceptable salt or prodrug thereof, is administered by systemic administration, e.g., via a non-parenteral route.

8. The method of claim 7, wherein said compound, or pharmaceutically acceptable salt or prodrug thereof, is administered orally.

9. The method of claim 1, wherein the subject undergoes concurrent treatment with enzyme replacement therapy (ERT), e.g., using a glucocerebrosidase (e.g., imiglucerase, velaglucerase, or taliglucerase).

10. The method of claim 1, wherein the subject is administered a daily dose of about 1 mg to about 50 mg of the compound, e.g., from 5 to 50 mg, or from 10 to 40 mg, or from 10 to 30 mg, or from 10 to 20 mg, or from 20 to 30 mg, or from 30 to 40 mg, or from 40 to 50 mg, or from 5 to 25 mg, or from 20 to 50 mg, or from 5 to 15 mg, or from 15 to 30 mg, or about 15 mg.

11. The method of claim 1, wherein the subject is administered a single daily dose of 15 mg (measured as the quantity of free base) of (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate in malate salt form.

12. The method of claim 1, wherein the compound is not administered concurrently with a strong or moderate inducer of CYP3A, e.g., rifampin, phenobarbital, or efavirenz.

13. The method of claim 1, wherein the subject is an adult or pediatric patient≥12 years of age with Gaucher disease Type 3 who is stabilized with enzyme replacement therapy (ERT) using imiglucerase for systemic conditions, and wherein the subject is administered a single daily dose of 15 mg (measured as the quantity of free base) of (S)-quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate in malate salt form.

14. The method of claim 1, wherein the method is effective to improve cognitive ability or reduce cognitive deficits, e.g., as measured by a reduction in the time taken to complete the trail-making test (TMT), TMT-A and/or TMT-B, a reduction in the difference between TMT-A time and TMT-B time (TMT-A−TMT-B), for example, a reduction of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% (e.g., wherein TMT-A decreases by 5-20%, and/or TMT-B decreases by 25-30%, and/or [TMT-A−TMT-B] decreases by 25-30%).

15. The method of claim 1, wherein the method results in increased blood flow in the brain (e.g., in one or more of the frontal, occipital, parietal, or temporal lobes), for example, as shown by fMRI imaging.

16. The method of claim 1, wherein the method results in increased nodal connectivity in the brain (e.g., between posterior and anterior aspects of the brain, and/or between occipital-parietal structures and frontal, temporal and/or limbic structures, for example, as shown by fMRI imaging).

17. The method of claim 1, wherein the method results in enhanced connectivity in brain regions associated with executive function.

18. The method of claim 1, wherein the method results in resting-state functional networks with improved connectivity between default mode and medial and frontal networks.

19. The method of claim 1, wherein the method results in enhanced connectivity between RSNs 1, 2 and 3 (perception-vision, cognition-language-orthography, cognition space) and RSNs 6, 7 and 8 (sensorimotor, auditory and executive control).

20. (canceled)

21. (canceled)

22. A method for increasing brain tissue volume, or preventing or delaying loss of brain tissue volume, in a subject, such as in a subject in need thereof, said method comprising administering to the subject an effective amount of a compound as defined in claim 1.

23. The method of claim 22, wherein the method results in an increase in brain tissue volume, or prevention or delay in loss of brain tissue volume, in one or more brain regions selected from: right accumbens, left putamen, left entorhinal cortex, right putamen, right postcentral lobe, left pericalcarine, right amygdala, left cuneus and left lingual.

24. The method of claim 22, wherein the method results in increased brain volume in one or more brain regions associated with executive function.

25. The method of claim 22, wherein the increase in brain volume in the one or more brain regions is accompanied by an enhancement in neuronal connectivity within the one or more brain regions, e.g., as shown using functional magnetic resonance imaging (fMRI).

26. The method of claim 22, wherein the method results in an increase in the whole brain tissue volume.

27. The method of claim 22, wherein the subject has Gaucher disease Type 3.

28. (canceled)

29. (canceled)

30. A method for monitoring the progression or regression of a neurological disorder associated with a lysosome storage disease in a subject, wherein the subject is undergoing a treatment which comprises administering to the subject an effective amount of a compound as defined in claim 1, said method comprising measuring brain tissue volume of the subject over a time period during the course of the treatment, e.g. using volumetric magnetic resonance imaging (vMRI), and assessing the extent of any change in brain tissue volume over said time period.

31. The method of claim 30, wherein said time period is from 3 months to 24 months, e.g., 3 months to 12 months, 3 months to 6 months, 6 months to 24 months, 6 months to 18 months, 6 months to 12 months, 12 months to 24 months, or 12 to 18 months.

32. The method of claim 30, wherein, if there is a decrease or absence of an increase in whole brain volume observed over said time period, the method further comprises modifying the treatment by increasing the dosage of the compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof, administered to the subject and reassessing the extent of any change in brain tissue volume after a further time period over the course of the modified treatment with the increased dosage.

33. The method of claim 301, wherein, if there is a decrease or absence of an increase in volumes in three or more of the following brain regions: right accumbens area, left putamen, left entorhinal cortex, right putamen, right postcentral lobe, left pericalcarine, right amygdala, left cuneus and left lingual, observed over said time period, the method further comprises modifying the treatment by increasing the dosage of the compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof, administered to the subject and reassessing the extent of any change in brain tissue volume after a further time period over the course of the modified treatment with the increased dosage.

34. The method of claim 30, wherein the subject has Gaucher disease Type 3.

35. (canceled)

36. (canceled)

37. A method for assessing the onset of a neurological disorder associated with a lysosomal storage disease in a subject at risk of developing said neurological disorder, said method comprising: a) measuring the brain tissue volume of the subject (e.g., using vMRI) and comparing against a reference standard to assess whether brain tissue volume is lower than the reference standard; b) where the brain tissue volume identified in step (a) is lower than the reference standard, identifying the onset of said neurological disorder; optionally further comprising: c) commencing treatment of the subject by administering to the subject an effective amount of a compound as defined in claim 1, or a pharmaceutically acceptable salt or prodrug thereof.

38. The method of claim 37, wherein the method further comprises administering to the subject the compound, or a pharmaceutically acceptable salt or prodrug thereof.

39. The method of claim 37, wherein the subject undergoes concurrent treatment with imiglucerase.

40. The method of claim 37, wherein the subject has been administered enzyme replacement therapy (e.g., imiglucerase, velaglucerase, and/or taliglucerase) prior to the initiation of any optional treatment with the compound.

41. The method of claim 37, wherein the subject has been administered imiglucerase therapy for at least 6 months (optionally at a stable dose) prior to beginning optional therapy with the compound.

42. The method of claim 37, wherein the method further comprises the step of transitioning the subject from ERT therapy (e.g., imiglucerase, velaglucerase, or taliglucerase) to the optional treatment with the compound.

43. The method of claim 37, wherein the subject has a hemoglobin level of at least 11 g/dL for females and at least 12 g/dL for males; a platelet count of at least 100,000/cubic millimeter; a splenic volume of less than 10 multiples of normal (MN); and/or a hepatic volume of less than 1.5 MN.

44. The method of claim 37, wherein measuring of brain tissue volume of the subject is by brain positron emission tomography (PET) or by volumetric magnetic resonance imaging (vMRI).

45. The method of claim 37, wherein the subject is found to have brain tissue volume lower than the reference standard.

46. The method of claim 37, wherein comparison against the reference standard indicates that the subject has a lower brain tissue volume in one or more brain regions selected from: right accumbens, left putamen, left entorhinal cortex, right putamen, right postcentral lobe, left pericalcarine, right amygdala, left cuneus, and left lingual.

47. The method of claim 37, wherein comparison against the reference standard indicates that the subject has a lower brain tissue volume in one or more brain regions associated with executive function.

48. The method of claim 37, wherein comparison against the reference standard indicates that the subject has a lower brain tissue volume in one or more brain regions where loss of neuronal connectivity is assessed to be present, e.g., as shown using functional magnetic resonance imaging (fMRI).

49. The method of claim 37, wherein comparison against the reference standard indicates that the subject has a lower whole brain tissue volume.

50. The method of claim 37, wherein brain tissue volume of the subject is measured a plurality of times, intermittently or routinely, e.g., weekly, monthly, every 2, 3, 4, 6, 9, 12 months, etc., after the treatment with the compound as is commenced to assess a change in brain tissue volume.

51. The method of claim 37, wherein the subject has Gaucher disease Type 3.

52. (canceled)

53. (canceled)

Description

[0573] Having been generally described herein, the follow non-limiting examples and appended FIGURE are provided to further illustrate this invention, wherein:

[0574] FIG. 1 is a graph showing change in mean whole brain volume (WBV) after 52 weeks of a treatment with a compound of Formula (I) for Group A patients and change in WBV for Patient 5, determined from vMRI measurements and TBM analysis according to Example 5 herein.

EXAMPLES

General Procedures for Chemical Synthesis

[0575] General Procedure A: Carbamate Formation with Triphosgene

[0576] To a suspension of amine hydrochloride (1 equivalent) and triethylamine (3-4 equivalents) in a THF (concentration ˜0.2M) at room temperature was added triphosgene (0.35 equivalents). The reaction mixture was stirred for 10 min and small amount of ether (1-2 mL) was added. The triethylammonium salt was filtered off to afford a clear solution of isocyanate in THF/ether.

[0577] To a solution of alcohol (1.5 equivalents) in THF (concentration ˜0.2M) at room temperature was added NaH [60%, oil] (1.5 equivalents). The reaction mixture was stirred for 15 min and the above solution (isocyanate in THF/ether) was added dropwise. In a standard workup, the reaction was quenched with brine. The solution was extracted with EtOAc and the organic layer was dried over Na.sub.2SO.sub.4, filtered, and concentrated. The crude material was purified on combiflash (SiO.sub.2 cartridge, CHCl.sub.3 and 2N NH.sub.3 in MeOH) to afford the corresponding carbamate.

General Procedure B: Alkylation with Organocerium

[0578] A suspension of CeCl.sub.3 (4 equivalents) in THF (concentration ˜0.2M) was stirred at room temperature for 1 h. The suspension was cooled to −78° C. and MeLi/Ether [1.6M] (4 equivalents) was added dropwise. The organocerium complex was allowed to form for a period of 1 h and a solution of nitrile (1 equivalent) in THF (concentration 2.0M) was added dropwise. The reaction mixture was warmed up to room temperature and stirred for 18 h. The solution was cooled to 0° C. and quenched with water (˜1 mL) followed by addition of 50% aqueous solution of ammonium hydroxide (˜3 mL) until precipitated formed and settled to the bottom of the flask. The mixture was filtered through a pad of celite and concentrated. The crude material was treated with a solution of HCl/dioxane [4.0M]. The intermediate arylpropan-2-amine hydrochloride was triturated in ether and used as is for the next step. Alternatively, the crude free base amine was purified on combiflash (SiO.sub.2 cartridge, CHCl.sub.3 and 2N NH.sub.3 in MeOH) to afford the corresponding arylpropylamine.

General Procedure C: Suzuki Coupling

[0579] To a solution of aryl halide (1 equivalent) in a mixture of DME/water [4:1] (concentration ˜0.2M) was added boronic acid (2 equivalents), palladium catalyst (0.1-0.25 equivalent), and sodium carbonate (2 equivalents). The reaction mixture was microwaved 25 min at 150° C. After filtering through a celite plug and concentrating, the crude product was purified on combiflash (SiO.sub.2 cartridge, CHCl.sub.3 and 2N NH.sub.3 in MeOH) to afford the corresponding coupling adduct.

[0580] Alternatively: To a solution of aryl halide (1 equivalent) in a mixture of toluene/water [20:1] (concentration ˜0.2 M) was added boronic acid (1.3-2.5 equivalents), palladium catalyst (0.05-0.15 equivalent), tricyclohexylphosphine (0.15-0.45 equivalent), and potassium phosphate (5 equivalents). The reaction mixture was microwaved 25 min at 150° C. After filtering through a celite plug and concentrating, the crude product was purified on combiflash (SiO.sub.2 cartridge, CHCl.sub.3 and 2N NH.sub.3 in MeOH) to afford the corresponding coupling adduct.

General Procedure D: Cyclopropanation

[0581] To a mixture of aryl nitrile (1 equivalent) and Ti(Oi-Pr).sub.4 (1.7 equivalents) stirring at −70° C., was added dropwise EtMgBr [3.0 M in ether] (1.1 equivalents). The reaction mixture was allowed to warm to 25° C. and stirred for 1 h. To the above mixture was added BF.sub.3.Et.sub.20 (3 equivalents) dropwise at 25° C. After the addition, the mixture was stirred for another 2 h, and then quenched with aqueous HCl [2M]. The resulting solution was then basified by adding aqueous NaOH [2M]. The organic material was extracted with ethyl ether. The organic layers were combined, dried over Na.sub.2SO.sub.4, filtered, and concentrated. The crude material was purified by silica gel column chromatography (eluting with petroleum ether/EtOAc: 10/1 to 1/1) to give the corresponding 1-aryl-cyclopropanamine.

General Procedure E: Biaryl coupling using Suzuki conditions

[0582] To a stirred solution of the aryl halide component (1 equivalent) in 5:1 (v/v) dioxane/water (˜0.15 M) or 5:1 (v/v) N,N-dimethylformamide (˜-0.15 M) was added the arylboronate or arylboronic acid component (1-1.5 equivalents), sodium carbonate (2-3 equivalents), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.05 equivalents). The mixture was heated (90° C.) overnight and then filtered through a plug of Celite. The Celite was rinsed with ethyl acetate and the combined filtrate was washed with brine, dried (Na.sub.2SO.sub.4), and concentrated. The residue was purified by flash chromatography over silica.

General Procedure F: Carbamate formation using an isocyanate generated via a mixed anhydride/Curtius Rearrangement route

[0583] To a stirred solution of the carboxylic acid component (1 equivalent) in tetrahydrofuran (˜0.1 M) was added triethylamine (2 equivalents). The reaction was cooled (0° C.) and treated with isobutyl chloroformate (1.5 equivalents). After 1 hour at 0° C., a solution of sodium azide (2 equivalents) in water (˜1 M) was added and the reaction was allowed to warm to room temperature. After overnight stirring, the reaction was diluted with water and extracted with ethyl acetate. The combined extracts were washed with aqueous sodium bicarbonate solution and brine, dried (Na.sub.2SO.sub.4), and concentrated. The crude acyl azide was further dried via coevaporation with toluene and then taken up in toluene (˜0.1 M). The stirred solution was refluxed for 2-2.5 hours, cooled, and treated with an alcohol component (1.25-2 equivalents). The reaction was heated at reflux overnight and then concentrated. The residue was taken up in either ethyl acetate or chloroform and washed with aqueous sodium carbonate (Na.sub.2SO.sub.4) and concentrated. The crude product was purified by flash chromatography over silica using chloroform/methanol (less polar carbamates) or chloroform/methanol/ammonia (more polar carbamates) solvent gradients.

Example 1: Synthesis of Quinuclidine Compounds

1-azabicyclo[2.2.2]oct-3-yl [2-(4′-fluorobiphenyl-3-yl)propan-2-yl]carbamate (Compound 1)

[0584] Using General Procedure C, 1-azabicyclo[2.2.2]oct-3-yl [2-(3-bromophenyl)propan-2-yl]carbamate (600 mg, 1.63 mmol), 4-fluorophenyl boronic acid (457 mg, 3.27 mmol), and palladium (II) acetate gave the title compound as a white solid (373 mg; 60%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.56 (s, 1H), 7.52 (dd, J=5.4, 8.4 Hz, 2H), 7.42-7.38 (m, 3H), 7.12 (m, 2H), 5.18 (5, 1H), 4.62 (s, 1H), 2.66 (m, 6H), 1.72 (s, 6H), 2.01-0.83 (m, 5H) ppm. .sup.13C NMR (100 MHz, CDCl.sub.3) δ 125.0, 124.0, 123.8, 116.0, 116.0, 71.3, 55.9, 55.5, 47.6, 46.7, 29.6, 25.6, 24.8, 19.8 ppm. Purity: 98.0% UPLCMS (210 nm); retention time 0.95 min; (M+1) 382.9. Anal. Calcd. for C.sub.23H.sub.27FN.sub.2O.sub.2.0.37 (CHCl.sub.3): C, 65.86; H, 6.47; N, 6.57. Found: C, 65.85; H, 6.69; N, 6.49.

(S)-quinuclidin-3-yl 2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-ylcarbamate (Compound 2)

[0585] To a stirred solution of 4-fluorothiobenzamide (8.94 g, 57.6 mmol) in ethanol (70 mL) was added ethyl 4-chloroacetoacetate (7.8 mL, 58 mmol). The reaction was heated at reflux for 4 hours, treated with an addition aliquot of ethyl 4-chloroacetoacetate (1.0 mL, 7.4 mmol), and refluxed for an additional 3.5 hours. The reaction was then concentrated and the residue was partitioned between ethyl acetate (200 mL) and aqueous NaHCO.sub.3 (200 mL). The organic layer was combined with a back-extract of the aqueous layer (ethyl acetate, 1×75 mL), dried (Na.sub.2SO.sub.4), and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate as a low melting, nearly colourless solid (13.58 g, 89%).

[0586] To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate (6.28 g, 23.7 mmol) in DMF (50 mL) was added sodium hydride [60% dispersion in mineral oil] (2.84 g, 71.0 mmol). The frothy mixture was stirred for 15 minutes before cooling in an ice bath and adding iodomethane (4.4 mL, 71 mmol). The reaction was stirred overnight, allowing the cooling bath to slowly warm to room temperature. The mixture was then concentrated and the residue partitioned between ethyl acetate (80 mL) and water (200 mL). The organic layer was washed with a second portion of water (1×200 mL), dried (Na.sub.2SO.sub.4) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate as a colourless oil (4.57 g, 66%).

[0587] To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate (4.56 g, 15.5 mmol) in 1:1:1 THF/ethanol/water (45 mL) was added lithium hydroxide monohydrate (2.93 g, 69.8 mmol). The reaction was stirred overnight, concentrated, and redissolved in water (175 mL). The solution was washed with ether (1×100 mL), acidified by the addition of 1.0 N HCl (80 mL), and extracted with ethyl acetate (2×70 mL). The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated to afford 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (4.04 g, 98%). This material was used in the next step without purification.

[0588] To a stirred and cooled (0° c.) solution of 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoic acid (4.02 g, 15.2 mmol) in THF (100 mL) was added trimethylamine (4.2 mL, 30 mmol) followed by isobutyl chloroformate (3.0 mL, 23 mmol). The reaction was stirred cold for another 1 hour before adding a solution of sodium azide (1.98 g, 30.5 mmol) in water (20 mL). The reaction was stirred overnight, allowing the cooling bath to slowly warm to room temperature. The mixture was then diluted with water (100 mL) and extracted with ethyl acetate (2×60 mL). The combined extracts were washed with aqueous NaHCO.sub.3 (1×150 mL) and brine (1×100 mL), dried (Na.sub.2SO.sub.4) and concentrated. After coevaporating with toluene (2×50 mL), the resulting white solid was taken up in toluene (100 mL) and refluxed for 4 hours. (S)-3-quinuclidinol (3.87 g, 30.4 mmol) was then added and reflux was continued overnight. The reaction was concentrated and the residue partitioned between ethyl acetate (100 mL) and aqueous NaHCO.sub.3 (150 mL). The organic layer was washed with water (1×150 mL), dried (Na.sub.2SO.sub.4), and concentrated. The resulting off-white solid was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford the title compound as a white solid (4.34 g, 73%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.96-7.88 (m, 2H), 7.16-7.04 (m, 3H), 5.55 (br s, 1H), 4.69-4.62 (m, 1H), 3.24-3.11 (m, 1H), 3.00-2.50 (m, 5H), 2.01-1.26 (m, 11H) ppm. .sup.13C NMR (400 MHz, CDCl.sub.3) δ 166.4, 165.1, 163.8 (d, J=250.3 Hz), 162.9, 155.0, 130.1 (d, J=3.3 Hz), 128.4 (d, J=8.5 Hz), 115.9 (d, J=22.3 Hz), 112.5, 71.2, 55.7, 54.2, 47.5, 46.5, 28.0, 25.5, 24.7, 19.6 ppm. Purity: 100% UPLCMS (210 nm & 254 nm); retention time 0.83 min; (M+1) 390.

(S)-quinuclidin-3-yl (2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 3)

[0589] Using General Procedure E and the reaction inputs ethyl 2-(4-bromophenyl)-2-methylpropanoate and 4-(2-methoxyethoxy)phenylboronic acid, ethyl 244′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoate was prepared as an off-white solid. To a stirred solution of this compound (3.01 g, 8.78 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (45 mL) was added lithium hydroxide monohydrate (1.47 g, 61.4 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was dissolved in water, treated with 1N hydrochloric acid (65 mL), and extracted with ethyl acetate. The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4), and concentrated to afford 2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoic acid as a white solid (2.75 g, 100%). This intermediate and (S)-quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a colourless, glassy solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.62-7.29 (m, 7H), 7.01 (d, J=8.9 Hz, 2H), 4.47-4.37 (m, 1H), 4.17-4.08 (m, 2H), 3.72-3.62 (m, 2H), 3.32 (s, 3H), 3.09-2.25 (m, 6H), 2.05-1.18 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 157.9, 154.5, 146.7, 137.4, 132.5, 127.5, 125.7, 125.2, 114.8, 70.4, 70.0, 66.9, 58.2, 55.4, 54.2, 46.9, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.87 min; (M+H.sup.+) 439.5.

1-azabicyclo[2.2.2]oct-3-yl [2-(biphenyl-3-yl)propan-2-yl]carbamate (Compound 4)

[0590] Using General Procedure C, 1-azabicyclo[2.2.2]oct-3-yl [2-(3-bromophenyl)propan-2-yl]carbamate (600 mg, 1.63 mmol), phenylboronic acid (398 mg, 3.27 mmol), and palladium (II) acetate gave the title compound as a white solid (379 mg, 64%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.61 (s, 1H), 7.56 (d, J=7.4 Hz, 2H), 7.50-7.38 (m, 4H), 7.34 (m, 2H), 5.16 (s, 1H), 4.63 (s, 1H), 3.39-2.09 (m, 6H), 1.72 (s, 6H), 2.02-0.73 (m, 5H) ppm. .sup.13C NMR (100 MHz, CDCl.sub.3) δ 154.8, 147.8, 141.6, 129.0, 129.0, 128.6, 127.5, 125.8, 125.0, 124.0, 71.6, 71.3, 55.9, 55.5, 47.6, 46.8, 31.5, 30.2, 30.0, 29.5, 25.6, 24.8, 19.8 ppm. Purity: 99% UPLCMS (210 nm); retention time 0.84 min; (M+1) 365.0. Anal. Calcd. for C.sub.23H.sub.28N.sub.2O.sub.2.29 (CHCl.sub.3): C, 70.02; H, 7.14; N, 7.01. Found: C, 70.02; H, 7.37; N, 6.84.

(S)-quinuclidin-3-yl 2-(biphenyl-4-yl)propan-2-ylcarbamate (Compound 5)

[0591] Using General Procedure B, bromobenzonitrile (2.00 g, 11.0 mmol) was converted to the corresponding 2-(4-bromophenyl)propan-2-amine (1.20 g, 51%) as a brown oil.

[0592] Using General Procedure A, 2-(4-bromophenyl)propan-2-amine (1.0 g, 4.7 mmol) and (S)-quinuclidin-3-ol gave (S)-quinuclidin-3-yl 2-(4-bromophenyl)propan-2-ylcarbamate (1.0 g, 58%) as a brown oil.

[0593] Using General Procedure C, the above bromide (200 mg, 0.540 mmol), phenylboronic acid (133 mg, 1.10 mmol), and [PdCl.sub.2(pddf)]CH.sub.2Cl.sub.2 gave the title compound as a white solid (70 mg, 35%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.60-7.53 (m, 4H), 7.47 (d, J=8.5 Hz, 2H), 7.42 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 1H), 5.26 (br s, 1H), 4.64 (m, 1H), 3.33-3.15 (m, 1H), 3.10-2.45 (m, 5H), 2.40-1.80 (m, 2H), 1.78-1.58 (m, 7H), 1.55-1.33 (m, 2H) ppm. .sup.13C NMR (125 MHz, CDCl.sub.3) δ 154.5, 146.1, 140.8, 139.5, 128.7, 127.2, 127.1, 127.1, 125.2, 70.9, 55.5, 55.1, 47.4, 46.4, 31.1, 29.5, 25.3, 24.5, 19.5 ppm. Purity: 100% LCMS (214 nm & 254 nm); retention time 1.56 min; (M+1) 365.

Quinuclidin-3-yl 1-(biphenyl-4-yl)cyclopropylcarbamate (Compound 6)

[0594] Using General Procedure D, bromobenzonitrile (3.00 g, 16.5 mmol) was converted to the corresponding 1-(4-bromophenyl)cyclopropanamine (1.80 g, 51%) as a yellow solid.

[0595] Using General Procedure A, 1-(4-bromophenyl)cyclopropanamine (1.0 g, 4.7 mmol) and quinuclidin-3-ol gave quinuclidin-3-yl 1-(4-bromophenyl)cyclopropyl-carbamate (1.3 g, 75%) as a white semi-solid.

[0596] Using General Procedure C, the above carbamate (400 mg, 1.12 mmol), phenylboronic acid (267 mg, 2.22 mmol), and [PdCl.sub.2(pddf)]CH.sub.2Cl.sub.2 the title compound as a viscous oil (100 mg, 25%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.47 (d, J=7.5 Hz, 2H), 7.43 (d, J=8.0 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.26-7.15 (m, 3H), 5.93 (br s, 0.6H), 5.89 (br s, 0.4H), 4.67 (m, 1H), 3.20-3.06 (m, 1H), 2.88-2.42 (m, 5H), 1.98-1.08 (m, 9H) ppm. .sup.13C NMR (125 MHz, CDCl.sub.3) δ 155.0, 141.0, 139.7, 138.2, 127.7, 126.1, 126.0, 124.8, 124.1, 70.0, 54.5, 46.3, 45.4, 34.1, 24.3, 23.2, 18.3, 17.0 ppm. Purity: 100% LCMC (214 nm & 254 nm); retention time 1.52 min; (M+1) 363.

(S)-quinuclidin-3-yl 1-(4′-fluorobiphenyl-4-yl)cyclopropylcarbamate (Compound 7)

[0597] Using General Procedure C, (S)-quinuclidin-3-yl 1-(4-bromophenyl)cyclopropyl carbamate, 4-F-phenylboronic acid, and [PdCl.sub.2(pddf)]CH.sub.2Cl.sub.2 gave the title compound as a white solid (45%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.06-7.83 (d, 1H), 7.69-7.66 (m, 2H), 7.59-7.55 (m, 2H), 7.29-7.22 (m, 4H), 4.56-4.54 (m, 1H), 3.13-2.32 (m, 6H), 1.91-1.19 (m, 9H) ppm. .sup.13C NMR (125 MHz, DMSO-d.sub.6) δ 163.2, 161.2, 156.4, 143.7, 136.9, 128.9, 128.8, 126.8, 125.6, 116.2, 116.0, 70.7, 55.8, 47.4, 46.4, 34.8, 25.7, 24.6, 19.6, 18.7, 18.6 ppm. Purity: >97% LCMS (214 nm & 254 nm); retention time 1.96 min; (M+1) 381.2.

(S)-1-azabicyclo[2.2.2]oct-3-yl [1-(2′,4′-difluorobiphenyl-4-yl)cyclopropyl]carbarnate (Compound 8)

[0598] Using General Procedure C, (S)-quinuclidin-3-yl 1-(4-bromophenyl)cyclopropylcarbamate (0.446 g, 1.22 mmol), 2,4-difluorophenyl boronic acid (0.386 g, 2.44 mmol) and Pd(OAc).sub.2 (0.015 g, 0.067 mmol) gave the title compound as a tan solid (0.111 g, 23%). .sup.1H NMR (CDCl.sub.3) δ 7.43 (dd, J=8.4, 1.6 Hz, 2H), 7.40-7.33 (m, 1H), 7.31 (d, J=7.7 Hz, 2H), 6.99-6.81 (m, 2H), 5.54 (d, J=48.0 Hz, 1H), 4.82-4.65 (m, 1H), 3.30-3.07 (m, 1H), 2.98-2.44 (m, 5H), 1.97 (d, J=32.7 Hz, 1H), 1.83 (d, J=10.3 Hz, 1H), 1.64 (s, 1H), 1.52 (s, 1H), 1.39 (s, 1H), 1.31 (d, J=6.8 Hz, 4H) ppm. .sup.13C NMR major rotomer (CDCl.sub.3) δ 162.2 (dd, J=12.8, 249.1 Hz), 159.8 (dd, J=11.8, 251.0 Hz), 156.9, 156.0, 142.6, 133.1, 131.3 (m), 128.9, 125.6, 124.9, 111.5 (dd, J=3.9, 21.2 Hz) 104.4 (dd, J=25.2, 29.4 Hz), 72.1, 71.6, 55.7, 47.4, 46.5, 35.7, 35.3, 25.5, 24.6, 24.4, 19.5, 18.1 ppm. Purity: LCMS>99.3% (214 nm & 254 nm); retention time 0.90 min; (M+1) 399.0.

1-azabicyclo[2.2.2]oct-3-yl [1-(4′-methoxybiphenyl-4-yl)cyclopropyl]carbamate (Compound 9)

[0599] Using General Procedure C, quinuclidin-3-yl 1-(4-bromophenyl)cyclopropylcarbamate (0.485 g, 1.33 mmol), 4-methoxyphenyl boronic acid (0.404 g, 2.66 mmol), and Pd(OAc).sub.2 (0.016 g, 0.071 mmol) gave the title compound as a grey solid (0.337 mg, 65%). .sup.1H NMR (CDCl.sub.3) δ 7.48 (dd, J=8.6, 5.5 Hz, 4H), 7.29 (d, J=7.6 Hz, 2H), 6.96 (d, J=8.8 Hz, 2H), 5.58 (d, J=48.7 Hz, 1H), 4.83-4.63 (m, 1H), 3.84 (s, 3H), 3.20 (dd, J=24.0, 15.5 Hz, 1H), 2.97-2.42 (m, 5H), 1.97 (d, J=30.9 Hz, 1H), 1.81 (s, 1H), 1.75-1.33 (m, 3H), 1.28 (d, J=6.8 Hz, 4H) ppm. .sup.13C NMR major rotomer (CDCl.sub.3) δ 159.1, 156.0, 141.4, 139.0, 133.4, 128.0, 126.7, 125.9, 114.2, 71.5, 55.7, 55.3, 47.4, 46.5, 35.3, 25.5, 24.6, 19.6, 17.8 ppm. Purity: LCMS>97.1% (214 nm & 254 nm); retention time 0.88 min; (M+1) 393.4.

Quinuclidin-3-yl 2-(5-(4-fluorophenyl)thiophen-3-yl)propan-2-ylcarbamate (Compound 10)

[0600] To a stirred and cooled (0° C.) solution of ethyl 5-bromothiophene-3-carboxylate (13.30 g, 56.57 mmol) in THF (100 mL) was added a solution of methylmagnesium bromide in diethyl ether [3.0 M] (55.0 mL, 165 mmol), dropwise over 20 minutes. After 2 hours, the reaction solution was concentrated. The residue was taken up in aqueous NH.sub.4Cl (200 mL) and extracted with ethyl acetate (2×100 mL). The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford 2-(5-bromothiophen-3-yl)propan-2-ol as a pale amber oil (8.05 g, 64%).

[0601] To a stirred solution of 2-(5-bromothiophen-3-yl)propan-2-ol (8.03 g, 36.3 mmol) in methylene chloride (80 mL) was added sodium azide (7.08 g, 109 mmol) followed by trifluoroacetic acid (8.0 mL; dropwise over 5-6 minutes). The thickening suspension was stirred for 1.5 hour before diluting with water (350 mL) and extracting with ethyl acetate (1×200 mL). The organic layer was washed with aqueous NaHCO.sub.3 (1×250 mL), dried (Na.sub.2SO.sub.4), and concentrated to afford the crude azide product. To a stirred solution of this material in THF (160 mL) was added water (11 mL) followed by triphenylphosphine (23.8 g, 90.7 mmol). The reaction was stirred for 2 days before concentrating. The resulting residue was dissolved in ethyl acetate (250 mL) and extracted with 1 N aqueous HCl (4×75 mL). The combined extracts were basified with concentrated NH.sub.4OH and extracted with ethyl acetate (2×100 mL). These extracts were, in turn, dried (Na.sub.2SO.sub.4), and concentrated. The resulting amber oil was purified by flash chromatography using a methylene chloride/methanol/ammonia gradient to afford a mixture of 2-(5-bromothiophen-3-yl)propan-2-amine and triphenylphosphine oxide (70/30 ratio) as a viscous amber oil (1.32 g, 17%).

[0602] To a stirred solution of 3-quinuclidinol (3.00 g, 23.6 mmol) in THF (100 mL) was added 4-nitrophenyl chloroformate (5.94 g, 29.5). After stirring for 4 hours, the precipitate was filtered off, rinsed with THF, and air dried on the frit under house vacuum. The filter cake was dissolved in ethyl acetate (150 mL) and washed with aqueous NaHCO.sub.3 (1×150 mL) and water (2×150 mL). The organic layer was dried (Na.sub.2SO.sub.4) and concentrated to afford crude 4-nitrophenyl quinuclidin-3-yl carbonate product, which was used in the next step without purification.

[0603] To a stirred solution of 2-(5-bromothiophen-3-yl)propan-2-amine (0.366 g, 1.66 mmol) in THF (10 mL) was added 4-nitrophenyl quinuclidin-3-yl carbonate (0.571 g, 1.95 mmol) and a few granules of 4-(dimethylamino)pyridine. The mixture was refluxed overnight, concentrated, and partitioned between ethyl acetate (50 mL) and aqueous NaHCO.sub.3 (50 mL). The organic layer was washed again with aqueous NaHCO.sub.3 (1×50 mL), dried (Na.sub.2SO.sub.4), and concentrated. The resulting dirty yellow gum was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford quinuclidin-3-yl (1-(5-bromothiophen-3-yl)cyclopropyl)carbamate as an off-white solid (0.305 g, 49%).

[0604] Using General Procedure C, quinuclidin-3-yl (1-(5-bromothiophen-3-yl)cyclopropyl)carbamate (0.227 g, 0.742 mmol), 4-fluorophenyl boronic acid (0.208 g, 1.49 mmol), tricyclohexylphosphine (0.021 g, 0.075 mmol), potassium phosphate (0.866, 4.08 mmol), and palladium acetate (8.0 mg, 36 μmol) gave the title compound as a grey solid (0.142 g, 49%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.60-7.45 (m, 2H), 7.24-7.19 (m, 1H), 7.10-6.97 (m, 3H), 5.23 (br s, 1H), 4.72-4.61 (m, 1H), 3.30-3.04 (m, 1H), 3.03-2.25 (m, 5H), 2.09-1.02 (m, 11H) ppm. .sup.13C NMR (400 MHz, CDCl.sub.3) δ 162.3 (d, J=247.1 Hz), 154.5, 149.8, 143.6, 130.7, 127.4 (d, J=8.1 Hz), 121.8, 118.9, 115.8 (d, J=21.6 Hz), 70.8, 55.5, 53.4, 47.3, 46.4, 29.0, 25.4, 24.4, 19.4 ppm. Purity: 95.8% UPLCMS (210 nm & 254 nm); retention time 0.90 min; (M+1) 389.

(S)-quinuclidin-3-yl 2-(3-(4-fluorophenyl)isothiazol-5-yl)propan-2-ylcarbamate (Compound 11)

[0605] To stirred solution of 2-(3-(4-fluorophenyl)isothiazol-5-yl)propan-2-amine (1.21 g, 5.12 mmol) in toluene was added a solution of phosgene in toluene [˜1.9 M] (10.8 mL, 20.5 mmol). The reaction was heated at reflux for two hours and then concentrated. The residue was co-evaporated with toluene (2×15 mL) to afford the crude isocyanate intermediate as golden oil. This material was taken up in toluene (10 mL) and treated with (S)-3-quinuclidinol (0.749 g, 5.89 mmol). The reaction was heated at reflux overnight and concentrated. The residue was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford the title compound as a white solid (0.971 g, 49%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.09-8.00 (m, 2H), 7.87 (br s, 1H), 7.75 (s, 1H), 7.35-7.25 (m, 2H), 4.54-4.45 (m, 1H), 3.14-2.92 (m, 1H), 2.87-2.17 (m, 5H), 1.98-0.98 (m, 11H) ppm. .sup.13C NMR (400 MHz, DMSO-d.sub.6) δ 180.1, 165.6, 162.6 (d, J=246.4 Hz), 154.7, 131.2 (d, J=3.0 Hz), 128.7 (d, J=8.4 Hz), 118.2, 115.7 (d, J=21.8 Hz), 70.6, 55.3, 52.8, 46.9, 45.9, 29.9, 25.2, 24.2, 19.2 ppm. Purity: 100% UPLCMS (210 nm & 254 nm); retention time 0.82 min; (M+1) 390.

(S)-quinuclidin-3-yl 2-(4-(4-fluorophenyl)thiazol-2-yl)propan-2-ylcarbamate (Compound 12)

[0606] To a stirred solution of ethyl 3-amino-3-thioxopropanoate (20.00 g, 135.9 mmol) in ethanol (120 mL) was added 2-bromo-4′-fluoroacetophenone (29.49 g, 135.9 mmol). The mixture was refluxed for 1 hour, concentrated, and partitioned between ethyl acetate (300 mL) and aqueous NaHCO.sub.3 (400 mL). The organic layer was combined with a back-extract of the aqueous layer (ethyl acetate, 1×100 mL), dried (Na.sub.2SO.sub.4), and concentrated. The resulting light brown solid was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)acetate as an off-white solid (29.92 g, 83%).

[0607] To a stirred and cooled (−78° C.) solution of ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)acetate (10.00 g, 37.69 mmol) in THF (250 mL) was added a solution of potassium t-butoxide in THF [1.0 M] (136 mL, 136 mmol), dropwise over 15 minutes, followed by 18-crown-6 (1.6 mL, 7.5 mmol). After an additional 30 minutes at −78° C., iodomethane (8.5 mL) was added, dropwise over 5 minutes. The reaction was stirred cold for another 2 hours before pouring into water (450 mL) and extracting with ethyl acetate (2×150 mL). The combined extracts were washed with brine (1×200 mL), dried (Na.sub.2SO.sub.4), and concentrated. The resulting brown oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoate as a pale amber oil (8.64 g, 78%).

[0608] To a stirred solution of ethyl 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoate (0.900 g, 3.07 mmol) in 1:1:1 THF/ethanol/water (15 mL) was added lithium hydroxide monohydrate (0.451 g, 10.7 mmol). After overnight stirring, the reaction was concentrated and redissolved in water (80 mL). The solution was washed with ether (1×50 mL), acidified with the addition of 1N HCl (15 mL), and extracted with ethyl acetate (2×50 mL). The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated to afford 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoic acid as a pale golden solid (0.808 g, 99%).

[0609] To stirred and cooled (0° C.) solution of 2-(4-(4-fluorophenyl)thiazol-2-yl)-2-methylpropanoic acid (0.784 g, 2.96 mmol) in THF (25 mL) was added triethylamine (0.82 mL, 5.9 mmol) followed by isobutyl chloroformate (0.58 mL, 4.4 mmol). The reaction was stirred cold for another 1 hour before adding a solution of sodium azide (0.385 g, 5.92 mmol) in water (7 mL). The reaction was stirred overnight, allowing the cooling bath to slowly warm to room temperature. The mixture was then diluted with water (100 mL) and extracted with ethyl acetate (2×60 mL). The combined extracts were washed with aqueous NaHCO.sub.3 (1×150 mL) and brine (1×100 mL), dried (Na.sub.2SO.sub.4), and concentrated. After coevaporating with toluene (2×30 mL), the resulting off-white solid was taken up in toluene (25 mL) and refluxed for 4 hours. (S)-3-quinuclidinol (0.753 g, 5.92 mmol) was then added and reflux was continued for 3 hours. The reaction was concentrated and the residue was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford the title compound as a white solid (0.793 g, 69%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90-7.81 (m, 2H), 7.32 (s, 1H), 7.14-7.05 (m, 2H), 5.76 (br s, 1H), 4.72-4.65 (m, 1H), 3.26-3.10 (m, 1H), 3.03-2.37 (m, 5H), 2.05-1.23 (m, 11H) ppm. .sup.13C NMR (400 MHz, CDCl.sub.3) δ 177.6, 162.6 (d, J=248.4 Hz), 154.8, 153.6, 130.8 (d, J=3.2 Hz), 128.1 (d, J=8.1 Hz), 115.9 (d, J=21.7 Hz), 112.2, 71.6, 55.7, 47.4, 46.5, 29.1, 25.4, 24.7, 19.6 ppm. Purity: 100% UPLCMS (210 nm & 254 nm); retention time 0.82 min; (M+1) 390.

Quinuclidin-3-yl (2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 13)

[0610] Using General Procedure F and the reaction inputs 2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoic acid (prepared as described in Example 3) and quinuclidin-3-ol, the title compound was generated as a colourless, glassy solid (23%). NMR data matched that of Example 3. Purity: 100%, 99.1% (210 & 254 nm) UPLCMS; retention time: 0.87 min; (M+H.sup.+) 439.0.

(S)-quinuclidin-3-yl (2-(3′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 14)

[0611] Exchanging 4-(2-methoxyethoxy)phenylboronic acid for 3-(2-methoxyethoxy)phenylboronic acid, the reaction sequence outlined in Example 3 was used to prepare 2-(3′-(2-methoxyethoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoic acid. This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a glassy, colourless solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.63-7.31 (m, 6H), 7.24-7.10 (m, 2H), 6.92 (dd, J=8.2, 1.9 Hz, 1H), 4.51-4.34 (m, 1H), 4.21-4.08 (m, 2H), 3.72-3.64 (m, 2H), 3.32 (s, 3H), 3.09-2.26 (m, 5H), 2.04-1.22 (m, 9H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 158.9, 154.6, 147.6, 141.5, 137.6, 129.9, 126.3, 125.2, 118.9, 113.2, 112.5, 70.4, 70.0, 66.9, 58.2, 55.4, 54.2, 46.9, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.91 min; 15 (M+H.sup.+) 439.4.

Quinuclidin-3-yl (2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-3-yl)propan-2-yl)carbamate (Compound 15)

[0612] Exchanging ethyl 2-(4-bromophenyl)-2-methylpropanoate for ethyl 2-(3-bromophenyl)-2-methylpropanoate, the reaction sequence outlined in Example 3 was used to prepare 2-(4′-(2-methoxyethoxy)-[1,1′-biphenyl]-3-yl)-2-methylpropanoic acid. This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.62-7.20 (m, 7H), 7.03 (d, J=8.7 Hz, 2H), 4.48-4.35 (m, 2H), 4.18-4.08 (m, 2H), 3.72-3.62 (m, 2H), 3.32 (s, 3H), 3.10-2.19 (m, 6H), 2.10-1.10 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 158.0, 154.6, 148.8, 139.5, 133.1, 128.5, 127.7, 123.8, 123.2, 122.7, 114.8, 70.4, 69.9, 67.0, 58.2, 55.3, 54.5, 47.0, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 97.4%, 94.6% (210 & 254 nm) UPLCMS; retention time: 0.88 min; (M+H.sup.+) 439.3.

Quinuclidin-3-yl (2-(4′-(3-methoxypropoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 16)

[0613] To a stirred solution of 4-iodophenol (10.05 g, 45.68 mmol) in acetonitrile (100 mL) was added potassium carbonate (6.95 g, 50.2 mmol) and 1-chloro-3-methoxypropane (6.4 mL, 57.1 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was taken up in water and extracted with ethyl acetate. The combined extracts were washed with aqueous sodium bicarbonate solution, dried (Na.sub.2SO.sub.4). and concentrated. The crude material was purified by flash chromatography over silica using a hexane/ethyl acetate eluent to afford 1-iodo-4-(3-methoxypropoxy)benzene as a colourless oil (4.39 g, 33%). This intermediate and ethyl 2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate were reacted according to General Procedure E to generate ethyl 2-(4′-(3-methoxypropoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoate. To a stirred solution of this compound (0.693 g, 1.94 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (10 mL) was added lithium hydroxide monohydrate (0.326 g, 7.77 mmol). The mixture was heated at reflux overnight and then concentrated. The residue was dissolved in water, treated with 1N hydrochloric acid (10 mL), and extracted with ethyl acetate. The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4), and concentrated to afford 2-(4′-(3-methoxypropoxy)-[1,1′-biphenyl]-4-yl)-2-methylpropanoic acid as a waxy, off-white solid (0.630 g, 99%). This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a glassy, colourless solid (62%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.61-7.29 (m, 7H), 7.00 (d, J=8.8 Hz, 2H), 4.47-4.36 (m, 1H), 4.05 (t, J=6.4 Hz, 2H), 3.48 (t, J=6.3 Hz, 2H), 3.26 (s, 3H), 3.10-2.25 (m, 6H), 2.04-1.74 (m, 4H), 1.65-1.23 (m, 9H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 158.0, 154.5, 146.7, 137.4, 132.4, 127.5, 125.7, 125.2, 114.8, 69.9, 68.5, 64.6, 57.9, 55.4, 54.2, 46.9, 46.0, 29.4, 29.0, 25.2, 24.1, 19.2 ppm. Purity: 97.7%, 98.2% (210 & 254 nm) UPLCMS; retention time: 0.96 min; (M+H.sup.+) 453.5.

Quinuclidin-3-yl (2-(4′-(hydroxymethyl)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 17)

[0614] Using General Procedure E and the reaction inputs ethyl 2-(4-bromophenyl)-2-methylpropanoate and 4-formylphenylboronic acid, ethyl 2-(4′-formyl-[1,1′-biphenyl]-4-yl)-2-methylpropanoate was prepared as a pale amber solid. This intermediate and quinuclidin-3-ol were reacted according to General Procedure F to generate quinuclidin-3-yl (2-(4′-formyl-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate as foamy, yellow solid. To a stirred solution of this material (0.755 g, 1.92 mmol) in 2:1 (v/v) tetrahydrofuran/ethanol (15 mL) was added sodium borohydride (0.073 g, 1.93 mmol). After 45 minutes, the reaction was diluted with water and extracted with chloroform. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated onto silica. Flash chromatography over silica using a chloroform/methanol/ammonia eluent provided the title compound as a white solid (0.323 g, 43%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.66-7.29 (m, 9H), 5.18 (t, J=5.7 Hz, 1H), 4.53 (d, J=5.7 Hz, 2H), 4.46-4.37 (m, 1H), 3.11-2.19 (m, 6H), 2.11-1.10 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 154.7, 147.3, 141.5, 138.4, 137.7, 127.0, 126.2, 126.1, 125.3, 70.0, 62.6, 55.4, 54.2, 46.9, 45.9, 29.4, 25.3, 24.2, 19.2 ppm. Purity: 97.5%, 99.1% (210 & 254 nm) UPLCMS; retention time: 0.73 min; (M+H.sup.+) 395.

Quinuclidin-3-yl (2-(4′-(2-hydroxyethyl)-[1,1′-biphenyl]1-4-yl)propan-2-yl)carbamate (Compound 18)

[0615] Using General Procedure E and the reaction inputs 1-(2-(benzyloxy)ethyl)-4-bromobenzene and ethyl 2-methyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate, ethyl 2-(4′-(2-(benzyloxy)ethyl)-[1,1′-biphenyl]-4-yl)-2-methylpropanoate was prepared as a colourless gum. To a stirred solution of this compound (1.34 g, 3.33 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (18 mL) was added lithium hydroxide monohydrate (0.698 g, 16.6 mmol). After heating at reflux overnight, the reaction was concentrated and partitioned between water and diethyl ether. The resulting emulsion was extracted repeatedly with 0.2 N aqueous sodium hydroxide solution (5×50 mL). The clear portion of the aqueous layer was removed each time. The combined aqueous layers were then treated with 1.0 N hydrochloric acid (80 mL) and the resulting suspension of white solid was extracted with ethyl acetate. The combined organic layers were dried (Na.sub.2SO.sub.4) and concentrated to afford 2-(4′-(2-(benzyloxy)ethyl)-[1,1′-biphenyl]-4-yl)-2-methylpropanoic acid as a white solid (1.20 g, 96%). This compound and quinuclidin-3-ol were reacted according to General Procedure F to generate quinuclidin-3-yl (2-(4′-(2-benzyloxyethyl)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate. To a stirred solution of this material (0.435 g, 0.806 mmol) in methanol was added 1.0 N hydrochloric acid (1 mL) and 10% palladium on carbon (50% water; 0.087 g). The mixture was cycled between vacuum and a nitrogen purge several times, refilling with hydrogen after the last evacuation. After 1.25 hours the reaction was filtered through Celite and concentrated. The residue was taken up in aqueous sodium carbonate solution and extracted with 4:1 (v/v) chloroform/isopropanol. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated onto silica. Flash chromatography over silica using a chloroform/methanol/ammonia gradient provided the purified title compound as a colourless solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.85-7.63 (m, 1H), 7.63-7.19 (m, 8H), 4.78-4.62 (m, 2H), 3.71-2.78 (m, 8H), 2.76 (t, J=6.8 Hz, 2H), 2.26-1.96 (m, 2H), 1.96-1.40 (m, 9H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 153.8, 146.8, 138.7, 137.9, 137.6, 129.4, 126.3, 126.1, 125.3, 66.2, 62.1, 54.4, 52.8, 45.4, 44.5, 38.6, 29.5, 29.2, 24.0, 19.9, 16.6 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.75 min; (M+H.sup.+) 409.

Quinuclidin-3-yl (2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate (Compound 19)

[0616] To a stirred suspension of 4-methoxythiobenzamide (9.99 g, 59.7 mmol) in ethanol (75 mL) was added ethyl 4-chloroacetoacetate (8.1 mL, 60 mmol). The mixture was heated at reflux for 4 hours before cooling, adding additional ethyl 4-chloroacetoacetate (0.81 mL, 6.0 mmol), and returning to reflux. After 4 more hours of heating the reaction was concentrated and partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic layer was combined with additional ethyl acetate extracts, dried (Na.sub.2SO.sub.4), and concentrated. The crude product was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-methoxyphenyl)thiazol-4-yl)acetate as a pale amber oil (14.51 g, 87%). To a stirred solution of this compound (14.48 g, 52.2 mmol) in N,N-dimethylformamide (125 mL) was added sodium hydride (60% dispersion in mineral oil; 6.27 g, 157 mmol), portion wise over 15 minutes. The resulting red suspension was cooled (0° C.) and treated, dropwise over 10 minutes, with iodomethane (9.80 mL, 157 mmol). The cooling bath was removed and the reaction was allowed to stir 4 hours before concentrating and partitioning the residue between ethyl acetate and water. The organic layer was washed twice more with water, dried (Na.sub.2SO.sub.4), and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-methoxyphenyl)thiazol-4-yl)-2-methylpropanoate as a pale amber oil (14.12 g, 89%). To a stirred solution of this intermediate (14.12 g, 46.24 mmol) in methylene chloride (250 mL) was added boron tribromide (11.0 mL, 116 mmol), dropwise over 5 minutes. After stirring overnight, the reaction was quenched by the slow addition of methanol (˜20 mL) and then concentrated. The residue was taken up in methanol (250 mL) and concentrated sulfuric acid (7.0 mL). The stirred solution was heated at reflux for 2 hours, concentrated, and partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic layer was combined with a second ethyl acetate extract of the aqueous layer, dried (Na.sub.2SO.sub.4), and concentrated to afford methyl 2-(2-(4-hydroxyphenyl)thiazol-4-yl)-2-methylpropanoate as a white solid (12.56 g, 98%). To a stirred solution of 1-bromo-3-methoxypropane (1.66 g, 10.8 mmol) in acetone (30 mL) was added the phenol intermediate (2.00 g, 7.21 mmol) and potassium carbonate (1.25 g, 9.04 mmol). The mixture was heated overnight at reflux, filtered, and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to afford methyl 2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)-2-methylpropanoate as a faint amber gum (2.47 g, 98%). To a stirred solution of this compound (2.45 g, 7.01 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (45 mL) was added lithium hydroxide monohydrate (1.47 g, 35.0 mmol). After overnight stirring, the reaction was concentrated and partitioned between water and diethyl ether. The aqueous layer was treated with 1.0 N hydrochloric acid (40 mL) and extracted with ethyl acetate. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated to afford 2-(2-(4-(3-methoxypropoxy)phenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (2.19 g, 40 93%). This compound and quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a soft, faint amber solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.82 (d, J=8.9 Hz, 2H), 7.36 (br s, 1H), 7.24 (br s, 1H), 7.03 (d, J=8.9 Hz, 2H), 4.49-4.41 (m, 1H), 4.07 (t, J=6.4 Hz, 2H), 3.48 (t, J=6.4 Hz, 2H), 3.26 (s, 3H), 3.09-2.26 (m, 6H), 2.02-1.91 (m, 2H), 1.91-1.03 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 165.8, 162.4, 160.0, 154.6, 127.5, 126.1, 114.9, 112.1, 70.1, 68.4, 64.8, 57.9, 55.4, 53.5, 46.9, 45.9, 28.9, 28.3, 25.2, 24.2, 19.2 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.87 min; (M+H.sup.+) 460.

Quinuclidin-3-yl (2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)propan-2-yl)carbamate (Compound 20)

[0617] To a stirred solution of 2-bromoethyl methyl ether (1.88 g, 13.5 mmol) in acetone was added methyl 2-(2-(4-hydroxyphenyl)thiazol-4-yl)-2-methylpropanoate (prepared as described in Example 19, 2.00 g, 7.21 mmol) and potassium carbonate (1.56 g, 11.3 mmol). After heating at reflux overnight, the mixture was treated with additional 2-bromo ethyl methyl ether (1.88 g, 13.5 mmol) and potassium carbonate (1.56 g, 11.3 mmol). The reaction was heated at reflux for a second night, filtered, and concentrated. The residue was purified by flash chromatography over silica using a hexane/ethyl acetate gradient to afford methyl 2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)-2-methylpropanoate as a white solid (2.71 g, 90%). To a stirred solution of this compound (2.71 g, 8.08 mmol) in 1:1:1 (v/v/v) tetrahydrofuran/ethanol/water (50 mL) was added lithium hydroxide monohydrate (1.70 g, 40.5 mmol). After overnight stirring, the reaction was concentrated and partitioned between water and diethyl ether. The aqueous layer was treated with 1.0 N hydrochloric acid (41 mL) and extracted with ethyl acetate. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated to afford 2-(2-(4-(2-methoxyethoxy)phenyl)thiazol-4-yl)-2-methylpropanoic acid as a white solid (2.57 g, 99%). This compound and quinuclidin-3-ol were reacted according to General Procedure F to generate the title compound as a pale amber solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.82 (d, J=8.8 Hz, 2H), 7.36 (br s, 1H), 7.24 (br s, 1H), 7.04 (d, J=8.8 Hz, 2H), 4.49-4.41 (m, 1H), 4.19-4.12 (m, 2H), 3.71-3.65 (m, 2H), 3.32 (s, 3H), 3.11-2.87 (m, 1H), 2.86-2.19 (m, 5H), 1.92-1.16 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 165.7, 162.9, 159.9, 154.6, 127.5, 126.2, 114.9, 112.2, 70.3, 70.1, 67.1, 58.2, 55.4, 53.5, 46.9, 45.9, 28.3, 25.2, 24.3, 19.2 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.85 min; (M+H.sup.+) 446.

Quinuclidin-3-yl 2-(5-(4-(2-methoxyethoxy)phenyl)pyridin-2-yl)propan-2-ylcarbamate (Compound 21)

[0618] Using General Procedure E and the reaction inputs 5-bromopicolinonitrile and 2-(4-(2-methoxyethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 5-(4-(2-methoxyethoxy)phenyl)picolinonitrile was prepared. Cerium trichloride (8.05 g, 21.6 mmol) was loaded into a flask and dried by heating (170° C.) under vacuum for 3 hours. The solid was taken up in tetrahydrofuran (20 mL) and stirred vigorously for 30 minutes. The suspension was cooled to −78° C. and treated, dropwise, with a 3.0 M solution of methyllithium in diethyl ether (7.2 mL, 21.6 mmol). Following addition, the reaction was stirred at −78° C. for 1 hour before adding a solution of the above aryl borate (1.83 g, 7.20 mmol) in tetrahydrofuran (20 mL). The mixture was maintained at −78° C. for 2 hours and then allowed to warm to room temperature. At this time, the reaction was quenched by the addition of aqueous ammonium hydroxide (10 mL) and filtered through a plug of Celite. The filtrate was extracted with ethyl acetate and the combined extracts were washed with brine, dried (Na.sub.2SO.sub.4), and concentrated. The residue was purified by flash chromatography over silica using ethyl acetate eluent to afford 2-(5-(4-(2-methoxyethoxy)phenyl)pyridin-2-yl)propan-2-amine as a yellow solid (0.800 g, 39%). To a stirred suspension of this intermediate (0.500 g, 1.75 mmol) in water (10 mL) and concentrated hydrochloric acid (0.44 mL) was added toluene (10 mL). The mixture was cooled (0° C.) and treated with, simultaneously over 1 hour, solutions of triphosgene (0.776 g, 2.62 mmol) in toluene (10 mL) and sodium bicarbonate (2.2 g, 26 mmol) in water (20 mL). Following the additions, the reaction was stirred for an additional 30 minutes before the upper toluene layer was removed and dried (Na.sub.2SO.sub.4). At the same time, a stirred solution of quinuclidin-3-ol (0.445 g, 3.64 mmol) in tetrahydrofuran (10 mL) was treated with sodium hydride (60% dispersion in mineral oil; 0.154 g, 3.85 mmol). This mixture was stirred for 5 minutes and then added to the solution of crude isocyanate in toluene. The reaction was stirred for 10 minutes, quenched with the addition of brine (5 mL), and extracted with ethyl acetate. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified by flash chromatography over reversed phase silica to afford the title compound as a light yellow solid (0.100 g, 13%). .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.70-8.70 (d, J=2.0 Hz, 1H), 7.83-7.81 (m, 1H), 7.49-7.47 (d, J=9.0 Hz, 2H), 7.45-7.43 (d, J=8.0 Hz, 1H), 7.03-7.01 (d, J=8.5 Hz, 2H), 6.63 (br s, 1H), 4.68-4.66 (m, 1H), 4.16 (t, J=5.0 Hz, 2H), 3.77 (t, J=5.0 Hz, 2H), 3.45 (s, 3H), 3.19-2.70 (m, 6H), 2.15-1.89 (m, 2H), 1.76 (s, 6H), 1.73-1.36 (m, 3H) ppm. .sup.13C NMR (125 MHz, CDCl.sub.3) δ 162.7, 158.9, 154.9, 145.9, 134.8, 134.3, 130.1, 128.1, 119.2, 115.2, 71.0, 70.8, 67.4, 59.2, 55.9, 55.7, 47.4, 46.5, 46.4, 27.9, 25.4, 24.6, 19.5 ppm. Purity: >99% (214 & 254 nm) LCMS; retention time: 1.32 min; (M+H.sup.+) 440.2.

Quinuclidin-3-yl (2-(4′-(3-cyanopropoxy)-[1,1′-biphenyl]-4-yl)propan-2-yl)carbamate (Compound 22)

[0619] To a stirred solution of 4-bromophenol (17.1 g, 98.8 mmol) in acetonitrile (150 mL) was added 1-bromobutylnitrile (12.3 mL, 124 mmol) and potassium carbonate (15.0 g, 109 mmol). The mixture was heated to reflux overnight, cooled, and concentrated. The residue was taken up in water and extracted with ethyl acetate. The combined extracts were dried (Na.sub.2SO.sub.4) and concentrated and the crude material was purified by flash chromatography over silica using a hexane/ethyl acetate eluent to afford 4-(4-bromophenoxy)butanenitrile as a white solid (20.8 g, 88%). To a stirred solution of this product in N,N-dimethylformamide (100 mL), was added bis(pinacolato)diboron (4.60 g, 18.1 mmol), potassium acetate (7.41 g, 75.5 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) complex with dichloromethane (0.616 g, 1.04 mmol). The mixture was heated to reflux overnight and then concentrated. The residue was taken up in ethyl acetate and washed with water and brine. The organic layer was dried (Na.sub.2SO.sub.4) and concentrated and the crude product was purified by flash chromatography over silica using a hexane/ethyl acetate eluent to afford 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butanenitrile as a white solid (3.43 g, 79%). This product and quinuclidin-3-yl (2-(4-bromophenyl)propan-2-yl)carbamate (prepared by reacting quinuclidin-3-ol and 2-(4-bromophenyl)propan-2-amine using General Procedure F) were reacted according to General Procedure E to generate the title compound as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.67-7.26 (m, 7H), 7.02 (d, J=8.8 Hz, 2H), 4.50-4.33 (m, 1H), 4.08 (t, J=6.0 Hz, 2H), 3.14-2.18 (m, 8H), 2.04 (quin, J=6.7 Hz, 2H), 1.94-1.70 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 157.7, 154.5, 146.8, 137.4, 132.7, 127.6, 125.7, 125.2, 120.2, 114.9, 70.0, 65.8, 55.4, 54.2, 46.9, 45.9, 29.4, 25.3, 24.7, 24.2, 19.2, 13.4 ppm. Purity: 100%, 98.9% (210 & 254 nm) UPLCMS; retention time: 0.88 min; (M+H.sup.+) 448.6.

Quinuclidin-3-yl (2-(4′-(cyanomethoxy)-[1,1′-biphenyl]4-yl)propan-2-yl)carbamate (Compound 23)

[0620] Using General Procedure E and the reaction inputs quinuclidin-3-yl (2-(4-bromophenyl)propan-2-yl)carbamate (prepared by reacting quinuclidin-3-ol and 2-(4-bromophenyl)propan-2-amine using General Procedure F) and 4-(cyanomethoxy)phenylboronic acid, the title compound was prepared as a pale amber solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.65 (d, J=8.2 Hz, 2H), 7.60-7.31 (m, 5H), 7.15 (d, J=8.9 Hz, 2H), 5.21 (s, 2H), 4.53-4.30 (m, 1H), 3.18-2.19 (m, 6H), 2.05-1.18 (m, 11H) ppm. .sup.13C NMR (100 MHz, DMSO-d.sub.6) δ 155.8, 154.6, 147.2, 137.2, 134.4, 127.8, 126.0, 125.3, 116.7, 115.3, 70.0, 55.4, 54.2, 53.5, 46.9, 45.9, 29.4, 25.2, 24.2, 19.2 ppm. Purity: 100%, 100% (210 & 254 nm) UPLCMS; retention time: 0.85 min; (M+H.sup.+) 420.3.

Example 2: Preparation of (S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate free base

[0621] Step 1: Dimethylation with Methyl Iodide

##STR00009##

[0622] A 3N round-bottom (RB) flask was equipped with a thermometer, an addition funnel, and a nitrogen inlet. The flask was flushed with nitrogen and potassium tert-butoxide (MW 112.21, 75.4 mmol, 8.46 g, 4.0 equiv., white powder) was weighed out and added to the flask via a powder funnel followed by the addition of THF (60 mL). Most of the potassium tert-butoxide dissolved to give a cloudy solution. This mixture was cooled in an ice-water bath to 0-2° C. (internal temperature). In a separate flask, the starting ester (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv.) was dissolved in THF (18 mL+2 mL as rinse) and transferred to the addition funnel. This solution was added dropwise to the cooled mixture over a period of 25-30 min, keeping the internal temperature below 5° C. during the addition. The reaction mixture was cooled back to 0-2° C. In a separate flask, a solution of methyl iodide (MW 141.94, 47.13 mmol, 6.7 g, 2.5 equiv.) in THF (6 mL) was prepared and transferred to the addition funnel. The flask containing the methyl iodide solution was then rinsed with THF (1.5 mL) which was then transferred to the addition funnel already containing the clear colorless solution of methyl iodide in THF. This solution was added carefully dropwise to the dark brown reaction mixture over a period of 30-40 min, keeping the internal temperature below 10° C. at all times during the addition. After the addition was complete, the slightly turbid mixture was stirred for an additional 1 h during which time the internal temperature dropped to 0-5° C. After stirring for an hour at 0-5° C., the reaction mixture was quenched with the slow dropwise addition of 5.0M aqueous HCl (8 mL) over a period of 5-7 min. The internal temperature was maintained below 20° C. during this addition. After the addition, water (14 mL) was added and the mixture was stirred for 2-3 min. The stirring was stopped and the two layers were allowed to separate. The two layers were then transferred to a 250 mL 1N RB flask and the THF was evaporated in vacuo as much as possible to obtain a biphasic layer of THF/product and water. The two layers were allowed to separate. A THF solution of the Step1 product was used in the next reaction.

Step 2: Hydrolysis of the Ethyl Ester with LiOH Monohydrate

##STR00010##

[0623] The crude ester in THF was added to the reaction flask. Separately, LiOH.H.sub.2O (MW 41.96, 75.0 mmol, 3.15 grams, 2.2 equiv.) was weighed out in a 100 mL beaker to which a stir bar was added. Water (40 mL) was added and the mixture was stirred till all the solid dissolved to give a clear colorless solution. This aqueous solution was then added to the 250 mL RB flask containing the solution of the ester in tetrahydrofuran (THF). A condenser was attached to the neck of the flask and a nitrogen inlet was attached at the top of the condenser. The mixture was heated at reflux for 16 hours. After 16 hours, the heating was stopped and the mixture was cooled to room temperature. The THF was evaporated in vacuo to obtain a brown solution. An aliquot of the brown aqueous solution was analyzed by HPLC and LC/MS for complete hydrolysis of the ethyl ester. Water (15 mL) was added and this aqueous basic solution was extracted with TBME (2×40 mL) to remove the t-butyl ester. The aqueous basic layer was cooled in an ice-water bath to 0-10° C. and acidified with dropwise addition of concentrated HCl to pH ˜1 with stirring. To this gummy solid in the aqueous acidic solution was added TBME (60 mL) and the mixture was shaken and then stirred vigorously to dissolve all the acid into the TBME layer. The two layers were transferred to a separatory funnel and the TBME layer was separated out. The pale yellow aqueous acidic solution was re-extracted with TBME (40 mL) and the TBME layer was separated and combined with the previous TBME layer. The aqueous acidic layer was discarded. The combined TBME layers are dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated in vacuo to remove TBME and obtain the crude acid as an orange/dark yellow oil that solidified under high vacuum to a dirty yellow colored solid. The crude acid was weighed out and crystallized by heating it in heptane/TBME (3:1, 5 mL/g of crude) to give the acid as a yellow solid.

Step 3: Formation of Hydroxamic Acid with NH.sub.2OH.HCl

##STR00011##

[0624] The carboxylic acid (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv.) was weighed and transferred to a 25 mL 1N RB flask under nitrogen. THF (5.0 mL) was added and the acid readily dissolved to give a clear dark yellow to brown solution. The solution was cooled to 0-2° C. (bath temperature) in an ice-bath and N, N′-carbonyldiimidazole (CDI; MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv.) was added slowly in small portions over a period of 10-15 minutes. The ice-bath was removed and the solution was stirred at room temperature for 1 h. After 1 h of stirring, the solution was again cooled in an ice-water bath to 0-2° C. (bath temperature). Hydroxylamine hydrochloride (NH.sub.2OH.HCl; MW 69.49, 37.7 mmol, 2.62 g, 2.0 equiv.) was added slowly in small portions as a solid over a period of 3-5 minutes as this addition was exothermic. After the addition was complete, water (1.0 mL) was added to the heterogeneous mixture dropwise over a period of 2 minutes and the reaction mixture was stirred at 0-10° C. in the ice-water bath for 5 minutes. The cooling bath was removed and the reaction mixture was stirred under nitrogen at room temperature overnight for 20-22 h. The solution became clear as all of the NH.sub.2OH.HCl dissolved. After 20-22 h, an aliquot of the reaction mixture was analyzed by High Pressure Liquid Chromatography (HPLC). The THF was then evaporated in vacuo and the residue was taken up in dichloromethane (120 mL) and water (60 mL). The mixture was transferred to a separatory funnel where it was shaken and the two layers were allowed to separate. The water layer was discarded and the dichloromethane layer was washed with 1N hydrochloride (HCl; 60 mL). The acid layer was discarded. The dichloromethane layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and the solvent evaporated in vacuo to obtain the crude hydroxamic acid as a pale yellow solid that was dried under high vacuum overnight.

Step 3 Continued: Conversion of Hydroxamic Acid to Cyclic Intermediate (not Isolated)

[0625] ##STR00012##

[0626] The crude hydroxamic acid (MW 280.32, 5.1 g) was transferred to a 250 mL 1N RB flask with a nitrogen inlet. A stir bar was added followed by the addition of acetonitrile (50 mL). The solid was insoluble in acetonitrile. The yellow heterogeneous mixture was stirred for 2-3 minutes under nitrogen and CDI (MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv.) was added in a single portion at room temperature. No exotherm was observed. The solid immediately dissolved and the clear yellow solution was stirred at room temperature for 2-2.5 h. After 2-2.5 h, an aliquot was analyzed by HPLC and LC/MS which showed conversion of the hydroxamic acid to the desired cyclic intermediate.

[0627] The acetonitrile was then evaporated in vacuo to give the crude cyclic intermediate as reddish thick oil. The oil was taken up in toluene (60 mL) and the reddish mixture was heated to reflux for 2 hours during which time, the cyclic intermediate released CO.sub.2 and rearranged to the isocyanate (see below).

##STR00013##

Step 3 Continued: Conversion of the Isocyanate to the Free Base

[0628] ##STR00014##

[0629] The reaction mixture was cooled to 50-60° C. and (S)-(+)-quinuclidinol (MW 127.18, 28.28 mmol, 3.6 g, 1.5 equiv.) was added to the mixture as a solid in a single portion. The mixture was re-heated to reflux for 18 h. After 18 h, an aliquot was analyzed by HPLC and LC/MS which showed complete conversion of the isocyanate to the desired product. The reaction mixture was transferred to a separatory funnel and toluene (25 mL) was added. The mixture was washed with water (2×40 mL) and the water layers were separated. The combined water layers were re-extracted with toluene (30 mL) and the water layer was discarded. The combined toluene layers were extracted with 1N HCl (2×60 mL) and the toluene layer (containing the O-acyl impurity) was discarded. The combined HCl layers were transferred to a 500 mL Erlenmeyer flask equipped with a stir bar. This stirring clear yellow/reddish orange solution was basified to pH 10-12 by the dropwise addition of 50% w/w aqueous NaOH. The desired free base precipitated out of solution as a dirty yellow gummy solid which could trap the stir bar. To this mixture was added isopropyl acetate (100 mL) and the mixture was stirred vigorously for 5 minutes when the gummy solid went into isopropyl acetate. The stirring was stopped and the two layers were allowed to separate. The yellow isopropyl acetate layer was separated and the basic aqueous layer was re-extracted with isopropyl acetate (30 mL). The basic aqueous layer was discarded and the combined isopropyl acetate layers were dried over anhydrous Na.sub.2SO.sub.4, filtered into a pre-weighed RB flask, and the solvent evaporated in vacuo to obtain the crude free base as beige to tan solid that was dried under high vacuum overnight.

Step 3 Continued: Recrystallization of the Crude Free Base

[0630] The beige to tan colored crude free base was weighed and re-crystallized from heptane/isopropyl acetate (3:1, 9.0 mL of solvent/g of crude free base). The appropriate amount of heptane/isopropyl acetate was added to the crude free base along with a stir bar and the mixture was heated to reflux for 10 min (free base was initially partially soluble but dissolved to give a clear reddish orange solution when heated to reflux). The heat source was removed and the mixture was allowed to cool to room temperature with stirring when a white precipitate formed. After stirring at room temperature for 3-4 h, the precipitate was filtered off under hose vacuum using a Buchner funnel, washed with heptane (20 mL) and dried under hose vacuum on the Buchner funnel overnight. The precipitate was the transferred to a crystallizing dish and dried at 55° C. overnight in a vacuum oven. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.04-7.83 (m, 2H), 7.20-6.99 (m, 3H), 5.53 (s, 1H), 4.73-4.55 (m, 1H), 3.18 (dd, J=14.5, 8.4 Hz, 1H), 3.05-2.19 (m, 5H), 2.0-1.76 (m, 11H) ppm. .sup.13C NMR (100 MHz, CDCl.sub.3) δ 166.38, 165.02, 162.54, 162.8-155.0 (d, C—F), 130.06, 128.43, 128.34, 116.01, 115.79, 112.46, 71.18, 55.70, 54.13, 47.42, 46.52, 27.94, 25.41, 24.67, 19.58 ppm.

Example 3: Preparation of Crystalline Forms of (S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate salts

[0631] Crystalline salts of (S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate may be formed from the free base prepared as described in Example 23.

[0632] For example, the free base of (S)-Quinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate (about 50 mmol) is dissolved IPA (140 ml) at room temperature and filtered. The filtrate is added into a 1 L RB flask which is equipped with an overhead stirrer and nitrogen in/outlet. L-malic acid (about 50 mmol) is dissolved in IPA (100+30 ml) at room temperature and filtered. The filtrate is added into the above 1 Liter flask. The resulting solution is stirred at room temperature (with or without seeding) under nitrogen for 4 to 24 hours. During this period of time, crystals form. The product is collected by filtration and washed with a small amount of IPA (30 ml). The crystalline solid is dried in a vacuum oven at 55° C. for 72 hours to yield the desired malate salt.

[0633] Crystal forms of other salts, e.g., acid addition salts with succinic acid or HCl, may be prepared in an analogous manner.

Example 4: In-Vitro GCS Inhibition (Compound 2 and Analogs)

[0634] Inhibition of glucosylceramide synthase activity can be measured with one or more assays. A first assay is a microsomal assay that directly measures the conversion of ceramide to glucosylceramide by HPLC. Microsomes are a source of glucosylceramide synthase activity in the microsomal assay. A second assay is a cell based, phenotypic assay that monitors cell surface expression of the downstream lipid GM3 by antibody mediated immunofluorescence. Specific protocols are provided below.

Glucosylceramide Synthase Activity Microsomal Assay:

[0635] An enzyme assay using microsomes as a source of glucosylceramide synthase activity. Fluorescent ceramide substrate is delivered to membrane-bound enzyme as a complex with albumin. After reaction, ceramide and glucosylceramide are separated and quantitated by reverse-phase HPLC with fluorescence detection. Enzymatic activity is assessed using a fluorescent labeled substrate and microsomes as a source of glucosylceramide synthase. C.sub.6-NBD-Ceramide is complexed with albumin for delivery to microsomes that are isolated according to the procedure described below. The final concentration of C.sub.6-NBD-Ceramide in the stock solution is 0.5 mM; the final concentration of BSA is 0.5 mM. Separation and quantitation of substrate and product (glucosylceramide) are achieved by reverse-phase HPLC with fluorescence detection.

Preparation of Microsomes from A375 Human Melanoma Cells;

[0636] Microsomes are isolated from A375 human melanoma cells. Eight to ten million cells are harvested by trypsinization and washed with ice cold PBS. Cells are resuspended in ice-cold lysis buffer containing protease inhibitors. Cell lysate is sonicated on ice using a probe sonicator. After sonication, the cell lysate is separated from debris by centrifugation at 10,000 g for 10 minutes at 4° C. The supernatant is removed and cleared by additional centrifugation at 100,000 g for 1 hour at 4° C. The pellet is then resuspended in the lysis buffer, aliquoted, and stored at −80° C. prior to use.

Glucosylceramide Synthase Assay

[0637] To determine glucosylceramide synthase inhibition, substrates at 2× of their Km (fluorescent ceramide and UDP-glucose, 3 μM and 4 μM respectively) and microsomes (1:50 dilution) are combined 1:1 and incubated at room temperature for 1 hour in the dark on a plate shaker. The reaction is stopped by the addition of 150 μL of 100 μM C.sub.8-ceramide in 50% aq. isopropanol; 10 μL of the final mix is analyzed on HPLC (with fluorescence detector). The mobile phase is 1% formic acid added to 81% methanol/19% water with flow rate 0.5 mL/min. Fluorescence is detected with λ.sub.ex=470 nm and λ.sub.em=530 nm. Under these conditions, NBD-C.sub.6-GluCer had a retention time of about 1.7 min and NBD-C.sub.6-Cer elutes from the column after about 2.1 min. Both peaks are separated from each other and the baseline and were integrated automatically by the HPLC software. The percent conversion of substrate to product is used as the readout for inhibitor testing.

GM3 Fluorescent-Linked Immunosorbent Assay (FLISA):

[0638] This is a phenotypic assay that measures GM3 expression in B16 mouse melanoma or C32 human melanoma cells following treatment with test compounds. Cell surface GM3 expression is determined by antibody mediated fluorescence.

[0639] Compounds are diluted in media and plated in 384 well plates in DMSO. B16 and C32 cells are assayed at densities of 20,000 cells/ml and 62,500 cells/ml, respectively, per well. Each titration curve contains 10 points that are assayed in duplicate on each test run. The plates are incubated for 48 hours at 37° C., 5% CO2, and are then washed once with TBS. Anti-GM3 antibody is added to each well and the plates are then incubated for an additional one hour at room temperature. Plates are subsequently washed twice and incubated for an additional hour with the labeled secondary antibody. Following the final incubation, the plates are washed twice and the fluorescence at λ.sub.ex=D640/20 nm and λ.sub.em=657 nm is detected on a fluorescent reader.

Assay Results

[0640] Individual assay results of certain exemplified compounds in these assays are presented in the Table below. The results of the microsomal assays are expressed as “GCS IC.sub.50,” which represents the concentration of the compound causing 50% inhibition of glucosylceramide synthase activity. The results of the cell-based assays are expressed as “GM3 B16 IC.sub.50” or “GM3 C32 IC.sub.50” for the B16 assay and the C32 assay, respectively. These values represent the concentration of the compound causing 50% inhibition of GM3 expression on the cell surface.

TABLE-US-00002 Compound GCS IC.sub.50 GM3 B16 GM3 C32 No. (mM) IC.sub.50 (mM) IC.sub.50 (mM) 1 0.0019 0.0156 0.0021 2 0.0601 0.1068 0.0096 3 0.00414 0.0437 0.00131 4 0.0015 0.0116 0.0008 5 0.0012 0.0193 0.0003 6 0.0028 0.0181 0.0006 7 0.0014 0.0081 0.0004 8 0.0010 0.0075 0.0004 9 0.0014 0.0168 0.0004 10 0.0064 0.0213 0.0022 11 0.0149 0.0819 0.0018 12 0.0203 0.0878 0.0037 13 0.0035 0.0386 0.0007 14 0.0104 0.1096 0.0053 15 0.0267 0.0295 0.0049 16 0.0024 0.0666 0.0016 17 0.4544 0.8786 0.0216 18 0.1480 0.6555 0.0223 19 0.1701 0.1972 0.0426 20 0.3601 0.1065 0.0198 21 0.0506 0.2658 0.0111 22 0.0096 0.0865 0.0032 23 0.0026 0.0477 0.0008

[0641] These comparative results demonstrate that compounds according to the present disclosure have comparable in-vitro activity as inhibitors of GCS, and as a result, are expected to demonstrate similar in-vivo benefits.

Example 5: Clinical Study of Compound 2 in GD-3 Patients

[0642] A 156-week, multi-part, open-label, multinational study of the safety, tolerability, pharmacokinetics, pharmacodynamics, and exploratory efficacy of Compound 2 in combination with imiglucerase in adult patients with Gaucher disease Type 3 stabilized with imiglucerase was initiated (called LEAP or LEAP2IT trial). Compound 2 is administered orally in the malate salt form (L-malic acid) at a dose of 15 mg/day (measured as the quantity of free base) in a single daily dose. The endpoint assessments are related to safety, CSF biomarkers, pharmacokinetics/pharmacodynamics, systemic disease, and neuroimaging and neurological function (CNS/neurological manifestations).

[0643] Patients 18 years of age or older with a clinical diagnosis of GD3 and documented deficiency of acid beta-glucosidase activity having received treatment with ERT for at least 3 years and with imiglucerase (Cerezyme) at a stable monthly dose for at least 6 months prior to enrollment were included in the study. Patients must have reached the following GD therapeutic goals: hemoglobin level of ≥11.0 g/dL for females and ≥12.0 g/dL for males; platelet count≥100 000/mm3; spleen volume<10 multiples of normal (MN), or total splenectomy (provided the splenectomy occurred>3 years prior to randomization); liver volume<1.5 MN; and no bone crisis and free of symptomatic bone disease such as bone pain attributable to osteonecrosis and/or pathological fractures within the last year. Patients must have GD3 featuring oculomotor apraxia (supranuclear gaze palsy) characterized by a horizontal saccade abnormality.

(A) 52-Week Interim Analysis (N=6)

[0644] An interim analysis was performed when 6 patients had completed 52 weeks of concurrent treatment with (1) imiglucerase (Cerezyme from Sanofi Genzyme) under each patient's established regimen, and (2) Compound 2 administered orally at 15 mg/day in a single dose. During the study patients were evaluated for safety and tolerability, CSF and plasma biomarkers (glucosylceramide, GL-1; glucosylsphingosine, lyso-GL1), pharmacokinetics, markers for systemic disease (spleen and liver volume measured by magnetic resonance imaging (MRI), platelet count, hemoglobin levels), indicia of interstitial lung disease (high resolution pulmonary computed tomography (CT)), and horizontal saccadic eye movement. In addition, exploratory biomarkers were quantified in CSF of GD3 patients: ceramide (the precursor of GL-1), chitotriosidase (CHITO), GM3, and GPNMB. Symptoms of ataxia were measured using the SARA scale, neurological symptoms were measured using the trail making test, and functional MRI was used to assess neural connectivity in the brain.

[0645] At baseline, five patients had mild neurological involvement and one had moderate neurological involvement, as measured using the Modified Severity Scoring Tool (mSST; see e.g., Davies, et al., J Inherit Metab Dis. (2011) 34(5), pp 1053-1059).

[0646] Analysis of plasma and CSF concentrations of Compound 2 shows that Compound 2 effectively cross the blood-brain barrier in all patients. Patient 5, however, is found to have about 50% lower concentrations of Compound 2 in plasma and CSF at Week 26, and undetectable concentrations at Week 52. It is believed that this is due to either compliance or dosing errors, and therefore, analysis is repeated without Patient 5's Week 26 and 52 data included. The data supports the conclusion that a steady state concentration of Compound 2 is reached in plasma and CSF at or prior to Week 4:

TABLE-US-00003 Compound 2 in plasma Day 1 (N = 6) AUC.sub.0-24, ng .Math. h/mL (mean ± SD) 729 ± 205 C.sub.max, ng/mL (mean ± SD) 49.1 ± 17.3 t.sub.max, h (median) 2.00

TABLE-US-00004 Day 1 Week 4 Week 26 Week 52 (N = 6) (N = 6) (N = 6) (N = 6) Compound 2 in Plasma Concentration 2-4 39.7 ± 12.6 92.3 ± 36.4 102.0 ± 49.5  69.8 ± 58.3 hours post dose, ng/mL (mean ± SD) Excluding Patient 5 112 84 Compound 2 in CSF Concentration 2-4 <LLOQ 4.56 ± 1.20 5.26 ± 2.49 4.43 ± 3.23 hours post dose, ng/mL (mean ± SD) Excluding Patient 5 6.13 5.32

[0647] At 52 weeks, the data further shows sustained significant improvements in plasma and CSF biomarkers for GD-3. Over all six GD3 patients, plasma and CSF GL-1 and lyso-GL-1 concentrations were as follows:

TABLE-US-00005 Lyso-GL-1 GL-1 Baseline 52-weeks Baseline 52-weeks Plasma 29.3 ng/mL 15.2 ng/mL 6.21 μg/mL 1.59 μg/mL  (6.3-159.0) (2.5-46.8) (4.2-8.3) (0.9-2.7) CSF 34.0 pg/mL 17.3 pg/mL 6.36 ng/mL 2.48 ng/mL (20.1-67.6) (5.8-37.4)  (4.4-11.1) (1.0-6.1)

[0648] Thus, at 52-weeks compared to baseline, plasma and CSF concentrations had changed as follows:

TABLE-US-00006 Lyso-GL-1 (% change) GL-1 (% change) Plasma Concentration −56.7% −71.6% CSF Concentration −55.9% −55.4%

[0649] In addition, exploratory biomarkers were quantified in CSF of GD3 patients: ceramide (the precursor of GL-1), chitotriosidase (CHITO; an enzyme known to be elevated in GD patients), GM3 (a glycosphingolipid marker known to be elevated in GD patients), and GPNMB (glycoprotein nonmetastatic melanoma protein B, reportedly a biomarker of neuropathic GD3). After 52 weeks of treatment, no significant changes were observed in CSF concentrations of ceramide, CHITO, or GPNMB. Four of the six patients had measurable concentrations of GM3 in CSF at baseline, and each of these patients was found to have undetectable GM3 in CSF at 4 weeks, 26 weeks, and 52 weeks.

[0650] In addition, at 52 weeks, 5 of 6 patients showed improvements in ataxia. The degree of ataxia at baseline and throughout the study was evaluated by the Scale for Assessment and Rating of Ataxia (SARA; Schmitz-Hübsch et al. [2006]), which assesses eight distinct attributes of cerebellar ataxia on a scale of 0-40. The eight attributes are gait, stance, sitting, speech disturbance, finger chase, nose-finger test, fast alternate hand movement, and heel-shin slide. SARA ataxia scoring results for all six patients is presented in the chart below:

TABLE-US-00007 SARA Cumulative Score At Screening Week 26 Week 52 Patient 1 3.0 1.0 0.0 Patient 2 3.0 2.0 1.5 Patient 3 3.5 0.0 0.0 Patient 4 3.0 5.0 4.5 Patient 5 0.5 0.0 0.0 Patient 6 4.0 2.0 3.0 Average Score 2.83 1.67 2.00 Average Score 2.80 1.00 0.90 excluding Patient 4

[0651] As shown in the table, five of the six patients were mildly ataxic at baseline, with the mean cumulative SARA score being 2.8 (SD=1.2). The most common deficits at baseline were gait disorders. Excluding Patient 5 due to the low level of Compound 2 exposure in this patient and the patient's substantially normal baseline ataxia score (only 0.5), then 4 out of 5 patients exhibited an improvement in ataxia at Week 52 (mean improvement=−0.9; SD=3.2). Patient 4 exhibited an increase in ataxia scoring, with the score at baseline being 3 and at Week 52, 7.5. It should be noted that this apparent deterioration was driven almost entirely by a change in the ‘stance’ scoring parameter (stance score at baseline and Week 26=1; score at Week 52=5) and that the patient was complaining of left knee pain at the time of the exam. Additionally, the subject had injured his left great toe prior to the exam; this injury was considered resolved 11 days after the exam. Excluding these outlier effect of Patient 4, treatment with Compound 2 resulted in a significantly decreased mean SARA Score by Week 26 which was further slightly improved upon by Week 52.

[0652] The trail making test (TMT) was used to evaluate cognitive function in the patients. The TMT is one of the most widely used neuropsychological tests and is included in most test batteries. The TMT is a diagnostic tool to assess general intelligence and cognitive dysfunctions (Tombaugh et al. [2004]; Cavaco et al. [2013]). In part A of the TMT (TMT-A), subjects are asked to connect a cluster of numbers in ascending order (Trail A). This task is a combination of visual search and general visual and motor processing speed. Part B (TMT-B) presents a sequence which alternates between numbers and letters (Trail B). Subjects must actively switch between both categories when connecting them in ascending, but alternating order. Hence, this task is considered to include an executive function component since the subject must actively switch between categories while connecting the symbols (MacPherson et al. [2017]).

[0653] TMT-A evaluates mainly perceptual and psychomotor speed. TMT-B assesses more specifically mental flexibility and shifting abilities. TMT-B minus TMT-A score is used to remove the variance attributable to the graphomotor and visual scanning components of TMT-A. This derived score reflects the unique task requirements of TMT-B.

[0654] In a study of normative data for TMT-A and TMT-B in community-dwelling individuals aged 18-89 years (n=911), mean (SD) values in the 18-24 years age group (n=155) were 22.9 s (6.9) for TMT-A and 49 s (12.7) for TMT-B (Tombaugh et al. [2004]). In contrast, the mean times taken to complete Trail A and Trail B for patients in the study were 67.8 s (SD=60.3 s) and 193.8 s (SD=197.0), respectively. At baseline, the mean difference in time taken to complete Trail B minus Trail A was 126.0 s (SD=142.9 s). This shows that the GD-3 patients in this study demonstrated some degree of cognitive dysfunction at baseline.

[0655] At Week 52, the mean period of time taken to complete Trail A was 56.5 s (SD=55.2 s) and Trail B was 122.7 s (SD=91.8 s). Four of six patients exhibited reduction in time taken to complete Trail A and six of six exhibited reduction in time taken to complete Trail B. Excluding Patient 5 due to the low level of Compound 2 exposure in this patient, four of five patients exhibited a TMT-A reduction and five of five patients exhibited a TMT-B reduction.

[0656] At Week 52, 5 of 6 patients exhibited a reduction in the (TMT-B−TMT-A) time. Individual results are shown in the table below.

TABLE-US-00008 Change (%) TMT-A (s) − from Baseline TMT-B (s) At Screening Week 26 Week 52 to Week 52 Patient 1 13 21 16 +23% Patient 2 71 37 57 −20% Patient 3 72 56 20 −72% Patient 4 116 (no data) 66 −43% Patient 5 74 60 72  −3% Patient 6 410 440 166 −60% Average 126 (n = 6) 123 (n = 5) 66 (n = 6) −29%

[0657] At 52 weeks, the mean difference in time taken to complete Trail B minus Trail A was 66.2 s (SD=54.3). Excluding Patient 5, four of five patients exhibited an improvement in Trail B minus Trial A at Week 52, with a mean improvement of −71.4 s (−31.6%) (SD 99.3 s (37.6%)).

[0658] Neurological function was further evaluated using functional magnetic resonance imaging (fMRI). Patient 2 was excluded because no fMRI data was collected at the Week 52 session. Resting-state fMRI screening sessions were performed at baseline screening, Week 26, and Week 52 visits. Connectivity estimates from four subjects (Patients 1, 3, 4, and 5) were entered into second-level analyses as a “compliant” group. Patient 5 was isolated due to likely non-compliance with study medication, as described above. Analyses were performed as described elsewhere (Smith et al. [2009]).

[0659] It was found that the compliant subjects demonstrate an enhanced connectivity between a more broadly distributed set of brain regions than the non-compliant subject, with increasing strength between posterior and anterior aspects as the most prominent feature. At the anatomic level, compliant subjects demonstrate a widespread and robust strengthening of connections between occipital-parietal structures and frontal, temporal, and limbic targets. Connectivity changes in Patient 5 were more modest and restricted within spatially proximal structures. At the functional level, enhanced connectivity between default mode and medial frontal networks is seen in every subject except Patient 5. This suggests signal within these disparate networks becomes more coherent, such that brain activity can be more efficiently transferred between cognitive reserve (posterior) and higher-order executive functions (anterior). A consistent reciprocal mapping of resting state networks (RSNs) 2 and 3 (“cognition-language-orthography” and “cognition-space”) to RSNs 8 and 9 (executive and left frontoparietal) is also evident. The spatial distribution of connectivity changes is much more focal for Patient 5, primarily reflecting overlap between medial-frontal and frontoparietal networks. Both perspectives suggest that patients who fully complied with the treatment protocol developed greater coherence between posterior and anterior aspects of the brain, such that the entire brain becomes amenable to efficient information transfer. Where apparent, altered connectivity for Patient 5 appears within a narrower set of anterior brain regions and represents less holistic evidence of therapeutic benefit.

[0660] The results are summarized in the table below. Spatial analysis of the connectivity between different anatomic regions of the brain is performed to define a correlation coefficient for regressed voxelwise mean intensity. The results show that connectivity between the default mode (resting) network and the executive function network increased in Patients 1, 3, 4, and 6, but decreased in Patient 5.

TABLE-US-00009 Patient 1 Patient 3 Patient 4 Patient 5 Patient 6 Change in +0.20 +0.20 +0.20 −0.13 +0.70 Correlation Coefficient

(B) 52-Week Interim Analysis (N=11)

[0661] An additional interim analysis was performed when 11 patients had reached the 52-week milestone. The results of this analysis confirm the observation made at the previous interim analysis.

[0662] The 11 patients at the second interim analysis included 7 men and 4 women. 8 patients were homozygous for the p.Leu483Pro (a.k.a. L444P) mutation in the beta-galactosidase gene, 2 were compound heterozygous for p.Phe523Ile/p.Leu483Pro variants, and one was compound heterozygous for p.Asp448His/p.Arg502Cys variants. Nine of the 11 patients were mildly ataxic at baseline, mostly with gait disorder. Two were considered not ataxic or very mildly ataxic. All 11 patients are continuing the study. No major adverse events are reported, the most frequent events being mild headache and back pain, and these were determined to be likely related to the lumbar puncture intervention for CSF sampling.

[0663] Plasma and CSF concentrations of the Compound 2 were measured as described above. As was previously observed, one patient (Patient 5) is an outlier demonstrating very low plasma and CSF levels of Compound 2. The data continues to support the conclusion that a steady state concentration of Compound 2 is reached in plasma and CSF at or prior to Week 4:

TABLE-US-00010 Compound 2 in plasma Day 1 (N = 11) AUC.sub.0-24, ng .Math. h/mL (mean ± SD) 851 ± 282 C.sub.max, ng/mL (mean ± SD) 58.1 ± 26.4 t.sub.max, h (median) 2.00

[0664] In the following chart, Patient 5 is excluded from the mean (N=10) values.

TABLE-US-00011 Compound 2 in Week 4 Week 26 Week 52 Plasma (N = 10) (N = 10) (N = 10) Concentration 2-4  116 ± 48.1  120 ± 40.1  114 ± 65.8 hours post dose, ng/mL (mean ± SD) Patient 5 Only, ng/mL 102 53.3 <LLOQ Week 4 Week 26 Week 52 Compound 2 in CSF (N = 10) (N = 8) (N = 10) Concentration 2-4 6.63 ± 2.42 6.77 ± 1.96 6.14 ± 3.44 hours post dose, ng/mL (mean ± SD) Patient 5 Only, ng/mL 3.05 1.77 <LLOQ

[0665] One patient (Patient 1) also experienced a significant drop in plasma and CSF concentrations of the compound beginning at the 52-week measurement, and this was traced to being likely a result of co-treatment with the CYP3A4 inducer rifampicin from Week 39 to Week 51 of the study. Because Compound 2 is suspected to be a CYP3A substrate, concomitant administration with CYP3A inducers is anticipated to result in a reduced systemic exposure.

[0666] At 52 weeks, the data further shows sustained significant improvements in plasma and CSF biomarkers for GD3, with the exception of Patient 5. Initial evidence of Compound 2 exposure correlated with reduction in lyso-GL1 and GL1 in CSF and plasma. Subsequently, reduced levels of Compound 2 exposure corresponded with an increase in both lyso-GL1 and GL1 in both the CSF and plasma. At Week 52, the CSF lyso-GL1 concentrations for Patient 5 were above the upper limit of quantification (ULOQ; >100 pg/mL), and biomarker results for CSF lyso-GL1 were therefore imputed using the exact ULOQ values.

[0667] Accordingly, in Patient 5, CSF lyso-GL1 and GL1 was found to increase by about 313% and 37%, respectively, at Week 52 compared to baseline, and levels of plasma lyso-GL1 and GL1 increased by about 43% and 14%, respectively, at Week 52 compared to baseline. Mean results for the other study subjects were as follows (N=10; Patient 5 excluded):

TABLE-US-00012 Lyso-GL-1 GL-1 Baseline 52-weeks Baseline 52-weeks Plasma 27.6 ng/mL 13.6 ng/mL 5.66 μg/mL 1.28 μg/mL  (5.7-79.8) (2.5-46.8) (4.2-7.9) (0.7-2.7) CSF 45.0 pg/mL 14.5 pg/mL 7.11 ng/mL 1.38 ng/mL (19.8-100)  (2.5-37.4)  (4.3-14.3) (1.0-3.7)

[0668] Thus, at 52-weeks compared to baseline, plasma and CSF concentrations had changed as follows (N=10; Patient 5 excluded):

TABLE-US-00013 Lyso-GL-1 (% change) GL-1 (% change) CSF Concentration −68% ± 17.2% −79% ± 12.1% Plasma Concentration −53% ± 13.5% −77% ± 11.7%

[0669] SARA ataxia scoring results for the five new patients is presented in the chart below:

TABLE-US-00014 SARA Cumulative Score At Screening Week 26 Week 52 Patient 7 5.5 7.5 5.0 Patient 8 2.0 0.5 0 Patient 9 0.0 0.0 0.0 Patient 10 2.0 2.0 1.0 Patient 11 3.0 3.0 2.0

[0670] As shown in the table, excluding Patient 9, who did not suffer from ataxia at baseline, 4 out of 4 patients exhibited an improvement in ataxia at Week 52. Patient 7 exhibited a transient increase in ataxia scoring, which resolved by Week 52.

[0671] To further demonstrate improvements in ataxia, two patients are videotaped at screening and at week 26 and week 52 timepoints attempting to walk along a straight line. A comparison of the video evidence demonstrates that compared to baseline, both patients show a steadier, better coordinated, and faster gait, with fewer touches against the nearby wall for support and fewer sidesteps.

[0672] TMT timing for the new patients is shown in the chart below.

TABLE-US-00015 Change (%) TMT-A (s) − from Baseline TMT-B (s) At Screening Week 26 Week 52 to Week 52 Patient 7 86 83 110 +28% Patient 8 100 54 59 −41% Patient 9 59 45 75 +27% Patient 10 73 33 6 −92% Patient 11 18 48 32 +78%

[0673] At 52 weeks, three of the new patients showed small increases in the (TMT-B−TMT-A) time of uncertain clinical significance, while two of the new patients showed a decrease in the (TMT-B−TMT-A) time, one patient showing an extremely large decrease (92% drop).

[0674] Neurological function was further evaluated in the new patients using functional magnetic resonance imaging (fMRI), as described above. fMRI results continue to indicate that patients with sufficient Compound 2 exposure develop greater coherence between posterior and anterior aspects of the brain, such that the entire brain becomes amenable to efficient information transfer. Where apparent, altered connectivity for the single patient with insufficient Compound 2 exposure (Patient 5) appears within a narrower set of anterior brain regions, and represents less holistic evidence of therapeutic benefit. This enhanced connectivity is seen in regions associated with executive function. Resting-state functional MRI demonstrates enhanced connectivity between default mode and medial frontal networks, suggesting that signaling within these disparate networks becomes more coherent, such that brain activity can be more efficiently transferred between cognitive reserve (posterior) and higher-order executive functions (anterior).

[0675] In particular, the results show enhanced connectivity between RSNs 1, 2 and 3 (perception-vision, cognition-language-orthography, and cognition space) and RSNs 6, 7 and 8 (sensorimotor, auditory, and executive control). At an anatomic level, there is a widespread and robust strengthening of connections between occipital-parietal structures and frontal, temporal, and limbic targets. This data is suggestive of increased functional connectivity that is most prominent within sensory, motor, and cerebellar networks which are thought to be disrupted in Gaucher disease.

[0676] The results are summarized in the table below for all patients for RSNs 3 and 6 (RSNs 4 and 8 are not included) for the change in correlation coefficient between baseline and 52 weeks (as noted supra, Patient 2 is excluded because of lack of data).

TABLE-US-00016 Patient 1 Patient 3 Patient 4 Patient 5 Patient 6 Change in 0.359 0.383 0.201 −0.173 0.779 Correlation Coefficient Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Change in −0.013 −0.058 −0.302 0.105 0.035 Correlation Coefficient

[0677] Statistical analysis of the SARA and TMT results compared to GL-1 concentration in GSF at 52 weeks is found to provide a good therapeutic correlation. For the SARA results, in which lower scores indicate therapeutic improvement, 8 of 11 patients show a positive correlation between reduction in CSF GL-1 (ng/mL) and SARA score (−5 to 5). For TMT, also in which shorter (Trail B minus Trail A) times indicate therapeutic improvement, 6 of 11 patients show a positive correlation between reduction of CSF GL-1 (ng/mL) and TMT time (seconds).

(C) Additional 52-Week Interim Analysis (N=9)

[0678] Neurological function was further evaluated using volumetric magnetic resonance imaging (vMRI). The vMRI data was collected at screening sessions at baseline and after 52 weeks of the treatment regime for eight patients (“Group A”) and an additional isolated patient (Patient 5). Group A corresponds to the eleven patients described above, excluding Patient 5 and two other patients lacking analyzable vMRI data. Patient 5 was isolated due to having plasma and CSF concentration of Compound 2, lower than the LLOQ after 52 weeks, suggesting failure to comply with the treatment regime.

[0679] vMRI data was obtained and subsequently analyzed using FreeSurfer anatomic parcellation and a Tensor-Based Morphometry (TBM) analysis cycle. FreeSurfer is an opensource software package for the analysis and visualization of structural and functional neuroimaging data developed by the Laboratory for Computational Neuroimaging. TBM analysis (also known as Jacobian Integration) consists in estimating the volume changes as captured within the deformation fields resulting from applying a symmetric deformable registration technique between a pair of MR scans (baseline and follow-up), using a non-linear symmetric log-demons deformation technique using robust cross-correlation metric to ensure invertibility of the transformation (symmetric process). The deformation field is then analyzed by computing the determinant of its Jacobian matrix, which is a measure of local volume change. An integration of the determinant over a region of interest provides an estimation of the change rate of the volume of this brain region over time.

[0680] The overall pipeline takes as input a baseline (BL) and a follow-up (FU) image and consists of the following steps: [0681] 1. Preprocessing and reformatting; [0682] 2. Segmentation of baseline (BL) and follow-up (FU) using FreeSurfer; [0683] 3. Multi-resolution rigid and affine registration of FU and BL to midspace; [0684] 4. Symmetric deformable nonlinear registration between FU and BL; [0685] 5. Jacobian image computation; [0686] 6. Voxel-wise volume change computation; [0687] 7. Region-wise volume change integration; and [0688] 8. Output for the changes for regions of interest specified for the study.

[0689] TBM is thus an image analysis technique that identifies regional structural differences from the gradients of the nonlinear deformation fields that align images to a common anatomical template. TBM is a well-known tool for analyzing brain vMRI data and further discussion of the application of the technique in that context is, for instance, provided in John Ashburner and Karl J Friston., Human Brain Function, Second edition, Academic Press 2004, Section 1, Chapter 6.3, pages 8 to 13, ISBN: 9780080472959; Moo K Chung., Computational Neuroanatomy, World Scientific 2012, Chapter 3, Pages 49 to 68, ISBN: 9789814472814. doi.org/10.1142/8036; and Thomson et al., Ann N Y Acad. Sci. 2007 February; 1097: 183-214. doi:10.1196/annals.1379.017.

[0690] vMRI data was collected for the whole brain tissue and analysis was able to quantify volume changes in individual regions of brain tissue. The results for Group A indicate that brain tissue volume is increased in numerous individual regions of the brain, giving rise to an increase in whole brain volume, after 52 weeks of the treatment regime outlined above. Brain regions with increased volume were also found to overlap with those regions exhibiting increased neuronal connectivity, as determined by fMRI discussed above. Results for Patient 5 conversely show evidence of brain atrophy and evidence of decreasing whole brain volume.

[0691] After 52 weeks of treatment, Group A showed increased mean volumes in at least the following brain regions: right accumbens area, left putamen, left entorhinal cortex, right putamen, right postcentral lobe, left pericalcarine lobe, right amygdala, left cuneus, and left lingual, as well as increased mean whole brain volume. These results are shown in the table below.

TABLE-US-00017 Group A Patient 5 mean change in change in volume (mm.sup.3) Group A volume (mm.sup.3) from Baseline to Standard from Baseline Brain Region Week 52 Deviation to Week 52 Right Accumbens Area +12.86 9.65 −52.1 Left Putamen +94.70 78.68 −51.7 Left Entorhinal Cortex +34.53 32.08 +41.4 Right Putamen +97.84 100.78 −63.1 Right Postcentral Lobe +91.66 99.96 −63.4 Left Pericalcarine Lobe +18.46 22.00 −29.2 Right Amygdala +60.78 74.88 −63.4 Left Cuneus +37.16 46.19 −23.9 Left Lingual +50.08 70.99 −60.0

[0692] It was also found that in Group A the treatment regime led to an overall increase in whole brain tissue volume, whereas Patient 5 conversely experienced a decrease in whole brain volume, as can be seen from FIG. 1. Patient 5, who did not successfully undergo the treatment regime, exhibited a relatively severe decrease in whole brain volume, as well as volume reductions in the majority of the above described individual brain regions. These results indicate that the treatment regime not only increased brain tissue volume in Group A, but also prevented and/or delayed the potential loss of brain tissue volume in Group A as a result of GD3 disease progression, as exhibited in Patient 5.

Example 6: Pharmacokinetics of Compound 2 in Healthy Human Volunteers

[0693] Two Phase 1 clinical studies were conducted to assess the pharmacokinetics, pharmacodynamics, safety, and tolerability of Compound 2 in healthy, human volunteers in the presence and absence of food. Compound 2 is also known as venglustat.

Study 1

[0694] Study 1 was a 2-part single-center trial in healthy adult male volunteers. Part 1 was a double-blind, randomized, placebo-controlled sequential ascending single-dose study of Compound 2 for safety, tolerability, and PK. Part 2 was an open-label, single-cohort, randomized, 2-sequence, 2-period, 2-treatment crossover study of Compound 2 for PK with and without a high-fat meal.

[0695] Part 1 of the study enrolled and randomized 55 healthy men (placebo, n=14; 2-, 5-, 15, 25-, 50, and 100-mg doses, n=6 each; 150-mg dose, n=5). Eight healthy men participated in Part 2.

[0696] In Part 1, the subjects were randomized to receive 2, 5, 15, 25, 50, 100, or 150 mg of Compound 2 (L-malic salt form, i.e., expressed under L-malic salt form) or matching placebo on the morning of the first day after at least a 10-hour fast. In Part 2, the subjects were randomized to receive a single oral dose of 5 mg Compound 2 either while fasting (at least 10 hours before and 4 hours after administration) or 30 minutes after a standardized high-fat breakfast (˜815 kcal). After a 7-day washout period, participants were crossed over to the other condition.

[0697] In Study 1, Part 1, blood was sampled for plasma concentrations of Compound 2 at the time of study drug administration (0 hour) and 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 48, 72, and 96 hours post-dose. Urine samples were collected for analysis of Compound 2 concentrations beginning 2 hours before study drug administration through 48 hours afterward.

[0698] In Study 1, Part 2, blood was sampled for plasma concentrations if Compound 2 at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, and 48 hours post-dose.

[0699] From Part 1, it was found that following single oral doses of 2 to 150 mg doses of Compound 2, maximal plasma concentration (C.sub.max) occurred at a median time of 3-5.5 hours before plasma concentrations began to decline exponentially, with a geometric mean t.sub.1/2 of 28.9 hours. Exposure increased close to dose-proportionally throughout the dose range: a 75-fold dose increase resulted in 97.3-, 89.2-, and 85.9-fold increases in geometric mean C.sub.max, AUC.sub.last, and AUC.sub.inf values, respectively. PK results are shown in the following table (AUC=area under the time concentration curve, either to last measurable concentration or extrapolated to infinity; t.sub.1/2=terminal half-life; CL/F=apparent total clearance from plasma; CV=coefficient of variation; SD=standard deviation; t.sub.max=time to C.sub.max; Vss/F=apparent volume of distribution at steady state):

TABLE-US-00018 2 mg 5 mg 15 mg 25 mg 50 mg 100 mg 150 mg Parameter (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 6) (N = 5) C.sub.max, ng/mL Mean (SD) 5.7 (1.2) 14.7 (1.61)  53.0 (16.7) 84.4 (31.8) 181 (56)  374 (38)  529 (109) Geometric  5.6 (21.4) 14.6 (10.9)  50.7 (31.5) 79.9 (37.7) 173 (31)    372 (10.3) 520 (21) mean (CV) t.sub.max, median 3.50 5.50 3.50 5.00 4.00 3.00 4.00 h (range) (3.00-8.00) (4.00-8.00) (2.00-5.00) (4.00-8.00) (3.00-6.00) (2.00-4.00) (1.00-8.00) AUC.sub.last, ng .Math. h/mL Mean (SD) 214 (52)  560 (71)  1,830 (520)  3,380 (1100)  6,310 (1880)  13,000 (2330)  18,600 (5480)  Geometric  209 (24.3)  556 (12.7) 1,760 (29)   3,240 (33)   6,070 (30)   12,800 (18)   18,000 (30)   mean (CV) AUC.sub.inf, ng .Math. h/mL Mean (SD) 243 (61)  652 (122) 2,070 (600)  3,810 (1,080) 7,130 (2,320) 14,400 (3,010) 20,600 (6,640) Geometric 237 (25)  643 (19)  1,990 (29)   3,690 (28)   6,800 (33)   14,100 (21)   19,900 (32)   mean (CV) t.sub.1/2, h Mean (SD) 29.2 (43)   33.3 (8.1)  29.7 (7.1) 30.2 (5.5)  28.9 (5.3)  27.8 (3.6) 26.9 (5.7) Geometric 28.9 (14.8) 32.5 (24.4)  29.0 (24.0) 29.8 (18.1) 28.5 (18.4)  27.6 (12.8)  26.4 (21.3) mean (CV) CL/F, L/h Mean (SD) 6.43 (1.41) 5.86 (1.01)  5.85 (1.89) 5.18 (1.31) 5.75 (2.01)  5.38 (1.25)  5.80 (1.55) Geometric  6.3 (22.0)  5.8 (17.3)  5.6 (32.2)  5.0 (25.3)  5.5 (34.9)  5.3 (23.4)  5.6 (26.7) mean (CV) V.sub.ss/F, L Mean (SD) 275 (54)  274 (30)  245 (81) 240 (78)  239 (62)  213 (22) 228 (50) Geometric 270 (20)  273 (11)  233 (33) 228 (33)  232 (26)  212 (10) 223 (22) mean (CV)

[0700] From Part 2, it was found that administration of a 5 mg dose with a high-fat meal had no effect on Compound 2 exposure compared with fasting conditions. Median t.sub.max was 6.00 hours whether fed or fasting. Fed/fasted geometric mean ratios were 0.92 and 0.91 for C.sub.max and AUC.sub.last, respectively. Within-subject variability (i.e., fed vs fasted) accounted for less than half the total subject variability.

Study 2

[0701] Study 2 was a single-center, double-blind, randomized, placebo-controlled, sequential ascending repeated-dose study of the safety, tolerability, PK, and pharmacodynamics of Compound 2 in healthy adult male and female volunteers.

[0702] The study enrolled and randomized 36 healthy adults (19 men and 17 women) (n=9 each to group). The subjects were randomized to receive once-daily doses of Compound 2 at 5, 10, or 20 mg (provided as 5-mg capsules of the L-malic salt form) or placebo for 14 days after at least a 10-hour fast.

[0703] Blood was sampled for plasma concentrations of Compound 2 as follows: Day 1 at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 16 hours post-dose; On Days 2-5, 8, 11, and 13, at 0 h; On Day 14, at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours post-dose; On Days 15-17, at 24, 48, and 72 hours, respectively, after the Day 14 dose. Urine samples were collected for analysis of Compound 2 concentrations on Day 1 (0 hours post-dose) and continuously on Day 14 from 0-24 hours post-dose. Pharmacodynamic endpoints (plasma GL-1, GL-3, and GM3 concentrations) were assessed on Days 1-5, 8, 11, 13, and 14, at 0 hours post-dose; and on Day 15, at 24 hours after the Day 14 dose.

[0704] It was found that in subjects receiving 5, 10, or 20 mg of Compound 2 once daily for 14 days, plasma C.sub.max occurred at a median time of 2-5 hours post-dose on Days 1 and 14. C.sub.trough values reached a plateau after Day 5. Compound 2 exposure increased close to dose-proportionally over the dose range of 5-20 mg: this 4-fold dose increase resulted in 3.76- and 3.69-fold increases in geometric mean C.sub.max and AUC.sub.0-24 values on Day 14, respectively. PK results from Study 2 are summarized in the following table:

TABLE-US-00019 Parameter 5 mg (N = 9) 10 mg (N = 9) 20 mg (N = 9) Day 1 C.sub.max, ng/mL Mean (SD) 18.5 (3.2)  38.5 (7.4)  68.0 (15.7) Geometric mean 18.2 (17.3)  37.8 (19.3)  66.5 (23.1) (CV) t.sub.max, median h 5.00 (2.00-8.17)  3.00 (2.00-5.00)  3.07 (2.00-6.00) (range) AUC.sub.0-24, ng .Math. h/mL Mean (SD)  296 (54)   635 (132) 1,100 (211) Geometric mean  292 (18)   623(21) 1,080 (19) (CV) Day 14 C.sub.max, ng/mL Mean (SD) 37.0 (6.4)  89.7 (29.1)   142 (40) Geometric mean 36.5 (17.2)  86.0 (32.5)   137 (28.3) (CV) t.sub.max, median h 3.00 (2.00-6.00)  2.00 (2.00-6.00)  3.00 (2.00-8.00) (range) AUC.sub.0-24, ng .Math. h/mL Mean (SD)  642 (121) 1,550 (464) 2,420 (705) Geometric mean  632 (19) 1,490 (30) 2,340 (29) (CV) C.sub.trough, ng/mL Mean (SD) 19.4 (4.0)  49.9 (19.3)  73.3 (24.4) Geometric mean 19.0 (20.5)  47.5 (38.7)  69.9 (33.2) (CV) t.sub.1/2, h Mean (SD) 29.3 (4.6)  31.3 (3.3)  35.0 (6.3) Geometric mean 29.0 (15.8)  31.2 (10.5)  34.5 (18.0) (CV) CL.sub.ss/F, L/h Mean (SD) 5.98 (1.17)  5.13 (1.25)  6.58 (1.70) Geometric mean (CV)  5.9 (19.5)  5.0 (24.4)  6.4 (25.8) CL.sub.R(0-24), L/h Mean (SD) 1.55 (0.68)  1.49 (0.41)  2.07 (0.58) Geometric mean NA.sup.a (44.0)  1.4 (27.7)  2.0 (28.0) (CV)

[0705] After 14 once-daily doses of Compound 2, its 24-hour unchanged urinary excretion fraction (mean fe.sub.0-24) ranged between 26.3% and 33.1% without any obvious dose-relatedness. Mean CL.sub.R(0-24) ranged between 1.49 L/h and 2.07 L/h, approximately 3.18-3.86-fold lower than observed plasma CL/F.

[0706] Plasma GL-1, GL-3, and GM3 in placebo recipients remained similar to baseline throughout, whereas plasma GL-1 and GM3 levels decreased from baseline time- and dose-dependently across the 3 Compound 2 dose groups, as shown in the following table (Point estimates of treatment ratios for glucosylceramide (GL-1), globotriaosylceramide (GL-3), and GM3 ganglioside (GM3) on Day 15 in the repeated ascending dose study):

TABLE-US-00020 90% Confidence Parameter Comparison Estimate Interval GL-1  5 mg vs placebo 0.39 0.29-0.50 10 mg vs placebo 0.32 0.25-0.42 20 mg vs placebo 0.23 0.17-0.30 GL-3  5 mg vs placebo 0.61 0.47-0.79 10 mg vs placebo 0.69 0.53-0.89 20 mg vs placebo 0.67 0.51-0.89 GM3  5 mg vs placebo 0.56 0.45-0.70 10 mg vs placebo 0.49 0.39-0.60 20 mg vs placebo 0.40 0.32-0.50

[0707] Maximal sustained effects on GL-1 occurred on Day 11 in the 5- and 10-mg groups and by Day 8 in the 20-mg group. Mean calculated GL-1 reductions from baseline at Day 15 were 41.9%, 69.6%, and 74.6% in the respective 5-, 10-, and 20-mg groups. GL-1 values were below the lower limit quantification (LLOQ) at baseline in 1 5-mg Compound 2 recipient and at Day 15 in 3, 5, and 9 subjects in the 5-, 10-, and 20-mg groups, respectively.

[0708] Maximal sustained GM3 decreases occurred across all Compound 2 dose groups starting on Day 13. Mean Day 15 plasma GM3 levels were 42.7%, 49.4%, and 57.8% of baseline for the 5-, 10-, and 20-mg dose groups, respectively. GM3 was below the LLOQ at Day 15 in 1 and 2 subjects in the 10- and 20-mg dose groups, respectively.

[0709] Plasma GL-3 also decreased with time in all Compound 2 dose groups, but variable and low baseline GL-3 values relative to LLOQ limited mean calculated GL-3 reductions. In the placebo, 5-, 10-, and 20-mg dose groups, GL-3 values were below LLOQ in 1, 3, 1, and 6 subjects, respectively, at baseline and in 4, 9, 7, and 9 subjects, respectively, at Day 15.

[0710] Mean estimated plasma GL-1 reductions from baseline (90% CI) attributable to Compound 2 C.sub.trough in the 5, 10, and 20 mg dose groups (19.0, 47.5, and 69.9 ng/mL, respectively) were 67.0% (54.4-79.7%), 74.4% (63.7-85.2%), and 76.3% (64.8-87.8%), respectively.

CONCLUSIONS

[0711] In these studies, Compound 2 exposure in healthy subjects (C.sub.max and AUC) was close-to-dose-proportional when administered as single doses ranging from 2-150 mg or as repeated, once-daily doses ranging from 5-20 mg for 14 days. Compared with fasting, a high-fat meal had no effect on exposure in subjects who received a single 5-mg dose. With repeated once-daily doses from 5-20 mg, steady state was achieved within 5 days; neither age nor gender affected accumulation. Pharmacodynamically, repeated once-daily doses of Compound 2 reduced plasma concentrations of GL-1 and GM3 in a time- and dose-dependent manner, consistent with Compound 2-mediated GCS inhibition, although baseline levels of GL-3 were too low to be useful as a pharmacodynamic biomarker. The dose-dependent GL-1 reduction corroborated the intended mechanism of action of Compound 2: inhibition of GL-1 formation from ceramide by GCS.

[0712] In all studies, safety profile was assessed by monitoring treatment-emergent adverse events (TEAEs) through 10 days after last dose of study medication, including serious adverse events [SAEs]), ECG monitoring, laboratory values, and physical examinations. There were no deaths, SAEs, severe TEAEs, or TEAEs leading to study discontinuation in any of the studies.

[0713] No clinically relevant hematologic or biochemical abnormalities were reported in any of the studies. Vital signs showed no relevant changes from baseline in any of the studies. ECG parameters showed no relevant changes in the single ascending dose and food effect studies; in the multiple ascending dose study no ECG parameters changed statistically significantly from average baseline versus placebo in recipients of Compound 2 at any dose. It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0714] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0715] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.