CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MODULATING AGENTS

Abstract

Compound (I), deuterated derivatives, and pharmaceutically acceptable salts of any of the foregoing are disclosed. Methods of treating cystic fibrosis using these compounds are also disclosed.

##STR00001##

Claims

1. A compound selected from Compound I: ##STR00154## deuterated derivatives of Compound I, and pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein the compound is Compound I.

3. The compound of claim 1, wherein the deuterated derivative of Compound I is selected from: ##STR00155## and pharmaceutically acceptable salts thereof.

4. The compound of claim 1, wherein the compound is a potassium salt.

5. The compound of claim 1, wherein the compound is a sodium salt.

6. The compound of claim 1, wherein the compound is a calcium salt.

7. A method of treating a CFTR-mediated disorder, comprising administering an effective amount of the compound of claim 1 to a patient in need thereof.

8. The method of claim 7, wherein the CFTR-mediated disorder is CF.

9. The method of claim 7, wherein the compound is Compound I or a pharmaceutically acceptable salt thereof.

10. The method of claim 7, further comprising administering one or more compounds selected from: ##STR00156## and pharmaceutically acceptable salts of Compounds II, III, III-d, and IV.

11. The method of claim 10, comprising administering Compound I and Compound III or Compound III-d.

12. The method of claim 10, comprising administering Compound I, Compound II, and Compound III or Compound III-d.

13. A process for preparing Compound I, comprising reacting compound 8: ##STR00157## with compound 3: ##STR00158## to produce Compound I.

14. A process for preparing Compound I, comprising: a) reacting compound 8 with compound 3, and subsequent treatment with HCl to produce compound 9: ##STR00159## and b) converting compound 9 into Compound I.

15.-20. (canceled)

21. A process for preparing Compound Ia, comprising converting compound 20: ##STR00160## into compound Ia.

22.-31. (canceled)

32. A process for preparing Compound Ib, comprising converting compound 22: ##STR00161## into compound Ib.

33. (canceled)

34. (canceled)

35. A process for preparing Compound Ic, comprising converting compound 23: ##STR00162## into compound Ic.

36. (canceled)

37. A process for preparing Compound Id: ##STR00163## comprising converting compound 35: ##STR00164## into compound Id.

38.-49. (canceled)

50. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.

51. The pharmaceutical composition of claim 50, wherein the compound is Compound I or a pharmaceutically acceptable salt thereof.

52. The pharmaceutical composition of claim 50, further comprising one or more compounds selected from: ##STR00165## and pharmaceutically acceptable salts of Compounds II, III, III-d, and IV.

53. The pharmaceutical composition of claim 52, wherein the pharmaceutical composition comprises Compound I and Compound III or Compound III-d.

54. The pharmaceutical composition of claim 52, wherein the pharmaceutical composition comprises Compound I, Compound II, and Compound III or Compound III-d.

55. A method of treating a CFTR-mediated disorder, comprising administering an effective amount of the pharmaceutical composition of claim 50 to a patient in need thereof.

56. A compound selected from compound 8: ##STR00166## deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing.

Description

EXAMPLES

Abbreviations

[0162] Boc anhydride ((Boc).sub.2O): di-tert-butyl dicarbonate [0163] DCM: dichloromethane [0164] DIEA (DIPEA): N,N-diisopropylethylamine [0165] DMAP: 4-Dimethylaminopyridine [0166] DMF: N,N-dimethylformamide [0167] DMSO: dimethyl sulfoxide [0168] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0169] COMU: (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate [0170] MeOH: methanol [0171] THF: tetrahydrofuran [0172] EtOAc: ethyl acetate [0173] Pd(dppf)Cl.sub.2: [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) [0174] MeTHF: 2-Methyltetrahydrofuran

General UPLC/HPLC Analytical Methods

[0175] LC Method A: Analytical reverse phase UPLC using an Acquity UPLC BEH Cis column (50×2.1 mm, 1.7 μm particle) made by Waters (pn: 186002350), and a dual gradient run from 1-99% mobile phase B over 3.0 min. Mobile phase A=H.sub.2O (0.05% CF.sub.3CO.sub.2H). Mobile phase B=CH.sub.3CN (0.035% CF.sub.3CO.sub.2H). Flow rate=1.2 mL/min, injection volume=1.5 μL, and column temperature=60° C.

[0176] LC Method B: Merck Millipore Chromolith SpeedROD C.sub.18 column (50×4.6 mm) and a dual gradient run from 5-100% mobile phase B over 6 min. Mobile phase A=water (0.1% CF.sub.3CO.sub.2H). Mobile phase B=acetonitrile (0.1% CF.sub.3CO.sub.2H).

[0177] LC Method C: Merck Millipore Chromolith SpeedROD C.sub.18 column (50×4.6 mm) and a dual gradient run from 5-100% mobile phase B over 12 min. Mobile phase A=water (0.1% CF.sub.3CO.sub.2H). Mobile phase B=acetonitrile (0.1% CF.sub.3CO.sub.2H).

Example 1: Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound I)

Synthesis of (2R)-4-Methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (3)

[0178] ##STR00096##

[0179] A mixture of spiro[2.3]hexan-5-one (100 g, 1.040 mol) and (2R)-2-amino-4-methyl-pentan-1-ol (123.5 g, 1.054 mol) in dichloroethane (DCE) (1.5 L) was stirred at ambient temperature for 1 h. To the mixture was added sodium triacetoxyborohydride (228 g, 1.076 mol) portionwise. The mixture was stirred at ambient temperature for 18 h. The reaction mixture was diluted with HCl (1.1 L of 2 M, 2.200 mol) until pH was ˜1. The aqueous phase was separated and the organic phase extracted with HCl (600 mL of 2 M, 1.200 mol). The organic phase was separated and the aqueous layer was basified with NaOH (550 g of 50% w/w, 6.875 mol) affording a solution at ˜pH 12. The mixture was extracted 2× with EtOAc (1 L) and the combined organic phases were washed with brine (150 mL), dried over MgSO.sub.4, filtered, and concentrated in vacuo to afford (2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (160.7 g, 78%) as a clear oil. Used without further purification. ESI-MS m/z calc. 197.17796, found 198.2 (M+1).sup.+; Retention time: 0.54 minutes (LC method A).

Synthesis of tert-Butyl N-tert-butoxycarbonyl-N-(4,6-dichloro-pyrimidin-2-yl)carbamate (5)

[0180] ##STR00097##

[0181] To a solution of 4,6-dichloropyrimidin-2-amine (300 g, 1.829 mol) in DCM (2.1 L) was added (Boc).sub.2O (838 g, 3.840 mol) followed by DMAP (5.6 g, 45.84 mmol). The mixture was stirred at ambient temperature for 6 h. Additional DMAP (5.6 g, 45.84 mmol) was added and the reaction was continued to stir at ambient temperature for 24 h. The mixture was diluted with water (2.1 L) and the organic phase separated. The organic phase was washed with water (2.1 L), 2.1 L of brine, dried over MgSO.sub.4, filtered over celite and concentrated in vacuo affording a light orange oil which had a silt in the slurry. The mixture was diluted with ˜500 mL of heptane and filtered using an M filter. The precipitate (starting material) was washed with 250 mL of heptane. The filtrate was concentrated in vacuo affording a thick orange oil which was seeded with solid from a previous experiment and crystallized on standing, to afford tert-butyl N-tert-butoxycarbonyl-N-(4,6-dichloropyrimidin-2-yl)carbamate (645 g, 97%) as a light orange hard solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.07 (s, 1H), 1.44 (s, 18H). ESI-MS m/z calc. 363.07526, found 364.1 (M+1).sup.+; Retention time: 2.12 minutes (LC method A).

Synthesis of tert-Butyl N-tert-butoxycarbonyl-N-[4-chloro-6-(2,6-dimethyl-phenyl)pyrimidin-2-yl]carbamate (6)

[0182] ##STR00098##

[0183] All solvents were degassed prior to use. To a slurry of tert-butyl N-tert-butoxycarbonyl-N-(4,6-dichloropyrimidin-2-yl)carbamate (88 g, 241.6 mmol), (2,6-dimethylphenyl)boronic acid (approximately 36.24 g, 241.6 mmol) and Cs.sub.2CO.sub.3 (approximately 196.8 g, 604.0 mmol) in DME (704 mL) and water (176 mL) were added. Pd(dppf)Cl.sub.2 (approximately 8.839 g, 12.08 mmol) was added and the mixture was vigorously stirred under N.sub.2 at 80° C. (reflux) for 1 h (no starting material remained). The reaction was cooled to ambient temperature and diluted with water (704 mL). The aqueous phase was separated and extracted with EtOAc (704 mL). The organic phase was washed with 700 mL of brine, dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude product was chromatographed on a 1500 g silica gel column eluting with 0-30% EtOAc/hexanes. The product fractions (eluted at 15% EtOAc) were combined and concentrated in vacuo affording tert-butyl N-tert-butoxycarbonyl-N-[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]carbamate (81.3 g, 78%) as a clear oil, which crystallized on standing. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.88 (s, 1H), 7.30 (dd, J=8.2, 7.0 Hz, 1H), 7.21-7.16 (m, 2H), 2.03 (s, 6H), 1.38 (s, 18H). ESI-MS m/z calc. 433.17682, found 434.1 (M+1).sup.+; Retention time: 2.32 minutes (LC method A).

Synthesis of 4-Chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (hydrochloride salt) (7.HCl)

[0184] ##STR00099##

[0185] tert-Butyl N-tert-butoxycarbonyl-N-[4-chloro-6-(2,6-dimethylphenyl) pyrimidin-2-yl]carbamate (514.8 g, 915.9 mmol) was dissolved in dichloromethane (4 L). Hydrogen chloride in p-dioxane (1 L, 4 mol) was added and the mixture was stirred overnight at ambient temperature. The resulting precipitate was collected by vacuum filtration and dried in vacuo to obtain 4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine hydrochloride (213.5 g, 64%) as a white solid (213.5 g, 82%). .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.45-6.91 (m, 3H), 6.73 (s, 1H), 2.08 (s, 6H). ESI-MS m/z calc. 233.072, found 234.1 (M+1).sup.+; Retention time: 2.1 minutes (LC Method C).

Synthesis of 4-Chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (7)

[0186] ##STR00100##

[0187] 4-Chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (Hydrochloride salt) (166 g, 614.5 mmol) and 4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (Hydrochloride salt) (30 g, 111.0 mmol) were suspended in DCM (2.5 L), treated with NaOH (725 mL of 1 M, 725.0 mmol) and stirred at ambient temperature for 1 h. The mixture was transferred into a separatory funnel and left standing overnight. The DCM phase was separated and the aqueous phase with insoluble material was extracted twice more with DCM (2×500 ml). The combined brown DCM phases were stirred with magnesium sulfate and charcoal for 1 h, filtered and the yellow solution concentrated to a volume of ˜500 mL. The solution was diluted with heptane (750 mL) and DCM was removed under reduced pressure at 60° C. to give a cream suspension. It was stirred at ambient temperature for 1 h, filtered, washed with cold heptane and dried to give 4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (157 g, 91%) as a cream solid.

[0188] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.28-7.14 (m, 3H), 7.10 (d, J=7.5 Hz, 2H), 6.63 (s, 1H), 2.06 (s, 6H). ESI-MS m/z calc. 233.07198, found 234.0 (M+1).sup.+; Retention time: 1.45 minutes (LC method A).

Synthesis of 3-[[4-Chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (8)

[0189] ##STR00101##

[0190] 4-Chloro-6-(2,6-dimethylphenyl)pyrimidin-2-amine (235 g, 985.5 mmol) was dissolved in MeTHF (2.3 L) and cooled in an ice bath under stirring and nitrogen. To the cold solution methyl 3-chlorosulfonylbenzoate (347 g, 1.479 mol) was added in one portion (seems slightly endothermic) and to the cold pale yellow solution a solution of 2-methyl-butan-2-ol (Lithium salt) (875 mL of 3.1 M, 2.712 mol) (in heptane) was added dropwise over 1.25 h (exothermic, internal temperature from 0° C. to 10° C.). The ice bath was removed and the greenish solution was stirred for 4 h at ambient temperature. To the greenish solution cold HCl (2 L of 1.5 M, 3.000 mol) was added, the phases separated and the organic phase was washed once with water (1 L) and once with brine (500 mL). The aqueous phases were back extracted once with MeTHF (350 mL) and the organic phases were combined. This yellow MeTHF solution of methyl 3-[[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoate (ESI-MS m/z calc. 431.07065, found 432.0 (M+1).sup.+; Retention time: 1.81 minutes) was treated with NaOH (2.3 L of 2 M, 4.600 mol) and stirred at ambient temperature for 1 h. The phases were separated and the NaOH phase was washed twice with MeTHF (2×500 mL) and the combined organic phases were extracted once with 2M NaOH (1×250 mL).

[0191] The combined NaOH phases were combined, stirred in an ice bath and slowly acidified by addition of HCl (416 mL of 36% w/w, 4.929 mol) while keeping the internal temperature between 10 and 20° C. At the end of the addition (pH ˜5-6) the final pH was adjusted to 2-3 by addition of solid citric acid. The formed yellow tacky suspension was stirred at ambient temperature over night to give a cream crisp suspension. The solid was collected by filtration, washed with plenty of water and sucked dry for 3 h. The solid was dried under reduced pressure with a nitrogen leak at 45-50° C. for 120 h. 3-[[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (395 g, 96%) was isolated as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.44 (s, 1H), 12.46 (s, 1H), 8.48-8.39 (m, 1H), 8.25-8.15 (m, 1H), 8.15-8.08 (m, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.31 (s, 1H), 7.28-7.18 (m, 1H), 7.10 (d, J=7.6 Hz, 2H), 1.84 (s, 6H). ESI-MS m/z calc. 417.055, found 418.0 (M+1).sup.+; Retention time: 1.56 minutes. (LC method A).

Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]-nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound I)

[0192] ##STR00102##

[0193] (2R)-4-Methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (1.42 g, 7.197 mmol), 3-[[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (3.077 g, 7.209 mmol), and sodium tert-butoxide (2.731 g, 28.42 mmol) were combined in THF (25 mL) and stirred at ambient temperature for 1 h (slight exotherm). After 1 h, the reaction mixture was added dropwise to a stirred solution of HATU (5.436 g, 14.30 mmol) in DMF (50 mL). The reaction was stirred an additional 16 h at ambient temperature. The reaction was evaporated to an oil. The resulting oil was partitioned between ethyl acetate (100 mL) and a 1M HCl solution (100 mL). The organics were separated, washed with additional 1M HCl (100 mL), then brine (100 mL). The organics were dried over sodium sulfate and evaporated.

[0194] The crude product was purified by silica gel chromatography eluting with 0-80% ethyl acetate in hexanes to give amorphous (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18), 15-hexaene-2,2,13-trione (1.73 g, 42%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.07 (s, 1H), 8.40 (s, 1H), 7.96-7.85 (m, 1H), 7.77-7.60 (m, 2H), 7.30-7.20 (m, 1H), 7.12 (d, J=7.6 Hz, 2H), 6.38 (s, 1H), 5.12 (dd, J=10.6, 4.2 Hz, 1H), 4.40 (t, J=11.1 Hz, 1H), 4.23 (p, J=8.5 Hz, 1H), 3.78-3.66 (m, 1H), 3.31-3.22 (m, 2H), 2.23-1.83 (m, 8H), 1.72-1.60 (m, 1H), 1.30 (s, 1H), 1.20-1.10 (m, 1H), 0.73 (d, J=6.7 Hz, 3H), 0.56-0.41 (m, 4H), 0.21 (d, J=6.3 Hz, 3H). ESI-MS m/z calc. 560.2457, found 561.4 (M+1).sup.+; Retention time: 2.01 minutes (LC method A).

Alternative Synthesis of Compound I

Synthesis of 3-[[4-(2,6-Dimethylphenyl)-6-[(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino) pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride Salt) (9)

[0195] ##STR00103##

[0196] To a solution of (2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (52.26 g, 264.9 mmol) and 3-[[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (103 g, 242.8 mmol) in MeTHF (700 mL) was added sodium tert-butoxide (93.4 g, 971.9 mmol) portion-wise keeping the reaction temperature <40° C. The addition is exothermic and the reaction temperature was controlled using an ice-water bath and addition rate of the base. The reaction was stirred for 2 h at ambient temperature. The reaction was quenched with the slow addition of HCl (1.2 L of 1 M, 1.200 mol) and stirred for 5 min. The mixture was transferred to a separatory funnel using MeTHF. The aqueous phase was separated and extracted with 250 mL of MeTHF. The combined organic phases were washed with 500 mL of brine, dried over MgSO.sub.4, filtered and concentrated in vacuo. The light yellow solid was slurried with EtOAc (200 mL) and stirred for 1 h. The solid was collected using a M frit and washing 3× with 10 mL of EtOAc. The off-white solid was air dried and dried in vacuo for 20 h to afford 3-[[4-(2,6-dimethylphenyl)-6-[(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (120 g, 76%). ESI-MS m/z calc. 578.2563, found 579.2 (M+1).sup.+; Retention time: 1.02 minutes. (LC method A).

Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound I)

[0197] ##STR00104##

[0198] 3-[[4-(2,6-Dimethylphenyl)-6-[(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (4.41 g, 7.169 mmol) and HATU (2.80 g, 7.364 mmol) were combined in DMF (100 mL) and triethylamine (3.0 mL, 21.52 mmol) was added. The reaction was stirred at ambient temperature for 5 h. The reaction mixture was poured into a stirred solution of water (150 mL) and HCl (35 mL of 1 M, 35.00 mmol). The mixture was stirred for 20 min and the resulting white solid was collected by filtration. The solid was dissolved in ethyl acetate (100 mL) and washed with 1M HCl (100 mL), brine (100 mL), then dried over sodium sulfate and evaporated. The crude product was purified by silica gel chromatography eluting with 0-70% ethyl acetate in hexanes to give amorphous (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (2.94 g, 73%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.07 (s, 1H), 8.40 (s, 1H), 7.91 (broad s, 1H), 7.68 (broad s, 2H), 7.25 (t, J=7.9 Hz, 1H), 7.12 (d, J=7.4 Hz, 2H), 6.38 (s, 1H), 5.12 (dd, J=10.7, 4.2 Hz, 1H), 4.40 (t, J=11.1 Hz, 1H), 4.23 (p, J=8.5 Hz, 1H), 3.79-3.65 (m, 1H), 3.31-3.22 (m, 2H), 2.21-1.84 (m, 8H), 1.72-1.61 (m, 1H), 1.36-1.23 (m, 1H), 1.21-1.10 (m, 1H), 0.73 (d, J=6.6 Hz, 3H), 0.55-0.41 (m, 4H), 0.21 (d, J=6.2 Hz, 3H). ESI-MS m/z calc. 560.2457, found 561.5 (M+1).sup.+; Retention time: 2.02 minutes (LC method A).

[0199] Salts of Compound I were prepared according to the following Examples.

Example 2: Synthesis of a Potassium Salt of Compound I

[0200] ##STR00105##

[0201] (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (2 g, 3.448 mmol) was dissolved in methanol (20 mL) and slowly treated with KOH (6.896 mL of 0.5 M, 3.448 mmol) (in methanol) under stirring. The clear solution was stirred at ambient temperature for 1 h, evaporated to give a glass and dried under house vacuum with nitrogen leak at 50-55° C. for 16 h to give amorphous (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]-nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Potassium Ion) (2.033 g, 98%) as an off-white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.40 (s, 1H), 7.82-7.74 (m, 1H), 7.53-7.46 (m, 2H), 7.12 (t, J=7.5 Hz, 1H), 7.03 (d, J=7.6 Hz, 2H), 5.78 (s, 1H), 5.09 (dd, J=10.5, 4.3 Hz, 1H), 4.17 (p, J=8.6 Hz, 1H), 4.06 (t, J=11.0 Hz, 1H), 3.88 (dq, J=11.1, 7.3, 5.5 Hz, 1H), 3.28 (dt, J=14.5, 9.3 Hz, 2H), 2.07 (dt, J=24.5, 9.2 Hz, 2H), 1.96 (s, 6H), 1.58 (ddd, J=14.0, 10.9, 2.8 Hz, 1H), 1.29 (ddt, J=13.3, 6.9, 2.8 Hz, 1H), 1.17 (ddd, J=13.7, 10.6, 2.9 Hz, 1H), 0.71 (d, J=6.6 Hz, 3H), 0.52 (dd, J=8.3, 5.6 Hz, 2H), 0.50-0.40 (m, 2H), 0.21 (d, J=6.4 Hz, 3H).

Example 3: Synthesis of a Sodium Salt of Compound I

[0202] ##STR00106##

[0203] (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (2 g, 3.448 mmol) was suspended in water (25 mL) and slowly treated with NaOH (3.448 mL of 1 M, 3.448 mmol) under stirring. The suspension was stirred at ambient temperature for 1.25 h to give a cloudy solution. The cloudy solution was filtered clear over a syringe filter (0.2 μm) and the clear colorless filtrate was lyophilized for two days to give amorphous (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Sodium salt) (2 g, 99%) as a colorless fluffy solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.40 (s, 1H), 7.78 (ddd, J=5.6, 3.5, 1.6 Hz, 1H), 7.52-7.45 (m, 2H), 7.12 (t, J=7.5 Hz, 1H), 7.02 (d, J=7.6 Hz, 2H), 5.77 (s, 1H), 5.09 (dd, J=10.5, 4.3 Hz, 1H), 4.16 (p, J=8.5 Hz, 1H), 4.06 (t, J=10.9 Hz, 1H), 3.89 (dq, J=11.3, 7.5, 5.6 Hz, 1H), 3.28 (dt, J=14.5, 9.3 Hz, 2H), 2.07 (dt, J=24.7, 9.3 Hz, 2H), 1.96 (s, 6H), 1.58 (td, J=11.3, 10.8, 5.7 Hz, 1H), 1.28 (ddd, J=9.6, 6.5, 3.0 Hz, 1H), 1.17 (ddd, J=13.6, 10.5, 2.8 Hz, 1H), 0.71 (d, J=6.6 Hz, 3H), 0.52 (dd, J=8.3, 5.6 Hz, 2H), 0.50-0.41 (m, 2H), 0.21 (d, J=6.4 Hz, 3H).

Example 4: Synthesis of a Calcium Salt of Compound I

[0204] ##STR00107##

[0205] (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Sodium salt) (500 mg, 0.8566 mmol) was stirred in water (5 mL) for 10 min to give a clear solution. A solution of CaCl.sub.2 (46.8 mg, 0.4217 mmol) in water (0.5 mL) was added resulting in the precipitation of a gelatinous solid to give a semi-solid mass. Another portion of water (5 mL) was added and the suspension stirred at ambient temperature for 23 h. The solid was collected by filtration, washed with plenty of water (˜3×5-10 mL) and dried under vacuum with a nitrogen bleed at 55-60° C. for 14 h to give amorphous (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo-[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Calcium salt (0.5)) (423 mg, 85%) as an off white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.40 (s, 1H), 7.78 (td, J=4.4, 1.6 Hz, 1H), 7.50 (d, J=4.7 Hz, 2H), 7.13 (t, J=7.5 Hz, 1H), 7.03 (d, J=7.5 Hz, 2H), 5.81 (s, 1H), 5.09 (dd, J=10.5, 4.3 Hz, 1H), 4.17 (p, J=8.6 Hz, 1H), 4.08 (t, J=11.0 Hz, 1H), 3.87 (dq, J=11.1, 7.3, 5.4 Hz, 1H), 3.28 (dt, J=14.2, 9.3 Hz, 2H), 2.07 (dt, J=24.3, 9.1 Hz, 2H), 1.99-1.95 (m, 6H), 1.59 (ddd, J=14.0, 10.8, 2.8 Hz, 1H), 1.28 (tt, J=6.4, 3.0 Hz, 1H), 1.17 (ddd, J=13.6, 10.4, 2.8 Hz, 1H), 0.71 (d, J=6.6 Hz, 3H), 0.55-0.40 (m, 4H), 0.21 (d, J=6.4 Hz, 3H).

[0206] ESI-MS m/z calc. 560.2457, found 561.0 (M+1).sup.+; Retention time: 2.1 minutes (LC method A).

Example 5: Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-[(1,1,2,2-tetradeutero)spiro[2.3]hexan-5-yl]-9-oxa-22λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound Ia)

Synthesis of [2-Bromo-1-(bromomethyl)ethoxy]methylbenzene (11)

[0207] ##STR00108##

[0208] To a solution of 2-benzyloxypropane-1,3-diol (24.3 g, 129.36 mmol) in anhydrous CH.sub.2C.sub.12 (350 mL) under argon atmosphere at 0° C. was added Ph.sub.3P (72.177 g, 272.43 mmol) followed by CBr.sub.4 (91.258 g, 272.43 mmol) (one portion addition caused a huge gas generation). The reaction mixture was stirred at 0° C. for 2.5 h. To the reaction mixture was added water (150 mL) and dichloromethane (600 mL). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. Then, 10% ether in hexane (500 mL) was added, sonicated and filtered. The solid cake was washed with 10% ether in hexane (300 mL). The solid was discarded and the combined filtrates were concentrated under reduced pressure to give the crude product. The crude product was purified by flash chromatography (loaded in CH.sub.2Cl.sub.2) (330 g SiO.sub.2, eluting 0 to 4% ether in hexane) to afford [2-bromo-1-(bromomethyl)ethoxy]methylbenzene (23.39 g, 59%) as a colorless liquid. .sup.1H NMR (500 MHz, Chloroform-d) δ 7.49-7.30 (m, 5H), 4.70 (s, 2H), 3.83 (p, J=5.2 Hz, 1H), 3.60 (d, J=5.2 Hz, 4H). ESI-MS m/z calc. 305.92548, NO MS was observed. Retention time: 5.47 minutes (LC Method C).

Synthesis of Diisopropyl 3-benzyloxycyclobutane-1,1-dicarboxylate (12)

[0209] ##STR00109##

[0210] NaH (6.42 g, 160.52 mmol, 60% oil suspension) was suspended in dry DMF (280 mL) under Argon. Diisopropyl propanedioate (26.49 g, 26.731 mL, 139.33 mmol) in anhydrous DMF (20 mL) was added dropwise while keeping the temperature around 20° C. On cessation of gas evolution, [2-bromo-1-(bromomethyl)ethoxy]-methylbenzene (21.334 g, 69.264 mmol) in anhydrous DMF (20 mL) was added. The reaction mixture was stirred at 140° C. for 26 h before being cooled to ambient temperature and poured into a saturated aqueous solution of NH.sub.4Cl (500 mL) to prevent emulsion formation. The solution was extracted with hexane (3×500 mL), washed with water (300 mL), brine (300 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to remove hexane. The crude product obtained was purified by flash chromatography (loaded in hexane) (330 g SiO.sub.2, eluting 0 to 15% ether in hexane) to afford diisopropyl 3-benzyloxycyclobutane-1,1-dicarboxylate (26.84 g, 90%) as a colorless liquid. Note: The product contained 22% of diisopropyl propanedioate. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 7.58-7.14 (m, 5H), 5.22-4.90 (m, 2H), 4.43 (s, 2H), 4.15 (p, J=7.2 Hz, 1H), 2.89-2.68 (m, 2H), 2.63-2.40 (m, 2H), 1.43-0.99 (m, 12H). ESI-MS m/z calc. 334.178, found 335.3 (M+1).sup.+; Retention time: 5.94 minutes (LC Method C).

Synthesis of [3-Benzyloxy-1-[dideuterio(hydroxy)methyl]cyclobutyl]-dideuterio-methanol (13)

[0211] ##STR00110##

[0212] To a solution of diisopropyl 3-benzyloxycyclobutane-1,1-dicarboxylate (26.84 g, 62.604 mmol) in anhydrous THF (250 mL) at 0° C. was added lithium tetradeuterioalumanuide (6.11 g, 145.55 mmol) portion by portion. After the addition was competed, the reaction was stirred at ambient temperature for 2 days before being re-cooled to 0° C. Water (6.2 mL) was added dropwise followed by 15% aqueous sodium hydroxide (6.2 mL) and water (18.6 mL). The resulting solution was stirred at ambient temperature for 30 min before being filtered through Celite and washed with THF. The filtrate was concentrated under reduced pressure.

[0213] The residue obtained was dissolved in ethyl acetate (450 mL), washed with water (100 mL×3) and brine (100 mL). The organic layer was separated, and the aqueous layer was extracted with ether (3×150 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to afford a crude white solid. The crude product was purified by flash chromatography (loaded in CH.sub.2Cl.sub.2) (330 g SiO.sub.2, eluting 0 to 50% acetone in hexane) to afford [3-benzyloxy-1-[dideuterio(hydroxy)methyl]cyclobutyl]-dideuterio-methanol (9.95 g, 68%) as a white solid. .sup.1H NMR (500 MHz, Chloroform-d) δ 7.40-7.32 (m, 4H), 7.32-7.28 (m, 1H), 4.42 (s, 2H), 4.17-3.99 (m, 1H), 2.48-2.05 (m, 4H), 1.87-1.79 (m, 2H). ESI-MS m/z calc. 226.1507, found 227.4 (M+1).sup.+, retention time 2.94 minutes (LC Method C).

Synthesis of [3,3-Bis[dideuterio(iodo)methyl]cyclobutoxy]-methylbenzene (14)

[0214] ##STR00111##

[0215] To a solution of [3-benzyloxy-1-[dideuterio(hydroxy)methyl]-cyclobutyl]-dideuterio-methanol (9.95 g, 43.967 mmol) in anhydrous CH.sub.2Cl.sub.2 (420 mL) was added Et.sub.3N (17.860 g, 24.6 mL, 176.50 mmol), then cooled to 0° C. Methanesulfonyl chloride (14.652 g, 9.9 mL, 127.91 mmol) was added dropwise, and the resulting solution was stirred at this temperature for 2 h before being quenched with saturated sodium bicarbonate solution (200 mL). Dichloromethane (400 mL) was added; the organic layer was separated, washed with brine (150 mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue obtained was placed under high vacuum to afford the bis-mesylate intermediate as a pale yellow liquid. The bis-mesylate was dissolved in anhydrous acetone (300 mL), and NaI (49.08 g, 327.43 mmol) was added. The reaction solution was heated at 50° C. for 5 days. The reaction solution was filtered and washed with acetone.

[0216] The filtrate was concentrated under reduced pressure. To the residue obtained was added ether (800 mL) and a 10% sodium sulfite solution (250 mL×2). The organic solution was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (loaded in CH.sub.2Cl.sub.2) (330 g SiO.sub.2, eluting 0 to 5% ether in hexane) to afford [3,3-bis[dideuterio(iodo)methyl]cyclobutoxy]methylbenzene (15.813 g, 81%) as a pale yellow oil. .sup.1H NMR (500 MHz, Chloroform-d) δ 7.42-7.28 (m, 5H), 4.42 (s, 2H), 3.93 (tt, J=7.2, 6.1 Hz, 1H), 2.40-2.23 (m, 2H), 2.07-1.92 (m, 2H).

[0217] ESI-MS m/z calc. 445.9542, found 464.3 (M+18).sup.+; Retention time: 6.53 minutes (LC Method C).

Synthesis of 5-Benzyloxy-1,1,2,2-tetradeuterio-spiro[2.3]hexane (15)

[0218] ##STR00112##

[0219] To a solution of [3,3-bis[dideuterio(iodo)methyl]cyclobutoxy]-methylbenzene (15.81 g, 35.441 mmol) in a mixture of ethanol (70 mL) and water (30 mL) was added Zn (9.96 g, 152.32 mmol). The resulting solution was refluxed for 2 h. The reaction solution was cooled to ambient temperature, and then diluted with dichloromethane (400 mL) and water (100 mL). The organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography (loaded in CH.sub.2Cl.sub.2) (220 g SiO.sub.2, eluting 0 to 4% ether in hexane) to afford 5-benzyloxy-1,1,2,2-tetradeuterio-spiro[2.3]hexane (6.77 g, 94%) as a colorless oil. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 7.60-7.09 (m, 5H), 4.45 (s, 2H), 4.30 (p, J=6.9 Hz, 1H), 2.41-2.22 (m, 2H), 2.22-1.99 (m, 2H). ESI-MS m/z calc. 192.14522, no MS was observed. Retention time: 5.62 minutes (LC Method C).

Synthesis of 1,1,2,2-Tetradeuteriospiro[2.3]hexan-5-ol (16)

[0220] ##STR00113##

[0221] To a solution of 5-benzyloxy-1,1,2,2-tetradeuterio-spiro[2.3]hexane (6.58 g, 34.219 mmol) in methyl acetate (35 mL) was added 5% Pd/C (1.01 g, 9.4907 mmol), and the mixture was stirred under hydrogen atmosphere for 20 h. Another 5% Pd/C (0.51 g, 4.7923 mmol) amount was added to the reaction mixture, and it was stirred under a hydrogen atmosphere for another 20 h. The catalyst was filtered off and rinsed with diethyl ether (30 ml). The solvent was distilled off slowly at atmospheric pressure. The residue was distilled under vacuum (Bp.sub.20=72° C.) to give 1,1,2,2-tetradeuteriospiro[2.3]hexan-5-ol (3.18 g, 91%) as a clear liquid.

[0222] .sup.1H NMR (250 MHz, CDCl.sub.3) δ 4.53 (q, J=6.5 Hz, 1H), 2.28-2.19 (m, 4H), 1.72 (d, J=5.7 Hz, 1H).

Synthesis of 1,1,2,2-Tetradeuteriospiro[2.3]hexan-5-one (17)

[0223] ##STR00114##

[0224] CrO.sub.3/pyridine complex was obtained by adding CrO.sub.3 (9.95 g, 99.506 mmol) in pyridine (95 mL). To this solution was added dropwise a solution of 1,1,2,2-tetradeuteriospiro[2.3]hexan-5-ol (3.16 g, 30.929 mmol) in pyridine (35 mL). The reaction mixture was stirred at ambient temperature for 21 h. After cooling down, water (50 mL) was added and then extracted with diethyl ether (200 mL×3). The organic phase was washed with 2N aqueous HCl until the aqueous phase reached pH=4, then washed with saturated aqueous NaHCO.sub.3 (200 mL) and water (200 mL). After being dried over sodium sulfate, the solvent was slowly removed by distillation at atmospheric pressure. The residue was distilled (Bp.sub.14=40-43° C.) to obtain 1,1,2,2-tetradeuteriospiro[2.3]hexan-5-one (2.36 g, 71%). .sup.1H NMR (500 MHz, Chloroform-d) δ 3.16 (s, 4H).

Synthesis of 3-[[4-[(2R)-2-(tert-Butoxycarbonylamino)-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (18)

[0225] ##STR00115##

[0226] To a stirring solution of (2R)-2-amino-4-methyl-pentan-1-ol (12.419 g, 105.97 mmol) in anhydrous THF (200 mL) at ambient temperature under nitrogen was added sodium tert-butoxide (15.276 g, 158.95 mmol). The reaction mixture was stirred for 10 min and 3-[[4-chloro-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (22.14 g, 52.983 mmol) was added. The reaction mixture was placed on a water bath preheated to 60° C. and stirred for 20 min. After cooling to ambient temperature, di-tert-butyl dicarbonate (69.381 g, 317.90 mmol) was added and the reaction mixture was stirred for 3 h. The reaction was quenched with saturated aqueous ammonium chloride (150 mL). Volatiles were removed under vacuum and the aqueous layer was acidified to pH ˜3 with 10% aqueous citric acid. The product was extracted with ethyl acetate (3×200 mL). Combined organic layers were washed with brine (80 mL), dried over anhydrous sodium sulfate and concentrated to a residual volume of ˜250 mL. The product was precipitated out into excess hexanes (750 mL) and collected by vacuum filtration. The obtained white solid was repurified by silica gel chromatography using 0-40% acetone (0.15% acetic acid buffer) gradient in hexanes (0.15% acetic acid buffer) to afford 3-[[4-[(2R)-2-(tert-butoxycarbonylamino)-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (20.73 g, 61%) as a white solid. ESI-MS m/z calc. 598.2461, found 599.4 (M+1).sup.+; Retention time: 5.85 minutes (LC Method C).

Synthesis of 3-[[4-[(2R)-2-Amino-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride Salt) (19)

[0227] ##STR00116##

[0228] To a stirring solution of 3-[[4-[(2R)-2-(tert-butoxycarbonylamino)-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (20.73 g, 34.624 mmol) in DCM (200 mL) at ambient temperature was added HCl (87 mL of 4 M solution in 1,4-dioxane, 346.24 mmol). The reaction mixture was stirred for 2 h. Volatiles were removed under vacuum and the obtained solid was triturated with diethyl ether (150 mL). After removal of the volatiles, the product was dried under vacuum to afford 3-[[4-[(2R)-2-amino-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (hydrochloride salt) (19.68 g, 100%) as a white solid.

[0229] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.56-8.27 (m, 4H), 8.14 (t, J=6.8 Hz, 2H), 7.70 (t, J=7.8 Hz, 1H), 7.34-7.18 (m, 1H), 7.17-7.02 (m, 2H), 6.31 (s, 1H), 4.42-4.23 (m, 1H), 4.23-4.06 (m, 1H), 3.5-3.4 (m, 1H, overlapped with water), 2.01 (s, 6H), 1.82-1.31 (m, 3H), 1.02-0.78 (m, 6H). ESI-MS m/z calc. 498.1937, found 499.3 (M+1).sup.+; Retention time: 1.63 minutes (LC Method B).

Synthesis of 3-[[4-(2,6-Dimethylphenyl)-6-[(2R)-4-methyl-2-[(1,1,2,2-tetradeuteriospiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]-benzoic acid (Hydrochloride Salt) (20)

[0230] ##STR00117##

[0231] A 4 mL vial was charged with 3-[[4-(2R)-2-amino-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (100 mg, 0.1869 mmol), 1,1,2,2-tetradeuteriospiro[2.3]hexan-5-one (53 mg, 0.5292 mmol), anhydrous DCM (0.30 mL) and sodium acetoxyborohydride (Sodium salt) (133 mg, 0.6275 mmol). The vial was briefly purged with nitrogen and the mixture was stirred at ambient temperature for 1.5 h at which time LCMS showed 87% conversion. More borohydride (90 mg) was added and the mixture was stirred for an additional 30 min. A bit of methanol was added and the mixture was concentrated and dissolved in DMSO (total final volume 3 mL). The mixture was purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. The pure fractions were collected and the solvents evaporated to give 3-[[4-(2,6-dimethylphenyl)-6-[(2R)-4-methyl-2-[(1,1,2,2-tetradeuteriospiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (93 mg, 80%) as a white solid. The product (94.5% pure, contains 5.5% of M+3=585.51 impurity) was used for the next step without any further purification. ESI-MS m/z calc. 582.2814, found 583.46 (M+1).sup.+; Retention time: 1.27 minutes (LC method A).

Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-(2-methylpropyl)-12-[(1,1,2,2-tetradeutero)spiro[2.3]hexan-5-yl]-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound Ia)

[0232] ##STR00118##

[0233] A 20 mL flask was charged under nitrogen with HATU (157 mg, 0.4129 mmol), anhydrous DMF (6 mL), and DIEA (0.15 mL, 0.8612 mmol). A solution of 3-[[4-(2,6-dimethylphenyl)-6-[(2R)-4-methyl-2-[(1,1,2,2-tetradeuteriospiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (93 mg, 0.1502 mmol) in anhydrous DMF (4 mL) was added dropwise through syringe over a period of 4 min. The mixture was stirred at ambient temperature for 17 h. The mixture was concentrated and diluted with DMSO (2 mL). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. Evaporation gave a residue that was triturated in DCM/hexanes. Evaporation of the solvents gave (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-[(1,1,2,2-tetradeutero)spiro[2.3]hexan-5-yl]-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (48 mg, 54%) as an off-white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.01 (broad s, 1H), 8.41 (s, 1H), 7.92 (d, J=7.5 Hz, 1H), 7.78-7.60 (m, 2H), 7.26 (t, J=7.6 Hz, 1H), 7.13 (d, J=7.6 Hz, 2H), 6.38 (s, 1H), 5.13 (dd, J=10.7, 4.2 Hz, 1H), 4.39 (t, J=11.1 Hz, 1H), 4.23 (p, J=8.5 Hz, 1H), 3.73 (t, J=11.6 Hz, 1H), 3.30-3.20 (m, 2H), 2.31-1.77 (m, 8H), 1.67 (t, J=13.9, Hz, 1H), 1.32-1.24 (m, 1H), 1.16 (ddd, J=13.7, 10.4, 2.7 Hz, 1H), 0.73 (d, J=6.6 Hz, 3H), 0.22 (d, J=6.3 Hz, 3H). ESI-MS m/z calc. 564.2708, found 565.46 (M+1).sup.+; Retention time: 2.05 minutes (LC method A).

[0234] Deuterium content was determined by liquid chromatography mass spectrometry using a Sciex triple quad instrument. About 0.1 mg/mL of the sample was dissolved in MeOH. 10 μL of the sample was diluted in 1 mL MeOH. 1 μL of the sample was injected in the instrument. Column: Phenomenex Synergy Fusion RP 4 μm, 80 A 50×2 mm. Flow rate: 0.5 mL/min. Gradient: 40% B to 95% B in 3.5 min. Mobile phase A: 0.1% Formic acid in water. B: acetonitrile. The selected ion monitoring method was used. The mass spectrometer was operated in a positive ionization mode with an ESI source. The percentage of each ion monitored was as follows: D.sub.4: 98.52%, D.sub.3: 1.45%, D.sub.2: 0.00%, D.sub.1: 0.02%; Do:0.02%.

Example 6: Synthesis of a Potassium Salt of Compound Ia

[0235] ##STR00119##

[0236] Compound Ia is dissolved in methanol and slowly treated with KOH (0.5 M in methanol) under stirring. The solution is stirred at ambient temperature for 1 h, evaporated, and dried under house vacuum with nitrogen leak at 50-55° C. for 16 h to give Compound Ia (Potassium Ion).

Example 7: Synthesis of a Sodium Salt of Compound Ia

[0237] ##STR00120##

[0238] Compound Ia is suspended in water and slowly treated with NaOH (1 M) under stirring. The suspension is stirred at ambient temperature for 1.25 h. The solution is filtered clear over a syringe filter (0.2 μm) and the clear filtrate is lyophilized for two days to give Compound Ia (Sodium salt).

Example 8: Synthesis of a Calcium Salt of Compound Ia

[0239] ##STR00121##

[0240] Compound Ia (Sodium salt) is stirred in water for 10 min. A solution of CaCl.sub.2 in water is added. Another portion of water is added and the suspension stirred at ambient temperature for 23 h. The solid is collected by filtration, washed with plenty of water, and dried under vacuum with a nitrogen bleed at 55-60° C. for 14 h to give Compound Ia (Calcium salt (0.5)).

Example 9: Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-isobutyl-2,2-dioxo-12-(4,4,5,6,6-pentadeuteriospiro[2.3]hexan-5-yl)-9-oxa-2-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6,8(19),14,16-hexaen-13-one (Compound Ib)

Synthesis of 4,4,6,6-Tetradeuteriospiro[2.3]hexan-5-one (21)

[0241] ##STR00122##

[0242] A mixture of spiro[2.3]hexan-5-one (2.17 g, 22.574 mmol) and potassium carbonate (8 g, 57.885 mmol) in triglyme (24 mL) and D.sub.2O (8 mL) was stirred at 70° C. for 24 h. After cooling to ambient temperature, the reaction was extracted with ether (5×16 mL). The combined ether layers were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under atmosphere pressure to remove diethyl ether, and then it was distilled under reduced pressure (50-60 mBar, 50 to 60° C.) to furnish 4,4,6,6-tetradeuteriospiro[2.3]hexan-5-one (1.07 g, 13%) as a clear liquid.

[0243] A second reaction was run. A mixture of spiro[2.3]hexan-5-one (2 g, 20.806 mmol) and potassium carbonate (7.0 g, 50.649 mmol) in triglyme (21 mL) and D.sub.2O (7 mL) was stirred at 70° C. for 24 h. After being cooled to ambient temperature, the solution was extracted with diethyl ether (5×14 mL). The combined ether layers were dried over anhydrous sodium sulfate. The ether was removed under atmosphere pressure. The residue was distilled under vacuum (50-60 mbar, 50-60° C.) to furnish 4,4,6,6-tetradeuteriospiro[2.3]hexan-5-one (1.222 g, 37%) as a clear liquid. Both batches of crude product were combined and subjected to another deuterium exchange reaction.

[0244] Into a solution of crude 4,4,6,6-tetradeuteriospiro[2.3]hexan-5-one (2.29 g, 8.4601 mmol) in triglyme (6 mL) was added a solution of potassium carbonate (2.9231 g, 21.150 mmol) in D.sub.2O (3 mL). The reaction mixture was stirred at 70° C. for 24 h. After being cooled to ambient temperature, the reaction was extracted with diethyl ether (3×10 mL). The combined ether layers were dried over anhydrous sodium sulfate and concentrated under atmosphere pressure. To the residue was added a saturated sodium bisulfite solution (20 mL), and it was stirred for 1 h. The aqueous layer was extracted with diethyl ether (3×20 mL). The organic layer was discarded. The aqueous layer was basified with 10% NaOH (aq), and then it was extracted with diethyl ether (3×20 mL). The ether was removed by distillation under 1 atm pressure to furnish a 9.01% solution of 4,4,6,6-tetradeuteriospiro[2.3]hexan-5-one (2.55 g, 27%) in triglyme and diethyl ether. This triglyme solution was used for the next step without any further purification.

[0245] .sup.1H NMR (250 MHz, Chloroform-d) δ 0.77 (d, J=0.3 Hz, 4H).

Synthesis of 3-[[4-(2,6-Dimethylphenyl)-6-[(2R)-4-methyl-2-[(4,4,5,6,6-pentadeuteriospiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride Salt) (22)

[0246] ##STR00123##

[0247] A 4 mL vial was charged under nitrogen with 3-[[4-[(2R)-2-amino-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (43 mg, 0.08037 mmol), 4,4,6,6-tetradeuteriospiro[2.3]hexan-5-one (157 mg of 9% w/w, 0.1411 mmol) (9% pure material in triglyme solution), and cyano(trideuterio)boranuide (Sodium salt) (19 mg, 0.2885 mmol). The vial was purged with nitrogen, capped and the resulting suspension was vigorously stirred at ambient temperature for 1 h and 15 min. The reaction was quenched by the addition of deuterated methanol-d.sub.4 (1 mL, 24.68 mmol). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. The pure fractions were collected and the solvents evaporated with a genevac to give 3-[[4-(2,6-dimethylphenyl)-6-[(2R)-4-methyl-2-[(4,4,5,6,6-pentadeuterio-spiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (24 mg, 48%). ESI-MS m/z calc. 583.28766, found 584.45 (M+1).sup.+; Retention time: 1.26 minutes. Significant presence of d4 product also visible (M+H=583) (LC method A).

Synthesis of (11R)-6-(2,6-Dimethylphenyl)-11-isobutyl-2,2-dioxo-12-(4,4,5,6,6-pentadeuteriospiro[2.3]hexan-5-yl)-9-oxa-2-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6,8(19),14,16-hexaen-13-one (Compound Ib)

[0248] ##STR00124##

[0249] A 100 mL flask was charged under nitrogen with 3-[[4-(2,6-dimethylphenyl)-6-[(2R)-4-methyl-2-[(4,4,5,6,6-pentadeuteriospiro[2.3]hexan-5-yl)amino]pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (24 mg, 0.03870 mmol), HATU (53 mg, 0.1394 mmol), anhydrous DMF (2.5 mL) and DIEA (40 μL, 0.2296 mmol). The mixture was stirred at ambient temperature for 2.5 days. The mixture was concentrated and diluted with DMSO (2 mL). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. Genevac evaporation gave (11R)-6-(2,6-dimethylphenyl)-11-isobutyl-2,2-dioxo-12-(4,4,5,6,6-pentadeuteriospiro[2.3]hexan-5-yl)-9-oxa-2λ.sup.6-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6, 8(19),14,16-hexaen-13-one (4.2 mg, 19%) as an off-white solid.

[0250] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.41-11.69 (broad m, 1H), 8.40 (s, 1H), 7.91 (d, J=7.4 Hz, 1H), 7.77-7.60 (m, 2H), 7.25 (t, J=7.7 Hz, 1H), 7.12 (d, J=7.6 Hz, 2H), 6.38 (s, 1H), 5.12 (dd, J=10.8, 4.2 Hz, 1H), 4.39 (t, J=11.1 Hz, 1H), 3.72 (td, J=11.4, 6.0 Hz, 1H), 2.25-1.85 (m, 6H), 1.66 (ddd, J=14.0, 10.6, 2.8 Hz, 1H), 1.39-1.25 (m, 1H), 1.15 (ddd, J=13.8, 10.5, 2.8 Hz, 1H), 0.72 (d, J=6.6 Hz, 3H), 0.54-0.36 (m, 4H), 0.21 (d, J=6.3 Hz, 3H). ESI-MS m/z calc. 565.2771, found 566.43 (M+1).sup.+; Retention time: 2.04 minutes. (LC method A).

[0251] Deuterium content was determined by liquid chromatography mass spectrometry using a Sciex triple quad instrument. About 0.1 mg/mL of the sample was dissolved in MeOH. 10 μL of the sample was diluted in 1 mL MeOH. 1 μL of the sample was injected in the instrument. Column: Phenomenex Synergy Fusion RP 4 μm, 80 A 50×2 mm. Flow rate: 0.5 mL/min. Gradient: 40% B to 95% B in 3.5 min. Mobile phase A: 0.1% Formic acid in water. B: acetonitrile. The selected ion monitoring method was used. The mass spectrometer was operated in a positive ionization mode with an ESI source. The percentage of each ion monitored was as follows: D.sub.5: 58.66%, D.sub.4: 34.01%, D.sub.3: 6.81%, D.sub.2: 0.47%, D.sub.1: 0.03%; D.sub.0: 0.02%.

Example 10: Synthesis of a Potassium Salt of Compound Ib

[0252] ##STR00125##

[0253] Compound Ib is dissolved in methanol and slowly treated with KOH (0.5 M in methanol) under stirring. The solution is stirred at ambient temperature for 1 h, evaporated, and dried under house vacuum with nitrogen leak at 50-55° C. for 16 h to give Compound Ib (Potassium Ion).

Example 11: Synthesis of a Sodium Salt of Compound Ib

[0254] ##STR00126##

[0255] Compound Ib is suspended in water and slowly treated with NaOH (1 M) under stirring. The suspension is stirred at ambient temperature for 1.25 h. The solution is filtered clear over a syringe filter (0.2 μm) and the clear filtrate is lyophilized for two days to give Compound Ib (Sodium salt).

Example 12: Synthesis of a Calcium Salt of Compound Ib

[0256] ##STR00127##

[0257] Compound Ib (Sodium salt) is stirred in water for 10 min. A solution of CaCl.sub.2 in water is added. Another portion of water is added and the suspension stirred at ambient temperature for 23 h. The solid is collected by filtration, washed with plenty of water, and dried under vacuum with a nitrogen bleed at 55-60° C. for 14 h to give Compound Ib (Calcium salt (0.5)).

Example 13: Synthesis of (11R)-12-(5-Deuteriospiro[2.3]hexan-5-yl)-6-(2,6-dimethylphenyl)-11-isobutyl-2,2-dioxo-9-oxa-2-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6,8(19),14,16-hexaen-13-one (Compound Ic)

Synthesis of 3-[[4-1(2R)-2-[(5-Deuteriospiro[2.3]hexan-5-yl)amino]-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride Salt) (23)

[0258] ##STR00128##

[0259] A 4 mL vial was charged with 3-[[4-[(2R)-2-amino-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (99 mg, 0.1850 mmol), spiro[2.3]hexan-5-one (50 mg, 0.5201 mmol), anhydrous DCM (0.30 mL) and sodium cyanoborodeuteride (36 mg, 0.5466 mmol). The vial was briefly purged with nitrogen and the mixture (thick suspension at the beginning) was vigorously stirred at ambient temperature for 2 h at which time LCMS showed about 50% conversion. Deuterated methanol-d.sub.4 (1 mL, 24.62 mmol) was added and the mixture was concentrated and dissolved in DMSO (total final volume 3 mL). The mixture was purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. The pure fractions were collected and the solvents evaporated to give 3-[[4-(2R)-2-[(5-deuteriospiro[2.3]hexan-5-yl)amino]-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)-pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (28 mg, 25%) as a white solid. ESI-MS m/z calc. 579.2626, found 580.42 (M+1).sup.+; Retention time: 1.42 minutes (LC method A).

Synthesis of (11R)-12-(5-Deuteriospiro[2.3]hexan-5-yl)-6-(2,6-dimethylphenyl)-11-isobutyl-2,2-dioxo-9-oxa-2-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6,8(19),14,16-hexaen-13-one (Compound Ic)

[0260] ##STR00129##

[0261] A 20 mL flask was charged under nitrogen with HATU (48 mg, 0.1262 mmol), anhydrous DMF (2 mL) and DIEA (45 μL, 0.2584 mmol). A solution of 3-[[4-[(2R)-2-[(5-deuteriospiro[2.3]hexan-5-yl)amino]-4-methyl-pentoxy]-6-(2,6-dimethylphenyl)pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (28 mg, 0.04544 mmol) in anhydrous DMF (1 mL) was added dropwise through syringe and the mixture was stirred at ambient temperature for 14 h. The mixture was concentrated and diluted with DMSO (2 mL). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. Evaporation gave a residue that was triturated in DCM/hexanes. Evaporation of the solvents gave (11R)-12-(5-deuteriospiro[2.3]hexan-5-yl)-6-(2,6-dimethylphenyl)-11-isobutyl-2,2-dioxo-9-oxa-2λ.sup.6-thia-3,5,12,19-tetrazatricyclo[12.3.1.14,8]nonadeca-1(18),4,6,8(19),14,16-hexaen-13-one (11 mg, 42%) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.52 (broad m, 1H), 8.40 (s, 1H), 7.90 (br s, 1H), 7.67 (br s, 2H), 7.24 (t, J=7.9 Hz, 1H), 7.12 (d, J=7.6 Hz, 2H), 6.37 (br s, 1H), 5.11 (dd, J=10.8, 4.2 Hz, 1H), 4.39 (t, J=11.0 Hz, 1H), 3.74 (d, J=11.4 Hz, 1H), 3.27 (dd, J=10.1, 5.0 Hz, 2H), 2.24-1.81 (m, 8H), 1.66 (t, J=11.8 Hz, 1H), 1.38-1.23 (m, 1H), 1.23-1.00 (m, 1H), 0.72 (d, J=6.6 Hz, 3H), 0.59-0.37 (m, 4H), 0.21 (d, J=6.2 Hz, 3H). .sup.1H NMR shows about 95% of D.sub.1 analog and about 5% of non-deuterated analog. ESI-MS m/z calc. 561.252, found 562.48 (M+1).sup.+; Retention time: 2.07 minutes (LC method A).

Example 14: Synthesis of a Potassium Salt of Compound Ic

[0262] ##STR00130##

[0263] Compound Ic is dissolved in methanol and slowly treated with KOH (0.5 M in methanol) under stirring. The solution is stirred at ambient temperature for 1 h, evaporated, and dried under house vacuum with nitrogen leak at 50-55° C. for 16 h to give Compound Ic (Potassium Ion).

Example 15: Synthesis of a Sodium Salt of Compound Ic

[0264] ##STR00131##

[0265] Compound Ic is suspended in water and slowly treated with NaOH (1 M) under stirring. The suspension is stirred at ambient temperature for 1.25 h. The solution is filtered clear over a syringe filter (0.2 μm) and the clear filtrate is lyophilized for two days to give Compound Ic (Sodium salt).

Example 16: Synthesis of a Calcium Salt of Compound Ic

[0266] ##STR00132##

[0267] Compound Ic (Sodium salt) is stirred in water for 10 min. A solution of CaCl.sub.2 in water is added. Another portion of water is added and the suspension stirred at ambient temperature for 23 h. The solid is collected by filtration, washed with plenty of water, and dried under vacuum with a nitrogen bleed at 55-60° C. for 14 h to give Compound Ic (Calcium salt (0.5)).

Example 17: Synthesis of (11R)-6-[2,6-di(trideutero)methylphenyl]-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound Id)

Synthesis of 2-Bromobenzene-1,3-dicarboxylic acid (25)

[0268] ##STR00133##

[0269] To a stirring solution of 2-bromo-1,3-dimethyl-benzene (35.25 g, 190.48 mmol) in a mixture of tert-butanol (150 mL) and water (150 mL) at ambient temperature was portion-wise added potassium permanganate (61 g, 385.99 mmol). After the addition was complete, the reaction mixture was heated to 85° C. for 3 h. After cooling to ambient temperature, another portion of potassium permanganate (62 g, 392.32 mmol) was added. The reaction mixture was then heated to 85° C. for 16 h. The reaction mixture was filtered hot through a pad of celite, and the filter cake was washed with a hot 1:1 mixture of tert-butanol and water (2×100 mL). The combined filtrates were concentrated under vacuum to a residual volume of ˜200 mL and acidified to pH ˜1 with concentrated sulfuric acid. The mixture was cooled to 0° C. and left standing for 2 h. The precipitated product was collected by filtration, and the aqueous filtrate was further extracted with ethyl acetate (2×80 mL). The combined organic layers were washed with brine (40 mL), dried over anhydrous sodium sulfate and concentrated. All product fractions were combined, washed with hexanes (2×100 mL), and dried under vacuum to afford 2-bromobenzene-1,3-dicarboxylic acid (24.86 g, 51%) as a white solid. The product was carried forward to the next step without any further purification.

[0270] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.71 (d, J=7.6 Hz, 2H), 7.53 (t, J=7.6 Hz, 1H).

[0271] ESI-MS m/z calc. 243.9371, found 245.3 (M+1).sup.+; Retention time: 1.63 minutes (LC Method C).

Synthesis of Dimethyl 2-bromobenzene-1,3-dicarboxylate (26)

[0272] ##STR00134##

[0273] To a stirring solution of 2-bromobenzene-1,3-dicarboxylic acid (25.15 g, 102.64 mmol) in anhydrous DMF (250 mL) at ambient temperature under nitrogen was added anhydrous K.sub.2CO.sub.3 (31.5 g, 227.92 mmol), followed by iodomethane (32.376 g, 14.2 mL, 228.10 mmol). The reaction mixture was stirred at this temperature for 24 h. The reaction mixture was poured into brine (750 mL), and the product was extracted with ethyl acetate (3×250 mL). The combined organic layers were washed with brine (450 mL), dried over anhydrous sodium sulfate and concentrated to afford dimethyl 2-bromobenzene-1,3-dicarboxylate (28.45 g, 94%) as an amber oil. The product was carried to the next step without further purification. .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.91-7.74 (m, 2H), 7.70-7.52 (m, 1H), 3.88 (s, 6H). ESI-MS m/z calc. 271.9684, found 273.4 (M+1).sup.+; Retention time: 4.23 minutes (LC Method C).

Synthesis of [2-Bromo-3-[dideuterio(hydroxy)methyl]phenyl]-dideuterio-methanol (27)

[0274] ##STR00135##

[0275] To a stirring suspension of lithium aluminum deuteride (200 mg, 5.0044 mmol) in anhydrous THF (3 mL) at 0° C. under nitrogen was dropwise added a solution of dimethyl 2-bromobenzene-1,3-dicarboxylate (585 mg, 2.1422 mmol) in anhydrous THF (1 mL). After the addition was complete, the reaction mixture was stirred at this temperature for 1 h. The reaction mixture was diluted with THF (10 mL) and quenched using a standard Fieser protocol. Salts were filtered off and washed with THF (2×10 mL). The combined filtrates were concentrated under vacuum to afford [2-bromo-3-[dideuterio(hydroxy)methyl]phenyl]-dideuterio-methanol (416 mg, 79%) as a white solid. The product was carried to the next step without further purification.

[0276] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.44-7.39 (m, 3H), 5.37 (s, 2H). ESI-MS m/z calc. 220.0037, found 203.3 (M+1-18).sup.+; Retention time: 1.8 minutes (LC Method C).

Synthesis of 2-Bromo-1,3-bis[bromo(dideuterio)methyl]benzene (28)

[0277] ##STR00136##

[0278] To a stirring suspension of [2-bromo-3-[dideuterio(hydroxy)methyl]phenyl]-dideuterio-methanol (12.56 g, 56.811 mmol) in anhydrous DCM (370 mL) at ambient temperature under nitrogen was added carbon tetrabromide (45.3 g, 136.60 mmol), followed by a portionwise addition of triphenylphosphine (35.8 g, 136.49 mmol) over a 15-minute period. After the addition was complete, the reaction mixture became a homogeneous solution and was stirred at this temperature for 1 h. The reaction mixture was concentrated under vacuum to a residual volume of ˜100 mL and poured into diethyl ether (600 mL). The white precipitate was filtered off and washed with diethyl ether (2×50 mL). The combined filtrates were collected and concentrated under vacuum to a residual volume of ˜100 mL and poured into hexanes (600 mL). The white precipitate was filtered off and washed with hexanes (2×100 mL). The combined filtrates were collected and concentrated under vacuum. The crude product was purified by silica gel chromatography using 0-10% ethyl acetate gradient in hexanes to afford 2-bromo-1,3-bis[bromo(dideuterio)methyl]benzene (15.56 g, 71%) as a white solid. .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.60 (d, J=7.6 Hz, 2H), 7.39 (t, J=7.5 Hz, 1H).

Synthesis of 2-Bromo-1,3-bis(trideuteriomethyl)benzene (29)

[0279] ##STR00137##

[0280] To a stirring suspension of lithium aluminum deuteride (3.65 g, 91.330 mmol) in anhydrous THF (70 mL) at 0° C. under nitrogen was added dropwise a solution of 2-bromo-1,3-bis[bromo(dideuterio)methyl]benzene (15.5 g, 40.216 mmol) in anhydrous THF (70 mL). After the addition was complete, the reaction mixture was stirred at this temperature for 1 h. The reaction mixture was diluted with THF (100 mL), and quenched using a standard Fieser workup protocol. Salts were filtered off and washed with THF (2×50 mL). The combined filtrates were concentrated under vacuum. The residue was re-dissolved in hexanes (200 mL), filtered and concentrated under vacuum to afford 2-bromo-1,3-bis(trideuteriomethyl)benzene (5.58 g, 68%) as a pale yellow oil. The product was carried to the next step without further purification.

[0281] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.23-7.11 (m, 3H).

Synthesis of [2,6-Bis(trideuteriomethyl)phenyl]boronic acid (30)

[0282] ##STR00138##

[0283] To a stirring suspension of magnesium turnings (1.8 g, 74.059 mmol) and iodine (15 mg, 0.0030 mL, 0.0591 mmol) in anhydrous THF (20 mL) at ambient temperature under nitrogen was slowly added a solution of 2-bromo-1,3-bis(trideuteriomethyl)benzene (9.89 g, 46.578 mmol) in anhydrous THF (50 mL). Once initiated, the reaction is exothermic and the rate of addition was adjusted to keep the reaction temperature below the boiling point of THF. After the addition was complete, the reaction mixture was stirred at 65° C. for 1 h. The prepared solution of Grignard reagent was cooled down to ambient temperature and added dropwise to a solution of trimethyl borate (24.232 g, 26 mL, 233.20 mmol) in anhydrous THF (60 mL) cooled to −78° C. under nitrogen. After the addition was complete, the reaction mixture was stirred at −78° C. for 3 h, then allowed to warm up to ambient temperature and stirred overnight. The reaction mixture was cooled to 0° C. and aqueous HCl (95 mL of 1 M, 95.000 mmol) was slowly added. After the addition was complete, the reaction mixture was warmed up to ambient temperature and stirred for 3 h. Water (100 mL) was added and the volatiles were removed under vacuum. The aqueous layer was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with brine (80 mL), dried over anhydrous sodium sulfate and concentrated. The obtained residue was triturated with hexanes (100 ml). The precipitated product was collected by filtration and dried under vacuum to afford [2,6-bis(trideuteriomethyl)phenyl]boronic acid (4.667 g, 61%) as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.11 (broad s, 2H), 7.06 (dd, J=7.9, 7.1 Hz, 1H), 6.91 (d, J=7.5 Hz, 2H).

Synthesis of tert-Butyl N-[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]-N-tert-butoxycarbonyl-carbamate (31)

[0284] ##STR00139##

[0285] A stirring solution of [2,6-bis(trideuteriomethyl)phenyl]boronic acid (1.565 g, 10.031 mmol) and tert-butyl N-tert-butoxycarbonyl-N-(4,6-dichloropyrimidin-2-yl)carbamate (5.48 g, 15.046 mmol) in a mixture of 1,2-dimethoxyethane (30 mL) and water (10 mL) at ambient temperature was degassed with nitrogen for 30 min. Under nitrogen, cesium carbonate (8.18 g, 25.106 mmol) and [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II) (735 mg, 1.0045 mmol) were added. The reaction mixture was heated to 80° C. for 3 h. After cooling to ambient temperature, the reaction mixture was diluted with water (120 mL), and the product was extracted with ethyl acetate (3×150 mL). The combined organic layers were washed with brine (50 ml), dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel chromatography using 0-10% diethyl ether gradient in hexanes to afford tert-butyl N-[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]-N-tert-butoxycarbonyl-carbamate (3.6919 g, 79%) as pale yellow oil. ESI-MS m/z calc. 439.2145, found 440.4 (M+1).sup.+; Retention time: 7.48 minutes (LC Method C).

Synthesis of 4-[2,6-Bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-amine (32)

[0286] ##STR00140##

[0287] To a stirring solution of tert-butyl N-[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]-N-tert-butoxycarbonyl-carbamate (3.66 g, 8.3188 mmol) in DCM (30 mL) at ambient temperature was added a solution of HCl (12 mL of 4 M, 48.000 mmol) in 1,4-dioxane. The reaction mixture was stirred at this temperature for 20 h. Volatiles were removed under vacuum, the obtained white solid was re-suspended in saturated aqueous NaHCO.sub.3 (150 mL) and stirred at ambient temperature for 15 min. The product was extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated to afford 4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-amine (1.842 g, 87%) as a white solid. ESI-MS m/z calc. 239.1096, found 240.3 (M+1).sup.+; Retention time: 4.44 minutes (LC Method C).

Synthesis of Methyl 3-[[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoate (33)

[0288] ##STR00141##

[0289] To a stirring solution of 4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-amine (1.8 g, 7.5083 mmol) and methyl 3-chlorosulfonylbenzoate (2.64 g, 11.250 mmol) in anhydrous THF (50 mL) at 0° C. under nitrogen was added dropwise a solution of lithium tert-amoxide (3.2120 g, 11 mL of 40% w/w, 13.656 mmol) in heptanes. After the addition was complete, the reaction mixture was stirred at this temperature for 2 h. The reaction mixture was quenched cold with 1 M aqueous HCl (120 mL), then warmed up to ambient temperature and volatiles were removed under vacuum. The product was extracted with ethyl acetate (3×150 mL). Combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel chromatography using 0-20% ethyl acetate gradient in hexanes to afford methyl 3-[[4-[2,6-bis(trideuterio-methyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoate (2.786 g, 80%) as a white solid. ESI-MS m/z calc. 437.1083, found 438.3 (M+1).sup.+; Retention time: 5.95 minutes Final purity was determined by reverse phase HPLC using LC Method C.

Synthesis of 3-[[4-[2,6-Bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoic acid (34)

[0290] ##STR00142##

[0291] To a stirring solution of methyl 3-[[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoate (2.751 g, 6.2818 mmol) in THF (40 mL) at ambient temperature was added aqueous NaOH (26 mL of 1 M, 26.000 mmol). The reaction mixture was stirred at this temperature for 2 h. Water (100 mL) was added, and volatiles were removed under vacuum. The residual aqueous layer was extracted with ethyl acetate (1×100 mL), and the organic layer was discarded. The aqueous layer was acidified with 2 M aqueous HCl to pH ˜1, and the product was extracted with ethyl acetate (3×100 mL). Combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated to afford 3-[[4-[2,6-bis(trideuterio-methyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoic acid (1.719 g, 62%) as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.43 (s, 1H), 12.44 (s, 1H), 8.44 (t, J=1.8 Hz, 1H), 8.18 (dt, J=7.8, 1.4 Hz, 1H), 8.13 (ddd, J=7.9, 2.0, 1.2 Hz, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.31 (s, 1H), 7.24 (dd, J=8.0, 7.2 Hz, 1H), 7.10 (d, J=7.6 Hz, 2H). ESI-MS m/z calc. 423.0927, found 424.0 (M+1).sup.+; Retention time: 2.28 minutes (LC Method B).

Synthesis of 3-[[4-[2,6-Bis(trideuteriomethyl)phenyl]-6-1(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride Salt) (35)

[0292] ##STR00143##

[0293] In a 20 mL flask, 3-[[4-[2,6-bis(trideuteriomethyl)phenyl]-6-chloro-pyrimidin-2-yl]sulfamoyl]benzoic acid (157 mg, 0.3704 mmol) and (2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (Hydrochloride salt) (90 mg, 0.3850 mmol) were charged under nitrogen with anhydrous THF (2 mL) (suspension). Sodium tert-butoxide (156 mg, 1.623 mmol) was added (slight exotherm and dissolution of solids). The suspension was stirred at ambient temperature for 5.5 h. The mixture was partitioned between ethyl acetate (30 mL) and aqueous 1M HCl (30 mL) and brine (20 mL). After separation, the aqueous phase was further extracted with EtOAc (2×30 ml). The combined extracts were dried over sodium sulfate and the solvents evaporated to give a crude material. The material was dissolved in DMSO (4 mL). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. Evaporation gave 3-[[4-[2,6-bis(trideuterio-methyl)phenyl]-6-[(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (135 mg, 59%) as a white solid. ESI-MS m/z calc. 584.29395, found 585.73 (M+1).sup.+; Retention time: 1.22 minutes (LC method A).

Synthesis (2R)-4-Methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (hydrochloride salt) (3.HCl)

[0294] ##STR00144##

[0295] HCl (354 mL of 4 M, 1.416 mol) (4 M in dioxane) was added to a stirring (mechanical) solution of (2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (254 g, 1.287 mol) in diethyl ether (2.286 L) in an ice/water bath over 20 minutes, keeping the internal temp between 10° C. and 22° C. After the addition was complete, the solution was stirred at ambient temperature for 1.5 h. The product was filtered out and rinsed with 2000 mL diethyl ether. The exact same process was repeated again on the exact same scale (a total of 508 g of amino alcohol starting material was used). The product was dried under vacuum at 35° C. overnight and gave 562.3 g. (2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentan-1-ol (Hydrochloride salt) (562.3 g, 93%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.17-8.84 (m, 2H), 5.38 (s, 1H), 3.99 (p, J=7.2 Hz, 1H), 3.70-3.60 (m, 1H), 3.55-3.45 (m, 1H), 3.03-2.91 (m, 1H), 2.63-2.54 (m, 2H), 2.20-2.05 (m, 2H), 1.73-1.60 (m, 1H), 1.60-1.48 (m, 1H), 1.43-1.30 (m, 1H), 0.93-0.83 (m, 6H), 0.55-0.45 (m, 2H), 0.45-0.36 (m, 2H).

Synthesis of (11R)-6-[2,6-di(trideutero)methylphenyl]-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.SUP.6.-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound Id)

[0296] ##STR00145##

[0297] A 20 mL flask was charged under nitrogen with COMU (244 mg, 0.5697 mmol), anhydrous DMF (8 mL) and DIEA (0.22 mL, 1.263 mmol). A solution of 3-[[4-[2,6-bis(trideuteriomethyl)phenyl]-6-[(2R)-4-methyl-2-(spiro[2.3]hexan-5-ylamino)pentoxy]pyrimidin-2-yl]sulfamoyl]benzoic acid (Hydrochloride salt) (135 mg, 0.2173 mmol) in anhydrous DMF (4 mL) was added dropwise through syringe over a period of 3 min. The mixture was stirred at ambient temperature for 17 h. The mixture was concentrated and diluted with DMSO (2 mL). The solution was microfiltered through a Whatman 0.45 μM PTFE syringe filter disc and purified by reverse phase preparative HPLC (C18) using a gradient of acetonitrile in water (1 to 99% over 15 min) and HCl as a modifier. Evaporation gave a residue that was triturated in DCM/hexanes. Evaporation of the solvents gave (11R)-6-[2,6-di(trideutero)-methylphenyl]-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ.sup.6-thia-3,5,12,19-tetraazatricyclo-[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (73.4 mg, 60%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 13.47-11.72 (broad m, 1H), 8.40 (s, 1H), 7.99-7.81 (m, 1H), 7.82-7.52 (m, 2H), 7.26 (t, J=7.7 Hz, 1H), 7.12 (d, J=7.6 Hz, 2H), 6.37 (br s, 1H), 5.12 (dd, J=10.7, 4.2 Hz, 1H), 4.39 (t, J=11.1 Hz, 1H), 4.23 (p, J=8.5 Hz, 1H), 3.72 (t, J=10.4 Hz, 1H), 3.30-3.22 (m, 2H, overlapped with water), 2.09 (dt, J=19.0, 9.4 Hz, 2H), 1.66 (t, J=12.5 Hz, 1H), 1.38-1.24 (br m, 1H), 1.15 (dd, J=14.0, 10.4 Hz, 1H), 0.72 (d, J=6.6 Hz, 3H), 0.61-0.35 (m, 4H), 0.21 (d, J=6.2 Hz, 3H). ESI-MS m/z calc. 566.2834, found 567.69 (M+1).sup.+; Retention time: 2.05 minutes (LC method A).

[0298] Deuterium content was determined by liquid chromatography mass spectrometry using a single quad instrument. About 0.1 mg/mL of the sample was dissolved in MeOH. 10 μL of the sample was diluted in 1 mL MeOH. 1 μL of the sample was injected into the instrument. Column: Agilent SB C18, 1.8 μm, 2.1×50 mm. Flow rate: 0.7 mL/min. Gradient: 40% B to 100% B in 2.5 min. Mobile phase A: 0.1% trifluoroacetic acid in water. B: acetonitrile. The selected ion monitoring method was used. The mass spectrometer was operated in a positive ionization mode with an ESI source. The percentage of each ion monitored was as follows: D.sub.6: 88.49%, D.sub.5: 8.14%, D.sub.4: 0.93%, D.sub.3: 0.52%, D.sub.2: 1.93%, D.sub.1: 0.00%; D.sub.0: 0.00%.

Example 18: Synthesis of a Potassium Salt of Compound Id

[0299] ##STR00146##

[0300] Compound Id is dissolved in methanol and slowly treated with KOH (0.5 M in methanol) under stirring. The solution is stirred at ambient temperature for 1 h, evaporated, and dried under house vacuum with nitrogen leak at 50-55° C. for 16 h to give Compound Id (Potassium Ion).

Example 19: Synthesis of a Sodium Salt of Compound Id

[0301] ##STR00147##

[0302] Compound Id is suspended in water and slowly treated with NaOH (1 M) under stirring. The suspension is stirred at ambient temperature for 1.25 h. The solution is filtered clear over a syringe filter (0.2 μm) and the clear filtrate is lyophilized for two days to give Compound Id (Sodium salt).

Example 20: Synthesis of a Calcium Salt of Compound Id

[0303] ##STR00148##

[0304] Compound Id (Sodium salt) is stirred in water for 10 min. A solution of CaCl.sub.2 in water is added. Another portion of water is added and the suspension stirred at ambient temperature for 23 h. The solid is collected by filtration, washed with plenty of water, and dried under vacuum with a nitrogen bleed at 55-60° C. for 14 h to give Compound Id (Calcium salt (0.5)).

Bioactivity Assays

[0305] Solutions

[0306] Base medium (ADF+++) consisted of Advanced DMEM/Ham's F12, 2 mM Glutamax, 10 mM HEPES, 1 μg/ml penicillin/streptomycin.

[0307] Intestinal enteroid maintenance medium (IEMM) consisted of ADF+++, 1×B27 supplement, 1×N2 supplement, 1.25 mM N-acetyl cysteine, 10 mM Nicotinamide, 50 ng/mL hEGF, 10 nM Gastrin, 1 μg/mL hR-spondin-1, 100 ng/mL hNoggin, TGF-b type 1 inhibitor A-83-01, 100 μg/mL Primocin, 10 μM P38 MAPK inhibitor SB202190.

[0308] Bath 1 Buffer consisted of 1 mM MgCl.sub.2, 160 mM NaCl, 4.5 mM KCl, 10 mM HEPES, 10 mM Glucose, 2 mM CaCl.sub.2.

[0309] Chloride Free Buffer consisted of 1 mM Magnesium Gluconate, 2 mM Calcium Gluconate, 4.5 mM Potassium Gluconate, 160 mM Sodium Gluconate, 10 mM HEPES, 10 mM Glucose.

[0310] Bath1 Dye Solution consisted of Bath 1 Buffer, 0.04% Pluronic F127, 20 μM Methyl Oxonol, 30 μM CaCCinh-A01, 30 μM Chicago Sky Blue.

[0311] Chloride Free Dye Solution consisted of Chloride Free Buffer, 0.04% Pluronic F127, 20 μM Methyl Oxonol, 30 μM CaCCinh-A01, 30 μM Chicago Sky Blue.

[0312] Chloride Free Dye Stimulation Solution consisted of Chloride Free Dye Solution, 10 μM forskolin, 100 μM IBMX, and 300 nM Compound III.

[0313] Cell Culture

[0314] Human intestinal epithelial enteroid cells were obtained from the Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands and expanded in T-Flasks as previously described (Dekkers J F, Wiegerinck C L, de Jonge H R, Bronsveld I, Janssens H M, de Winter-de Groot K M, Brandsma A M, de Jong N W M, Bijvelds M J C, Scholte B J, Nieuwenhuis E E S, van den Brink S, Clevers H, van der Ent C K, Middendorp S and M Beekman J M. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 2013 July; 19(7):939-45.

[0315] Enteroid Cell Harvesting and Seeding

[0316] Cells were recovered in cell recovery solution, collected by centrifugation at 650 rpm for 5 min at 4° C., resuspended in TryPLE and incubated for 5 min at 37° C. Cells were then collected by centrifugation at 650 rpm for 5 min at 4° C. and resuspended in IEMM containing 10 μM ROCK inhibitor (RI). The cell suspension was passed through a 40 μm cell strainer and resuspended at 1×106 cells/mL in IEMM containing 10 μM RI. Cells were seeded at 5000 cells/well into multi-well plates and incubated for overnight at 37° C., 95% humidity and 5% CO.sub.2 prior to assay.

[0317] Membrane Potential Dye Assay

[0318] Enteroid cells were incubated with test compound in IEMM for 18-24 h at 37° C., 95% humidity and 5% CO.sub.2. Following compound incubations, a membrane potential dye assay was employed using a FLIPR Tetra to directly measure the potency and efficacy of the test compound on CFTR-mediated chloride transport following acute addition of 10 μM forskolin and 300 nM N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide. Briefly, cells were washed 5 times in Bath 1 Buffer. Bath 1 Dye Solution was added and the cells were incubated for 25 min at ambient temperature. Following dye incubation, cells were washed 3 times in Chloride Free Dye Solution. Chloride transport was initiated by addition of Chloride Free Dye Stimulation Solution and the fluorescence signal was read for 15 min. The CFTR-mediated chloride transport for each condition was determined from the AUC of the fluorescence response to acute forskolin and 300 nM N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide stimulation. Chloride transport was then expressed as a percentage of the chloride transport following treatment with 1 μM (14S)-8-[3-(2-{Dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ.sup.6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]-tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione, 3 μM ((R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide and 300 nM acute N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide triple combination positive control (% Activity). Table 3 provides CFTR modulating activity for Compound I and exemplary deuterated derivatives of Compound I.

TABLE-US-00003 TABLE 3 CFTR Modulating Activity for Compound I and Exemplary Deuterated Derivatives of Compound I Compound Chemical Structure Max. Activity EC.sub.50 Compound I [00149] +++ +++ Compound Ia [00150] +++ +++ Compound Ib [00151] +++ +++ Compound Ic [00152] +++ +++ Compound Id [00153] +++ +++ Max Activity: +++ is >60%; ++ is 30-60%; + is <30%. EC.sub.50: +++ is <1 μM.

Other Embodiments

[0319] The foregoing discussion discloses and describes merely exemplary embodiments of this disclosure. One skilled in the art will readily recognize, from such discussion and from the accompanying claims, that various changes, modifications, and variations can be made therein without departing from the spirit and scope of this disclosure as defined in the following claims.