OXYSTEROLS AND METHODS OF USE THEREOF

Abstract

Compounds are provided according to Formula (I) and pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof; wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.8 are as defined herein. Compounds of the present invention are contemplated useful for the prevention and treatment of a variety of conditions.

##STR00001##

Claims

1.-63. (canceled)

64. A compound selected from the group consisting of: ##STR00054## or a pharmaceutically acceptable salt thereof.

65. (canceled)

66. A pharmaceutical composition comprising a compound of claim 64, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

67. A method of inducing sedation or anesthesia comprising administering to a subject an effective amount of a compound of claim 64, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

68. A method for treating or preventing a disorder described herein, comprising administering to a subject in need thereof an effective amount of a compound of claim 64, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

69. The method according to claim 68, wherein the disorder is a gastrointestinal (GI) disorder e.g., constipation, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (e.g., ulcerative colitis, Crohn's disease), a structural disorder affecting the GI tract, an anal disorder (e.g., hemorrhoids, internal hemorrhoids, external hemorrhoids, anal fissures, perianal abscesses, anal fistula), colon polyps, cancer, diabetes, a sterol synthesis disorder, or colitis.

70. The method according to claim 69, wherein the disorder is inflammatory bowel disease.

71. The method according to claim 69, wherein the disorder is cancer, diabetes, or a sterol synthesis disorder.

72. A method for treating or preventing a CNS-related condition comprising administering to a subject in need thereof an effective amount of a compound of claim 64, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

73. The method according to claim 72, wherein the CNS-related condition is an adjustment disorder, an anxiety disorder (including obsessive-compulsive disorder, posttraumatic stress disorder, and social phobia), a cognitive disorder (including Alzheimer's disease and other forms of dementia), a dissociative disorder, an eating disorder, a mood disorder (including depression (e.g., postpartum depression), bipolar disorder, dysthymic disorder, suicidality), schizophrenia or an other psychotic disorder (including schizoaffective disorder), a sleep disorder (including insomnia), a substance-related disorder, a personality disorder (including obsessive-compulsive personality disorder), an autism spectrum disorder (including those involving mutations to the Shank group of proteins (e.g., Shank3)), a neurodevelopmental disorder (including Rett syndrome, Tuberous Sclerosis complex), multiple sclerosis, a sterol synthesis disorder, pain (including acute and chronic pain), encephalopathy secondary to a medical condition (including hepatic encephalopathy and anti-NMDA receptor encephalitis), a seizure disorder (including status epilepticus and monogenic forms of epilepsy such as Dravet's disease), stroke, traumatic brain injury, a movement disorder (including Huntington's disease and Parkinson's disease), vision impairment, hearing loss, or tinnitus.

74. The method according to claim 73, wherein the disorder is a sterol synthesis disorder.

Description

EXAMPLES

[0203] In order that the invention described herein may be more fully understood, the following examples are set forth. Synthetic methods or intermediates may be found, for example in WO2014/160480*. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

[0204] The stereochemistry assigned herein (e.g., the assignment of “R” or “S” to the C24 position of the steroid) may be tentatively (e.g., randomly) assigned. For example, a C24 position may be drawn in the “R” configuration when the absolute configuration is “S.” A C24 position may also be drawn in the “S” configuration when the absolute configuration is “R.”

Example 1. Synthesis of Compound 1

[0205] ##STR00028##

[0206] Synthesis of Compound 1-2. To a solution of Compound 1-1 (0.3 g, 0.58 mmol; synthesized as described in Takahashi et al., Tetrahedron Letters, 2003, 44(2), 341-344) in DCM (10 mL) was added DMAP (7.08 mg, 0.058 mmol), TEA (95.7 mg, 0.87 mmol), DCC (179 mg, 0.87 mmol) and 2-((tert-butoxycarbonyl)amino)acetic acid (203 mg, 1.16 mmol). The mixture was stirred at 15° C. for 16 hours, at which point an additional aliquot of DMAP (7.08 mg, 0.058 mmol), TEA (95.7 mg, 0.87 mmol), DCC (179 mg, 0.87 mmol) and 2-((tert-butoxycarbonyl)amino)acetic acid (203 mg, 1.16 mmol) were added. The mixture was stirred at 25° C. for 16 hours, then the mixture was filtered and the filtrate was diluted with aqueous sat. NH.sub.4Cl (30 mL) and extracted with DCM (10 mL×2). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered, and concentrated to give the crude product Compound 1-2, which was used in the next step directly without purification. .sup.1H NMR indicated an estimated yield of 45%.

[0207] Synthesis of Compound 1-3. Compound 1-2 (350 mg, 0.519 mmol) was dissolved in TBAF (5.18 mL, 5.18 mmol, 1M in THF) and the mixture was stirred at 15° C. for 16 hours. The mixture was quenched with saturated NH.sub.4Cl(10 mL) and extracted with EtOAc (5 mL×2). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated, and purified by silica gel (PE: EtOAc=10:1) to give the crude product Compound 1-3 (250 mg, Compound 1-2/Compound 1-3=0.55/0.45) product as a colorless oil.

[0208] Synthesis of Compound 1. Compound 1-3 was dissolved in HCl/dioxane (4 N, 5 mL), and the mixture was stirred at 15° C. for 30 minutes. Then MTBE (5 mL) was then added to the mixture to form a precipitate, which was filtered and purified by prep-HPLC(column: Phenomenex Synergi C18 150*30 mm*4 um, gradient: 36-66% B (A=0.05% HCl-ACN, B=acetonitrile) flow rate: 30 mL/min)to afford the HCl salt of Compound 1 (6 mg, 2.92%) as an off white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.36-5.35 (m, 1H), 3.91-3.81 (m, 2H), 3.43-3.38 (m, 1H), 2.68-2.25 (m, 2H), 2.10-0.94 (m, 37H), 0.74 (s, 3H). LCMS MS ESI calcd. for C.sub.29H.sub.49NO.sub.3Na [M+Na].sup.+ 482, found 482.

Example 2. Synthesis of Compound 2

[0209] ##STR00029##

[0210] Synthesis of Compound 2-2. To a solution of Compound 1-1 (0.3 g, 0.58 mmol) in DCM (10 mL) was added DMAP (21.2 mg, 0.174 mmol), TEA (191 mg, 1.74 mmol), DCC (452 mg, 1.74 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanoic acid (378 mg, 1.74 mmol). The mixture was stirred at 25° C. for 16 hours. The mixture was filtered, the filtrate was concentrated and purified by combi-flash (PE: EA=100%-95%) to give Compound 2-2 (380 mg, 92%) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.31-5.19 (m, 1H), 5.03-4.99 (m. 1H), 4.74-4.72 (m, 1H), 4.23-4.20 (m, 1H), 3.50-3.44 (m, 1H), 2.29-2.17 (m, 3H), 1.85-0.85 (m, 60H), 0.66 (s, 3H), 0.05 (s, 6H).

[0211] Synthesis of Compound 2-3. Compound 2-2 (380 mg, 0.53 mmol) in TBAF (15.9 mL, 15.9 mmol, 1M in THF) was stirred at 15° C. for 16 hours. The mixture was quenched with aqueous sat, NH.sub.4Cl (20 nL) and extracted with EtOAc (10 mL×2) The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated, and purified by combi-flash (PE: EA=100%-90%) to give Compound 2-3 as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5 35-5 29 (m, 1H), 5.01-4.99 (m, 1H), 4.75-4.73 (m, 1H), 4.23-4.20 (m, 111), 3.52-3.50 (m, 1H), 2.29-2.17 (m, 3H). 1.85-0.85 (m, 52H), 0.66 (s, 3H).

[0212] Synthesis of Compound 2. Compound 2-3 (50 mg, 0.083 mmol) was dissolved in HCl/dioxane (1 mL) and stirred at 15° C. for 30 minutes. Then MTBE (1 mL) was then added to the mixture and a precipitate was formed, which was filtered to afford the HCl salt of Compound 2 (5 mg, 12.0%) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.36-5.30 (m, 1H), 3.97-3.80 (m, 1H), 3.50-3.40 (m, 1H), 2.50-2.40 (m, 3H), 2.35-0.85 (m, 46H), 0.74 (s, 3H). LCMS MS ESI calcd. for C.sub.32H.sub.56NO.sub.3 [M+H].sup.+ 502, found 502.

Example 3. Synthesis of Compound 3

[0213] ##STR00030##

[0214] Synthesis of 3-2. Iodine (1.17 g, 4.63 mmol) was added to a solution of trimethyl phosphite (526 mg, 4.24 mmol) in CH.sub.2Cl.sub.2 (20 mL) at 0° C. After stirring for 5 minutes, the clear colorless solution was allowed to warm to 15° C. The phosphorylating agent was added dropwise to a solution of Compound 1-1 (2.0 g, 3.86 mmol) and pyridine (1.21 g, 15.4 mmol) in CH.sub.2Cl.sub.2 (30 mL) at 0° C. The resulting mixture was stirred at 0° C. for 1 h, then cooled to 15° C. and stirred for another 1 h. The reaction mixture was treated water (50 mL), extracted with CH.sub.2Cl.sub.2 (50 mL×2). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue was purified by column chromatography on silica gel (PE/EtOAc=10/1 to 3/1) to afford Compound 3-2 (1.8 g, 75%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.35-5.30 (m, 1H), 4.20-4.17 (m, 1H), 3.76 (s, 3H), 3.73 (s. 3H), 3.48-3.46 (m. 1H), 2.26-2.15 (m, 2H), 1.93-1.61 (m. 7H), 1.55-0.88 (m. 38H), 0.66 (s, 3H), 0.05 (s, 6H).

[0215] Synthesis of Compound 3. To a solution of Compound 3-2 (300 mg, 480 μmol) in CH.sub.2Cl.sub.2 (5 mL) was added bromotrimethylsilane (220 mg, 1.44 mmol) at 15° C. and the reactions was stirred for 12 h. The reaction mixture was adjusted to pH=8 with aq. NaOH (1.44 mL, 1.44 mmol, 1 M in H.sub.2O) and a precipitate was formed, which was then filtered, washed with CH.sub.2Cl.sub.2 (2 mL) and water (2 mL), and dried under vacuum to afford Compound 3 (30 mg, 12%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 5.24-5.23 (m, 1H), 3.87-3.85 (m, 1H), 3.24-3.21 (m, 1H), 2.13-2.06 (m, 2H), 1.91-1.60 (m, 6H), 1.50-0.80 (m, 33H), 0.62 (s, 3H). LCMS MS ESI calcd. for C.sub.27H.sub.44 [M+H—H.sub.2O—Na.sub.2O.sub.4P].sup.+ 367, found 367. HRMS MS ESI calcd. for C.sub.27H.sub.46O.sub.5P [M−H].sup.− 481.3088, found 481.3105.

Example 4. Synthesis of Compound 4

[0216] ##STR00031##

[0217] Synthesis of Compound 4-2. To a solution of Compound 4-1* (2 g, 5.01 mmol) and Pd/C (200 mg, 10%) in THF (30 mL) was hydrogenated under 15 psi of hydrogen at 25° C. for 3 h. The mixture was filtered through a pad of celite and the filtrate was concentrated in vacuum to afford Compound 4-2 (1.8 g, crude) as an off-white solid.

[0218] Synthesis of Compound 4-3. To a solution of Compound 4-2 (1.8 g, 4.47 mmol) in THF (25 mL) was added a solution LiAlH.sub.4 (339 mg, 8.94 mmol) in THF (5 mL) drop wise below 15° C. The solution was stirred at 15° C. for 2 h. The reaction was quenched by the addition of saturated aqueous NH.sub.4Cl (20 mL) at 0° C. The resulting mixture was extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (2×30 mL) and concentrated in vacuum to afford Compound 4-3 (1.6 g, crude) as a light yellow solid.

[0219] Synthesis of Compound 4-4. A mixture of Compound 4-3 (1.6 g, 4.27 mmol) in DCM (10 mL) and THE (10 mL) was added PCC (2.27 g, 10.6 mmol) at 25° C. The reaction was stirred at 25° C. for 3 hrs. The solution was filtered and the filter cake was washed with DCM (25 mL). The combined filtrate was concentrated in vacuum. The residue was purified by silica gel colum, eluting with PE/EtOAc=8/1 to give Compound 4-4 (0.9 g, 54%) as an off-white solid.

[0220] Synthesis of Compound 4-5. To a solution of Compound 4-4 (0.9 g, 2.41 mmol) in THF (30 mL) was added drop wise isopropyl magnesium chloride (3.61 mL, 7.23 mmol, 2M in THF) at −78° C. The mixture was stirred at −78° C. for 2 hrs. Then, the mixture was allowed to warm up to 25° C. and stirred for 3 hrs. The reaction was poured into water (100 mL) and extracted with EtOAc (2×30 mL). The combined organic layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuum. The residue was purified by silica gel colum, eluting with PE/EtOAc=5/1 to afford Compound 4-5 (0.6 g, 57%) as an off-white solid.

[0221] Synthesis of Compound 4. To a solution of Compound 4-5 (200 mg, 479 μmol) in pyridine (3 mL) was added SO.sub.3-Py (76.1 mg, 4.79 mmol), and the mixture was stirred at 40° C. for 16 hours. Pyridine was removed under reduced pressure and the residue was diluted with NaOH (3% in water, 20 mL), then extracted with BuOH (10 mL×3). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated, and purified by combi-flash (DCMin MeOH=100%-75%) to give Compound 4 (6 mg, 2%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 5.25-5.19 (m, 1H), 4.35 (brs, 1H), 3.85-3.75 (m, 1H), 1.95-0.79 (m, 41H), 0.65 (m, 3H). LCMS MS ESI calcd. for C.sub.28H.sub.47O.sub.4S [M+H—H.sub.2O].sup.+ 479, found 479.

Example 5. Synthesis of Compound 5

[0222] ##STR00032##

[0223] Synthesis of Compound 5-2A and Compound 5-2B. Molecular iodine (1.10 g, 4.34 mmol) was added to a solution of trimethyl phosphite (584 mg, 4.71 mmol) in CH.sub.2Cl.sub.2 (3 mL) at 0° C. After stirring for 5 minutes, the clear, colorless solution was warmed to 20° C. and added dropwise to a solution of Compound 5-1 (500 mg, 1.24 mmol) and pyridine (783 mg, 9.92 mmol) in CH.sub.2Cl.sub.2 (2 mL) at 0° C. The resulting mixture was stirred at 0° C. for 1 h, then warmed to 20° C. and stirred for another 1 h. The reaction mixture was treated with water (20 mL) and extracted with CH.sub.2Cl.sub.2 (20 mL×2). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, concentrated under vacuum, and purified on silica gel (PE/EtOAc=8/1 to 3/1) to afford Compound 5-2A (200 mg, 32%) as an off-white solid and Compound 5-2B (50 mg, 7%) as a light yellow oil. Compound 5-2A: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.39-5.35 (m, 1H), 4.30-4.18 (m, 2H), 3.77 (s, 6H), 3.74 (s, 6H), 2.44-2.40 (m, 2H), 1.98-1.57 (m, 8H), 1.48-0.92 (m, 28H), 0.67 (s, 3H). Compound 5-2B: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.39-5.36 (m, 1H), 4.25-4.18 (m, 1H), 3.77 (s, 3H), 3.74 (s, 3H), 3.35-3.25 (m, 1H), 2.44-2.40 (m, 2H), 1.99-1.57 (m, 8H), 1.57-0.88 (m, 29H), 0.68 (s, 3H).

[0224] Synthesis of Compound 5. To a solution of Compound 5-2A (50 mg, 80.8 μmol mol) in CH.sub.2Cl.sub.2 (3 mL) was added TMSBr (74.1 mg, 484.8 μmolμmol) at 20° C. and the mixture stirred for 12 hrs, at which point TLC analysis indicated the starting material was consumed completely. The reaction mixture was adjusted to pH=8 with aq. NaOH (484 uL, 484 μmol, 1 M in H.sub.2O) and a precipitate was formed. The white solid was filtered and washed with CH.sub.2Cl.sub.2 (2 mL) and water (2 mL), and dried under vacuum to afford Compound 5 (9.8 mg, 19%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 5.30-5.25 (m, 1H), 3.95-3.80 (m, 2H), 2.37-2.26 (m, 1H), 2.26-2.15 (m, 1H), 1.96-1.77 (m, 6H), 1.55-0.79 (m, 30H), 0.62 (s, 3H). LCMS MS ESI calcd. for C.sub.27H.sub.43 [M+H-2H.sub.3PO.sub.4].sup.+ 367, found 367. HRMS MS ESI calcd. for C.sub.27H.sub.47O.sub.8P.sub.2 [M−H].sup.− 561.2752, found 561.2760.

Example 6. Synthesis of Compound 6

[0225] ##STR00033##

[0226] Synthesis of Compound 3-2. Iodine (1.17 g, 4.63 mmol) was added to a solution of trimethyl phosphite (526 mg, 4.24 mmol) in CH.sub.2Cl.sub.2 (20 mL) at 0° C. After stirring for 5 minutes, the clear, colorless solution was allowed to warm to 15° C., after which it was added dropwise to a solution of Compound 1-1 (1.21 g, 15.4 mmol) in CH.sub.2Cl.sub.2 (30 mL) at 0° C. The resulting mixture was stirred at 0° C. for 1 h and then warmed to 15° C. and stirred for another 1 h. The reaction mixture was treated water (50 mL), extracted with CH.sub.2Cl.sub.2(50 mL×2), and the combined organic phase was washed with brine (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue was purified on silica gel (PE/EtOAc=10/1 to 3/1) to afford Compound 3-2 (1.8 g, 75%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.35-5.30 (m, 1H), 4.20-4.18 (m, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.48-3.46 (m, 1H), 2.26-2.17 (m, 2H), 1.95-1.61 (m, 7H), 1.48-0.88 (m, 41H), 0.66 (s, 3H), 0.05 (s, 6H).

[0227] Synthesis of Compound 6-3A and Compound 6-3B. Compound 3-2 (1.5 g, 2.40 mmol) was dissolved in MeOH (20 mL) and separated by supercritical fluid chromatography (SFC) to afford Compound 6-3A (490 mg, 33%) and Compound 6-3B (400 mg. 27%) as off-white solids. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.25 (m, 1H), 4.20-4.18 (m, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.48-3.44 (m, 1H), 2.30-2.10 (m, 2H), 1.98-1.57 (m, 7H), 1.53-0.88 (m, 38H), 0.67 (s, 3H), 0.05 (s, 6H). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.25 (m, 1H), 4.20-4.17 (m, 1H), 3.76 (s, 3H), 3.73 (s, 3H), 3.50-3.44 (m, 1H), 2.26-2.10 (m, 2H), 1.98-1.62 (m, 11H), 1.48-0.88 (m, 34H), 0.67 (s, 3H), 0.05 (s, 6H). For 6-3B, 91% de was obtained that was not subjected to deprotection.

[0228] Synthesis of 6. To a solution of Compound 6-3A (100 mg, 160 μmol) in CH.sub.2Cl.sub.2 (3 mL) was added TMSBr (97.9 mg, 640 μmol) and the reaction was stirred f at 20° C. or 12 h. The reaction mixture was adjusted to pH=8 with aq. NaOH (640 uL, 640 μmol, 1 M in H.sub.2O) and the solid was precipitated. The white solid was filtered and washed with CH.sub.2Cl.sub.2 (2 mL), water (2 mL), dried by vacuum to provide Compound 6 (45.7 mg, 54%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 5.30-5.20 (m, 1H), 3.89-3.80 (m, 1H), 3.26-3.20 (m, 1H), 2.15-1.60 (m, 8H), 1.55-0.80 (m, 30H), 0.63 (s, 3H). LCMS Rt=1.330 min in 2.0 min chromatography, 10-80 AB, MS ESI calcd. for C.sub.27H.sub.43 [M+H—H.sub.3PO.sub.4—H.sub.2O].sup.+ 367, found 367. HRMS MS ESI calcd. for C.sub.27H.sub.46O.sub.5P [M−H].sup.− 481.3088, found 481.3085.

Example 7. Synthesis of Compound 7

[0229] ##STR00034##

To a solution of Compound 4-1 (0.1 g, 0.239 mmol) in pyridine (2 mL) was added dihydrofuran-2,5-dione (71.7 mg, 0.717 mmol) and DMAP (14.5 mg, 0.119 mmol). The mixture was stirred at 15° C. for 16 hours. To the reaction was added aqueous sat. NH.sub.4Cl (10 mL) and extracted with EtOAc (2×5 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (PE: EA=100%-70%, DCM: MeOH=100%-95%) to give Compound 7 (40 mg) as colorless oil, which was washed with PE (5 mL) and filtered to give Compound 7 (10 mg, yield 8%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.29 (m, 1H), 4.73-7.70 (m, 1H), 2.67-2.63 (m, 4H), 2.45-2.35 (m, 1H), 1.99-1.95 (m, 3H), 1.85-0.80 (m, 39H), 0.66 (s, 3H). LCMS t.sub.R=1.591 min in 2 min chromatography, 10-80AB_ELSD, MS ESI calcd. for C.sub.32H.sub.52O.sub.5Na [M+Na].sup.+ 539, found 539.

Example 10. Synthesis of Compound 10

[0230] ##STR00035##

To a solution of Compound 10-1 (50 mg, 0.124 mmol; synthesized as described in Upasani et al., WO2013/36835, [00375]) in pyridine (2 mL) was added dihydrofuran-2,5-dione (37.2 mg, 0.372 mmol) and DMAP (7.57 mg, 0.062 mmol). The mixture was stirred at 15° C. for 16 hours and then another batch of dihydrofuran-2,5-dione (37.2 mg, 0.372 mmol) and DMAP (7.57 mg, 0.062 mmol) was added. The mixture was stirred at 15° C. for 16 hours. The reaction solution was quenched with aqueous sat. NH.sub.4Cl (5 mL) and extracted with EtOAc (3 mL×2). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated. The residue was purified by combi-flash (0˜30% of EtOAc in PE) to give 50 mg of impure Compound 10. The mixture was further purified by prep-HPLC (column: Gemini 150*25 5u, gradient: 65-65% B (A=0.05% HCl-ACN, B=acetonitrile) to give Compound 10 (5 mg, 8% yield) as an off-white solid. .sup.1H NMR (400 MHz, CDCl3) δ 5.35-5.25 (m, 1H). 2.60-2.55 (m, 5H), 2.45-2.35 (m, 2H), 2.10-0.80 (m, 39H), 0.67 (s, 3H). LCMS t.sub.R=1.523 min in 2 min chromatography, 10-80AB_ELSD, MS ESI calcd. for C.sub.31H.sub.50O.sub.5Na [M+Na].sup.+ 525, found 525.

Example 11. Synthesis of Compound 11

[0231] ##STR00036##

Synthesis of Compound 11-2. To a solution of Compound 5-1 (300 mg, 745 μmol) and (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (423 mg, 2.41 mmol) in DCM (5 mL) was added DCC (767 mg, 3.72 mmol), DMAP (45 mg, 368 μmol), TEA(452 mg, 4.47 mmol). The mixture was stirred at 30° C. for 3 hours. The mixture was washed by brine (10 mL) and extracted by EtOAc (20 mL×2). The combined organic layer was dried by Na.sub.2SO.sub.4, filtered and evaporated in vacuum. The residue was purified by silica gel chromatography (PE/EtOAc=5/1) to afford Compound 11-2 (300 mg, 54% yield) as a yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.40-5.37 (m, 1H), 5.17-5.04 (m, 2H), 4.76-4.61 (m, 2H), 4.30-4.17 (m, 2H), 2.36-2.30 (m, 2H), 2.00-1.95 (m, 2H), 1.88-1.77 (m, 4H), 1.61-0.88 (m, 54H), 0.67 (s, 3H).
Synthesis of Compound 11. Compound 11-2 (150 mg, 209 μmol) was added to HCl/dioxane (3 mL, 4M). The mixture was stirred at 25-27° C. for 1 hour. The mixture was filtered and washed with MTBE (5 mL×2), dried in vacuum to give an off-white solid. The solid was dissolved in water/MeCN (5 mL/1 mL) and lyophilized 3 times to remove residual solvent to afford Compound 11 (71 mg, yield 43% yield) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) δ5.45-5.44 (m, 1H), 4.74-4.66 (m, 1H), 4.17-4.05 (m, 2H), 2.44-2.37 (m, 2H), 2.09-1.87 (m, 6H), 1.72-0.95 (m, 37H), 0.75 (s, 3H). LCMS Rt=0.831 min in 2.0 min chromatography, 30-90 AB, MS ESI calcd. for C.sub.33H.sub.57N.sub.2O.sub.4 [M+H].sup.+ 545, found 545.5.

Example 12. Synthesis of Compound 12

[0232] ##STR00037##

Synthesis of Compound 12-2. To a solution of Compound 1-1 (500 mg, 967 μmol) in DCM (5 mL) was added (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (586 mg, 3.10 mmol), DCC (764 mg, 3.71 mmol), TEA (376 mg, 3.72 mmol), DMAP (151 mg, 1.24 mmol). The mixture was stirred at 30° C. for 3 hours. The mixture was washed by water (10 mL) and extracted with EtOAc (20 mL×2). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated to give a residue, which was diluted with water (15 mL). The suspension was heated at 60° C. for 30 minutes. The mixture was filtered to give Compound 12-2 (579 mg, 1.00 mmol) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.31 (d, 1H), 5.19 (s, 1H), 5.08 (d, 1H), 4.79-4.62 (m, 1H), 4.38-4.06 (m, 1H), 3.56-3.39 (m, 1H), 2.35-2.10 (m, 2H), 2.08-1.90 (m, 2H), 1.90-1.66 (m, 5H), 1.65-1.32 (m, 21H), 1.30-0.79 (m, 28H), 0.66 (s, 3H), 0.17-0.01 (m, 6H).
Synthesis of Compound 12-3. To a solution of Compound 12-2 (300 mg, 435 μmol) was added TBAF/THF (5 mL, 1 M). The mixture was stirred at 23-25° C. for 30 minutes. The mixture was diluted with water (20 mL) and extracted by EtOAc (20 mL×2). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated to give a residue, which was purified by SFC ((Column: AD (250 mm*30 mm, 5 um), Condition: Base-ETOH, Begin: B 25% FlowRate(ml/min): 70) to give Compound 12-3 (392 mg) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.53-5.20 (m, 1H), 5.09 (d, 1H), 4.86-4.57 (m, 2H), 4.30 (t, 1H), 3.84-3.35 (m, 1H), 2.52-2.17 (m, 2H), 2.04-1.92 (m, 2H), 1.89-1.75 (m, 4H), 1.69-0.82 (m, 42H), 0.67 (s, 3H).
Synthesis of Compound 12. Compound 12-1 (300 mg, 522μmol) was added to HCl/dioxane (3 ml, 4M). The mixture was stirred at 25-27° C. for 1 hour. The mixture was filtered and washed with MTBE (5 ml×2), dried in vacuum to give an off-white solid. The residue was dissolved in water/MeCN (5 mL/1 mL) and lyophilized 3 times to removal residual solvents to give Compound 12-2 (90 mg, 34% yield) as an off white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.36 (d, 1H), 4.95-4. 4.70 (m, 3H), 3.80-3.70 (m, 1H), 3.46-3.36 (m, 1H), 2.28-2.16 (m, 2H), 2.10-1.75 (m, 6H), 1.76-1.61 (m, 2H), 1.62-1.39 (m, 11H), 1.38-1.25 (m, 1H), 1.25-1.07 (m, 4H), 1.04 (s, 3H), 1.01-0.91 (m, 10H), 0.74 (s, 3H). LCMS Rt=0.991 min in 2.0 min chromatography, 30-90 AB, MS ESI calcd. for C.sub.30H.sub.51NO.sub.3Na [M+Na].sup.+ 496.39, found 496.3.

Example 13. Synthesis of Compound 13

[0233] ##STR00038##

Step 1. To a solution of 13-1 (7.0 g, 16.2 mmol) in THF (70 mL) was added dropwise ethylmagnesium bromide (26.9 mL, 80.9 mmol, 3M in Et.sub.2O) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at 25° C. for 12 h. TLC showed the starting material was consumed completely. The mixture was quenched with saturated aqueous NH.sub.4Cl (100 mL) and extracted with EtOAc (200 mL×2). The combined organic phase was washed with brine (200 mL), dried over anhydrous Na.sub.2SO.sub.4, concentrated in vacuum. The residue was purified by column chromatography on silica gel (PE/EtOAc=20/1) to afford 13-2 (500 mg, 7.7%) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 5.30-5.28 (m, 1H), 2.42-2.39 (m, 5H), 1.98-1.63 (m, 8H), 1.53-1.25 (m, 16H), 1.23-0.84 (m, 28H), 0.66 (s, 3H).
Step 2. To a solution of 13-2 (500 mg, 1.24 mmol) in MeOH (10 ml) was added NaBH.sub.4 (93.8 mg, 2.48 mmol) in portions. The reaction mixture was stirred at 25° C. for 2 h. After TLC showed the starting material was consumed and a new spot was produced. The reaction mixture was quenched with saturated aqueous NH.sub.4Cl (10 mL), extracted with EtOAc (30 mL×2). The combined organic phase was washed with brine (30 mL), concentrated in vacuum. The residue was purified by column chromatography on silica gel (PE/EtOAc=20/1) to afford 13-3 (500 mg, crude), which was purified by prep-HPLC to afford the pure 13-3 (60 ng, 12%) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 5.29 (d, J=4.4 Hz, 1H), 3.48-3.47 (m, 1H), 2.42 (d, J=12.8 Hz, 1H), 2.02-1.57 (m, 12H), 1.57-0.92 (m, 26H), 0.67 (s, 3H).
Step 3. To a solution of Compound 13-3 (200 mg, 496 μmol) in pyridine (2 mL) was added DMAP (30.3 mg, 248 μmol) and dihydrofuran-2,5-dione (199 mg, 1.98 mmol). The mixture was stirred at 25-27° C. for 16 hr. The reaction mixture was washed by aqueous sat.NH.sub.4Cl(3 ml) and extracted with ethyl acetate (2 mL×2). The combined organic layer was concentrated in vacuum. The residue was purified by silica gel chromatography (PE/EtOAc=1/1) to afford Compound 13 (42 mg, 17% yield for mixture of diastereomers) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δδ 5.35-5.22 (m, 1H), 4.87-4.70 (m, 1H), 2.79-2.54 (m, 4H), 2.42 (d, 1H), 2.09-1.91 (m, 3H), 1.89-1.64 (m, 3H), 1.64-1.30 (m, 13H), 1.32-0.76 (m, 21H), 0.67 (s, 3H). LCMS Rt=2.265 min in 3.0 min chromatography, 10-80 AB, MS ESI calcd. for C.sub.31H.sub.50O.sub.5Na [M+Na].sup.+ 525.37, found 525.3.

Example 14. Synthesis of Compound 14

[0234] ##STR00039##

[0235] Synthesis of Compound 14-2. To a solution of Compound 1-1 (0.3 g, 0.58 mmol) in pyridine (2 mL) was added Py-SO.sub.3 (276 mg, 1.74 mmol). The mixture was stirred at 40° C. for 16 hours. The mixture was concentrated and the residue was diluted with NaOH (20 mL, 3% in water). The mixture was extracted with PE (10 mL) and the organic layer was separated. The aqueous layer was extracted with BuOH (2×15 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated to give Compound 14-2 (400 mg, crude) as yellow solid. .sup.1H NMR (400 MHz, MeOD) δ 5.34-5.33 (m, 1H), 4.14-4.11 (m, 1H), 3.58-3.54 (m, 1H), 2.30-0.85 (m, 48H), 0.74 (s, 3H), 0.08 (s, 6H).

Synthesis of Compound 14. To a solution of Compound 14-2 (200 mg, 0.335 mmol) in DCM (5 ml) was added HF-Py (2 mL). The mixture was stirred at 15° C. for 16 hours. The reaction mixture was diluted with NaOH solution (10 mL, 3% in water) and extracted with DCM (2×5 mL). The combined organic layer was washed with NaOH solution (10 mL, 3% in water), separated, dried over Na.sub.2SO.sub.4, filtered and concentrated to give a residue, which was purified by combi-fish (PE: EA=100%-60%, DCM: MeOH=100%-90%) to give 50 mg of Compound 14 as colorless oil. The oil was crystallized from H.sub.2O to give Compound 14 (10 mg, 6%) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.36-5.35 (m, 1H), 4.63 (s, 1H), 4.17-4.13 (m, 1H), 3.43-3.38 (m, 1H), 2.24-2.20 (m, 2H), 2.15-0.85 (m, 37H), 0.74 (s, 3H). LCMS t.sub.R=1.271 min in 2 min chromatography, 10-80AB_ELSD, MS ESI calcd. for C.sub.27H.sub.45O.sub.4S [M+H—H.sub.2O].sup.+ 465, found 465.

Example 16. Synthesis of Compounds 16 and 17

[0236] ##STR00040##

Synthesis of Compound 5-3. To a solution of Compound 5-1 (1.5 g, 3.72 mmol), Boc-Ala-OH (703 mg, 3.72 mmol), DMAP (45.4 mg, 0.37 mmol), TEA (376 mg, 3.72 mmol) in DCM (20 mL) was added DCC (767 mg, 3.72 mmol) at 15° C. The mixture was stirred at 15° C. for 20 hrs. The reaction mixture was filtered and the filtrate was concentrated in vacuum to give a crude product, which was purified by flash column (EtOAc in PE, 0-30% in 60 minutes) to give Compound 5-3 (600 mg, 28%).
Synthesis of Compounds 16 and 17. To a solution of Compound 5-3 (600 mg) in dioxane (4 mL) was added HCl (2 mL, 4 M in dioxane) at 15° C. The mixture was stirred at 15° C. for 16 hrs. MTBE (15 mL) was added and an off-white solid was produced. The mixture was filtered. The filtered cake was washed with MTBE, concentrated in vacuum to give an off-white solid (600 mg), which was purified by prep. HPLC (Column: Phenomenex Gemini 150*25 mm*10um; condition: water (0.05% HCl)-ACN, 50-60% B in 10 mins, 100% B Hold Time(min): 4; FlowRate(ml/min): 25) to give Compound 16 (9 mg 11%) and Compound 17 (33 mg, 40%).
Compound 16: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.40-8.30 (br, 3H), 5.45-5.30 (m, 1H), 4.70-4.50 (m, 1H), 4.23-4.10 (m, 1H), 4.10-3.98 (m, 1H), 3.10-3.00 (m, 1H), 2.40-2.20 (m, 2H), 2.03-1.71 (m, 5H), 1.67-10.88 (m, 28H), 0.86-0.78 (m, 6H), 0.66 (s, 3H). LCMS Rt=1.014 min in 2.0 min chromatography, 30-90 AB_E, MS ESI calcd. for C.sub.27H.sub.45O [M+H-AlaOH].sup.+ 385, found 385.
Compound 17: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.40-8.30 (br, 3H), 5.45-5.30 (m, 1H), 4.70-4.50 (m, 1H), 4.234.10 (in, 1H), 4.10-3.98 (m, 1H), 3.10-3.00 (m, 1H), 2.40-2.20 (m, 2H), 2.03-1.71 (m, 5H), 1.67-0.88 (m, 28H), 0.86-0.78 (m, 6H), 0.66 (s, 3H). LCMS Rt=1.011 min in 2.0 min chromatography. 30-90 AB_E, MS ESI calcd. C.sub.27H.sub.45O [M+H-AlaOH].sup.+ 385, found 385.

Example 17. Synthesis of Compound 18

[0237] ##STR00041##

Synthesis of Compound 5-4. To a solution of Compound 5-1 (100 mg, 248 μmol) in DCM (2 mL) at 13˜18° C. was added N,N-dimethylpyridin-4-amine (3 mg, 24.5 μmol), triethylamine (25 mg, 247 μmol), (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (47 mg, 248 μmol) and N,N′-methanediylidenedicyclohexanamine (51 mg, 247 μmol). The reaction was stirred over 16 hrs at 20° C. The reaction was filtered and filtrate was concentrated. The residue was purified by silica gel chromatography (PE/EtOAc=20/1) to give the desired product (45 mg, 24%) as a solid.
Synthesis of Compound 18. To a solution of Compound 5-4 (45 mg, 78.4 μmol) in dioxane (1 mL) was added HCl/dioxane (4 M, 1 mL) at 15-28° C. The reaction mixture was stirred for 3 hrs. and then 5 mL of sat. NaHCO.sub.3 was added so the reaction was at pH=9. The mixture was extracted with EtOAc (3×10 mL). The combined organic layer was washed with brine (10 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH=15/1) to give Compound 18 (3.2 mg, 8%) as an off-white solid. .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 5.40-5.39 (m, 1H), 4.67-4.64 (m, 1H), 3.56-3.54 (m, 1H), 3.34-3.33 (m, 1H), 2.35-2.33 (m, 2H), 2.05-1.90 (m, 2H), 1.88 (m, 3H), 1.67-0.91 (m, 37H), 0.70 (s, 3H). LCMS Rt=1.866 min in 3.0 min chromatography, 10-80 AB, MS ESI calcd. for C.sub.27H.sub.45O [M+H-AlaOH].sup.+ 385, found 385.

Example 18. Synthesis of Compound 19

[0238] ##STR00042##

Synthesis of Compound 18-2. To a solution of Compound 18-1 (100 mg, 0.225 mmol; synthesized as described in Martinez et al., WO2014/160480) in DCM (1 mL) was added DMAP (8.2 mg, 0.0675 mmol), Boc-Vla-OH (146 mg, 0.675 mmol), TEA (68.3 mg, 0.675 mmol) and DCC (139 mg, 0.675 mmol). The mixture was stirred at 25° C. for 16 hours. The mixture was concentrated under vacuum, purified by column chromatography on silica gel (PE/EtOAc=15:1) to give Compound 18-2 (80 mg, 55%) as an off-white solid.

[0239] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.35-5.20 (m, 2H), 5.00-4.90 (m, 1H), 4.38-4.25 (m, 1H), 2.48-2.37 (m, 1H), 2.25-2.15 (m, 1H), 2.02-1.90 (m, 3H), 1.85-0.85 (m, 46H), 0.67 (s, 3H).

Synthesis of Compound 19. To a solution of Compound 18-2 (70 mg, 0.124 mmol) in dioxane (0.5 mL) was added HCl/dioxane (1 mL, 4 M). The mixture was stirred at 25° C. for 2 hours and an off-white solid was formed. To the reaction mixture was added MTEB (10 mL) and the reaction was filtered. The solid was washed with MTEB (10 mL) and then dissolved in MeOH (10 mL). The MeOH solution was concentrated in vacuum to give Compound 19 (55.6 mg, 77%) as an off-white solid. .sup.1H NMR (400 MHz, methanol-d4) δ 5.60-5.50 (m, 1H), 5.34-5.25 (m, 1H), 4.16 (d, J=4.0 Hz, 1H), 2.48-2.30 (m, 2H), 2.05-1.40 (m, 16H), 1.35-0.90 (m, 23H), 0.74 (s, 3H). LCMS Rt=0.970 min in 2.0 min chromatography. 30-90 AB, MS ESI calcd. for C.sub.31H.sub.51F.sub.3NO.sub.3 [M+H].sup.+ 542, found 542.

Example 19. Synthesis of Compound 20

[0240] ##STR00043##

To a solution of Compound 18-1 (50 mg, 0.122 mmol) in pyridine (1 mL) was added DMAP (6.8 mg, 0.056 mmol) and dihydrofuran-2,5-dione (33.6 mg, 336 mmol). The mixture was stirred at 25° C. for 16 hours. The mixture was concentrated under vacuum, purified by column chromatography on silica gel (PE/EtOAc=1:1) to give Compound 20 (14.1 mg, yield 23%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.35-5.25 (m, 2H), 2.78-2.65 (m, 4H), 2.46-2.37 (m, 1H), 2.05-1.90 (m, 4H), 1.70-0.90 (m, 30H), 0.67 (s, 3H). LCMS Rt=1.226 min in 2.0 min chromatography, 30-90 AB, MS ESI calcd. for C.sub.30H.sub.44F.sub.3O.sub.4 [M+H—H.sub.2O].sup.+ 525, found 525.

Example 20. Synthesis of Compound 21

[0241] ##STR00044##

Synthesis of Compound 20-2. To a solution of Compound 18-1 (100 mg, 225 μmol) in DCM (3 mL) was added DMAP (2.22 mg, 67.5 μmol), TEA (68.3 mg, 675 μmol), DCC (17.5 mg, 675 mol) and (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (127 mg, 675 μmol). The mixture was stirred at 25° C. for 16 hours. The mixture was filtered, the filtrated was conentrated and purified by combi-flash (PE: EA=100%-95%) to give Compound 20-2 (120 mg, 87%) as an off-white solid. .sup.1H NMR CDCl.sub.3 400 MHz 6 5.33-5.30 (m, 2H), 2.46-2.43 (m, 1H), 2.07-0.87 (m, 48H), 0.70 (s, 3H).
Synthesis of Compound 21. To a solution of Compound 20-2 (120 mg, 195 μmol) was added HCl/dioxane (4N, 1 mL). The mixture was stirred at 25° C. for 0.5 hour. To the mixture was added MTBE (3 mL) and stirred at 25° C. for 10 minutes. The mixture was filtered. The solid was washed with MTBE (3 mL), dried in vacuum to give Compound 21 (60 mg, 56%). .sup.1H NMR (400 MHz, MeOD) δ 5.52-5.51 (m, 1H), 5.32-5.31 (m, 1H), 4.30-4.26 (m, 1H), 2.46-2.43 (m, 1H), 2.05-1.00 (m, 36H), 0.76 (s, 3H). LCMS R.sub.t=0.940 min in 2 min chromatography, 30-90AB, MS ESI calcd. for C.sub.29H.sub.47F.sub.3NO.sub.3 [M+H].sup.+ 514, found 514.

Example 21. Synthesis of Compound 22

[0242] ##STR00045##

Step 1. To a mixture of Compound 18-1 (50 mg, 112 μmol) and DMAP (6.84 mg, 56.0 μmol) in pyridine (2 mL) was added pivalic anhydride (104 mg, 560 μmol) in one portion at 20° C. The mixture was stirred at 60° C. for 16 hrs. The resulting mixture was concentrated to give a residue, which was diluted with water (30 mL) and extracted with EtOAc (2×15 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum to give a crude product, which was purified by silica gel chromatography (PE/EtOAc=8/1) to give Compound 22 (14 mg, 24%) as an off-white solid. The reaction was conducted for a second time to give 30 mg of impure product. The 2 batches of product (30 mg) were combined and triturated with hexane (5 mL) to give Compound 22 (23 mg, 52%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.33-5.19 (m, 2H), 2.45-2.38 (m, 1H), 2.03-1.91 (m, 3H), 1.86-0.80 (m, 40H), 0.60 (s, 3H). LCMS t.sub.R=1.575 min in 2 min chromatography, 30-90AB_ELSD, MS ESI calcd. for C.sub.31H.sub.4s F.sub.3O.sub.2 [M+H—H.sub.2O].sup.+ 509, found 509.

Example 21. Synthesis of Compound 23

[0243] ##STR00046##

Step 1. To a solution of Compound 18-1 (100 mg, 0.225 mmol) in THF (5 mL) was added NaH (22.3 mg, 0.562 mmol, 60%) under N.sub.2 at 0° C. The mixture was stirred at 20° C. for 30 minutes. Acetyl chloride (35.3 mg, 0.45 mmol) was added. The reaction solution was stirred at 20° C. for 30 minutes. The mixture was quenched with Sat. NH.sub.4Cl(10 mL) and extracted with MTBE (3×5 mL). The combined organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-8% of EtOAc in PE) to give Compound 23 (39 mg, 35%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.29 (m, 2H), 2.48-2.38 (m, 1H), 2.14 (s, 3H), 2.05-1.95 (m, 3H), 1.94-1.56 (m, 6H), 1.54-1.47 (m, 8H), 1.45-0.92 (m, 17H), 0.59 (s, 3H). LCMS t.sub.R=1.383 min in 2 min chromatography, 30-90AB_ELSD, MS ESI calcd. for C.sub.28H.sub.42 F.sub.3O.sub.2 [M+H—H.sub.2O].sup.+ 467, found 467.

Example 22. Synthesis of Compound 24

[0244] ##STR00047##

Step 1. To a solution of Compound 22-1 (500 mg, 1.09 mmol; synthesized as described in Martinez et al., WO2014/160480, [00199]) in pyridine (3 mL) was added SO.sub.3—Py (519 mg, 3.27 mmol). The mixture was stirred at 50° C. for 1h. The mixture was diluted with water (10 mL) and extracted with DCM (2×10 mL). The combined organic phase was washed with water (10 mL). To the organic phase was added dropwise NaOH solution (2 mL, 3% in water) with stirring and some white solid appeared. The solid was collected by filtration and washed with DCM (10 mL) and water (10 mL). The solid was purified by preparative-HPLC ((column: DuraShell 150*25 mm*5 um, gradient: 3B (water(10 mM NH.sub.4HCO.sub.3)), flow rate: 30 mL/min). After the purification was finished, to the combined eluent was added DCM (10 mL) and added dropwise NaOH solution (2 mL, 3% in water). Compound 24 (62 mg, 10%) was obtained by collection and lyophilization to give an off-white solid. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 5.36-5.35 (m, 1H), 2.88-2.85 (m, 1H), 2.35-2.31 (m, 1H), 2.21-2.01 (m, 2H), 2.00-1.75 (m, 4H), 1.70-1.43 (m, 10H), 1.41 (s, 3H), 1.38-1.30 (m, 1H), 1.27 (s, 3H), 1.22-1.10 (m, 5H), 1.09-1.02 (m, 4H), 1.00-0.92 (m, 4H), 0.75 (s, 3H). LCMS Rt=1.683 min in 2 min chromatography, 10-80CD_ELSD, MS ESI calcd. for C.sub.27H.sub.42F.sub.3O.sub.5S [M].sup.− 535, found 535.

Example 23. Synthesis of Compound 25

[0245] ##STR00048##

Step 1. To a solution of Compound 22-1 (500 mg, 1.09 mmol) in THF (10 mL) was added NaH (108 mg, 2.72 mmol, 60%) under N.sub.2 at 0° C. The mixture was stirred at 20° C. for 30 minutes. Then acetyl chloride (171 mg, 2.18 mmol) was added. The reaction solution was stirred at 20° C. for 1 h. The mixture was quenched with Sat. NH.sub.4Cl (10 mL) and extracted with EtOAc (3×10 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-30% of EtOAc in PE, 60 mins) to give Compound 25 (26 mg, 5%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.35-5.25 (m, 1H), 2.50-2.40 (m, 1H), 2.30-2.10 (m, 1H), 2.06 (s, 3H), 2.00-1.60 (m, 12H), 1.53-1.35 (m, 7H), 1.30-0.90 (m, 17H), 0.67 (s, 3H). LCMS Rt=3.743 min in 7.0 min chromatography, 50-100 AB_E, MS ESI calcd. for C.sub.29H.sub.44F.sub.3O.sub.2[M+H—H.sub.2O].sup.+ 481, found 481.

Example 24. Synthesis of Compound 26

[0246] ##STR00049##

Step 1. To a solution of Compound 24-1 (100 mg, 239 μmol; synthesized as described in Martinez et al., WO2014/160480, [00210]) in pyridine (2 mL) was added DMAP (30.5 mg, 478 μmol) and dihydrofuran-2,5-dione (119 mg, 1.19 mmol). The mixture was stirred at 60° C. for 40 hrs. The reaction mixture was washed by Sat.NH.sub.4Cl (3 ml) and extracted with ethyl acetate (2×2 ml). The combined organic layer was concentrated in vacuum. The residue was purified by silica gel chromatography (petroleum PE:EtOAc=10:1) to afford Compound 26 (35 mg, yield 28%) as an off-white solid. .sup.1H NMR CDCl.sub.3 400 MHz 8 5.35-5.30 (m, 1H), 2.64-2.61 (m, 5H), 2.58-2.56 (m, 1H), 2.01-0.86 (m, 42H), 0.67 (s, 3H). LCMS R.sub.t=1.341 min in 2 min chromatography, 30-90AB, MS ESI calcd. for C.sub.28H.sub.45 [M+H—H.sub.2O—HOOCCH.sub.2CH.sub.2COOH].sup.+ 381, found 381.

Example 25. Synthesis of Compounds 27 and 28

[0247] ##STR00050##

Step 1. To a solution of Compound 25-1 (900 mg, 2.23 mmol) in pyridine (20 mL) was added dihydrofuran-2,5-dione (1.11 g, 11.1 mmol) and DMAP (272 mg, 2.23 mmol). The mixture was stirred at 80° C. for 96 hrs. The reaction solution was quenched with sat.NH.sub.4Cl (25 mL) and extracted with EtOAc (3×10 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated. The reaction mixture was separated by combi-flash (0-10% of (MeOH: EtOAc, v: v=1:20) in DCM: PE, v: v=1:2) and the recycled starting material was reused in this experiment (this procedure was repeat for 4 times). The combined impure product was purified by preparative-HPLC (column: Boston Green ODS 150*30 5u), gradient: 65-95% B (A=0.05% HCl/H.sub.2O, B=MeCN), flow rate: 75 mL/min) to give Compound 27 (40 mg, 34%) and Compound 28 (55 mg, 4%) as off-white solids.
27: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.29 (m, 1H), 2.65-2.55 (m, 4H), 2.45-2.35 (m, 1H), 2.05-1.92 (m, 3H), 1.90-1.76 (m, 3H), 1.75-1.53 (m, 5H), 1.52-1.46 (m, 3H), 1.45-1.40 (m, 7H), 1.39-1.30 (m, 2H), 1.29-1.20 (m, 1H), 1.19-1.15 (m, 2H), 1.14-1.10 (m, 4H), 1.09-1.02 (m, 2H), 1.01-0.94 (m, 4H), 0.93-0.86 (m, 5H), 0.67 (s, 3H). LCMS Rt=1.268 min in 2.0 min chromatography, 30-90AB_ELSD, MS ESI calcd. for C.sub.27H.sub.43 [M+H—H.sub.2O—(CH.sub.2COOH).sub.2].sup.+ 367, found 367. HRMS MS ESI calcd. for C.sub.31H.sub.49O.sub.5[M−H].sup.− 501.3585, found 501.3575. 28: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.30-5.29 (m, 1H), 2.66-2.52 (m, 5H), 2.38-2.32 (m, 1H), 2.08-1.77 (m, 6H), 1.76-1.71 (m, 1H), 1.70-1.52 (m, 3H), 1.51-1.40 (m, 10H), 1.39 (s, 3H), 1.38-1.18 (m, 6H), 1.17-1.11 (m, 4H), 1.10 (s, 3H), 1.09-0.95 (m, 1H), 0.94-0.88 (m, 3H), 0.68 (s. 3H). LCMS Rt=1.280 min in 2.0 min chromatography, 30-90AB_ELSD, MS ESI calcd. for C.sub.27H.sub.43 [M+H—H.sub.2O—(CH.sub.2COOH).sub.2].sup.+ 367, found 367. HRMS MS ESI calcd. for C.sub.31H.sub.49O.sub.5 [M−H].sup.− 501.3585, found 501.3597.

Example 26. Synthesis of Compound 29

[0248] ##STR00051##

Step 1. To a solution of Compound 29-1 (1.5 g, 3.72 mmol) in DCM (30 mL) was added DMAP (226 mg, 1.86 mmol) and Py (8.65 g, 111 mmol), followed by adding bimethyl phosphorochloridate (2.68 g, 18.6 mmol) dropwise under N.sub.2. The reaction was stirred at 20° C. for 2 hrs. The mixture was quenched with sat.NaHCO.sub.3 (100 mL) and extracted with DCM (3×40 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-50% of EtOAc in PE/DCM (v/v=2/1)) to give Compound 29-2 (620 mg, 27%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.40-5.37 (m, 1H), 4.35-4.15 (m, 2H), 3.77 (s, 6H), 3.74 (s, 6H), 2.45-2.35 (m, 2H), 2.05-1.87 (m, 4H), 1.86-1.78 (m, 2H), 1.77-1.58 (m, 2H), 1.56-1.33 (m, 7H), 1.30-1.15 (m, 4H), 1.14-1.04 (m, 3H), 1.02 (s, 3H), 1.01-0.88 (m, 11H), 0.67 (s, 3H).
Step 2. To a solution of Compound 29-2 (200 mg, 0.323 mmol) in DCM (5 mL) was added TMSBr (246 mg, 1.61 mmol) under N.sub.2. The mixture was stirred at 20° C. for 16 hrs. The reaction mixture was adjusted to pH=9 with aq. NaOH (1.9 mL, 1 M) and the solid precipitated. The white solid was filtered and washed with CH.sub.2Cl.sub.2 (2 mL), water (2 mL), dried and lyophilized to give Compound 29 (68 mg, 37%) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.40-5.37 (m, 1H), 4.10-3.90 (m, 2H), 2.48-2.30 (m, 2H), 2.08-1.85 (m, 7H), 1.70-1.58 (m, 10H), 1.35-1.25 (m, 1H), 1.20-1.04 (m, 5H), 1.02 (s, 3H), 1.00-0.96 (m, 7H), 0.95-0.88 (m, 3H), 0.71 (s, 3H). LCMS t.sub.R=0.207 min in 3 min chromatography, 10-80CD_ELSD, MS ESI calcd. for C.sub.27H.sub.47O.sub.8P.sub.2 [M-4Na+4H—H].sup.− 561, found 561.

Example 27. Synthesis of Compound 30

[0249] ##STR00052##

Step 1. To a solution of 29-1 (1.5 g, 3.72 mmol) in DCM (30 mL) was added DMAP (226 mg, 1.86 mmol) and Py (8.65 g, 111 mmol), followed by adding bimethyl phosphorochloridate (2.68 g, 18.6 mmol) dropwise under N.sub.2. The reaction was stirred at 20° C. for 2 hrs. The mixture was quenched with sat.NaHCO.sub.3 (100 mL) and extracted with DCM (3×40 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-50% of EtOAc in PE/DCM (v/v=2/1)) to give 27-1 (700 mg, 37%) as an off-white solid.

[0250] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.40-5.37 (m, 1H), 4.28-4.15 (m, 1H), 3.80-3.70 (m, 6H), 3.35-3.25 (m, 1H), 2.45-2.40 (m, 2H), 2.02-1.90 (m, 3H), 1.89-1.76 (m, 2H), 1.75-1.58 (m, 2H), 1.56-1.33 (m, 8H), 1.30-1.15 (m, 7H), 1.14-0.96 (m, 5H), 0.95-0.88 (m, 10H), 0.67 (s, 3H).

Step 2. To a solution of 27-1 (200 mg, 0.391 mmol) in DCM (5 mL) was added TMSBr (298 mg, 1.95 mmol). The mixture was stirred at 20° C. for 16 hrs. Another TMSBr (596 mg, 3.9 mmol) was added at 0° C. The mixture was stirred at 20° C. for 16 hrs. The mixture was adjusted with NaOH (1M in H.sub.2O) to pH=9 and the solid was precipitated. The white solid was filtered and washed with CH.sub.2Cl.sub.2 (2 mL), water (2 mL), dissolved in MeOH (6 ml) and concentrated to give Compound 30 (16 mg, 8%) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) δ 5.42-5.29 (m, 1H), 4.04-3.82 (m, 1H), 3.25-3.15 (m, 1H). 2.63-2.41 (m. 1H), 2.35-2.11 (m, 1H), 2.10-1.80 (m, 4H), 1.79-1.37 (m, 10H), 1.37-1.27 (m, 2H), 1.26-1.05 (m, 6H), 1.02 (s, 3H), 0.99-0.77 (m, 13H), 0.72 (s, 3H). LCMS t.sub.R=1.311 min in 3 min chromatography, 10-80CD_ELSD, MS ESI calcd. for C.sub.27H.sub.46O.sub.5 P [M-2Na+2H—H].sup.− 481, found 481.

Example 28. Alternative Synthesis of Compound 6

[0251] ##STR00053##

Step 1. To a solution of 29-1 (1.4 g, 3.47 mmol) in DCM (25 mL) was added imidazole (471 mg, 6.94 mmol). A solution of TBSCl (1.39 g, 10.4 mmol) in DCM (5 mL) was added dropwise under N.sub.2. The reaction was stirred at 20° C. for 16 hrs. The mixture was quenched with water (30 mL) and extracted with DCM (2×15 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-5% of EtOAc in PE) to give 28-1 (1.5 g, 84%) as an off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.33-5.30 (m, 1H), 3.52-3.43 (m, 1H), 3.34-3.27 (m, 1H), 2.31-2.13 (m, 2H), 2.04-1.92 (m, 2H), 1.90-1.76 (m, 2H), 1.75-1.57 (m, 4H), 1.54-1.37 (m, 7H), 1.34-1.19 (m, 4H), 1.18-1.01 (m, 5H), 0.99 (s, 3H), 0.96-0.89 (m, 9H), 0.88-0.85 (m, 10H), 0.67 (s, 3H), 0.05 (s, 6H).
Step 2. To a solution of 28-1 (900 g, 1.74 mmol) in THF (30 mL) was added n-BuLi (1.04 mL, 2.61 mmol, 2.5M in hexane) dropwise at −70° C. under N.sub.2. The reaction solution was stirred at −70° C. for 30 minutes. Then dimethyl phosphorochloridate (502 mg, 3.48 mmol) was added dropwise. After addition, the mixture was stirred at 20° C. for 2 hrs. The reaction solution was quenched with sat.NH.sub.4Cl (50 mL) and extracted with DCM (2×30 mL). The combined organic phase was dried over Na.sub.2SO.sub.4, filtered, concentrated and purified by combi-flash (0-20% of EtOAc in PE) to give 6-3A (540 mg, 50%) as colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.33-5.29 (m, 1H), 4.23-4.15 (m, 1H), 3.81-3.71 (m, 6H), 3.53-3.43 (m, 1H), 2.32-2.22 (m, 1H), 2.18-1.64 (m, 9H), 1.60-1.33 (m, 11H), 1.31-1.06 (m, 5H), 0.99 (s, 3H), 0.97-0.90 (m, 10H), 0.89-0.85 (m, 8H), 0.67 (s, 3H), 0.05 (s, 6H).
Step 3. To a solution of 6-3A (180 mg, 0.288 mmol) in DCM (5 mL) was added TMSBr (220 mg. 1.44 mmol). The mixture was stirred at 20° C. for 16 hrs. The mixture was adjusted with NaOH (1M in H.sub.2O) to pH=9 and the solid was precipitated, which was filtered and washed with CH.sub.2Cl.sub.2 (2 mL), water (2 mL). The filter cake was dissolved in MeOH (6 mL) and concentrated to give Compound 6 (35 mg, 25%) as an off-white solid. TH NMR (400 MHz, MeOD) δ 5.37-5.31 (m, 1H), 4.01-3.89 (m, 1H), 3.45-3.35 (m, 1H), 2.29-2.14 (m, 2H), 2.09-1.84 (m, 5H), 1.82-1.72 (m, 1H), 1.66-1.38 (m, 10H), 1.37-1.26 (m, 1H), 1.24-1.04 (m, 5H), 1.03-1.00 (m, 4H), 0.99-0.93 (m, 7H), 0.92-0.85 (m, 4H), 0.72 (s, 3H). LCMS t.sub.R=1.370 min in 3 min chromatography, 10-80CD_ELSD, MS ESI calcd. for C.sub.27H.sub.46O.sub.5 P [M-2Na+2H—H].sup.− 481, found 481.

Materials and Methods

[0252] The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures, for example, as described in WO 2013/036835 and WO 2014/160480. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization.

[0253] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[0254] The compounds provided herein may be isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography, HPLC, or supercritical fluid chromatography (SFC). The compounds provided herein may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis. Exemplary chiral columns available for use in the separation/purification of the enantiomers/diastereomers provided herein include, but are not limited to, CHIRALPAK® AD-10, CHIRALCEL® OB, CHIRALCEL® OB-H, CHIRALCEL® OD, CHIRALCEL® OD-H, CHIRALCEL® OF, CHIRALCEL® OG, CHIRALCEL® OJ and CHIRALCEL® OK.

[0255] .sup.1H-NMR reported herein (e.g., for the region between δ (ppm) of about 0.5 to about 4 ppm) will be understood to be an exemplary interpretation of the NMR spectrum (e.g., exemplary peak integrations) of a compound. Exemplary general method for preparative HPLC: Column: Waters RBridge prep 10 μm C18, 19*250 mm. Mobile phase: acetonitrile, water (NH.sub.4HCO.sub.3) (30 L water, 24 g NH.sub.4HCO.sub.3, 30 mL NH.sub.3.H.sub.2O). Flow rate: 25 mL/min

[0256] Exemplary general method for analytical HPLC: Mobile phase: A: water (10 mM NH.sub.4HCO.sub.3), B: acetonitrile Gradient: 5%-95% B in 1.6 or 2 min Flow rate: 1.8 or 2 mL/min; Column: XBridge C18, 4.6*50 mm, 3.5 μm at 45 C.

OTHER EMBODIMENTS

[0257] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0258] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0259] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0260] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.