SCYX-7158

[4-fluoro-N-(1-hydroxy-3,3-dimethyl-1,3-dihydro-benzo[c]oxaborol-6-yl-2-trifluoromethyl benzamide]

• C₁₇H₁₄BF₄NO₃
• Average mass 367.103 Da

Human African trypanosomiasis (HAT) is an important public health problem in sub-Saharan Africa, affecting hundreds of thousands of individuals. An urgent need exists for the discovery and development of new, safe, and effective drugs to treat HAT, as existing therapies suffer from poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. We have discovered and optimized a novel class of small-molecule boron-containing compounds, benzoxaboroles, to identify SCYX-7158 as an effective, safe and orally active treatment for HAT.

The presence of a boron atom in the heterocyclic core structure has been found essential for trypanocidal activity of orally active series of benzoxaborole-6-carboxamides in murine models of human African trypanosomiasis. SCYX-7158  has been identified as an effective, safe and orally active treatment for human African trypanoso-miasis to enter preclinical studies, with expected progression to phase 1 clinical trials in 2011 ………http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764666/

A drug discovery project employing integrated biological screening, medicinal chemistry and pharmacokinetic characterization identified SCYX-7158 as an optimized analog, as it is active in vitro against relevant strains of Trypanosoma brucei, including T. b. rhodesiense and T. b. gambiense, is efficacious in both stage 1 and stage 2 murine HAT models and has physicochemical and in vitro absorption, distribution, metabolism, elimination and toxicology (ADMET) properties consistent with the compound being orally available, metabolically stable and CNS permeable.

In a murine stage 2 study,SCYX-7158 is effective orally at doses as low as 12.5 mg/kg (QD×7 days). In vivo pharmacokinetic characterization of SCYX-7158 demonstrates that the compound is highly bioavailable in rodents and non-human primates, has low intravenous plasma clearance and has a 24-h elimination half-life and a volume of distribution that indicate good tissue distribution.

Most importantly, in rodents brain exposure of SCYX-7158 is high, with C_max >10 µg/mL and AUC_{0–24 hr} >100 µg*h/mL following a 25 mg/kg oral dose. Furthermore, SCYX-7158 readily distributes into cerebrospinal fluid to achieve therapeutically relevant concentrations in this compartment.

Medicinal Chemistry Synthesis of SCYX-7158  SCHEME1

While the original route was eff ective for producing multi-gram quantities of the API, it was not amenable to scale-up. The route started with 2, a relatively expensive aryl boronic acid. This was protected as borocan 3 and halogen-lithium exchange followed by reaction with acetone and subsequent deprotection provided the oxaborole 4. This protection/alkylation/deprotection sequence added two steps to the overall synthesis and the metalation was not reliable. However, the biggest concern in the sequence was nitration of 4 to give 5. This was accomplished by adding a concentrated solution of 4 to cold fuming nitric acid. Besides the signifi cant safety considerations, the reaction did not scale well. Reduction of the nitro group to give aniline 6 was followed by amide formation to provide 1. While this end game was effi cient, the material produced was dark in color. The colored impurities were not removed by crystallization of 1 and furthermore a mixture of two polymorphs was formed under the original conditions.

The process chemistry route to SCYX-7158 is shown in Scheme 2. When considering alternative routes to 1, the readily available and inexpensive methyl 2-bromobenzoate (8) was identifi ed as an attractive starting point. Gratifyingly, treatment of 8 with methylmagnesium bromide aff orded 2-bromocumyl alcohol (9) in high yield using simple operating conditions. Lithiumhalogen exchange followed by reaction with triisopropyl borate and acidic work-up provided benzoxaborole 4, along with cumyl alcohol (10). While this conversion was not completely atom-effi cient, it was easily scalable and several strategies are available to suppress the by-product in the future.

With benzoxaborole 4 in hand, attention turned to the introduction of a nitrogen-linked amide at the C(6) position. This was accomplished using the same nitration/reduction/acylation strategy used in Scheme 1. Yet signifi cant changes to the chemistry were required for safety and reliability reasons. The fi rst task was introduction of the nitrogen. Nitration was demonstrated using acetic anhydride/nitric acid. However, due to slow rates of nitration and potential for accumulation of a reactive intermediate, alternative conditions had to be identifi ed. These limitations were overcome by use of trifl uoroacetic anhydride/nitric acid, which provided a more reactive nitrating intermediate, thus improving the rate of nitration and aff ording a process in which nitric acid was slowly added until 4 was consumed. Full safety assessment of the nitration reaction, including extensive calorimetry studies, demonstrated the safety of this reaction. This process was used to prepare kilogram quantities of 5.

Following reduction of nitrobenzoxaborole 5 to aniline 6 under standard catalytic hydrogenation conditions, acylation with 7 provided the fi nal drug candidate in high chemical yield. Two challenges remained which needed to be addressed through further optimization of the process. The fi rst challenge was color and purity of the API, which derived from a highly colored impurity generated in the nitration reaction which carried through to fi nal product and was not removed by crystallization. The second challenge was to consistently obtain a single polymorph of the API. Both challenges were addressed by isolation of crystalline isopropyl boronate 11 which rejected colored impurities, followed by regeneration of 1 through addition of water and azeotropic removal of isopropanol. This crystallization provided the API as a single polymorph. The API was isolated in good yield, very high purity and was white in color.

PATENT

https://www.google.co.in/patents/WO2011019616A1?cl=en

N-(3,3-Dimethyl-l-phenyl-2,3-dihvdro-lH-benzotblborol-6-yl)-4-fluoro-2- trifluoromethylbenzatnide

HNO₃

This is not the compd, see precursor

To a suspension of 2-bromophenylboronic acid (75.Og, 373.4 mmol) in toluene (525 niL) was added JV-butyldiethanolamine (64.ImL, 392.1 mmol, 1.05 equiv.) via a syringe. The mixture was heated at 50 ⁰C for two hours. After cooling to room temperature, the toluene was evaporated under reduced pressure and the remaining clear colorless oil was treated with heptanes (500 mL). The heptanes mixture was then sonicated for 5 min and the resulting suspension was allowed to stand at room temperature overnight. The solid that precipitated was collected by filtration, washed with heptanes, and dried in a vacuum oven overnight to yield 2-(2′- bromophenyl)-6-butyl[l,3,6,2]dioxazaborocan as a white solid. Data: ¹H NMR (400 MHz, CHLOROFORM-^) δ ppm 0.86 (t, J=7.4 Hz, 3 H) 1.14 – 1.25 (m, 2 H) 1.51 – 1.62 (m, 2 H) 2.61 – 2.70 (m, 2 H) 3.01 – 3.11 (m, 2 H) 3.26 – 3.37 (m, 2 H) 4.09 – 4.26 (m, 4 H) 7.10 (td, J=7.6, 2.0 Hz, 1 H) 7.24 (td, J=7.3, 1.1 Hz, 1 H) 7.51 (d, J=7.9 Hz, 1 H) 7.81 (dd, J=IA, 1.9 Hz, 1 H). Amount obtained, 123.7 g (98.6% yield).

To a solution of 2-(2′-bromophenyl)-6-butyl[l,3,6,2]dioxazaborocan (30.0g, 89.2 mmol) in THF (740 mL) at -78 ⁰C was added /?-BuLi (42.8 mL, 2.5M in hexane, 107.0 mmol, 1.2 equiv.) dropwise via a syringe over a period of 10 min while maintaining reaction temperature at -78 ⁰C. After the addition the reaction solution was stirred for 20 min at -78 ⁰C before acetone (7.5 mL, 124.8 mmol, 1.4 equiv.) was added dropwise via a syringe over a period of 10 min while maintaining the reaction temperature at -78 ⁰C. The resulting mixture was allowed to stir for 20 min at -78 ⁰C then warm to room temperature gradually. Once the reaction vessel reached room temperature, 6N HCl solution (150 mL) was added and the mixture was stirred for an additional 30 min. The mixture was extracted with EtOAc (3X). The EtOAc extracts were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The light yellow oil was then subjected to flash chromatography (Isco Companion, 8Og SiO₂ cartridge, solid loaded SiO₂, neat heptanes to 20:80 EtOAc gradient at 60 ml/min for 90 min). 3,3-Dimethyl-3H-benzo[c][l,2]oxaborol-l-ol was recovered as clear colorless oil. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.44 (s, 6 H) 7.31 (d, J=Ll Hz, 1 H) 7.38 – 7.47 (m, 2 H) 7.66 (d, J=7.2 Hz, 1 H) 8.99 (s, 1 H). Amount obtained: 9.4O g (65.2 % yield).

To 60 mL fuming HNO₃ at -45 ⁰C was slowly added a solution of 3,3- dimethyl-3H-benzo[c][l,2]oxaborol-l-ol (9.4 g, 58.0 mmol) in 11.9 mL nitrobenzene via a syringe while maintaining the reaction temperature between -40 to -45 ⁰C. Once the addition was complete the resulting solution was allowed to stir at -45 ° C for an additional 45 min before poured into crushed ice. The ice mixture was allowed to melt and the aqueous solution was extracted with DCM (3X). The combined DCM extracts were dried over Na₂SO₄ then evaporated. The crude oil remaining was mixed with one liter 1 : 1 DCM/heptanes. The volume of the solution was reduced under reduced pressure by half and the resulting solution was allowed to stand overnight in a -20 ⁰C freezer. The precipitate formed was filtered out, washed with heptanes and vacuum dried to give 3,3-dimethyl-6-nitro-3H-benzo[c][1.2]oxaborol-l-ol as a white solid. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.46 (s, 6 H) 7.69 (d, J=8.4 Hz, 1 H) 8.28 (dd, J=8.4, 2.3 Hz, 1 H) 8.48 (d, J=2.2 Hz, 1 H) 9.41 (br. s., 1 H). Amount obtained: 7.31 g (60.4 % yield).

To a solution of 3,3-dimethyl-6-nitro-3H-benzo[c][l .2]oxaborol-l-ol (6.98 g, 33.3 mol) in THF ( 277 mL) was added 6N HC1( 16.6 mL, 100.2 mmol, 3.0 equiv.). The vessel was vacuum/N₂ purged three times and 5% Pd/C (3.5 g) was added. The mixture was again vacuum/N₂ purged three times then vacuum purged again. H₂ was then introduced from a balloon and the reaction was allowed to stir at room

temperature over night. The reaction solution was filtered through a short pad of celite and the filtrate was evaporated to yield 6-amino-3, 3 -dimethyl -3H- benzo[c][l,2]oxaborol-l-ol HCl salt as a dark brown foamy solid. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.36 (s, 6 H) 4.94 (s, 2 H) 6.66 (dd, J=8.1, 2.2 Hz, 1 H) 6.79 (d, J=2.0 Hz, 1 H) 7.01 (d, J=8.1 Hz, 1 H) 8.72 (s, 1 H). Amount obtained: 8.29 g (100% yield).

To a solution of 6-amino-3, 3 -dimethyl -3H-benzo[c][l,2]oxaborol-l-ol HCl salt (8.29 g, 33.3 mmol) in DCM (170 mL) was added Et₃N (11.6 mL, 83.2 mmol, 2.5 equiv.). The mixture was cooled to 0 ⁰C and 2-trifluoromethyl-4- fluorobenzoyl chloride (6.1 mL, 39.9 mmol, 1.2 equiv.) was added slowly via a syringe. The resulting solution was allowed to warm to room temperature gradually and stir for 2 hours. The reaction solution was diluted with DCM, washed with IN HCl, H₂O, brine and then dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure to give an off- white solid. The solid was recrystallized from DCM/heptanes to give 4-fluoro-N-(l-hydroxy-3,3-dimethyl-l,3-dihydro- benzo[c][l,2]oxaborol-6-yl-2-trifluoromethyl benzamide as a white solid. LCMS (M/Z) : 368 (M+H); ¹H NMR (DMSO-d₆) δ: 10.58 (s, IH), 9.11 (s, IH), 8.02 (d, J = 1.7 Hz, IH), 7.75 – 7.83 (m, 2H), 7.60 – 7.71 (m, 2H), 7.38 (d, J = 8.2 Hz, IH), 1.44 (s, 6H). Amount obtained: 11.7 g (96% yield)………IS SCYX 7158

BELOW NOT SCYX 7158

The title compound was prepared using a similar procedure to that of N-(I- phenyl- 1 ,3 -dihydrobenzo [c] [ 1 ,2]oxaborol-6-yl)-2-trifluoromethylbenzamide with phenyl magnesium bromide replacing p-to IyI magnesium bromide and 4-fluoro-iV-(l- hydroxy-3,3-dimethyl-2,3-dihydro-lH-benzo[b]borol-6-yl)-2-trifluoromethyl benzamide replacing N-(I -hydroxy-1 ,3-dihydrobenzo[c] [ 1 ,2]oxaborol-6-yl)-2- trifiuoromethylbenzamide. Data: LCMS m/e: 428 (M+H); ¹H NMR (400 MHz, DMSO-J6) δ ppm 1.59 (s, 6 H) 7.46 – 7.62 (m, 4 H) 7.71 (td, J=8.5, 2.7 Hz,l H) 7.77 – 7.90 (m, 3 H) 8.00 – 8.09 (m, 2 H) 8.39 (d, J=2.0 Hz, 1 H) 10.66 (s, 1 H). 10 N-fl-p-Tolyl-lJ-dihydro-benzofcIflJIoxaborol-ό-vD-benzatnide

PATENT

82 4-Fluow-N-(l-hydwxy-3,3-dimethyl-l,3-dihydw-benzofcIfl,2Ioxabowl-6- yl-2-trifluoromethyl benzamide

To a suspension of 2-bromophenylboronic acid (10. Og, 49.7 mmol) in toluene (70 niL) was added N-butyldiethanolamine (8.5 mL, 52.2 mmol, 1.05 equiv.) via a syringe. The mixture was heated at 50 ⁰C for two hours. After cooling to room temperature, the toluene was evaporated under reduced pressure and the remaining clear colorless crude oil was treated with heptanes (~ 500 mL). The heptanes mixture was then sonicated ~ 5 min and the resulting suspension was allowed to stand at room temperature overnight. The solid that precipitated was collected by filtration, washed with heptanes, and dried in a vacuum oven overnight to yield a white solid as the titled compound. ¹U NMR (400 MHz, CHLOROFORM-J) δ ppm 0.86 (t, J=7.4 Hz, 3 H) 1.14 – 1.25 (m, 2 H) 1.51 – 1.62 (m, 2 H) 2.61 – 2.70 (m, 2 H) 3.01 – 3.11 (m, 2 H) 3.26 – 3.37 (m, 2 H) 4.09 – 4.26 (m, 4 H) 7.10 (td, J=7.6, 2.0 Hz, 1 H) 7.24 (td, J=7.3, 1.1 Hz, 1 H) 7.51 (d, J=7.9 Hz, 1 H) 7.81 (dd, J=IA, 1.9 Hz, 1 H). Amount obtained, 16.0 g, (98 % yield).

To a solution of 2-(2′-bromophenyl)-6-butyl[l,3,6,2]dioxazaborocan (3.0g, 9.2 mmol) in THF (76 mL) at -78 ⁰C was added /?-BuLi (4.4 mL, 2.5M in hexane, 11.0 mmol, 1.2 equiv.) dropwise via a syringe over a period of 10 min while maintaining reaction temperature at -78 ⁰C. After the addition the reaction solution was stirred 20 min at -78 ⁰C before acetone (946 μL, 12.8 mmol, 1.4 equiv.) was added dropwise via a syringe over a period of 10 min while maintaining the reaction temperature at -78 ⁰C. The resulting mixture was allowed to stir for 20 min at -78 ⁰C then warm to room temperature gradually. Once the reaction vessel reached room temperature, 6M HCl solution (30 mL) was added and the mixture was stirred for 30 min. The mixture was extracted with EtOAc (3X). The EtOAc extracts were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude slightly yellow in color residual oil remaining was then subjected to flash chromatography (Isco Companion, 8Og SiO₂ cartridge, solid loaded SiO₂, neat heptane to 20:80 EtOAc gradient at 60 ml/min for 90 min). The product was recovered as clear colorless oil. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.44 (s, 6 H) 7.31 (d, J=Ll Hz, 1 H) 7.38 – 7.47 (m, 2 H) 7.66 (d, J=7.2 Hz, 1 H) 8.99 (s, 1 H). Amount obtained: 1.76 g (61%).

To 14.2 ml fuming HNO₃ at -45 ⁰C was added a solution of 3,3-dimethyl- 3H-benzo[c][l,2]oxaborol-l-ol (2.28 g, 14.1 mmol) in 3.0 ml nitrobenzene slowly via a syringe while maintaining the reaction temperature between -40 to -45 ⁰C. Once the addition was complete the resulting solution was allowed to stir at -45 ° C for an additional 45 min before poured into crushed ice (600 g). The ice mixture was allowed to melt and the aqueous solution was extracted with dichloromethane. The combined dichloromethane extracts were dried over Na₂SO₄ then evaporated. The crude oil remaining was mixed with one liter 1 : 1 DCM:heptane. The volume of the solution was reduced on a rotovap by half and the resulting solution was allowed to stand overnight in a -20 ⁰C freezer overnight. The precipitate formed was filtered out, washed with heptanes and vacuum dried to give the titled compound as a white solid. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.46 (s, 6 H) 7.69 (d, J=8.4 Hz, 1 H) 8.28 (dd, J=8.4, 2.3 Hz, 1 H) 8.48 (d, J=2.2 Hz, 1 H) 9.41 (br. s., 1 H). Amount obtained: 2.01 g (68%).

To a solution of 3,3-dimethyl-6-nitro-3H-benzo[c][1.2]oxaborol-l-ol (790 mg, 3.8 mmol) in THF ( 20 mL) was added HOAc (1.7 mL, 30 mmol). The vessel was vacuum/N₂ purged three times and 5% Pd/C (200 mg) was added. The mixture was again vacuum/N₂ purged three times then vacuum purged again. H₂ was then introduced from a balloon and the reaction was allowed to stir for 2.5 hours. The reaction solution was filtered through a short pad of celite and the filtrate was evaporated to yield the title compound as a dark brown foamy solid. ¹H NMR (400 MHz, DMSO-J₆) δ ppm 1.36 (s, 6 H) 4.94 (s, 2 H) 6.66 (dd, J=8.1, 2.2 Hz, 1 H) 6.79 (d, J=2.0 Hz, 1 H) 7.01 (d, J=8.1 Hz, 1 H) 8.72 (s, 1 H). Amount obtained: 670 mg (89%). [0382] To a solution of 6-amino-3, 3 -dimethyl -3H-benzo[c][l,2]oxaborol-l-ol acetate salt (100 mg, 0.42 mmol) in DCM (2 niL) was added Et₃N ( 117.3 μL, 0.84 mmol). The mixture was cooled to 0 ⁰C and the 2-trifluoromethyl-4-fluorobenzoyl chloride (70.0 μL, 0.46 mmol) was added slowly via a syringe. The resulting solution was allowed to warm to room temperature gradually and stir for 2 hours. The reaction solution was diluted with DCM, washed with IN HCl, H₂O and then dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure and the crude material was subjected to flash chromatography (Isco Companion, 4 g SiO₂ cartridge, SiO₂ solid load, neat heptanes to neat EtOAc gradient over 45 min, flow rate = 18 ml/min). The title compound was recovered as a white foam. LCMS (M/Z) : 368 (M+H); 1H NMR (DMSO-d₆) δ: 10.58 (s, IH), 9.11 (s, IH), 8.02 (d, J = 1.7 Hz, IH), 7.75 – 7.83 (m, 2H), 7.60 – 7.71 (m, 2H), 7.38 (d, J = 8.2 Hz, IH), 1.44 (s, 6H). Amount obtained: 144.6 mg (93% yield).

Alternate Synthesis

82e

82b

A 500 mL round-bottomed-flask equipped with a magnetic stir bar and ice- H₂O bath was charged with 82a (18.4g, 85.5 mmol) and anhydrous THF (200 mL). MeMgCl (68 mL, 3.0M in 2-methylTHF) was added dropwise through an additional funnel. The mixture was allowed to warm to rt. gradually and stirred overnight. After cooling back to 0 ⁰C, the white milky suspension was carefully treated with HCl (3M) until the upper layer turned clear with white precipitate at the bottom of the flask (pH = 6). The upper clear solution was decanted into a separatory funnel. The precipitate was rinsed with methyl tert-butyl ether (MTBE) (100 mL) 3 times. Combined MTBE with the clear solution and the mixture was washed with H₂O (100 mL) 3 times, brine (100 niL), dried over MgSO₄, filtered and concentrated under reduced pressure to give 82b as a light yellow oil (20.2g, 100%).

82c

A 50 mL round-bottomed-flask equipped with a magnetic stir bar and ice- H₂O bath was charged with 82b (860 mg, 4.0 mmol) and anhydrous THF (20 mL). MeMgBr (1.3 mL, 2.0 M in THF) was slowly added via a syringe. The mixture was stirred at 0 ⁰C for 10 minute and the ice bath was replaced with a dry ice-acetone bath at -40 ⁰C. BuLi (1.9 mL, 2.5 M in hexanes) was added dropwise via a syringe. The resulting mixture was stirred at -40 ⁰C for another 2h before B(O-ipr)₃ (1.4 mL, 4.8 mmol) was added dropwise. The mixture was allowed to warm up to rt gradually and stirred overnight. After carefully quenched the reaction with H₂O (1 mL), HCl (3M, 10 mL) was added and the mixture was stirred at rt for Ih. The mixture was extracted with EtOAc (20 mL) 3 times. Combined extracts was washed with H₂O (20 mL), brine (20 mL), dried over MgSO₄, filtered and concentrated under reduced pressure to give a clear oil. The oil solidified overnight to give 82c as a pale yellow waxy solid (544mg, 82.4%).

82d

A 3 L round-bottomed-flask equipped with a mechanical stirrer, thermocouple and ice bath was charged with 82c (86.2 g of 58 wt%, 309 mmol) and trifluoroacetic acid (259 mL). Trifluoroacetic anhydride (129 mL, 926 mmol) was added in one portion. An exotherm of 18 ⁰C was observed. The solution was again cooled to 0 ⁰C and 90% nitric acid (18.0 mL, 386 mmol) was added via syringe pump over 2 h. After the addition was complete, the solution was aged for 1 h. Water (1.75 L) was added. Note: Initially the quench is quite exothermic. Add the water in 5 mL aliquots until the exotherm subsides. The resulting suspension was stirred for 16 h while warming to rt. The solids were collected on a frit, rinsed with water (2 x 500 mL), and air dried to constant weight to provide 50.3 g of crude 82d as a free-flowing orange solid. Note: the crude 82d can be carried forward without recrystallization. The solid was charged to a IL three-necked round-bottomed-flask equipped with a nitrogen inlet adapter, thermocouple, heating mantle and mechanical stirrer.

Isopropylacetate (IPAc, 75 mL) was added and the resulting slurry was warmed to 75 ⁰C and heptanes (250 mL) was added over 15 min while maintaining an internal temp of > 65 ⁰C. The slurry was allowed to cool to rt over night. The solids were collected on a frit and rinsed with 10% IP Ac/heptanes (100 mL) and then heptanes (100 rnL). The product was air dried to constant weight to provide a tan solid (31.7 g, 58%).

82e

A 500 mL round-bottomed-flask equipped with a magnetic stir bar, thermocouple and septum was charged with 82d (29.7 g, 192 mmol) and THF (150 mL, anhydrous stabilizer free). The vessel was inerted by cycling vacuum the nitrogen three times and 5% Pd/C (6.0 g, 50% wet, Degussa type NO/W) was added. The vessel was again inerted by cycling vacuum then nitrogen three times. A hydrogen filled balloon was attached via needle and the atmosphere was changed by cycling vacuum the hydrogen three times. The slurry was stirred vigorously for 16 h. The atmosphere was changed again to nitrogen by cycling vacuum then nitrogen three times. The mixture was filtered through a 1″ pad of celite and the cake was rinsed with THF (50 mL). Concentration in vacuo provided a light tan powder (26.82 g). In a 500 mL round bottomed-flask, the solids were slurried in IPAc (50 mL) and warmed in an 80 ⁰C water bath. Heptanes (150 mL) were added over 10 min. The resulting slurry was allowed to cool to rt and stir for 16 h. The solids were collected on a frit, rinsed with heptanes (50 mL) and air dried to provide an off- white solid (24.39 g, 96%).

4-Fluoro-N-(l-hvdroxy-3,3-dimethyl-l,3-dihvdro-benzofcJfl,2Joxaborol-6-yl-2- triβuoromethyl benzatnide

A lL three-necked round-bottomed-flask equipped with a nitrogen inlet adapter, mechanical stirrer and thermocouple was charged with 82e (15.7g, 88.4 mmol), THF (160 mL, anhydrous, stabilizer free) and K2CO3 (14.7g, 106 mmol). The suspension was stirred at rt and 4-fluoro-2-(trifluoromethyl)benzoyl chloride (22.Og, 97.3 mmol) was added over 10 min. The resulting suspension was aged for 24 h at rt. Water (80 mL) and isopropyl acetate (160 mL) were added and the phases were partitioned. The organic phase was further extracted with water (80 mL) and then brine (50 mL). The organic phase was dried over MgSO₄ (20 g) and concentrated in vacuo to provide a tan solid (34.26 g). The solid was dissolved with acetone (195 mL) and transferred to a mechanically stirred IL round-bottomed-flask. Distilled water (113 mL) was added in one portion and the mixture was stirred for 30 min to produce a seed bed and then additional distilled water (60 mL) was added over 30 min. The suspension was stirred at rt overnight and the solids were collected on a frit. The cake was rinsed with 1 : 1 acetone/water (100 rnL) and air dried to constant weight to provide an off-white solid (30.5 g, 94%).

Alternate Synthesis

HNO₃ CF₃CO₂H (CF₃CO)₂O

to RT

1-1

A 72 L round-bottomed-flask was equipped with a cold bath, mechanical stirrer, nitrogen inlet adaptor, oxygen sensor, thermowell and 2 L dropping funnel. The flask was charged with methyl 2-bromobenzoate (2513 g, 11.7 mol) and the system was flushed with nitrogen to <0.1% O₂. THF (18L, anhydrous, inhibitor free) was added and the cold bath was charged with ice and acetone. When the internal temp reached -4 ⁰C, MeMgBr (11.6 L of a 3M solution in ether, 34.8 mol) was added via dropping funnel over 3 h. The internal temp was maintained below 15 ⁰C throughout. At the end of addition, the cold bath was drained and the reaction was aged overnight at ambient temperature. The bath was again charged with ice and acetone and the suspension cooled to below 15 ⁰C. HPLC indicated incomplete conversion (92:8 product, starting ester), so additional MeMgBr (2.3L of a 3M solution in ether) was added. After Ih, HPLC showed the conversion to be >99: 1. The reaction was quenched by slow addition of IN HCl (42 L) keeping the internal temp below 15 ⁰C throughout. At the end of the quench, the pH was adjusted to 6 with IN HCl. The mixture was extracted with MTBE (10 L then 2x5L). The combined organic phases were dried over MgSO₄, filtered and concentrated via rotary evaporation to provide 2482 g of 2-(2-bromophenyl)-propan-2-ol as a pale yellow oil. ¹H NMR (CHLOPvOFORM-d) δ: 7.62 – 7.67 (m, IH), 7.53 – 7.58 (m, IH), 7.24 – 7.30 (m, IH), 7.03 – 7.10 (m, IH), 1.70 – 1.75 (m, 6H). 1-2

A 72L round-bottomed-flask was equipped with a mechanical stirrer, O₂ sensor, thermowell, 2L dropping funnel, N₂inlet adaptor, and cold bath. The vessel was inerted to 0.01% O₂ and charged with THF (27L, anhydrous, inhibitor free). The resulting solution was cooled to -70 ⁰C using dry ice and acetone and n-BuLi (8.2 L of a 2.5M solution in heptane, 20.5 mol) was added over Ih. 2-(2-Bromophenyl)- propan-2-ol (1994 g, 9.27 mol) was dissolved in THF (9L) and the solution was added to the BuLi via dropping funnel over 2h, keeping the internal temp below -70 ⁰C. The resulting thin yellow suspension was aged for 30 min then B(OiPr)₃ (244 Ig, 13.0 mol) was added rapidly via addition funnel. The cold bath was drained and the misture was allowed to warm to room temperature while aging over night. HPLC analysis shows an 81 :19 ratio of desired product: 2-phenyl-2-propanol. The mixture was cooled to -10 ⁰C and 2N HCl (9.3 L) was added via dropping funnel over 30 min, keeping the reaction mixture below 10 ⁰C. After 3 h, the pH was adjusted to 4 with additional HCl. The reaction mixture was extracted with MTBE (2 x 4L). The combined organic phases were concentrated to provide 2028 g of a heavy oil. The oil was dissolved in MTBE (14L) and extracted with IN NaOH (4.6, then 5, then 4L). The aqueous phases were combined and acidified with 2N HCl (6.8 L) to a pH of 4-5. The mixture was extracted with MTBE (5L). The organic phase was dried over MgSO₄ (282 g) and concentrated to provide 1450 g (ca 60 wt%) of 3,3-dimethyl-3H- benzo[c][l,2]oxaborol-l-ol as a waxy white solid. LC/MS: m/z 163 (M+H)⁺; ¹H NMR (DMSO-de) δ: 8.96 (br. s., IH), 7.62 (d, J = 7.2 Hz, IH), 7.33 – 7.45 (m, 2H), 7.25 – 7.30 (m, IH), 1.40 (s, 6H).

1-3

A 22 L round-bottomed-flask equipped with a mechanical stirrer, thermocouple, 2 L dropping funnel and cold bath was charged with 3,3-dimethyl-3H- benzo[c][l,2]oxaborol-l-ol (508 g, 300 g contained, 1.85 mol) and trifluoroacetic acid (1.54 L). The solution was cooled to 5 ⁰C. Trifluoroacetic anhydride (722 mL, 5.56 mol, 3.00 eq) was added via dropping funnel over 15 min. After aging at 0 – 3 ⁰C for 30 min, nitric acid (90% fuming, 108 mL, 2.31 mol, 1.5 eq) was added dropwise over 2h 50 min keeping the internal temp below 5 ⁰C. After aging for 1 h, icewater (10.4L) was added over 50 min maintaining the reaction temp below 15 ⁰C to provide a slurry. The slurry was aged at 0 ⁰C overnight to provide an orange suspension. The solids were collected on a frit, rinsed with cold water (5L) and air dried under a stream of air to constant weight (ca 24h) to provide 364 g of 3,3- dimethyl-6-nitro-3H-benzo[c][l,2]oxaborol-l-ol as a 92.4 wt% pure solid (88%). LC/MS : m/z 208 (M+H)⁺; ¹H NMR (DMSO-d₆) δ: 8.52 (d, J = 2.2 Hz, IH), 8.32 (dd, J = 8.4, 2.2 Hz, IH), 7.74 (d, J = 8.4 Hz, IH), 1.50 (s, 6H)

1-4

A 2 gallon stirred pressure vessel was charged with 3,3-dimethyl-6-nitro- 3H-benzo[c][l,2]oxaborol-l-ol (966 g, 812 g corrected, 3.92 mol), 5% Pd/C (193 g, 50% wet, Degussa type 101 NO/W) and THF (4.83 L, inhibitor free). The vessel was sealed, the atmosphere was changed to H₂ (5 psi) and the reaction was fun for 16 h. An exotherm to 30 ⁰C was observed over about 30 min. The vessel was purged with N₂, and completion of reaction was determined by HPLC. The reaction was vacuum filtered through a pad of celite (very slow filtration) and the filter cake was rinsed with THF (2L). The filtrate was concentrated via rotary evaporation to provide 982 g of a dark brown solid. This was transferred to a 22L round-bottomed-flask and warmed to 80 ⁰C in iPAc (1.83 L) to provide a dark brown slurry. The slurry was cooled to 60 ⁰C and heptanes (5.49L) were added over 2 h. The slurry was allowed to age with stirring over night while cooling to room temperature. The solids were collected on a frit, rinsed with heptanes (4L) and air dried to provide a dark brown solid (747 g).

The solids (747 g) were transferred to a 22L rbf and slurried in iPAc (3 L) at 70 ⁰C. The batch was allowed to cool to 40 ⁰C and heptanes (3L) were added over 5 h. The slurry was aged at room temperature over night and the solids were collected on a frit, rinsed with 1 : 1 iP Ac/heptanes (2L) then heptanes (IL) and air dried to provide 554 g of 6-amino-3,3-dimethyl-3H-benzo[c][l,2]oxaborol-l-ol as a brown solid. ¹H NMR (400 MHz, DMSO-d6) δ ppm 1.36 (s, 6 H) 4.94 (s, 2 H) 6.66 (dd, J=8.1, 2.2 Hz, 1 H) 6.79 (d, J=2.0 Hz, 1 H) 7.01 (d, J=8.1 Hz, 1 H) 8.72 (s, 1 H). 4-Fluow-N-(l-hvdwxy-3,3-dimethyl-l,3-dihvdw-benzofcJfl,2Joxabowl-6-yl)-2- triβuoromethyl benzatnide

A 22L four-necked round-bottomed-flask equipped with a nitrogen inlet adapter, mechanical stirrer and thermocouple was charged with 6-amino-3,3- dimethyl-3H-benzo[c][l,2]oxaborol-l-ol (554g, 3.13 mol), THF (5.5 L, anhydrous, stabilizer free) and K₂CO₃ (865 g, 6.26 mol). The suspension was stirred at room temperature for 30 min and 4-fluoro-2-(trifluoromethyl)benzoyl chloride (780 g, 3.44 mol) was added over 30 min. The resulting suspension was aged for 24 h at room temperature. HPLC showed unreacted 6-amino-3,3-dimethyl-3H-benzo[c][l,2] oxaborol-1-ol so an additional 42 niL of the acid chloride was added. After 30 min, water (2.8 L) and isopropyl acetate (5.5 L) were added and the phases were partitioned. The organic phase was further extracted with water (2.8 L) and then brine (2.8 L). The organic phase was dried over MgSO₄ and concentrated in vacuo to provide a tan solid. The solid was dissolved with acetone (3.0 L) and transferred to a mechanically stirred 5OL round-bottomed-flask. Distilled water (2.0 L) was added in one portion and the mixture was stirred for 30 min to produce a seed bed and then additional water (1.0 L) was added over 30 min. The suspension was stirred at room temperature overnight and the solids were collected on a frit. The cake was rinsed with 1 : 1 acetone/water (1.0 L) and air dried to constant weight to provide 4-fluoro-N- ( 1 -hydroxy-3 ,3 -dimethyl- 1 ,3 -dihydro-benzo [c] [ 1 ,2]oxaborol-6-yl)-2-trifluoromethyl benzamide as a dark tan solid (1.3 kg).

Recrystallization of4-Fluoro-N-(l-hydroxy-3,3-dimethyl-l,3-dihvdro- benzotcl t 1,21 oxaborol-6-yl)-2-trifluoromethyl benzamide

A 22 L round-bottomed-flask was charged with the dark tan crude 4- fluoro-N-(l -hydroxy-3, 3 -dimethyl- 1 ,3-dihydro-benzo[c] [ 1 ,2]oxaborol-6-yl)-2- trifluoromethyl benzamide (1.3 kg), acetone (8L) and Darco G-60 (55 g, 400 mesh) and water (5.3L). The resulting suspension was stirred for 15 min, filtered through a pad of celite (ca 500 g) to provide a brown solution. The celite pad was washed with 60% acetone/water (8L). The combined filtrate and rinse were transferred to a 50 L round-bottomed-flask and water (2L) was added. The solution was seeded (5 g) to initiate crystallization and additional water (2.2 L) was added slowly via addition funnel. After aging at room temperature overnight, the solids were collected and the filter cake was rinsed with 30% acetone/water (4L). The solids were air dried for 24 h then dried in a room temperature vacuum oven for 5 days to constant weight to provide 969 g (72% recovery) of 4-fluoro-N-(l-hydroxy-3,3-dimethyl-l,3-dihydro- benzo[c][l,2]oxaborol-6-yl)-2-trifluoromethyl benzamide as a light tan solid.

LC/MS: m/z 368 (M+H)⁺;

¹H NMR (400 MHz, DMSO-d6) δ ppm 1.44 (s, 5 H) 1.49 (s, 2 H) 7.39 (d, J=8.2 Hz, 1 H) 7.61 – 7.76 (m, 2 H) 7.77 – 7.84 (m, 2 H) 7.86 – 7.90 (m, 0 H) 8.03 (d, J=I.7 Hz, 1 H) 9.09 (s, 1 H) 10.58 (s, 1 H).

POTASSIUM SALT

Formation of potassium salt

To a 50OmL three-neck flask fitted with a mechanical stirrer was charged KOH (1.51 g, 26.9 mmol, 1.0 eq.). Under a nitrogen atmosphere, anhydrous acetone (140 mL) and H₂O (2.5 mL, 5 eq.) were added via syringe. A solution of 4-fluoro-N- (l-hydroxy-3,3-dimethyl-l,3-dihydro-benzo[c][l,2]oxaborol-6-yl-2-trifluoromethyl benzamide (10.0 g, 27.2 mmol, 1.0 eq.) in anhydrous acetone (60 mL) was added to the flask with vigorous stirring. The resulting clear solution was stirred at room temperature. The potassium salt precipitated from the solution over ca. 4 hours to afford a thick suspension. The precipitate was collected by filtration, washed with acetone (200 mL) and dried in a vacuum oven overnight to afford a white solid (10.6g, 91.9% yield). ¹H NMR (methanol-d₄) δ: 7.70 – 7.76 (m, IH), 7.53 – 7.60 (m, 2H), 7.47 – 7.53 (m, IH), 7.33 – 7.36 (m, IH), 7.01 – 7.06 (m, IH), 1.46 (s, 6H); M.P. (range) 197 – 200 ⁰C; Elemental analysis: Theory: C 48.25%, H 3.57%, N 3.31%, K 9.24%; Found: C 48.70%, H 3.41%, N 3.25%, K 9.19%.

REFERENCES

http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0001151

• touguia J, Costa J (1999) Therapy of human African trypanosomiasis: current situation. Mem Inst Oswaldo Cruz 94: 221–224
• Barrett MP, Boykin DW, Brun R, Tidwell RR (2007) Human African trypanosomiasis: pharmacological re-engagement with a neglected disease. Br J Pharmacol 152: 1155–1171.

1. 1986. Epidemiology and control of African trypanosomiasis. Report of a WHO expert committee. World Health Organization. Geneva, Switzerland. Technical Report Series, No. 739. 126 pp.
2. Benzoxaboroles: a new class of potential drugs for human African trypanosomiasis. Robert T Jacobs, Jacob J Plattner, Bakela Nare, Stephen A Wring, Daitao Chen, Yvonne Freund, Eric G Gaukel, Matthew D Orr, Joe B Perales, Matthew Jenks, Robert A Noe, Jessica M Sligar, Yong-Kang Zhang, Cyrus J Bacchi, Nigel Yarlett, and Robert Don. Future Medicinal Chemistry. August 2011. Vol. 3, No. 10. Pages 1259-1278.

http://www.swisstph.ch/fileadmin/user_upload/Pdfs/Events/2010_09_Jacobs.pdf  ……….POWERPOINT

Lead optimization investigation of oxaboroles for the treatment of human African trypanosomiasis
238th Am Chem Soc (ACS) Natl Meet (August 16-20, Washington) 2009, Abst MEDI 345

LINK

https://www.acsmedchem.org/ama/orig/abstracts/mediabstractf2009.pdf

Robert Jacobs, bob.jacobs@scynexis.com

Daitao Chen1 , Matt Orr1 , Jessica Sligar1 , Matt. Jenks1 , Andy Noe1 , Bakela Nare2 , Luke T. Mercer2 , Tana S. Bowling2 , Cindy Rewerts1 , Stephen Wring1 , Cyrus Bacchi3 , Nigel Yarllet3 , Charles Ding4 , Yvonne Freund5 , Kurt Jarnagin5 , Jacobs Plattner5 , and Robert Don6 . (1) Scynexis Inc, Duhram, NC 27713, (2) SCYNEXIS, Inc, Research Triangle Park, NC 27709-2878, (3) Pace University, New York, NY, (4) Anacor Pharmaceuticals, Inc, Palo Alto, CA, (5) Anacor Pharmaceuticals, Inc, (6) Drugs for Neglected Diseases initiative, Geneva, Switzerland

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