Preparation of a Key Intermediate En Route to the Anti-HIV Drug Lenacapavir

A very efficient four-step synthesis of the main fragment of Gilead’s anti-HIV drug lenacapavir is described. The route showcases a 1,2-addition to an intermediate aldehyde using an organozinc halide derived from a commercially available difluorobenzyl Grignard reagent. This sets the stage for the oxidation of the resulting secondary alcohol to the desired ketone, which relies solely on catalytic amounts of TEMPO together with NaClO as the terminal oxidant, affording the targeted ketone in 67% overall yield.


Reagents:
Reagents were purchased from Sigma-Aldrich, Reike Metals, Combi-Blocks and were used without further purification unless noted otherwise.
Note: Although the concentration of 3,5-difluorobenzylmagnesium bromide is predetermined by the vendor, it is highly recommended that the Grignard solution be titrated prior to use to determine its exact concentration.

Chromatography:
Silica gel TLC plates (UV 254 indicator, thickness 200 mm standard grade, glass backed and 230-400 mesh from Merck) were used.The developed TLC plate was analyzed by a UV lamp (254 nm).The plates were further analyzed with the use of an aqueous ceric ammonium molybdate stain or ethanolic vanillin and developed with a heat gun.Flash chromatography was performed using Silicycle Silicaflash® P60 unbonded grade silica.

HPLC:
HPLC-grade solvents were obtained from Fisher Scientific.HPLC analysis was performed on an Agilent 1260 series HPLC with a Kromasil 100-5-C18 4.6×50 mm column at the flow-rate of 1mL/min using gradient solvent starting from 40% v/v acetonitrile/water to 98% v/v acetonitrile/water over the course of 12-15 min.

Procedure for a 20 mmol scale reaction:
To an oven dried 250 mL round bottom flask (RBF) equipped with a large magnetic stir was added 2,5-dibromo-6-methylpyridine 7 (5.0184g, 20 mmol, 1 equiv), then capped with a rubber septum.The RB was then filled with argon and evacuated (3x).Anhydrous 2-MeTHF (50 mL) was then added via syringe while the flask was maintained under a stream of argon.The solution was then cooled to 0 o C followed by the addition of t-butyl nitrite (2.68 g, 26 mmol, 1.3 equiv, 3.1 mL) by syringe.A solution of potassium t-butoxide in anhydrous 2-MeTHF (1.74 M, 17.24 mL, 30 mmol t-BuOK) was added dropwise into the RBF while stirring and maintaining the temperature at 0 o C. The reaction was then stirred at 0 o C for 3 h.After 3 h, the reaction was diluted with 2-MeTHF (15 mL) and quenched with sat.NH4Cl aqueous solution (25 mL).The 2-MeTHF was then removed in vacuo with an 89% recovery of 2-MeTHF.Once all the 2-MeTHF was removed a light-yellow precipitate was left behind in the aqueous phase.The light-yellow solid was then filtered and the filter cake was washed with water (2 x 25 mL) and allowed to dry overnight to afford a light yellow solid (96% yield, 5.347 g, 87% purity by 1 H NMR). Oxime 8 was used directly in the next step without further purification.
Note: It is important to use a large enough RBF and stir bar to ensure proper stirring as the product begins to precipitate out shortly after the addition of t-BuOK.Preparation of t-BuOK solution in 2-MeTHF: Inside an argon filled glove-box, to a 20 mL oven dried volumetric flask was added 3.905 g of t-BuOK.The flask was capped with a septum and removed from the glove-box.To the flask was then added anhydrous 2-MeTHF to the volumetric line.To assure complete dissolution the volumetric flask was sonicated for a period of 5 min.

Figure S1. Synthesis of 8
Table S2.Optimization of step 2

General procedure for hydrolysis of 8
To an appropriately sized flask equipped with a magnetic stir bar was added the acidic reaction medium warmed to 80 o C using an oil bath for a period of ca. 10 min.Oxime 8 was then added in one portion.The heterogenous mixture was then stirred un a water jacketed reflux condenser for a period of 3 h.Procedure for a 17.5 mmol scale reaction under optimized conditions: To 250 mL RBF equipped with a large magnetic stir bar was added a 50% w/w solution of glyoxylic acid in H2O (48.5 mL).The solution was warmed to 80 o C using an oil bath for ca. 10 min.To the warm solution was then added crude oxime 8 (17.5 mmol, 4.89 g) in one portion via an addition funnel.The yellow suspension was stirred vigorously at 80 o C using an oil bath under a water jacketed condenser for a period of 3 h.The suspension was then cooled to rt and then to 0 o C. The precipitate was vacuum filtered and washed with cold water (25 mL x 3) and dried overnight in vacuo to afford a light yellow/golden crystalline solid 9 (3.712 g, 80% yield, 99% pure by 1 H NMR).This material was used in the next step without any further purification.The yield represented in Scheme S2 is the average of entries 6 and 7 in Table S2.

Procedure for one-pot synthesis of aldehyde 9
To an oven dried 6-dram vial equipped with large magnetic stir bar was added 7 (500 mg, 2 mmol).
The vial was capped with a septum, filled with argon and evacuated 3 times.Under a stream of argon was added anhydrous 2-MeTHF (5 mL) followed by the addition of t-butylnitrite (1.3 equiv, 2.6 mmol, 309 µL).The solution was cooled to 0 o C in an ice bath and from a previously made stock solution in 2-MeTHF (1.76 M) (see procedure for step 1, above) t-BuOK (1.5 equiv, 1.74 M, 1.72 mL) was added dropwise.The reaction mixture was stirred at 0 o C using an ice bath for 3 h.The yellow heterogeneous mix was diluted with 2-MeTHF (3 mL) and quenched with sat.aq NH4Cl (5 mL).The 2-MeTHF was removed by rotary evaporation to afford a yellow precipitate in the aqueous layer.The vial was centrifuged for a period of 2 min.The supernatant was removed by syringe.To the vial was then added water (1.5 mL), centrifuged and the water was removed by syringe.This process was repeated 5 times to afford a yellow residue, crude 8.
To the same vial containing the crude material from the last step was added water (2 mL) and warmed to 80 o C using an aliminum heating block for a period of ca. 5 min.To the warm mixture was added a 50% w/w solution of glyoxylic acid in water (5.6 mL) dropwise via syringe.The mixture was then stirred at 80 o C for a period of 3h.The reaction mixture was then cooled to rt then to 0 o C. The heterogeneous mixture was centrifuged and the remaining acidic solution was removed by syringe.To the vial was then added water (1.5 mL), stirred, and centrifuged following removal of the supernatant.The process was repeated 4 more times.The light yellow/golden material was then dried at 40 o C using an aluminum heating block overnight to afford aldehyde 9 (320 mg, 61% yield).
Note: Due to the small scale associated with this two step one-pot sequence it is suspected that some material was lost during the aqueous washes at both steps.However, this demonstrates the potential to carry out an efficient one-pot sequence to synthesize aldehyde 9 from the corresponding dibromopicoline 7 when run at a larger scaled using the appropriate reactors.
Table S3.Optimization of step 3

Procedure using ZnBr2:
To an oven-dried 6-dram vial equipped with a magnetic stir bar was added ZnBr2 (0.24 mmol, 54 mg, 1.2 equiv) in an argon filled glove box.The vial was sealed with a rubber septum, then removed from the glove box.To the vial containing ZnBr2, a Grignard solution of 13 in 2-MeTHF (0.25 M, 1.2 equiv, 0.96 mL) was added.The solution was stirred at 0 o C using ice bath for 2 h or until complete consumption of ZnBr2 was observed.In a separate oven-dried 2-dram vial equipped with a magnetic stir bar was added 3,6-dibromopicolinaldehyde 9 (1 equiv, 0.2 mmol, 53 mg).The vial was sealed with a rubber septum and filled with argon and evacuated three times.Under a stream of argon 50 µL of anhydrous 2-MeTHF was added to the vial.This solution was then cooled to 0 o C in an ice bath and the ZnBr2 / Grignard solution was added dropwise.The vial was then removed from the ice bath and stirred at 23 0 o C for a period of at least 12 h.Upon completion the reaction was cooled to 0 o C then quenched with saturated aqueous NH4Cl solution (1 mL).The organic layer was then separated and the aqueous layer was extracted with diethyl ether (1 mL x ).The organic layers were combined, dried, concentrated in vacuo.The crude material was then subjected to quantitative GC-MS analysis using naphthalene as an internal standard to determine a yield of 88%.

Procedure using ZnCl2:
To an oven-dried 6-dram vial equipped with a magnetic stir bar was added ZnCl2 (1.3 equiv, 0.33 mmol, 44.3 mg) in an argon filled glove box.The vial was sealed with rubber septum then removed from the glove box.To the vial containing ZnCl2, a Grignard solution of 13 in 2-MeTHF (0.25 M, 1.3 equiv, 1 mL) was added.The solution was stirred at 0 o C for 2 h or until complete consumption of ZnCl2 was observed.In a separate oven dried 2-dram vial equipped with a magnetic stir bar was added 3,6-dibromopicolinaldehyde 9 (1 equiv, 66.2 mg).The vial was sealed with a rubber septum and filled with argon and evacuated three times.Under a stream of argon 0.5 mL of anhydrous 2-MeTHF was added to the vial.The 6-dram vial containing the ZnCl2 Grignard solution was cooled to 0 o C and the solution of 2-MeTHF containing 9 was added via syringe dropwise while under a stream of argon.Anhydrous 2-MeTHF (0.5 mL) was then added to the vial in which the 3,6dibromo-picolinaldehyde 9 and then transferred to the reaction vial containing the ZnCl2 / Grignard solution.The vial was removed from the ice bath and stirred at 23 0 o C for a period of 16 h.Upon completion the reaction was cooled to 0 o C then quenched with saturated aqueous NH4Cl solution (1 mL).The organic layer was then separated and the aqueous layer was extracted with diethyl ether (4 x 1 mL).The organic layers were combined, dried, concentrated in vacuo and purified by column chromatography using a gradient of 10-15% EtOAc/hexanes to afford the alcohol 5a as a white solid (87 mg, 89% yield).
The organic layer was collected, dried over anhydrous Na2SO4, and concentrated under vacuum.
Then the yield was obtained by qNMR (1,3,5-trimethoxybenzene as internal standard), resulting in no conversion; no product detected.
Note: Oxone was ground with a mortar and pestle into a fine powder prior to use. 7Optimization towards the oxidation of alcohol 11 to ketone 6 was done in parallel with the use of a nitrosyl-like catalyst.Although this method proceeds to be efficient for the oxidation, the focus was turned to using a potentially more cost-effective set of conditions (NaOCl and a nitrosyl-like catalyst).

Optimization of step 4 using a nitroxyl radical catalyst
General procedure for oxidation of 11 to 6 using TEMPO as catalyst 8 (1.5 mmol): To a 100 mL RBF equipped with a magnetic stir bar, 1-(3,6-dibromopyridin-2-yl)-2-(3,5difluorophenyl)ethan-1-ol, 11 (589.5 mg, 1.5 mmol), TEMPO (23.4 mg, 10 mol%), TBAB (24.15 mg, 5 mol %) and KBr (35.7 mg, 20 mol %) were added followed by addition of saturated aq.solution of NaHCO3 (1.8 mL) and then, toluene (7.5 mL, 0.2 M).The reaction mixture was then pre-mixed for 5 min at 0 o C. While maintaining the reaction mixture at 0 o C, a premixed cooled solution (0 o C) of 10-15% aq.NaOCl: sat.aq NaHCO3 (1:1.4,1.21 mL:1.7 mL : 2.9 mL) was added to the reaction flask at which it was stirred for 10 min.HPLC analysis of the corresponding reaction showed the presence of 11 (10%) in the crude reaction mixture.Accordingly, an additional NaOCl (0.1 equiv, 0.092 mL) was added and the reaction was stirred for another 10 min.Another HPLC sample was prepared and the analysis indicated 97% conversion of 11 to 6.To avoid product decomposition (over-oxidation), no further NaOCl was added and the reaction was quenched with sat.Na2S2O3 (1 mL), after which the crude reaction mixture was extracted with EtOAc (5 mL x 3).The organics were combined and concentrated in vacuo, and then subjected to qNMR and HPLC analysis (90% yield, 91% purity).For further purification, the crude product was subjected to recrystallization.

Figure S3. Oxidation of 5a using TEMPO followed by recrystallization
Recrystallization procedure for 6: To a 100 mL RBF equipped with a magnetic stir bar, the collected crude sample of 6 (585 mg) and acetonitrile (11.70 mL, 20 volume/g of a sample) was added while mixing (medium speed) at rt.Once a clear solution was observed, DI water (2.92 mL, 5 volume/g of a sample) was added to the flask.The solution was stirred for 10 min followed by the addition of another portion of DI water (2.92 mL, 5 volume/g of a sample).Once the tiny crystals had formed, the dispersion was stirred for 1 h at a low stir rate (ca.200 rpm).After ca. 30 min, another portion of DI water (5.85 mL, 10 volume/g of a sample) was added to the solution and solution was mixed for 30 min.After 30 min, the final portion of DI water (5.85 mL, 10 volume/g of a sample) was added and the dispersion was stirred for another 30 min.The solution was vacuum filtered and washed with DI water (3 x 2 mL) and dried to afford 6 (440 mg, 75% mass recovered) with 98% purity based on HPLC peak area.
Note: Because of its crystalline nature, 6 can easily undergo recrystallization as a preferred method for purification (as described above).Due the small academic scale on which this work was done, the mass recovery of the pure product was ca.75%.Table S5.Preliminary screening of different oxidizing agents Table S6.Preliminary screening of NaClO loading in DCM as the organic solvent Note: Initial screenings were done in DCM to gain insight into the efficiency of this transformation.Once a set of conditions were established, a solvent screening was carried out to find a nonchlorinated and potentially recoverable solvent (see Table S7).S7.Solvent screening Table S8.Screening of NaClO equivalents in Toluene as the organic solvent Note: NaClO typically comes in a solution with a range of concentration (10-15%).For this study, no titration was done although it is strongly recommended that the accurate concentration of the active chloride be determined prior to usage.It is worth mentioning that knowing this value would help to get highly pure product (6) to the extent that no purification might be needed.

Table S9. Correlation of HPLC purity and practical yield
Note: qNMR and isolated yield were derived from crude product and results indicated that 6 was formed in 97% yield in both cases.This yield was within 1% deviation from HPLC purity taken initially.This result was an indication that there is a close correlation between HPLC purity and actual yield as seen in the Table above.Consequently, HPLC was conveniently used to monitor the efficiency of all oxidation reactions that were run using N-oxyl catalyst,s unless noted otherwise.To a 1-dram vial equipped with a magnetic stir bar, 1-(3,6-dibromopyridin-2-yl)-2-(3,5difluorophenyl)ethan-1-ol, 11 (98.7 mg, 0.25 mmol), 0.10 mL of Azado stock solution (1.9 mg dissolved in 0.5 mL of toluene; 1 mol %) and toluene (1.15 mL, 0.2 M) were added.Then, TBAB (4.1 mg, 5 mol %) and KBr (6.0 mg, 20 mol %) were added to the reaction mixture followed by addition of saturated aq.solution of NaHCO3 (0.3 mL).The reaction mixture was pre-mixed for 1 min at 0 o C and while maintaining the reaction mixture at 0 o C, a pre-mixed cool solution (0 o C) of 10-15% aq.NaOCl: sat.aq.NaHCO3 (1:1.4)(0.21mL:0.294mL) was added to the reaction vial which was stirred for 10 min at (0 o C).The crude reaction mixture was extracted with EtOAc (3 x 1 mL) and the organic layers were combined and a small portion (15 µL) was subjected to HPLC analysis (98% HPLC peak area purity).The collected organic phase was concentrated in vacuo affording a total mass of 98.9 mg of crude sample.Quantitative NMR analysis of the crude sample gave 97% yield of 6.The sample was then prepared for recrystallization.

Synthesis of Weinreb amide 15
Table S11.Synthesis of Weinreb amide

Procedure using SOCl2:
Step 1: To an oven-dried 25 mL RBF equipped with a dry magnetic stir bar was added 3,6dibromopicolinic acid 14 (1 equiv, 1.5 mmol, 421 mg).The RBF was flushed with argon followed by the addition of SOCl2 (4.5 mL).The mixture was stirred at rt for a 15 min.then at 80 o C for a 3 h.The RBF was then cooled to rt and a reduced pressure distillation apparatus was attached to the RBF.The mixture was warmed to ca. 65 o C and the excess SOCl2 was distilled off.To the RBF was then added anhydrous THF (5 mL) and distilled off to afford the crude acid chloride as a yellow viscous oil.

Step 2:
To an oven-dried 6-dram vial equipped with a dry magnetic stir bar was added N,Odimethylhydroxylamine HCl (1.2 equiv, 175.6 mg), dry triethylamine (2 equiv, 3 mmol, 417 µL), and anhydrous THF (3 mL).The suspension was then cooled to 0 o C. Using an oven-dried glass syringe the acid chloride from step 1 was transferred in ca. 1 mL of anhydrous THF and added dropwise.The yellow suspension was stirred at 0 o C to rt for a period of 3 h.Upon completion the dark red reaction mixture was diluted with EtOAc (5 mL), washed with water (2 mL x 2), and the organics were then combined and washed with 1 M NaOH (2 mL), 1 M HCl (2 mL), then brine (2 mL), dried over anhydrous Na2SO2, filtered, concentrated in vacuo and purified by flash chromatography (10-30% EtOAc/hexanes) to afford and off white solid (375 mg, 77% yield).To an oven-dried 50 RBF equipped with a magnetic stir bar was added 3,6-dibromopicolinic acid 14 the RBF was then capped with a rubber septum.The RBF was filled with argon and evacuated 3 times.Under a pressure of argon was added anhydrous THF (10.7 mL) and DIPEA (3 equiv, 16.02 mmol, 2.79 mL) the clear faint yellow solution was stirred for 10 min then cooled to -10 o C.Then, dropwise was added a 50% wt solution of T3P in EtOAc (2 equiv, 10.68 mmol, 6.78 mL).The suspension was stirred at -10 o C for 1 h.The reaction mix was warmed to 0 o C, the septum was removed and N,O-dimethylhydroxylamine HCl (1.5 equiv, 8.01 mmol, 781 mg) was quickly added in 2 portions while maintaining stirring.The mixture was then stirred for 30 min at 0 o C then warmed to rt for 4.5 h monitored by TLC.Upon completion the reaction was diluted with EtOAc (10 mL), quenched with water (8 mL) and then stirred for ca. 10 min.The aqueous was extracted with EtOAc (10 mL x 3), the organics were combined and washed with 1 M HCl (8 mL x 3), washed with brine then dried over anhydrous MgSO4, filtered, and purified by flash chromatography (10-30% EtOAc/hexanes) to afford an off white solid (1.4381 g, 83% yield).

Scheme S1: Retrosynthesis of ketone 6 Scheme
Scheme S1: Retrosynthesis of ketone 6 Dropwise addition of a solution of t-BuOK in 2-MeTHF at 0 o C. b) Reaction mixture at 3 h.c) Reaction mixture after quenching with Sat.aqueous NH4Cl.d) Reaction mixture after removing 2-MeTHF.e) Filtration of product.

13 0
keep at 0 °C add aldehyde dropwise into Grignard reagent mix 1 equiv CuCN with Grignard, then use 0.8 equiv 11 keep at 0 °C mix 1 equiv ZnBr 2 with Grignard, then use 0.8 equiv mixture 35 43 °C to rt mix 1 equiv ZnBr 2 with Grignard, then use 1.0 equiv mixture, 12 h 80 to rt mix 1 equiv ZnBr 2 with Grignard, then use 0.8 equiv mixture, 12 h 62 4 2 20 a) Reactions run on a 0.2 mmol scale.b) Run on a 1.5 mmol scale.c) Isolated yield d) Yield determined by GC-MS using naphthalene as internal std.e)Reaction was run on a 0.25 mmol scale.15 0 °C to rt mix 1 equiv ZnCl 2 with Grignard, then use 1.3 equiv mixture, 20 h
a) Reaction mixture prior to adding NaOCl.b) 10 min.into reaction.c) 20 min.into reaction.d) Recrystallization of 6a.e) Filtration of crystals.
3,5-trimethoxybenzene (CAS:621-23-8) was used as an internal standard b) 10 mol% KBr, 5 mol% TBAB and saturated aqueous solution of NaHCO 3 was used.c)20 mol% KBr, 5 mol% TBAB and saturated aqueous solution of NaHCO 3 was used KBr/TBAB/NaHCO 3 of 11 based on HPLC area peak b) Conversion of 11 towards 6 based on HPLC area peak c) Conversion of 11 towards side product/s based on HPLC area peak d) The reaction was setup at - of 11 based on HPLC area peak b) Conversion of 11 towards 6 based on HPLC area peak c) Conversion of 11 towards side product/s based on HPLC area peak of 11 based on HPLC area peak b) Conversion of 11 towards 6 based on HPLC area peak c) Conversion of 11 towards side product/s based on HPLC area peak d) the reaction was performed in 0.), Sat.aq.NaHCO 3 /NaClO(equiv)

Table S1 .
Optimization of step 1Synthesis of aldehyde 9 from dibromopicoline 7 has been adapted from literature known procedure1

Table S12 .
Cost analysis of catalyst for oxidationNote: The prices listed above are a comparison of the prices available to us on an academic level.The cost per gram was estimated based on the available prices.The cost per gram will vary depending on the size of the container purchased.On an industrial scale the cost per gram may differ.Worth noting, however, highly encouraged by the cost of TEMPO even at this scale it can serve as a potential and efficient alternative to AZADO for this oxidation.