7‐Step Flow Synthesis of the HIV Integrase Inhibitor Dolutegravir

Abstract Dolutegravir (DTG), an important active pharmaceutical ingredient (API) used in combination therapy for the treatment of HIV, has been synthesized in continuous flow. By adapting the reported GlaxoSmithKline process chemistry batch route for Cabotegravir, DTG was produced in 4.5 h in sequential flow operations from commercially available materials. Key features of the synthesis include rapid manufacturing time for pyridone formation, one‐step direct amidation of a functionalized pyridone, and telescoping of multiple steps to avoid isolation of intermediates and enable for greater throughput.


Methods:
Stainless steel syringes were used to transfer air-and moisture-sensitive liquids. Reactions were monitored by thinlayer chromatography (TLC) on Silica Gel 60 F254 plates (EMD) under UV light (254 nm) or visualized with KMnO4 upon heating. Flash chromatography was performed using Silica Gel 60 (230-400 mesh, EM Science), SiliaFlash P60 (230-400 mesh, SiliCycle), or 200-400 mesh silica gel (Sorbent Technologies). Organic solutions were concentrated under reduced pressure on a Büchi or Heidolph rotary evaporator. Analytical thin-layer chromatography (TLC) was accomplished with UV light (254 nm), and column chromatography was mediated on a Biotage Isolera flash chromatography system using SNAP KP-Sil or Ultra-Sil columns (silica gel, average particle size 50 µM).
Continuous flow equipment was assembled from commercially available components supplied from IDEX Health & Science Technologies, and backpressure regulators were purchased from Zaiput Flow Technologies. Reactors were constructed from high-purity perfluoroalkoxy (PFA) tubing with a 1/16" outside diameter and either 0.02″ or 0.04″ inside diameter as well as stainless steel (SS) tubing with a 1/16" outside diameter and 0.04″ inside diameter, with complementary PEEK fittings. Harvard Apparatus PhD Ultra syringe pumps were used to infuse solutions contained within 8 mL high-pressure stainless steel syringes with 1/16" SWAGELOCK ® fitting. The check valves used had a CV inlet 1/4-28 (Perfluor), T-mixers were IDEX Tee Assy 1/8" PEEK 0.050 through, and ferrules were SF, SS Ring, 1/16", TZ.

Materials and reagents:
Commercial reagents were purchased from Millipore Sigma, Alfa Aesar, Strem, Oakwood Chemical, Acros Organics, Fisher Scientific, Matrix Scientific, or TCI and used without purification unless otherwise indicated.
Methyl-4-methoxyacetoacetate (97%) was distilled prior to use in the first step of the flow synthesis. Sodium methoxide solution in methanol was purchased from Alfa Aesar and used as received. Where specified, CH3CN, CH2Cl2, toluene, THF, and 1,4-dioxane (PURE SOLV) were dried by passing through columns of activated alumina.
Deuterated solvents CDCl3, CD3OD, and DMSO-d6 (Cambridge Isotope Laboratories or Millipore Sigma) were used without purification. Extraction and chromatography solvents were reagent grade and used without purification (VWR, Fisher Scientific or Millipore Sigma). Molecular sieves (4Å) were activated by heating in vacuo and stored in a vial in a dessicator. HPLC solvents were used without purification (VWR, Fisher Scientific).

Instrumentation:
Proton nuclear magnetic resonance ( 1 H NMR) spectra and proton-decoupled carbon nuclear magnetic resonance

(E)-Methyl 2-(((2,2-dimethoxyethyl)amino)methylene)-4-methoxy-3-oxobutanoate 6:
The Vapourtec R series flow reactor equipment consisting of two Knauer type HPLC pumps for reagent/solvent delivery and 2 x 10 mL PFA reactors (1/16" O.D. x 0.04" I.D.) was utilized in the flow chemistry experiment. Before the start of the actual experiment, all reactors were primed with PhCH3. Reactor I was pre-heated to 85 °C and Reactor II was pre-heated to 85 °C. The Vapourtec Pump A was used to pump the neat stock of methyl-4-methoxyacetoacetate (M4MAA) 12 (50 mL, 1 equiv) and Vapourtec Pump B was used to pump the neat stock of dimethyl formamide dimethylacetal (DMF-DMA) 13 (60 ml, 1.6 equiv). Solutions A and B were mixed at a T-piece (M1) (IDEX Health and Science, P-726 used with blanking plugs) and pumped through Reactor I. A 40 psi back pressure regulator (BPR) (IDEX Health and Science, P-785) was connected after Reactor I (Step 1). The output from Reactor I was connected to a second T-piece (M2) with an incoming neat solution of aminoacetaldehyde dimethylacetal (AADMA) 15 (60 ml, 1 equiv) that was pumped using a Syrris ASIA Pump C. The collective flow stream was allowed to pump into Reactor II. A 100 psi back pressure regulator (BPR) (IDEX Health and Science, P-787) was connected after Reactor II. After approximately three total system residence times, the output flow from Reactor II was collected for 15 mins (19.8 mL, 32.9 mmol of rate limiting material). The reactant mixture was concentrated in vacuo and purified using flash chromatography (50-100% EtOAc/hexanes) to give pure 6 (7.16 g, 27.4 mmol, 95%).
Harvard syringe pumps were set to infuse at 25 µL min -1 (total flow rate = 50 µL min -1 ), and the solution collected at the outlet of the continuous flow system for an equilibration period of 360 min. At this time, the reactant mixture was collected for 60 min (3.0 mL, 0.69 mmol of rate limiting material) into a vial; when 8 mL stainless steel syringes were empty, the syringes were recharged with 6 mL of dry PhCH3 as a carrier solvent. The reactant mixture was concentrated in vacuo and purified using flash chromatography (10-80% EtOAc/hexanes) to give pure 17 (290 mg, 0.66 mmol, 96% yield). connected to a T-mixer into PFA tubing 1/16" x 0.04" x 10'. Two solutions were prepared in 10 mL vials, and 6 mL of the resulting solutions were loaded into dry stainless steel Harvard syringes.
Harvard syringe pump with solutions 1 and 2 was set to infuse at 25 µL min -1 (total flow rate in 1st reactor = 50 µL min -1 ), Harvard syringe pump with solutions 3 and 4 was set to infuse at 20 µL min -1 (total flow rate in 2nd reactor = 70 µL min -1 , total flow rate in 3rd reactor = 90 µL min -1 ) and the solution collected at the outlet of the continuous flow system for an equilibration period of 540 min. At this time, the reactant mixture was collected for 60 min (5.4 mL, 0.62 mmol of rate limiting material) into a vial with sat. NaHCO3; when 8 mL stainless steel syringes were empty, the syringes were recharged with 6 mL of dry PhCH3 as a carrier solvent and pumping was resumed. The aqueous layer was extracted with EtOAc and the reactant mixture was concentrated in vacuo and purified using flash chromatography (20-100% EtOAc/hexanes) to give pure 19 (131 mg, 0.32 mmol, 48% yield). Diastereomeric

Pictures of Flow Setup Steps 1-3 Telescope
Step 4 Amidation