Green Chemistry CHM 459 Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Ian Joyce Abstract: Eli Lilly Laboratories has developed a novel synthesis that utilizes the Twelve Principles of Green Chemistry to prevent waste, decrease worker exposure, use safer solvents, apply a biocatalyst and provide inherently safer chemical synthesis in their production of the experimental drug LY300164 (Talampanel). The new synthesis prevents heavy metal Chromium waste by utilizing compressed air as an inherently safer chemical. By eliminating the heavy metal waste, they reduced worker exposure to the toxic chemical, and they prevented it from entering and damaging the environment. In doing this they are being economically responsible as well as environmental stewards. Through the use of a biocatalyst, they increased the atom economy significantly and converted the reaction over to an aqueous based solvent, rather than using harsh organic solvents. On top of all these improvements, they increased the percent yield of the total synthesis three-fold from 16 to 55%. Introduction In 1999, Eli Lilly Research Laboratories won the Chromium waste for every pound of the experimental Greener Synthetic Pathways Award for developing an drug produced. 1 The new synthesis helped reduce the effective, low-waste synthesis of the experimental number of isolation steps and increased the percent anticonvulsive drug LY300164, also known as yield from 16 to 55 percent.1 Talampanel. The drug was designed for the treatment Often times, a major drawback of of epilepsy and other neurodegenerative disorders.1 pharmaceutical syntheses is that a large amount of The new synthetic method addressed several of the waste is generated in the synthetic process, usually Twelve Principles of Green Chemistry, including the use from excess reagents or solvents. 1 2 Lilly Research Zygosaccharomyces rouxii yeast as a biocatalyst in Laboratory was able to create a novel synthesis method order to reduce waste and increase the atom economy that was economically viable, environmentally of the synthetic process. The researchers also utilized responsible, and less hazardous to all patrons involved safer aqueous solvents as opposed to harsh organic in its synthesis. 1 ones. They prevented the use of toxic heavy metal The new method of synthesis allows for the reagents which limited the amount of exposure to the yeast to perform a stereo-selective reduction of a workers in the factory, thereby providing an inherently ketone to form an enantiomerically pure alcohol.3 This safer chemical synthesis.1 The new synthetic method new method is effective; however, the activity of the helped eliminate forty-one gallons of solvent and yeast becomes limited at higher concentrations of approximately three pounds of potentially toxic products.4 To solve this problem, two major Winter 2016 | 1 Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459 innovations had to be made. First, the researches had Sodium Hydroxide, and compressed air to oxidize a to develop a method that converted the initial starting carbon alpha to two aromatic rings. 1 Additionally, this ketone to an aqueous slurry and then perform the new method proved more selective and increased the reaction in the presence of a polymeric resin. 4 As the percent yield.1 reaction proceeds, the final product adheres to the This new method of synthesis proved to be a resin, thus drawing the desired product out of the far more effective strategy for the production of the aqueous slurry to drive the reaction forward.4 Second, experimental drug LY300164.5 This impressive and the new synthesis aimed to eliminate the use of innovative new method utilizes a variety of the Twelve Chromium as an oxidizing reagent. The new synthesis Principles of Green Chemistry in order to benefit all utilized Dimethylsulfoxide, Dimethylformamide, parties involved in the manufacturing of the drug.6 7 Experimental Reagent Name A 3,4-methylenedioxyphenyl acetone Biotransformation with B Zygosaccharomyces rouxii C p-NO2PhCHO D HCl E Compressed Air (as O2 source) F NaOH G H2NNHAc H MSCl I Et3N J Pd/C, H2 K 1,5-bromobenzo[d][1,3]dioxole L n-Butyl Lithium M Methyloxirane Solvent Name S1 Glucose S2 Na2HPO4 S3 H3PO4 S4 H2O S5 PhMe S6 DMSO S7 DMF S8 EtOH S9 CH2Cl2 S10 Ethyl Acetate S11 Brine S12 Saturated Ammonium Chloride S13 Sodium Bicarbonate S14 Lithium tert-Butoxide S15 HCl S16 Diethyl Ether Scheme 1: Novel synthesis using the biocatalyst Z. rouxii as well as an alternative oxidizing agent to Chromium Trioxide. Winter 2016 | 2 Green Chemistry CHM 459 Practical Application of a Biocatalyst in Pharmaceutical Manufacturing It is important to note that the majority of the synthetic 3. The product was combined with 731 mL of DMSO procedure is similar for both the new and the old and 3L of DMF and cooled at 8-12°C. Compressed air pathway. The major steps that differ are the first step was then passed over the mixture. 117.5 mL of 50% and the third step of the synthesis. After the novel aqueous NaOH was added and stirred for 4.5 hours. synthesis is discussed, steps one and three of the old The mixture was transferred by cannula over a 30-60 synthesis will be explained. The novel synthesis of the min interval into an 8.25 L solution containing HCl at experimental drug LY300164 is described by the 10-15°C. The precipitate was filtered and washed with 8 following experimental procedure. H2O and left to air dry- Epimeric mix-(5RS,7S)-7,8- dihydro-7-methyl-5-(4-nitrophenyl)-5H-1,3-dioxoIo[4,5- 1. 60 g of 3,4-methylenedioxyphenyl acetone, 21 g G][2]benzopyran-5-ol (5). disodium phosphate 0.3 mL of phosphoric acid, 750 mL XAD-7 Resin and 350 mL H20 were mixed together for 4. This product was then added to 2.3 L ethanol, 94.5 g 40 minutes at 20-25°C. 105 g of Glucose and 225 g the of acetic hydrazide and 1 mL of concentrated HCl and prepared Zygosaccharomyces rouxii wet cell paste the mixture was refluxed for 2.5 h. After the solution were added to the solution. The mixture was diluted to was cooled, it was concentrated using a rotary 1.5 L and stirred for 12 hours at 33-35°C. After this, the evaporator and then dissolved in 4.9 L of ethyl acetate. reaction mixture was filtered using a 150 micron This solution was then washed with 1.5 L of saturated stainless steel screen. The product and resin remained sodium bicarbonate then 1.5 L of brine, and then dried on the screen and were washed with water. Acetone with sodium sulfate. After the product was filtered it was then used to wash the product from the resin. The showed a 91% yield- (S)-acetic acid-[[6-(2- acetone was then evaporated, leaving a residue. The hydroxypropyl)-l,3-benzodioxol-5-yl](4-nitrophenyl)m residue was dissolved in toluene and concentrated to ethylene]hydrazide (6). yield 53 g of a yellow, viscous oil. The total yield was 5. The product was then dissolved in 2.38L of 96% - (S)-a-methyE-1,3 benzodioxole-5-ethanol (2). methylene chloride and cooled to 0 to -10°C. 187 mL of trimethylamine and 78.2 mL of methanesulfonyl 2. 125 mL of toluene was added to the product along chloride were added and the solution was stirred for 30 with 14.15 g of 4-nitrobenzaldehyde. 10 mL of minutes. 510 mL of H2O was added and the organic concentrated HCl was used to dissolve the mixture and phase was washed with 460 mL of HCl and then 500 mL then it was heated at 60°C for 2 hours. The solution of brine. After this, the methylene chloride solution was then heated to distill off some of the solvent and was heated to 45°C and 4.8 L of hexanes was added then cooled to room temperature. Ethanol was added over 90 minutes. The product was then slowly cooled to form a slurry and then evaporated off. The addition down to 5°C to crystallize. It was then collected by and distillation of ethanol was performed three more vacuum filtration and dried in an oven at 50°C to give times to dissolve al solids and purify the solution. The an 87% yield- (S)-acetic acid[[6-[2- solution was stirred for an hour at 25°C and then the (methanesulfonyl)oxy]propyl]-1,3-benzodioxol-5-yl](4- final product was collected via vacuum filtration at an nitrophenyl)methylene]hydrazide (7) 88% yield- (5RS,7S)-7,8-dihydro-7-methyI-5-(4- nitrophenyl)-5H-1,3 dioxolo-[4,5-G][2] benzopyran (4). Winter 2016 | 3 Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459 6. The product was then added to 45 mL of THF and 1. 2 g 1,5-bromobenzo[d][1,3]dioxole was dissolved in 0.89 g of Lithium tert-butoxide at 0°C. The mixture was 25 mL of dry Et2O and cooled to -78 °C. 5 mL of n-Butyl warmed to 25°C and stirred for 3 hours. Saturated Lithium was added dropwise over twenty minutes. The ammonium chloride solution was then added to solution was stirred for thirty minutes and 0.75 g of 2- quench the reaction. The solution was diluted with methyloxirane was added. The temperature was ethyl acetate and wasted with H2O and brine to give a gradually increased to room temperature and the 92% yield- (R)-7-acetyl-8,9-dihydro-8-methyl-5-(4- reaction was stirred for one hour. The organic phase nitrophenyI)-7H-1,3-dioxolo[4,5-h][2,3]benzodiazepme was poured into 100 mL of saturated aqueous solution (8 ).