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 ). of NH4Cl and the product was extracted with four 30 mL portions of Ethyl Acetate. The organic layer was 7. The product was placed in 500 mL flask that dried with Magnesium Sulfate and filtered and a rotary contained a mechanical stirrer, a nitrogen inlet and a evaporator was used to obtain the final residual vacuum. 200 mL of ethanol was added and 2.12 g of product. 10% Pd/C was added to the flask. The flask was evacuated with nitrogen gas and then filled with The third step of the new synthetic method utilizes hydrogen gas from a balloon. The evacuation and compressed air as a source of oxygen, along with NaOH filling with hydrogen was done two more times at room in order to oxidize the α-benzylic carbon on product temperature. After stirring for 90 minutes the balloon number three. The old synthesis uses Chromium was removed and again the flask was purged with Trioxide along with NaOH to perform a partial oxidation nitrogen gas. The mixture was filtered using celite and and ultimately the same product, as displayed below.1 8 the remaining mixture was concentrated using a rotary evaporator. The residue was then dissolved in a 50:50 ethanol/water solution and cooled to 25°C. The solution was recrystallized and washed with cold ethanol and allowed to dry in the oven to give an 87% yield- (R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8- methyl-7H-1,3-Dioxolo[4,5-h][2,3]benzodiazepme, (LY300164).

The first step of the old synthesis utilized different starting reagents in order to try to obtain the chiral Reaction 2: Chromic Trioxide oxidation. alcohol product after the first step. The final product for this synthesis was racemic. The synthesis is as follows.9

Reaction 1: Initial synthesis of the chiral alcohol.

Winter 2016 | 4

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459

Discussion

Benzodiazepines such as the LY300164 are As shown in Reaction 1, one way to achieve this generally used for the treatment of chirality is to react 1,5-bromobenzo[d][1,3]-dioxole neurodegenerative disorders such as epilepsy.10 with (S)-2-methyloxirane in the presence of n- The drug acts as an AMPA receptor antagonist, Butyl Lithium. 11 The reaction goes on to produce which, essentially, means it dampens the extent of 91% the desired (-) chiral product, but also electrical impulses in specific regions of the synthesizes about 9% of the (+) isomer as well. 9 brain.10 The 1,4-benzodiazepine ring system is Synthesis using a Magnesium Gringard reagent in crucially important to achieve the anticonvulsant the presence of a Copper-Iodide catalyst also effect.10 yielded about 91% of the desired chiral product. 10 Another method involved using Sodium Originally, the drug was synthesized and tested on Borohydride as a reducing agent in a reaction with mice as a racemic mixture, however, it was found 3,4-methylenedioxyphenyl acetone. This proved to that the (-) isomer of LY300164 was significantly be less selective and inefficient in producing the stronger than the (+) isomer.10 This meant that desired product. 10 the researchers needed a synthetic method that produced a chiral product. The necessary chirality The synthesis method using n-Butyl Lithium is of the final product is produced early in the effective, however, it does pose a few issues, synthetic process. It is important to note that this including the disposal of Lithium waste and the discussion will only address a few of the many large amount of solvent necessary to complete the synthetic strategies that were used to achieve the reaction.9 The Process Flow Sheet 1 displays exact stereochemistry of this drug. Reaction 1 in more detail. The E-factor for this

2 Kg Reagent K Reaction 1 (old) 3.4 Kg Reagent L -78°C 0.62 Kg Reagent M 82% yield 17.8 Kg Solvent S16 2h

557=E-factor Stir 56% Atom Economy Warm to 25°C

108 kg Ethyl Acetate Extraction

153 kg Sat. NH4Cl 0°C 284.31 kg Byproducts

MgSO4 drying agent and solvent

0.51 kg product Process Flow Sheet 1: Old Synthesis using n-Butyl Lithium.

Winter 2016 | 5

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459 reaction is a staggering 557. The E-factor In comparison, the new synthesis is far more represents the amount unit of waste per unit of effective than the old synthesis and is displayed by final product. 12 Although this number is large, it is Process Flow Sheet 2. First, the E-factor is 71.8, not out of the ordinary for pharmaceutical which is not stellar, but it is almost six times less syntheses to utilize excess reagents and solvents. than the original method. Second, the Atom 13 The atom economy is 36% for this reaction. Economy for this reaction is listed as catalytic. This Atom economy is a measurement of how many of means that the reagents in solution are necessary the atoms in the reagents are actually part of the for the enzyme to catalyze the reduction reaction, final product.12 This step also involves an they are not necessarily reactants; therefore, the extraction in order to isolate the final product.8 atom economy is not listed as it would be This isolation step exposes employees to normally. The reagents present in solution are potentially hazardous chemicals and should be glucose, Na2HPO4, and H3PO4. The small amount avoided if possible. Another disadvantage to this of Na2HPO4, and H3PO4 are present as a buffer to synthesis method is that the Diethyl-Ether solution stabilize the yeast solution and avoid sharp that the reaction takes place in needs to be chilled changes in pH.14 The glucose is used to nourish to -78°C, which requires a lot of energy. The the yeast and provide energy to allow them to reaction takes a relatively short amount of time, catalyze the enzymatic reaction.15 about two hours and it has an 82% yield. The new synthesis takes about twelve hours to Additionally, the starting product (s)-2- complete and needs to be incubated at 33-35°C.8 methyloxirane must be chiral in order to achieve This reaction gives a 96% yield and is extremely the desired configuration. 8

60 Kg Reagent A Reaction 1 (new) 225 Kg Reagent B 33-35°C 0.787 Kg Resin 12 hours 105 Kg Solvent S1 1 ATM 21 Kg Solvent S2 0.6 Kg Solvent S3 96% yield 1500 Kg Solvent S4

1686 kg total 71.8 = E-factor Catalytic

1400 kg Water Washing Resin 1107 kg Acetone 0°C 4135 kg Byproducts and solvent

57.6 kg product Process Flow Sheet 2: New synthesis using biocatalyst.

Winter 2016 | 6

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459

reaction would occur and the percent yield would decrease. 16

This new extraction method is extremely innovative and it is important to note that it takes place in an aqueous solution.8 The old synthetic method used harsh organic solvents such as ether and ethyl acetate to achieve the final product.9 The step in Process Flow Sheet 2 involving acetone is the retrieval step, which means the final product Conversion Chart 1: Toxic limit for is about 6 g/L of final is washed off the resin and collected. The XAD-7 product. resin can be reused three times before there is a selective, producing almost all of the desired (-) loss in performance. 16 isomer with virtually no (+) isomer. 8 Conversion Chart 1 displays the enzyme effectiveness at The third step of the synthesis involves a carbon converting reactants to products at various oxidation alpha to the two aromatic rings on 8 concentrations.10 It was found that at about 6 g/L molecule 3, as depicted in Reaction 2. This of final alcohol product, the solution became toxic reaction was originally carried out using Chromium and the enzymes activity decreased dramatically.14 Trioxide as an oxidizing agent along with Sodium 9 This posed a problem for the synthetic chemists Hydroxide and a few organic solvents. The because they needed the drug to be made on a synthesis is a delicate one because it requires one large industrial scale. If the yeast couldn’t handle equivalent of Chromium Trioxide in order to high concentrations of product, it posed a successfully complete the partial oxidation of the problem. A synthetic resin was the key to solve α-benzylic carbon, as depicted in Scheme 2. If the this problem. The resin acts like the organic layer compound becomes too oxidized, a new (less in a traditional extraction.16 The final organic favorable) synthetic route must be taken to 10 product diffuses out of the aqueous layer and continue manufacturing the final product. adheres to the organic XAD-7 resin, as portrayed 16 by Isolation Step 1. The selection of the XAD-7 O resin was a key factor in maximizing the synthesis OH yield. If the organic components of the solution NO associated too strongly to the resin, an incomplete O 2 O

O O O

NO O 2 O NO2

O O

Scheme 2: Partial and full oxidation with Chromium Trioxide.

Isolation Step 1: Final product adheres to resin, afterwards the resin is removed with the product

Winter 2016 | 7

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459

The utilization of Chromium Trioxide to perform a partial oxidation on carbon alpha to two benzylic the oxidation was favorable to the synthetic rings; however, the new synthesis utilizes chemists because it posed both a health and an compressed air as an O2 source to partially oxidize environmental hazard.10 Several different the same carbon.8 The oxygen was passed combinations of reagents were tested in its place through the DMF/substrate solution and, and yielded unsuccessful results. Oxidizing agents combined with the effects of ultraviolet light, was such as Potassium Permanganate and other able to covalently bond to the carbon of interest to halogen based oxidizers proved inadequate form a peroxide, as depicted in scheme 3.10 After because they cleaved the substrate, generated the performing a work up with Sodium Hydroxide in di-ketone, or reacted with the aromatic ring.10 DMSO, the desired product was obtained.10 After extensive tests, the use of Chromium Trioxide proved to be the most favorable synthetic reagent.9

Although the Chromium reagent was effective, it 1 was unable to be recovered after the reaction. Scheme 3: Compressed air oxidation. For every one equivalent of the LY300164 product produced, three equivalents of Chromium waste were generated.1 On an industrial scale, this chromium waste built up quickly and the disposal The information necessary to provide a process became an issue that had to be addressed. flow sheet for the use of the Chromium Trioxide synthesis could not be obtained; however, a To solve this problem, chemists used an process flow sheet for the new synthesis using unorthodox, but ingenious method of oxidation. compressed air was obtained and is displayed in In the old synthesis, Chromium Trioxide performed

N/A Kg Reagent E Reaction 3 (new) 250 Kg Reagent F 8-12°C 804 Kg Solvent S6 Reagent E passed 2832 Kg Solvent S7 over for 4.5h 3000 Kg Solvent S4 3:1 Epimer Mix

6886 kg shit 384 kg product

50.1 = E-factor 86% Atom Economy

Neutralization 12750 kg 1M HCl 10-15°C 19252 kg Byproducts and solvent

Washing Resin 3000 kg water 0°C 384 kg product

Process Flow Sheet 3: Partial oxidation using compressed air. Winter 2016 | 8

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459

Process Flow Sheet 3.10 Although the two utilizing an inherently safer chemical synthesis methods of this synthesis cannot be numerically with compressed air. Additionally, by eliminating compared, the more important fact is that the use the heavy metal waste, they reduced worker of toxic Chromium reagent was eliminated upon exposure to the toxic chemical, and prevented it adoption of this novel synthetic method. The new from damaging the environment. In doing this method has an atom economy of about 86%, and they are being fiscally responsible as well as the E-factor is 50.1. Compressed air is also a environmental stewards. Through the use of a favorable reagent because it is cheap and if it goes biocatalyst, they increased the atom economy unreacted in this solution, there is minimal harm in significantly, and if this were not enough, the first releasing it to the atmosphere.12 This new step of the synthesis also utilizes a safer aqueous synthetic method showed an 86% yield.10 based solvent, rather than the harsh organic ether and ethyl acetate solvents. On top of all these The popularity of Talampanel as a treatment for improvements, they increased the percent yield several neurodegenerative disorders led to the three-fold. reevaluation of the synthetic manufacturing of this drug. In particular, the first step of the reaction to This award displays the power of green chemistry. generate the chiral alcohol was inefficient and Green chemistry is not so much of an individual proved economically unfavorable due to the field of chemistry as it is a philosophy. By pushing expensive reagents.10 Additionally, the partial the researchers at Eli Lilly laboratories to make the Chromium Trioxide oxidation was inefficient and synthesis better, the results were beneficial for all was a significant health hazard to both the workers parties involved. Reduced synthesis cost means and the environment. An alternative synthetic the manufacturing company needs less money to route was absolutely necessary to be both make the drug. The patient being prescribed the economically efficient and to be an environmental medication can then reap the benefits of these steward. The new method of synthesis not only lower syntheses costs and pay less for their reduced the amount of waste, but also increased prescription. All this occurs while protecting the the overall yield three times over, from 16 to 55%. environment and reducing the amount of waste and exposure to harmful chemicals. This award Conclusion displays several impressive innovations that every This particular Green Chemistry award has most chemical company and every business should take certainly earned its place as one of the top notice of. By striving to do better, Eli Lilly innovative solutions to a synthetic chemistry laboratories have truly achieved something problem. The researchers at Eli Lilly Laboratories special. addressed several of the Twelve Principles of

Green Chemistry when developing this new synthesis. Ultimately, the new synthetic method prevented heavy metal Chromium waste by

Winter 2016 | 9

Practical Application of a Biocatalyst in Pharmaceutical Manufacturing Green Chemistry CHM 459

References (1) Anastas, P. T.; Hammond, D. G. Inherent Safety at Chemical Sites: Reducing Vulnerability to Accidents and Terrorism Through Green Chemistry; Elsevier Science, 2015; Vol. 16. (2) Koenig, S. G. Scalable Green Chemistry; Pan Stanford Publishing, 2013. (3) Erdélyia, Szabóa, Birincsika, H. J. Mol. Catal. B Enzym. 2004, 29 (1-6), 195. (4) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B. Nature 2001, 409 (6817), 258. (5) Patel, R. N.; Banerjee, A.; Szarka, L. J. J. Am. Oil Chem. Soc. 1995, 72 (11), 1247. (6) Panke, S.; Held, M.; Wubbolts, M. Curr. Opin. Biotechnol. 2004, 15 (4), 272. (7) Ran; Zhao; Chen; Tao. Green Chem. 2008, 10 (4), 361. (8) Anderson, B. A.; Hansen, M. M.; Harkness, A. R.; Henry, C. L.; Vicenzi, J. T.; Zmijewski, M. J. J. Am. Chem. Soc. 1995, 117 (49), 12358. (9) Anderson, B. A.; Hansen, M. M.; Vicenzi, J. T.; Varie, D.; Zmijewski, M. J.; Harkness, A. R. Old_Synthesis_Papers.Pdf. 95306048.0, 1995. (10) Gadamasetti, K. G. Process Chemistry in the Pharmaceudical Industry; Marcel Dekker Inc., 1999. (11) Simon, R. C.; Busto, E.; Richter, N.; Belaj, F.; Kroutil, W. European J. Org. Chem. 2014, 2014 (1), 111. (12) Tundo, P.; Anastas, P. Green Chemistry: Challenging Perspectives; Oxford University Press Inc., 2000. (13) Cue, B. W.; Zhang, J. Green Chem. Lett. Rev. 2009, 2 (March 2015), 193. (14) Vicenzi, J. T.; Zmijewski, M. J.; Reinhard, M. R.; Landen, B. E.; Muth, W. L.; Marler, P. G. Enzyme Microb. Technol. 1997, 20 (7), 494. (15) Zaks, A. Curr. Opin. Chem. Biol. 2001, 5 (2), 130. (16) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations; Wiley-VCH, 2000.

Winter 2016 | 10