Method for the Production of Thiocarbamate Derivatives A2AR Inhibitors

Abstract

The present disclosure relates to synthesis of enantiomerically rich key drug intermediates as a means for manufacturing of thiocarbamate derivatives as A2A adenosine receptor (A2AR) inhibitors. More particularly, the present disclosure provides a viable efficient technology using enzymatic biotransformation process which utilizes cheaper substrate for production of high value key intermediates for A2AR inhibitors.

Claims

1. A process for preparing (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine (Intermediate B), or a pharmaceutically acceptable salt or solvate thereof, ##STR00060## comprising the step of contacting 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine, or a pharmaceutically acceptable salt thereof: ##STR00061## with an enzyme in a solvent.

2. The process of claim 1, wherein the enzyme is a monooxygenase.

3. The process of any of claims 1-2, wherein the enzyme is enzyme 3 (Accession number: ABG97104.1).

4. The process of claims 1-3, wherein the step further comprises the addition of an additional enzyme.

5. The process of claim 4, wherein the additional enzyme is a crude alcohol dehydrogenase.

6. The process of claim 4-5, wherein the additional enzyme is a ketoreductase (KRED).

7. The process of any of claims 4-6, wherein the additional enzyme is Enzyme 9 (Accession number: CAA46053.1).

8. The process of any of claims 1-7, wherein the solvent comprises an alcohol.

9. The process of any of claims 1-8, wherein the solvent comprises isopropyl alcohol.

10. The process of any of claims 1-9, wherein the solvent comprises water.

11. The process of any of claims 1-10, wherein the process affords Intermediate B in at least 99% ee.

12. The process of any of claims 1-10, wherein the process affords Intermediate B in at least 99.9% ee.

13. A compound of formula Intermediate B: ##STR00062## or a pharmaceutically acceptable salt thereof obtained by the step of contacting 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine, or a pharmaceutically acceptable salt thereof: ##STR00063## with an enzyme in a solvent.

14. The compound of claim 13, wherein the enzyme is a monooxygenase.

15. The compound of any of claims 13-14, wherein the enzyme is enzyme 3 (Accession number: ABG97104.1).

16. The compound of any of claims 13-15, wherein the step further comprises the addition of an additional enzyme.

17. The compound of any of claims claims 13-16, wherein the additional enzyme is a ketoreductase (KRED).

18. The compound of any of claims 13-17, wherein the additional enzyme is Enzyme 9 (Accession number: CAA46053.1).

19. A process for preparing compund 3A ##STR00064## comprising the step of reacting Intermediate B with intermediate A: ##STR00065## wherein Intermediate B was prepared using a process of any of claims 1-10.

20. The process of claim 19, wherein the step comprises addition of a base.

21. The process of claim 20, wherein the base is selected from the group of triethylamine (TEA), diisoproylethylamine (DIPEA), DEA, DIPA, and pyridine.

22. The process of claim 21, wherein the base is DIPEA.

23. The process of any of claim 19-22, wherein the step is performed in a solvent.

24. The process of claim 23, wherein the solvent is anisole.

Description

BRIEF DESCRIPTION OF FIGURES

[0358] FIG. 1: Schematic representation of protocol for large scale synthesis of intermediate compound B by enzymatical transformation

EXAMPLES

[0359] The present disclosure will be better understood with reference to the following examples. These examples are intended to representative of specific embodiments of the disclosure, and are not intended as limiting the scope of the disclosure.

[0360] The following abbreviations are used: [0361] CHMO: monooxygenase enzyme [0362] ADH: alcohol dehydrogenase enzyme [0363] IPA: Isopropyl alcohol [0364] DCM: Dichloromethane [0365] Wt: weight percentage [0366] DIPEA: N,N-Diisopropylethylamine, or Hnig's base [0367] HPLC: High-Pressure Liquid Chromatography [0368] GMP: Good Manufacturing Practice [0369] ND: Not Detected

Example 1

Selection and Optimization of Enzymes

1.1 Enzyme Screening and Selection of Best Candidate

[0370] 138 monooxygenases were screened to identify a suitable enzyme for the enzymatic synthesis of (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine (compound 3a in Table 1) which is a key intermediate of a series of thiocarbamate derivatives. The enzyme screening tests for were conducted and eight enzymes derived from 5 different organisms shown in Table 2 were identified based on several different criteria such as enantiomeric excess (ee), conversion efficiency, and chemoselectivity (oxidation only at the desired site e.g. on sulfur atom). Among eight enzymes identified during screening process, enzyme 3 from Rhodococcus jostii showed highest ee value without obvious impurities such as sulfone formation; therefore, it was selected for further optimization. After optimization process, the intrinsic ee value of enzyme 3 was roughly 96%, but the ee value increased to >99% along with the overoxidation of opposite enantiomer (R).

Overall Synthetic Route for Enzymatic Synthesis of (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine:

##STR00049##

[0371] Reaction Conditions for Screening Process

[0372] Reaction Condition 1: 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (20 mg, 0.07 mmol), isopropanol (100 L, 10% v/v), NADP+ (2 mg, 0.0024 mmol), NAD+ (2 mg, 0.003 mmol), crude monooxygenase (14 mg enzyme powder) in buffer (200 mM, PB8.0), crude alcohol dehydrogenase (20 mg enzyme solution), the final volume was 1 mL, 30 C., 200 rpm.

TABLE-US-00002 TABLE 2 Product Product Substrate (0.568 By- (0.670 Enzyme Enzyme Number Source notes min) products min) ee % cyclohexanone Enzyme 1 Wild type 13.02 0 86.98 27.64 monooxygenase from enzymes Arthrobacter sp. cyclopentadecanone 1,2- Enzyme 2 10.03 0 89.97 7.98 monooxygenase from Pseudomonas sp. monooxygenase from Enzyme 3 6.95 0 93.05 72.08 Rhodococcus jostii cyclohexanone Enzyme 4 Variants 45.28 0 54.72 93.74 monooxygenase variant from Brachymonas Enzyme 5 38.23 0 61.77 89.8 petroleovorans Enzyme 6 35 0 65 89.94 cyclohexanone Enzyme 7 Variants 5.64 0 94.36 41.66 monooxygenase variant from Rhodococcus sp. Enzyme 8 5.38 0 94.62 19.76

1.2 Preliminary Reaction Optimization for Enzyme 3

[0373] Reaction Condition 2: 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (20 mg, 0.07 mmol), isopropanol (100 L, 10% v/v), PEG400 (100 L), NADP+ (2 mg, 0.0024 mmol), NAD+ (2 mg, mmol), crude monooxygenase (20 mg enzyme powder) in buffer (200 mM, PB8.0), crude alcohol dehydrogenase (20 mg enzyme solution), the final volume was 2 mL, 30 C., 200 rpm.

[0374] Reaction Condition 3: 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (20 mg, 0.07 mmol), isopropanol (100 L, 5% v/v), Tween-80 (100 L, 5% v/v), NADP+ (2 mg, 0.0024 mmol), crude monooxygenase (20 mg enzyme powder) in buffer (200 mM, PB8.0), crude alcohol dehydrogenase (20 mg enzyme solution), the final volume was 2 mL, 30 C., 200 rpm.

[0375] A preliminary reaction optimization was conducted to synthesize (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine. Optimization conditions were summarized in Table 3. Lower IPA concentration (5%) was found to give better result. Adding 5% surfactant such as tween 80 was helpful for the reaction. The optimal cofactor was determined to be NADP+. With optimized reaction condition using enzyme 3, the conversion rate of starting material to (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine reached 60.01% without obvious impurities, while the ee value reached 94.91% (Table 3).

TABLE-US-00003 TABLE 3 Product Reaction condition Product Impurity Substrate ee Same as condition 2 11.17 0 88.83 n. d. Same as condition 2 9.46 0 90.54 n. d. Same as condition 2 10.20 0 89.80 n. d. Condition 2 and pH = 8.5 10.06 0 89.94 n. d. Condition 2 and pH = 9.0 3.02 0 96.98 n. d. Condition 2 and T = 25 C. 7.27 0 92.73 n. d. Condition 2 and T = 35 C. 3.26 0 96.74 n. d. Condition 2 and T = 40 C. 3.29 0 96.71 n. d. Condition 2 and IPA = 5% 16.43 0 83.57 n. d. Condition 2 and 14.12 0 85.88 n. d. IPA = 7.5% Condition 2 and 9.28 0 90.72 n. d. IPA = 12.5% Condition 2 and add 5% 8.27 0 91.73 n. d. methanol Condition 2 and add 5% 11.85 0 88.15 n. d. PEG 400 Condition 2 and add 5% 21.20 0 78.80 n. d. Tween-80 Condition 2 and add 5% 18.59 0 81.41 n. d. Triton-100 Condition 2 and add 5% 2.11 0 97.89 n. d. MTBE Condition 2 without NAD.sup.+ 13.75 0 86.25 n. d. Condition 2 without 3.27 0 96.73 n. d. NADP.sup.+ Same as condition 3 0 0 98.46 n. d. Same as condition 3 60.01 0 39.99 94.91 Same as condition 3 26.68 69.30 4.02 85.77 Same as condition 3 0 100 0 n. d. Same as condition 3 59.31 39.50 1.19 69.46 Same as condition 3 78.47 16.00 5.53 62.64 n. d. = not determined

1.3 Further Reaction Optimization for Enzyme 3

[0376] Enzymatic catalytic reaction was further optimized by altering quantity of enzyme and of cosolvent and volume of reaction relative to substrate amount.

[0377] Lower IPA concentration (2%) was found to give better result. Adding 15% surfactant such as tween-80 was helpful for the reaction. The optimum ADH dosage was determined to be 0.5 wt. Then, the reaction was amplified up to 100 mg scale, 2 wt CHMO gave the best result, with a residual substrate 1.56% and ee value>99% (Table 4). The intrinsic ee value was roughly estimated as 96% (Conv.=70.73%, ee=96.02%), but the ee value increased along with the overoxidation of opposite enantiomer (R). Then, the reaction volume was reduced to 60 V, 4 wt enzyme loading gave a good result with a residual substrate 1.12%, and the ee value>99% (Table 4).

[0378] Reaction Condition 4: 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (20 mg, 0.07 mmol), isopropanol (100 L, 5% v/v), Tween-80 (100 L, 5% v/v), NADP+ (4 mg, 0.0024 mmol), crude monooxygenase solution (10 wt) in buffer (200 mM, PB8.0), crude alcohol dehydrogenase solution (2 wt), the final volume was 2 mL, 30 C., 200 rpm.

[0379] Reaction Condition 5: 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (100 mg, 0.35 mmol), isopropanol (0.2 mL, 2% v/v), Tween-80 (1.5 mL, 15% v/v), NADP+ (20 mg, 0.012 mmol), crude monooxygenase solution (8 wt) in buffer (200 mM, PB8.0), crude alcohol dehydrogenase solution (0.5 wt), 200 rpm.

TABLE-US-00004 TABLE 4 Product Reaction condition Product Impurity Substrate ee Same as condition 4 50.35 0 49.65 n. d. CHMO (10 wt) Same as condition 4 40.81 0 59.19 n. d. CHMO (8 wt) Same as condition 4 30.89 0 69.11 n. d. CHMO (6 wt) Same as condition 4 20.68 0 79.32 n. d. CHMO (4 wt) Same as condition 4 17.68 0 82.32 n. d. CHMO (3 wt) Same as condition 4 10.96 0 89.04 n. d. CHMO (2 wt) Same as condition 4 5.44 0 94.56 n. d. CHMO (1 wt) Same as condition 4 31.55 0.04 68.40 n. d. ADH (2 wt) Same as condition 4 32.96 0.09 66.95 n. d. ADH (1 wt) Same as condition 4 38.17 0.08 61.75 n. d. ADH (0.5 wt) Same as condition 4 34.52 0.06 65.41 n. d. ADH (0.3 wt) Same as condition 4 41.36 0.11 58.53 n. d. Tween-80 (100 L) Same as condition 4 70.10 1.0 28.90 96.88 Tween-80 (200 L) Same as condition 4 78.88 1.83 19.30 98.13 Tween-80 (300 L) Same as condition 4 92.01 5.90 2.09 >99 IPA (40 L) Same as condition 4 90.45 2.12 7.43 98.18 IPA (60 L) Same as condition 4 94.12 3.31 2.56 >99 IPA (80 L) Same as condition 4 70.73 1.07 28.20 96.02 IPA (100 L) Same as condition 5 74.87 24.72 0.41 >99 CHMO (8 wt) Same as condition 5 79.71 19.80 0.49 >99 CHMO (6 wt) Same as condition 5 89.97 10.54 0.50 >99 CHMO (4 wt) Same as condition 5 94.08 4.36 1.56 >99 CHMO (2 wt) Same as condition 5 73.18 26.62 0.20 >99 Volume (100 V) Same as condition 5 75.45 24.34 0.22 >99 Volume (80 V) Same as condition 5 88.27 11.56 0.17 >99 Volume (60 V) Same as condition 5 82.02 15.05 0.63 >99 Volume (60 V, 8 wt) Same as condition 5 86.68 9.25 0.17 >99 Volume (60 V, 6 wt) Same as condition 5 93.31 4.36 1.12 >99 Volume (60 V, 4 wt) Same as condition 5 76.10 1.51 21.91 n. d. Volume (40 V, 8 wt) Same as condition 5 58.92 0.73 39.98 n. d. Volume (40 V, 6 wt) Same as condition 5 50.36 0.35 49.28 n. d. Volume (40 V, 4 wt) n. d. = not determined

[0380] After screening and validation, enzyme 3 (Table 7, Accession number: ABG97104.1) were selected to be used in a large scale synthesis of (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine. On large scale, synthesis parameters such as airflow, stirrer rate etc. were changed. Additionally, enzyme 3 catalyzed the conversion of sulfoxide to sulfone impurity which is a good way to improve stereoselectivity by over oxidizing the other enantiomer (R) and then removing the sulfone impurity from sulfoxide product without losing yield.

Example 2

Large-Scale Synthesis of Compound 3A

2.1 Synthesis of Intermediate A

[0381] ##STR00050##

Step 1:

[0382] 4,6-dichloropyrimidin-2-amine (82.0 kg, 1 wt, 1.0 eq) and Acetonitrile (660 kg, 8.05 wt) were charged to the reactor, then 2-methoxyethan-1-amine (57.0 kg, 0.70 wt, 1.5 eq) and DIPEA (78.8 kg, 0.96 wt, 1.2 eq) were added. The temperature was adjusted to 55-65 C. and the reaction was stirred until it was completed. The reaction end point was checked by HPLC. The reaction mixture was cooled to 5-15 C. over 6 h and stirred for 2 h. The solids were isolated by centrifuge and the wet cake was washed with Acetonitrile (150.0 kg, 1.83 wt). The wet cake was dried at 45-55 C. to give 6-chloro-N4-(2-methoxyethyl)pyrimidine-2,4-diamine (92.25kg, 91.1% yield).

Step 2:

[0383] KSCN (87.0 kg, 1.05 wt, 2.2 eq) and propionic acid (497 kg, 5.99 wt) were charged to the reactor and the temperature was adjusted to between minus 25 C. to minus 10 C. (25 to 10 C.). Br.sub.2 was added (0.88 wt, 1.1 eq) into the reactor maintaining a temperature below minus 10 C. (10 C.).

[0384] A filtered solution of 6-chloro-N4-(2-methoxyethyl) pyrimidine-2,4-diamine (83.0 kg, 1 wt, 1.0 eq) were dissolved in propionic acid (332.8 kg, 3.85 wt) and added to the reactor while maintaining the reactor contents below minus 5 C. (5 C.). The reaction was stirred at minus 15 to minus 5 C. (15 to 5 C.) until the reaction is complete. Aqueous ammonia (25% w/w, 917 kg, 11.05 wt) were then added while maintaining the reactor contents below 0 C. Once the addition was complete, the reaction temperature was adjusted to 45-55 C. and the reaction was stirred until the reaction is complete (Confirmed by HPLC). The reaction temperature was adjusted to 15-25 C. over 2 h and it was stirred for further 2 h. The solids were then separated by centrifuge and the wet cake was transferred into another reactor. Process water (498 kg, 6.00 wt) was added into the reactor and the temperature was adjusted to 15-25 C., then the reaction was stirred for 4 h. The wet cake was washed with H.sub.2O (321.2 kg, 3.87 wt) and then it was dried at 50-70 C. to obtain the final product of step 2 (90.4 Kg, 85% yield).

Step 3:

[0385] The final product of step 2 was charged into the reactor C19031849-B (90.4 kg, 1 wt, 1.0 eq) and dioxane (2,189 kg, 24.19 wt) was added and it was stirred at 20-40 C. for 3 h. The reaction was then filtered and washed with dioxane (101 kg, 1.12 wt). The reaction was transferred into another reactor, and H2O (95 kg, 1.05 wt) and TFA (99.0 kg, 1.09 wt, 2.5 eq) was added. The temperature as adjusted to 90-100 C. and stirred until the reaction was completed (Confirmed by HPLC). The reaction was concentrated to 5-7V below 55 C. The temperature was adjusted to 45-55 C. and H2O (900 kg, 9.94 wt) was added at 45-55 C. The temperature then adjusted to 55-65 C. and it was stirred over the course of 2 hours. Then, the reaction temperature was adjusted to 5-15 C. and stirred for 6 h. Solids were isolated by centrifuge, and the wet cake was washed with water (509 kg, 5.62 wt). The wet cake then was dried at 45-55 C. to give final product of step 3 (80.32 kg, 88.5% yield).

Step 4:

[0386] ##STR00051##

[0387] Final product of step 3 (37.3 Kg, 1.00 eq) and ethanol (20 rel. vol) were added to the reactor and the reactor was heated and stirred at 75-85 C., then the reaction was cooled to 20-30 C. and 2-furoic acid hydrazide (27.1 Kg, 1.5 eq) were added. After 10-15 minutes of stirring, 5-6 N HCl in 2-propanol (78 Kg, 3.0 eq) was added and the reactor contents were again heated to 75-85 C. The reaction was stirred for at least 18 hours until the reaction is complete (Confirmed by HPLC). Then, it was cooled to 20-30 C. and it was stirred for 2 hours. The solids were isolated by filtration and the isolated solids were washed with Methyl tert-Butyl Ether (110 Kg, 4.0 rel. vol). The solids were dried at 20-30 C. to give final product of step 4. (40.8 Kg, 79.8% yield)

Step 5 & 6:

[0388] ##STR00052##

Step 5:

[0389] The final product of step 4 (40.8 Kg, 1.00 eq), Hexamethyldisilazane (314 Kg, 10 rel. vol) and N,O-Bis(trimethylsilyl)acetamide (170.4 Kg, 5.0 rel. vol) were added to the reactor and heated to 115-125 C. over 2 hours while stirring. Then the reaction was allowed to be stirred for 20 hours sampling to test conversion. When reaction was complete, it was cooled to 45-55 C. Dose ethanol, abs. (325.8 Kg, 10.0 rel. vol) was added to the reactor at 45-55 C. over 2 hours and stirred for 1 hour, then the reaction was cooled to 15-25 C. The solids were isolated by filtration and washed with ethanol 70%. Finally, the solid product was dried at 20-30 C. to give final product of step 5 (24.9 Kg, 84.6% yield).

Step 6:

[0390] The final product of step 5 (24.9 Kg 1.00 eq) and dichloromethane (661 Kg, 20 rel. vol) were added to the reactor and stirred at 30-35 C. Dose boron tribromide 1.0 M in dichloromethane (329 Kg, 3.00 eq) was added at 30-35 C. over 1-2 h, then allowed stirring for at least 4.5 hours at 35-40 C. Then, the reaction was cooled to 20-30 C. The reactor temperature then was adjusted to 25-30 C. and dose methanol (15 rel. vol) was at 25-30 C. over 3 hours. The reactor was again heated to 35-45 C. and it was distilled off approximately 20 relative volumes solvent at reflux until 25 relative volumes remain.

[0391] Methanol (15 rel. vol) was added to the reactor and the reactor was heated to 40-50 C., then distilled off approximately 15 relative volumes solvent at reflux until 25 relative volumes remain.

[0392] Purified water (20 rel. vol) was added at 40-45 C. and distiled off approximately 25 relative volumes solvent at 30-50 C. and reduced pressure until 20 relative volumes remain.

[0393] Then, the reactor was cooled to 15-25 C. and pH was adjusted to 110.5 pH using 2 M sodium hydroxide while keeping the temperature at 20-30 C., then it was stirred for 1 hour.

[0394] The solids were isolated by filtration and the solids were slurry washed on the filter with purified water (4.0 rel. vol) at 15-25 C. for 15-20 mins, after removing the filtrate repeat the slurry wash a further three times.

[0395] The solids were slurry washed on the filter with methanol (4.0 rel. vol) at 15-25 C. for 15-20 mins, after removing the filtrate repeat the slurry wash once more. Dry the remaining solids at 35-45 C. to give final product of step 6 (21.3Kg, 89.3%).

Step 7:

[0396] ##STR00053##

[0397] Final product of step 6 (20.8 Kg, 1.00 eq) and pyridine (104 Kg, 5.0 rel. vol) were stirred at 15-25 C. Then, dose methanesulfonyl chloride (9.14 Kg, 1.2 eq) was added to the reactor at 15-25 C. over 1 hour, then the reaction was stirred for at least 6 hours at 15-25 C., monitoring conversion by HPLC. When the reaction was complete, dose purified water (211 Kg, 10 rel. vol) was added to the reactor at 15-25 C. over 1.50.5 hours and stirred for 1.50.5 hours.

[0398] The solids were isolated by filtration and it was washed three times with purified water (each 84 Kg, 4.0 rel. vol), then washed three times with tetrahydrofuran (each 75 Kg, 4.0 rel. vol).

[0399] The solids were dried at 15-25 C. for 12 hours to give intermediate A (23.2 Kg, 89.7% yield).

2.2 Synthesis of Intermediate (B) Using Enzymatic Transformation

2.2.1 Synthesis of Starting Material for Enzymatic Transformation

[0400] ##STR00054##

Step 1:

[0401] Tert-butyl 4-(2,4-difluoro-5-hydroxyphenyl)piperazine-1-carboxylate (134.5 Kg, 1.00 eq.) and acetonitrile (1,085 Kg, 8.0 rel. vol) were added to the reactor and stirred. Then, K2CO3 (94.2 Kg, 1.6 eq.) were added and continued to stir for 30 min at 20-30 C. Then, 1-chloro-2-methylsulfanyl-ethane (51.7 Kg, 1.1 eq.) were added to the reaction and the temperature was adjusted to 78-84 C. and stirred for 8-10 hours. After 8-10 hours, the temperature was cooled to 20-30 C. and the reaction was checked for completion (by HPLC). The solids were removed by filtration, and washed with acetonitrile (165 Kg). Then, the filtrate and washings were combined in another reactor, then concentrate to 270-540 L at 50 C. under reduced pressure, then n-heptane (1,598 Kg) was charged into the reactor. The resulting solids were isolated by centrifuge to give tert-butyl 4-(2,4-difluoro-5-(2-(methylthio)ethoxy)-phenyl)piperazine-1-carboxylate (135.6 Kg, 82% yield)the final product of step 1.

Step 2:

[0402] Tert-butyl 4-(2,4-difluoro-5-(2-(methylthio)ethoxy)-phenyl)piperazine-1-carboxylate (91 Kg, 1.00 eq.) and methanol (364 Kg, 4.0 rel. vol) was added to the reactor and stirred for 15-30 mins while cooling to 5-15 C. slowly. Dose 4 M HCl/MeOH solution (100 kg) were added with stirring while maintaining the temperature at 5-15 C., then reaction was stirred at this temperature for a further 20-24 hours. Once reaction was complete (confirmed by HPLC), 9% NaOH solution (355 Kg) were slowly added in portions at 25 C. The mixture was concentrated at 45 C. under vacuum to 315-405 L and the aqueous layer was extracted twice with DCM (2400 Kg) and the organic layers were combined in a clean reactor. N-heptane (1,077 Kg) was added and the mixture was concentrated at 45 C. under vacuum to 450-540 L. The solids were isolated by centrifuge and washed with n-heptane (46 Kg), dry at 50-60 C. for 18-25 h to give 1-(2,4-difluoro-5-(2-(methylthio)ethoxy)phenyl)piperazine (51.5 Kg, 79% yield)starting material for enzymatic reaction.

2.2.2 Synthesis of Intermediate (B) by Enzymatic Transformation

[0403] ##STR00055##

[0404] 29.5 kg 1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine-intermediate B was manufactured under GMP conditions using above illustrated biotransformation synthetic route with using monooxygenase enzyme 3 (Table 7, Accession number: ABG97104.1) and KRED (Enzyme 9, Table 7, Accession number: CAA46053.1) 49.7 kg starting material was used. After the work up process, intermediate B was obtained in 56.2% yield with 98.6% LCAP (Liquid Chromatography Area Percent), 97.4% assay and 100.0% ee. Summary of the reaction yield can be found in Table 5. Protocol for the procedure is summarized in Table 6.

TABLE-US-00005 TABLE 5 Scale Starting Crude (Reactor Material, Intermediate B, kg Purity Assay Yield Size), L kg Theoretical Actual (%) (%) (%) 8000 49.7 52.5 29.5, 28.7 98.6 97.4 56.2 corrected

[0405] Procedure (In 8000 L Reactor)

TABLE-US-00006 TABLE 6 Procedure Note 1. Exchange A clean and dry 8000 L stainless steel reactor was Oxygen content 0.8% evacuated to P 0.07 MPa then filled with N2 to normal pressure. This operation was repeated for 3 times until oxygen content was 1.0%. Dissolved oxygen electrode calibration was corrected. 2. Charging Purified water (2011.9 kg), disodium phosphate Charging temperature 23.8~25.0 C. dodecahydrate (82.8 kg), monosodium phosphate Purified water 2638.6 kg (3.4 kg) were added into above reactor at 15~30 C. Monosodium phosphate 82.8 kg The solid addition funnel and pipe were rinsed with Disodium phosphate dodecahydrate 3.4 kg purified water (626.7 kg), the mixture was stirred for Stirring time 34 min 0.5~1 h until the solid dissolved completely by pH 8.2 visual check. The mixture was sampled for pH analysis to make sure pH = 7.7~8.3. 3. Adding Isopropanol (78.7 kg) and starting material for Adding temperature 23.7~25.1 C. enzymatic reaction (SME) (49.7 kg) were added into Isopropanol 78.7 kg mixture at 15~30 C.The mixture was stirred for SME 49.7 kg 10~20 min. The mixture was sampled for pH Stirring time 15 min analysis to make sure pH = 8.0~9.0. pH 8.2 4. Adjusting temperature The mixture was adjusted to 15~20 C. Temperature 20.0 C. 5. Adding While maintaining the temperature at 10~20 C., Adding temperature 17.3~17.9 C. nicotinamide adenine dinucleotide phosphate Nicotinamide adenine 1.0 kg (1.0 kg), monooxygenase-Enzyme 3 (198.3 kg) and dinucleotide phosphate ethanol dehydrogenase-Enzyme 9 (24.6 kg) were Monooxygenase-Enzyme 3 198.3 kg added into mixture. Ethanol dehydrogenase-Enzyme 9 24.6 kg 6. Ventilation Air was ventilated into mixture at a reference rate of 3.0~5.0 m 3/h. Then nitrogen was ventilated into mixture at a reference rate of 6.0~10.0 m 3/h. 7. Reaction The mixture was stirred for reaction at 15~20 C. The Reaction temperature 17.3~19.0 C. dissolved oxygen value of the mixture was recorded Reaction time 34 h 30 min every 1 h. 2 h later, the mixture was sampled for Monooxygenase-Enzyme 3 18.4 kg HPLC analysis every 1~3 h until area % of SME 1.3% SME 1.5%, Sulfone 5.0% and ee 99.0%. Sulfone 2.8% The SME >1.5 area % and difference between two ee 100.0% consecutive samples was 0.5%, then monooxygenase 507047 (18.4 kg) was added and continued to react until area % of SME 1.5%, Sulfone 5.0% and ee >99.0%. Sampling method: Take 5 ml mixture, quench into 10 ml mixture of acetonitrile and purified water (The volume ratio is 1:1), the resulting homogeneous mixture was submitted for test. 8. Adjusting pH While maintaining the temperature at 10~25 C., Temperature 18.9~19.2 C. hydrochloric acid (88.4 kg) was added into mixture Hydrochloric acid 88.4 kg at a rate of 30~60 kg/h. After adding, the mixture pH 2 was sampled for pH analysis. 9. Adding & Filtration Celite (25.0 kg) was added into mixture, and stirred Celite 25.0 kg for 1~2 h. Then the mixture was filtered with a Stirring time 1 h 1 min plastic-lined centrifuge. The filtrate was discharged Centrifugation time 88 h 30 min to a 5000 L reactor through in-line filter. 10. Nanofiltration The nanofiltration parameter was set as below: Pump frequency 30.9~34.4 Hz Size of membrane: 5000 Da; Pump frequency: Membrane circulation flow 0.868~1.008 m 3/h 30~35 Hz; membrane pressure: 0.5~0.9 MPa. Then Membrane pressure 5.2~9.0 bar the mixture was concentrated until 100~200 kg left. Purified water by two batches (126.4 kg + 141.4 kg) Purified water 267.8 kg was added into the retentate and then continue to Protein content <50 ppm concentrate. The permeates were combined and Nanofiltration time 137 h sampled for protein content analysis to make sure it was 50 ppm. 11. Adjusting temperature The mixture was adjusted to 20~35 C. under stirring. Temperature 23.4 C. 12. Adding While maintaining the temperature at 20~35 C., L- Temperature 23.4~27.9 C. cysteine (10.0 kg) and anhydrous sodium carbonate L- cysteine 10.0 kg (361.1 kg) was added into mixture. Anhydrous sodium carbonate 361.1 kg 13. Decomposition of Sulfone The mixture was stirred for reaction at 20~35 C. 2 h Reaction temperature 27.5~28.0 C. later, the mixture was sampled for HPLC analysis Reaction time 6 h 30 min every 1~3 h until area% of Sulfone was 0.2%. Area % of Sulfone ND Sampling method: Take 5 ml mixture and submit for analysis testing. 14. Extraction While maintaining the temperature at 20~30 C., the Isopropyl acetate 436.3 kg mixture was extracted with isopropyl acetate by two 1st Stirring time 21 min times (217.6 kg + 218.7 kg). The mixture was stirred 1st Settling time 53 min for 15~30 min and then settled before separation. 2nd Stirring time 30 min The aqueous phase was sampled for purity to make 2nd Settling time 3 h 10 min sure SME 0.2 area %. Area % of SME ND 15. Adding While maintaining the temperature at 20~30 C., Adding temperature 20.2~24.5 C. anhydrous sodium carbonate (360.4 kg) was added Anhydrous sodium carbonate 360.4 kg portion-wise into aqueous phase at the interval of Stirring time 38 min 30~60 min, the mixture was stirred for 0.5~1 h. 16. Extraction While maintaining the temperature at 20~30 C., the Temperature 22.6~24.2 C. mixture was extracted with DCM five times DCM 1247.6 kg (249.9 kg + 250.0 kg + 249.3 kg + 249.1 kg + 249.3 kg). 1st Stirring time 17 min the mixture was stirred for 15~20 min every time 1st Settling time 45 min and then settled before separation. 2nd Stirring time 20 min 2nd Settling time 45 min 3rd Stirring time 18 min 3rd Settling time 2 h 44 min 4th Stirring time 17 min 4th Settling time 2 h 43 min 5th Stirring time 20 min 5th Settling time 2 h 25 min 17. Washing Sodium carbonate solution which was prepared with Temperature 13.9~15.2 C. sodium carbonate (19.3 kg + 19.2 kg) and purified Sodium carbonate 38.5 kg water (124.8 kg + 122.1 kg) was added into organic Purified water 246.9 kg phase at 10~30 C. The organic phase was sampled for HPLC analysis to make sure that the sample is free from large quantity of sulfone degradation products and other impurities. 18. Concentration The mixture was concentrated at Tjacket 45 C. Jacket temperature 35.6~39.2 C. under reduced pressure (P 0.05 MPa) until Concentration pressure 0.09~0.08 MPa 175~240 L left. 19. Concentration The mixture was transfer to 2000 L reactor through Isopropyl acetate 611.3 kg in-line filter. Isopropyl acetate two Concentration temperature 19.4~33.6 C. times(305.8 kg + 305.5 kg) was added into mixture Concentration pressure 0.096~0.080 MPa through in-line filter and stirred for 10~15 min. Then DCM residual 0.5% the mixture was concentrated at 45 C. under reduced pressure (P 0.08 MPa) until 170~200 L left. The mixture was sampled for DCM residual to make sure DCM 1.0% 20. Cooling& Maintaining The mixture was cooled to 15~20 C at a reference Maintaining temperature 18.5~19.0 C. rate of 10~15 C., and then it was maintained and Maintaining time 50 min stirred for 0.5~1 h at 15~20 C. 21. Adding n-Heptane (117.0 kg) was added into mixture n-Heptane 117.0 kg through in-line filter at a reference rate of 30~40 kg/h. 22. Crystallization The mixture was cooled to 0~10 C and maintained Cooling temperature 9.4 C for crystallization. After 2 h, the mixture was Crystallization temperature 4.7~9.4 C sampled for HPLC analysis every 1~3 h until the Crystallization time 6 h 47 min difference between two consecutive samples of The difference between two 0.56% intermediate B assay was 2.0% consecutive samples of intermediate B 23. Filtration The mixture was filtered with stainless steel nutsche n-Heptane 35.1 kg filter. The reactor wall or bottom was rinsed with n- Area % of SME ND heptane (35.1 kg), and then the n-heptane rinsed the filter cake. The filter cake was sampled for HPLC analysis to make sure that the sample is free from large quantity of impurities. 24. Drying The solid was dried at 15~25 C. 8 h later, the Drying temperature 15.0~24.6 C. mixture was sampled for analysis every 4~8 h until Drying time 65 h 7 min DCM residual 600 ppm; isopropyl acetate residual DCM residual 543 ppm 5000 ppm; n-heptane residual 5000 ppm. During Isopropyl acetate residual Less than 3339 ppm drying, the solid was turned over every 4~8 h. n-Heptane residual Less than 3339 ppm 25. Product - Intermediate B (+)-1-(2,4-difluoro-5-(2-(methylsulfinyl)ethoxy)phenyl)piperazine Appearance: Off-white solid Quantity: 29.5 kg, 28.7 kg corrected Purity: 98.6% Assay: 97.4% Yield: 54.8%

[0406] The detailed protocol is illustrated in FIG. 1. Materials used in the manufacturing process is summarized in Table 7.

TABLE-US-00007 TABLE 7 Mol. Quantity Molar Weight Material Wt (kg) Mol. Ratio Ratio Spec SME 288.4 49.7 172.3 1.0 1.0 Identification: Conforms to structure; Assay 97% Monosodium 82.8 1.7 Characteristics: White solid; phosphate Assay 98.0%; pH: 4.2~4.6; Identification for Sodium: Conforms; Identification for phosphate: Conforms Disodium phosphate 3.4 0.1 Characteristics: White solid; dodecahydrate pH: 8.8~9.2; Identification for Sodium: Conforms; Identification for phosphate: Conforms Purified water 3153.3 63.4 Appearance: Clear and colorless liquid and odorless; Conductivity (25 C.): Acceptance criteria <1.3 s/cm; TOC: Acceptance criteria <500 ppbC; Microbial: Purified water: Total microbial: Acceptance criteria <100 CFU/ml; Objectionable Organism: Should not be detected; test methods refer to USP (Current Version); Endotoxin: The acceptance criterion is NMT 1 EU/ml, test methods refer to ChP (Current Version); Other: Ammonia, pH, nitrate, nitrite, and non-volatile substance tests Ammonia, pH, nitrate, nitrite, and non-volatile substance tests: it should be passed refers to testing methods of CP (current version) Isopropanol 78.7 1.6 Characteristics: Colorless liquid; Purity 99.0%; Water content 0.2% w/w; Identification: Conform with standard spectrum Nicotinamide 1.0 0.02 White to off-white solid; adenine dinucleotide Assay 80%; Identification: phosphate Conform to structure Monooxygenase 216.7 4 + 0.4 Residue 1-(2,4-difluoro-5-(2- Enzyme 3 (methylthio) ethoxy)phenyl) piperazine of use-test 1.5% Ethanol 24.6 0.5 Residue 1-(2,4-difluoro-5-(2- dehydrogenase (methylthio) ethoxy)phenyl) Enzyme 9 piperazine of use-test 1.5% Hydrochloric acid 88.4 1.8 Characteristics: Colorless to light yellow liquid; Assay: 36% w/w Celite 25.0 0.5 Exemption L-Cysteine 10.0 0.2 White to off-white solid; Assay 90%; Identification: Conform to structure Anhydrous sodium 106.0 760.0 7169.8 41.6 15.3 Characteristics: White solid; carbonate Total alkali content (by Na2CO3) 98.0%; Identification for Sodium: Conforms; Identification for Carbonate: Conforms Isopropyl acetate 1047.6 21.0 Characteristics: Colorless liquid; Purity 99.0%; Water content 0.1% w/w; Identification: Conform with standard spectrum Dichloromethane 1247.6 25.1 Characteristics: Colorless liquid; Purity 99.0%; Water content 0.05%w/w; Identification: Conform with standard spectrum n-Heptane 117.0 2.4 Colorless liquid, Purity 97.0%, KF 0.1%, Identification: Conform with standard spectrum

2.3 Synthesis of Intermediate (B) Using Conventional Synthetic Route

[0407] ##STR00056## ##STR00057##

[0408] To a mixture of Compound 1 (60 kg, 191 mol, 1.0 eq) in acetonitrile (600 L) was added sodium hydroxide (22.9 kg, 573 mol) and 2-chloroethyl-methylsulfide (25.4 kg, 229 mol, 22.6 L) at 15 C. The mixture was stirred at 82 C. for 16 h. The reaction mixture was cooled to 1520 C. and then the reaction mixture was centrifuged and the filtrate was collected. The filter residue was stirred in ethylacetate (200 L) at 3540 C. for 2 h and then centrifuged and the filtrate was combined. The combined filtrate was decolorated with active carbon and filtered. The filter was concentrated until the residual solvent was 150 L. Then n-heptane (400 L) was added into the mixture and the mixture was concentrated until the solvent residual was 150 L. The residue was centrifuged and dried by drying oven to give Compound 2 (51 kg, 131 mol, 68.8% yield) as a light brown solid.

[0409] To a mixture of Compound 2 (50.5 kg, 130 mol, 1 eq) in acetic acid (200 L) was added aqueous hydrogen peroxide (17.7 kg, 156 mol, 15 L, 30% purity, 1.20 eq) at 1015 C. The mixture was stirred at 1015 C. for 3 h. The reaction was quenched with aq.Math.Na.sub.2S.sub.2O.sub.4 (25 Kg/200 L). The reaction mixture was then extracted with ethyl acetate (200 L3). The pH value of the organic layer was adjusted to 8 by adding saturated aqueous sodium hydroxide (250 L). The combined organic phase was washed with brine (100 L) and concentrated at 45 C./0.1 MPa until the residual solvent was 150 L. Then n-heptane (400 L) was added into the mixture and the mixture was concentrated until the solvent residual was 150 L. The residue was centrifuged and dried by drying oven to give a racemic mixture of enantiomers 3 and 4 (50.9 kg, 125 mol, 96.5% yield, 99.7% purity) as an off-white solid.

[0410] The racemic mixture of enantiomers 3 and 4 (29.52 Kg) was dissolved in methanol (628 L) and the coloured solution was decolorized with activated charcoal. The feed solution was then subjected to simulated moving bed chromatography, using as chiral stationary phase Chiralpak AD CSP (20 m particle size, 1 Kg), packed in 8 columns at 5 cm ID and 10 cm length evenly. Methanol was used as mobile phase. Enantiomer 3 was collected as the first eluting enantiomer and the corresponding fractions were concentrated under reduced pressure to yield compound 3 (20.1 Kg, enantiomeric excess of 98.1% and 99.9% HPLC purity).

[0411] Compound 3 (9.06 Kg, 22.7 mol) was treated with zinc bromide (12.8 Kg; 56.8 mol), ethyl acetate (34.4 Kg) and isopropanol (7.4 Kg). The mixture is heated at 75-80 C. for 6 hours and after completion of the reaction, the reaction was cooled (precipitation) and aged. The crude product was filtered, washed with ethyl acetate and dried at ambient temperature. The crude was dissolved in a mixture of dichloromethane, 20 wt % aqueous sodium carbonate solution and 28-30 wt % aqueous ammonium hydroxide solution. The phases were allowed to separate, the lower organic layer containing the product was separated off and the aqueous layer was re-extracted with dichloromethane. The combined organic layers were concentrated by distillation at reflux, cooled, passed through a 1-micron filter then charged back to the reactor. The solution containing the product was solvent exchanged from dichloromethane to isopropyl acetate under reduced pressure and constant volume, cooled to ambient, n-heptane was added and the mixture cooled further to complete crystallization. The product Intermediate B was filtered, washed with n-heptane then dried (4.57 Kg, 67%).

2.4 Synthesis of A2A Inhibitors Through Coupling Reaction Between Intermediate (A) and Intermediate (B)

[0412] ##STR00058## ##STR00059##

[0413] Intermediate A (23.2 Kg, 1.0 eq), Intermediate B (24.8 Kg, 1.4 eq) and anisole (93 Kg, 4.0 rel. vol) were added to the reactor and stirred. Then, N,N-diisopropylethylamine (11.5 Kg, 1.5 eq) and anisole (23.3 Kg, 1.0 rel. vol) were added and the reaction was heated to 105-115 C., and continued stirring for at least 20 hours. When reaction complete (confirmed by HPLC), the reactor contents was cooled to 70-80 C. Dose acetonitrile (128 Kg, 5.5 rel. vol) was added at 755 C. over at least 30 minutes, then it was cooled to 15-25 C. and stirred for 1 hour. The solids were isolated by filtration and washed with two portions of acetonitrile (each 55 Kg, 3.0 rel. vol), followed by two portions of methyl tert-butylether (each or 53 Kg, 3.0 rel. vol). The solids then were dried at 15-25 C. to give crude Compound 3A (29.1 Kg, 82.1% yield).

[0414] Crude compound 3a (22 Kg, 1.0 eq) and dimethyl sulfoxide (218 Kg, 9.0 rel. vol) were added to the reactor and stirred at 65-75 C. until fully dissolved. After 2 hours, the reaction was cooled to 45-50 C. and the contents were transferred through a polishing filter into a clean reactor, and stirred and heated to 55-65 C. Then, dose methanol (173 Kg, 10 rel. vol) whilst maintaining temp at 55-65 C. was added. After 2-4 hours stirring at 55-65 C., the reaction was cooled to 05 C. over a period of 3.5-4.5 hours, then stirred for a further 12-16 hours. The solids were isolated by filtration and washed twice with methanol (each 70 Kg, 4.0 rel. vol), then twice with methyl tert-butylether (each 65 Kg, 4.0 rel. vol). Finally, the solids were dried for at least 4 hours at 45-55 C. to yield Compound 3a (16.8 kg, 76% yield).

3. Enzymatic Biotransformation vs. Conventional Synthesis of Intermediate B

[0415] One of the important advantages of the enzymatic process is that it removes the necessity for a chiral separation using a chromatography on a chiral phase (SMB). Therefore, enzymatic biotransformation can produce the A2A inhibitors mentioned in this disclosure at a relatively higher yield in a cost-efficient way.

[0416] Conventional synthetic pathway for synthesis of Intermediate B showed that the SMB has a yield of approximately 40% (the other 40% is the other isolated enantiomer and 20% is lost as mixed fraction), whereas the enzymatic process has a yield of approximately 55% with high ee>99%.

[0417] There is also a clear cost advantage. For example, cost of chiral separation per kg of enantiomer produced by SMB is about 26,000 USD (based on 10 kg scale). On the other hand, cost of chiral oxidation per kg of enantiomer produced by enzymatic transformation is approximately 10,000 USD (based on 62 Kg scale). In addition, the cost per kg of the enzymatic process should decrease at higher scale, whereas the cost per kg of the SMB process does not really decrease much. A skilled person in the art knows that the cost of the processes can hugely vary depending on the various factors even global economy trends, however; costs calculated for the present disclosure should be evaluated under the time of filing.

[0418] Therefore, these two advantages mentioned above enhances industrial acceptance and suitability of the enzymatic transformation process of the present disclosure.

4. Sequences of the Lead Enzymes

[0419]

TABLE-US-00008 TABLE7 Accession SEQ Enzyme number IDNO Aminoacidsequence Monooxygenase ABG97104.1 1 MTTSMKAANPMNFPSTSDTGI from VDVLGVGAGFSGLYLSHRLTT Rhodococcus AGWTFAGFEAGPSVGGTWFW jostii NTYPGARCDVESIYYSYSFDEA (Enzyme3) LQQEWTWSQRFAPQAEILSYIN HVADRFDLRKHFTFNTRVVGA TWNAAERLWEVQLDNGETRR GRYLISGAGGLSTPKDFDVPGL GNFTGLQVSTSRWNISLDDLA GKRVAVIGTGSSGVQAIPLIAE VAEHVTVFQRTPNYVMPARN AELPLERVDSIKDDYPAIREEC RHSPGGIPDRPVTDKAFDVSAE ERQRRYEAAYERSGFNGVGGE FADLLTDVEANRTASEFIHDKI REIVEDPATAELLVPRYHPLGA KRSVFGTDYYETYNRPNVSLV SLRDEPIETMTANAIVTSKGTY EADAVVLAIGFDAFTGPLYGL GLTGASGRKLQETWQDGIRTY LGMMTTDFPNFFMVAGPQSPA LASNVVMTIEQAVDWIADLIE HARDSGATLVEATPEGQNDW VDITEETVAQTLYATTDSWYR GSNVEGKPNTFMGYVGGVGK YRRMCTEIAKRGYPGVRIDGE TESPHLGPIHREIS Ethanol CAA46053.1 2 MKGFAMLSIGKVGWIEKEKPAP Dehydrogenase GPFDAIVRPLAVAPCTSDIHTVFE from GAIGERHNMILGHEAVGEVVEV Thermoanaerobium GSEVKDFKPGDRVVVPAITPDWR brockii TSEVQRGYHQHSGGMLAGWKFS NVKDGVFGEFFHVNDADMNLA HLPKEIPLEAAVMIPDMMTTGFH GAELADIELGATVAVLGIGPVGL MAVAGAKLRGAGRIIAVGSRPV CVDAAKYYGATDIVNYKDGPIES QIMNLTEGKGVDAAIIAGGNADI MATAVKIVKPGGTIANVNYFGE GEVLPVPRLEWGCGMAHKTIKG GLCPGGRLRMERLIDLVFYKRVD PSKLVTHVFRGFDNIEKAFMLMK DKPKDLIKPVVILA

INCORPORATION BY REFERENCE

[0420] The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entireties for all purposes.

OTHER EMBODIMENTS

[0421] The disclosure set forth above may encompass multiple distinct disclosures with independent utility. Although each of these disclosures has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the disclosures includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Disclosures embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different disclosure or to the same disclosure, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the disclosures of the present disclosure.