TELAPREVIR

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Mar 292015

TELAPREVIR
MF C36H53N7O6
MolWeight: 679.85
CAS No.: 402957-28-2

NMR……….http://www.abmole.com/download/vx-950-hnmr.pdf
AND
http://file.selleckchem.com/downloads/nmr/S153802-Telaprevir-NMR-Selleck.pdf

1H NMR

13C NMR

Chem. Commun., 2010,46, 7918-7920

DOI: 10.1039/C0CC02823A
http://pubs.rsc.org/en/Content/ArticleLanding/2010/CC/c0cc02823a#!divAbstract‘

A very short and efficient synthesis of the important drug candidate telaprevir, featuring a biocatalytic desymmetrization and two multicomponent reactions as the key steps, is presented. The classical issue of lack of stereoselectivity in Ugi- and Passerini-type reactions is circumvented. The atom economic and convergent nature of the synthetic strategy require only very limited use of protective groups.

Graphical abstract: A highly efficient synthesis of telaprevir by strategic use of biocatalysis and multicomponent reactions

Telaprevir (1). 250 l of saturated K2CO3 was added to a solution of 14 (0.514 g, 0.75 mmol) in MeOH (20 ml) at room temperature. The reaction mixture was stirred for 30 minutes at room temperature resulting in a pale yellow suspension. After full conversion (as judged by TLC analysis), the reaction mixture was washed with 20 ml brine, the aqueous layer was washed again with 10 ml CH2Cl2 (2x). The organic layers were collected, dried with MgSO4 and concentrated in vacuo, to yield a pale yellow solid. The yellow solid was dissolved in CH2Cl2 (10 ml) and Dess-Martin periodinane (0.650 g, 1.532 mmol) was added at room temperature. The reaction mixture was stirred overnight before adding saturated NaHCO3 solution (10 ml) and saturated Na2S2O3 solution (10 ml). This mixture was stirred for 10 minutes, separated and the aqueous layers were washed with EtOAc (2 x 10 ml). The organic layers were collected, dried with MgSO4 and concentrated in vacuo to give the crude product as an 83:13:4 mixture of diastereomers. After silica gel flash chromatography (1% MeOH in CH2Cl2), 1 (0.412 mg, 0.61 mmol, 80%) was obtained as a white solid.

1H NMR (500.23 MHz, DMSO-d6): δ = 9.19 (d, J = 1.4 Hz, 1H), 8.91 (d, J = 24.5 Hz, 1H), 8.76 (dd, J = 1.5, 2.5 Hz, 1H), 8.71 (d, J = 5.3 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.21 (d, J = 8.9 Hz, 1H), 4.94 (m, 1H), 4.68 (dd, J = 6.5, 9.0 Hz, 1H), 4.53 (d, J = 9.0 Hz, 1H), 4.27 (d, J = 3.5 Hz, 1H), 3.74 (dd, J = 8.0, 10 Hz, 1H), 2.74 (m, 1H), 3.64 (d, J = 3.5 Hz, 1H), 0.92 (s, 9H), 0.87 (t, 3H), 0.84-1.40 (m, 23H), 0.65 (m, 2H), 0.56 (m, 2H);

13C NMR (125.78 MHz, CDCl3): δ = 197.0 (C), 171.8 (C), 170.4 (C), 169.0 (C), 162.1 (C), 161.9 (C), 147.9 (CH), 144.0 (C), 143.4 (CH), 56.4 (CH), 56.3 (CH), 54.2 (CH), 53.4 (CH), 42.3 (CH), 41.3 (CH), 32.1 (CH), 31.8 (CH), 31.6 (CH), 29.1 (CH), 28.0 (CH), 26.4 (CH3);

max (cm -1 ): 3302 (m), 2928 (m), 2858 (w), 1658 (s), 1620 (s), 1561 (s), 1442 (m);

HRMS (ESI, 4500 V): m/z calcd. for C36H53N7O6Na + ([M + Na] + ) 702.3950, found 702.3941.

SYN

Reference:

WO2011/103932 A1, ; Page/Page column 50; 51 ;

WO2011/103932 A1, ;

AND

WO2013/135870 A1, ;

WO2011/103932 A1, ;

……………….

http://www.google.com.ar/patents/WO2011103932A1?cl=en

t mpound XVIII,

(XVIII).

This compound, which also known as Telaprevir, could be prepared in higher yields and with higher efficiency than any previously disclosed processes. Furthermore, the chiral information used for the preparation was derived from readily available simple building blocks, making the process a highly effective approach to such prolyl dipeptides and similar peptidomimetics.

EXAMPLE 22

(5)-Methyl 2-cyclohexyl-2-(pyrazine-2-carboxamido)acetate (9).

Pyrazinecarboxylic acid (2.72 g, 21.9 mmol) was added to a solution of L- cyclohexylglycine methyl ester (4.13 g, 19.9 mmol) in CH₂CI₂ (100 ml) at room temperature under N₂, forming a white suspension. Triethylamine (6.33 ml, 4.62 g, 45.8 mmol) was added, followed by benzotriazol-l-yloxy-tris-(dimethylamino)- phosphonium hexafluorophosphate (BOP; 9.69 g, 21.9 mmol), which turned the reaction mixture from purple to an orange solution. After two days of stirring at room temperature the reaction mixture was washed two times with 50 ml saturated Na₂CC>3, followed by the washing of the aqueous layers with CH₂CI₂ (2 ^χ 50 ml). The organic layers were collected and dried with MgSC , followed by concentration in vacuo. Purification by silica gel flash chromatography (c-Hex:EtOAc = 2: 1 with 0.5% triethylamine) afforded 9 (5.28 g, 19.03 mmol, 96%) as a yellow oil that solidified upon standing to give a white solid.

[a f° = +42.5 (c= 1.13, CHC1₃); *H NMR (250.13 MHz, CDCI3) δ = 9.39 (d, J= 1.25 Hz, 1H), 8.76 (d, J = 2.5 Hz, 1H), 8.57 (t, J = 1.5 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 4.74 (dd, J= 5.5, 9.3 Hz, 1H), 3.78 (s, 3H), 1.96 (m, 1H), 1.77 (m, 5H), 1.24 (m, 5H); ¹³C NMR (62.90 MHz, CDCI3): δ= 172.0 (C), 162.8 (C), 147.4 (CH), 144.5 (CH), 144.1 (C), 142.7(CH), 57.0 (CH), 52.3 (CH₃), 41.2 (CH), 29.7 (CH₂), 28.4 (CH₂), 26.0 (CH₂); IR (neat): v^cm ) = 3374 (m), 2920 (s), 2845 (w), 1740 (s), 1665 (s); HRMS (ESI, 4500 V): m/z calcd. for Ci₄Hi₉N₃03Na⁺ ([M + Na]⁺) 300.1319, found 300.1319.

Example 23 :

(5)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetic acid (10). A solution of 1 M NaOH (12 ml, 12 mmol) was added to a solution of 9 (2.77 g, 10 mmol) in THF (25 ml) at 0°C. MeOH was added to the formed suspension, to give a clear, colorless solution. The reaction mixture was stirred overnight at room temperature, followed by concentration in vacuo. The pH of the aqueous layer was set on 3.5 with a 1 M KHSO₄ solution and was extracted with EtOAc (2 ^χ 25 ml). The mixture was dried with Na₂S04, filtered, and concentrated in vacuo, to give 10 (2.49 g, 9.45 mmol, 95%) as a white solid.

[a £° = +50.9 (c= 1.06, CHC1₃); H NMR (250.13 MHz, CDCI3): δ = 9.38 (d, J = 1.5

Hz, 1H), 8.78 (d, J= 2.5 Hz, 1H), 8.58 (dd, J= 1.5, 2.5 Hz, 1H), 8.27 (d, J= 9.0, 1H), 4.77 (dd, J = 4.3, 5.0 Hz, 1H), 2.00 (m, 1H), 1.76 (m, 5H), 1.37 (m, 5H); ¹³C NMR (62.90 MHz, CDCI3): δ = 175.7 (C), 163.0 (C), 147.2 (CH), 144.3 (CH), 144.2 (C), 142.0 (CH), 56.9 (CH), 40.9 (CH), 29.7 (CH₂), 28.1 (CH₂), 25.9 (CH₂); IR (neat): v_max (cm⁴) = 3383 (m), 2928 (s), 2852 (w), 1713 (m), 1676 (s), 1518 (s); HRMS (ESI, 4500 V): m/z calcd. For Ci₃H₁₇N₃0₃Na⁺ ([M + Na]⁺) 286.1162, found 286.1158.

Example 23 :

(S)-methyl 2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)-acetamido)-3,3- dimethylbutanoate (11). 10 (0.653 g, 4.5 mmol) was added to a solution of H-Tle- OMe (0.653 g, 4.5 mmol) in DMF (40 ml). l-Ethyl-3-(3-dimethylaminopropyl)- carbodiimide-HCl (EDOHC1; 0.919 g, 6.75 mmol) was added to this colorless solution followed by 1 -hydroxy-7-azabenzotriazole (HOAt; 1.035 g, 5.4 mmol) giving a bright yellow solution. The reaction mixture was stirred for 3 days and afterwards concentrated in vacuo. The formed yellow solid was dissolved in EtOAc, washed with 40 ml saturated aqueous ammonium chloride solution and 40 ml of saturated aqueous NaHCC>3 solution. The organic layers were collected, dried with MgSC^ and concentrated in vacuo to give 11 (1.48 g, 3.78 mmol, 84%) as a white solid.

[a f° = -2.0 (c= 1.0, CHC1₃); ¾ NMR (250.13 MHz, CDC1₃): δ = 9.39 (d, J = 1.5 Hz, 1H), 8.76 (d, J = 2.3 Hz, 1H), 8.55 (dd, J = 2.4, 1.8 Hz, 1H), 8.29 (d, J = 8.1, 1H), 6.40 (d, J= 9.3 Hz, 1H), 4.46 (m, 2H), 3.74 (s, 3H), 1.81 (m, 1H), 1.76 (m, 4H), 1.24 (m, 6H), 0.96 (s, 12H); ¹³C NMR (62.90 MHz, CDC1₃): δ = 171.7 (C) , 170.4 (C), 163.0 (C), 147.5 (CH), 144.5 (CH), 144.2 (C), 142.7 (CH), 60.2 (CH₃), 58.4 (CH), 51.9 (CH), 40.5 (CH), 31.7 (C), 29.7 (CH₂), 28.7 (CH₂), 26.6 (CH₃), 25.9 (CH₂); IR (neat): v„(cm^J) = 3350 (m), 2928 (m), 2853 (w), 1738 (s), 1686 (s), 1640 (s), 1520 (s); HRMS (ESI, 4500 V): m/z calcd. for C₂oH₃oN₄04Na⁺ ([M + Na]⁺) 413.2159, found 413.2169.

Example 24:

(S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3,3- dimethylbutanoic acid (2). A solution of 1 M NaOH (0.94 ml, 0.94 mmol) was added to a solution of 11 (0.31 g, 0.78 mmol) in THF (3 ml) at 0°C. MeOH was added to the formed suspension, to give a clear and colourless solution. The reaction mixture was stirred overnight at room temperature, followed by concentration in vacuo. The pH of this aqueous layer was set to 3.5 with 1 M KHSO₄ and subsequently extracted with EtOAc (2 ^χ 10ml). The mixture was dried with Na₂S0₄, filtered, and concentrated in vacuo, to give 2 (0.28 g, 0.75 mmol, 95%) as a white solid.

[a f° = +21.7 (c= 1.015, CHC1₃); *H NMR (250.13 MHz, CDC1₃): δ = 9.39 (d, J= 1.3

Hz, 1H), 8.77 (d, J = 2.5 Hz, 1H), 8.57 (dd, J = 1.5, 2.5 Hz, 1H), 8.35 (d, J = 9 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 4.45 (t, J = 8.8 Hz, 1H), 4.42 (d, J = 9.2 Hz, 1H), 1.94 (m, 1H), 1.71 (m, 5H), 1.20 (m, 5H), 1.01 (s, 9H); ¹³C NMR (62.90 MHz, CDCI3): δ = 173.4 (C), 170.5 (C), 163.3 (C), 147.4 (CH), 144.4 (CH), 144.2 (C), 142.8 (CH), 58.4 (CH), 51.9 (CH), 40.4 (CH), 34.7 (C), 29.8 (CH₂), 28.6 (CH₂), 26.6 (CH₃), 25.8 (CH₂); IR (neat): v„(cm ) = 3335 (w), 2930 (m), 1726 (m), 1663 (s), 1514 (s); HRMS (ESI, 4500 V): m/z calc. for Ci₉H₂₉N₄0₄Na⁺ ([M + Na]⁺) 399.2003, found 399.2013.

Example 25:

(5)-2-formamido-l-pentanol (12). (5)-2-amino-l-pentanol (1.00 g, 9.7 mmol) was dissolved in ethylformate (7.84 ml, 7.19 g, 97 mmol). This reaction mixture was refluxed at 80 °C for 4 hours, followed by stirring overnight at room temperature. The colourless solution was concentrated in vacuo and stirred for 1 hour in a 10 mol% K₂C0₃ in MeOH (25 ml). Afterwards, the pH was set to 7 with DOWEX 50wx8, followed by filtration and concentration in vacuo to give 12 (1.26 g, 9.61 mmol, 99%). [a f° = -29.6 (c = 1.15, CHCI₃); H NMR (250.13 MHz, CDC1₃): δ = 8.20 (s, 1H), 5.81 (bs, 1H), 4.04 (m, 1H), 2.11 (b, 1H), 1.47 (m, 4H), 0.94 (t, J = 7.0 Hz, 3H); ¹³C NMR (62.90 MHz, CDCI3): 161.8 (C), 65.1 (CH₂), 50.6 (CH), 33.2 (CH₂), 19.2 (CH₂), 13.9 (CH₃); IR (neat): v_max (cm ) = 3248 (s), 2957 (m), 1651 (s), 1528 (m), 1381 (m); HRMS (ESI, 4500 V): m/z calcd. for C₆Hi₃N0₂Na⁺ ([M + Na]⁺) 154.0838, found 154.0835.

Example 26:

(5)-2-formamidopentanal. (7). Dess-Martin periodinane (5.514 g, 13 mmol) was added to a solution of (5)-2-formamido-l-pentanol (12, 1.31 g, 10 mmol) in CH₂C1₂ (100 ml) at room temperature. The white suspension was stirred for 2 days and subsequently 35 ml MeOH was added and stirred for 30 minutes. The resulting suspension was filtrated and the filtrate was concentrated in vacuo. The crude product was purified by silica gel flash chromatography (cHex:EtOAc = 1 :4) to give 7 (1.08 g,

8.29 mmol, 83%) as a white solid. NMR analysis indicates that 7 is in equilibrium with its cyclic dimer.

[a f° = +37.6 ( c= 0.745, CHC1₃); ^lU NMR assigned to the monomer (250.13 MHz, CDCI₃): δ = 8.22 (s, 1H), 7.84 (s, 1H), 7.10 (m, 1H), 5.31 (m, 1H), 1.52 (m, 4H), 0.95 (m, 3H); ¹³C NMR assigned to the monomer (100.61 MHz, CDC13): 198.8 (CH), 161.7 (CH), 57.4 (CH), 30.8 (CH₂), 18.4 (CH₂), 13.7 (CH₃); ^lH NMR assigned to the dimer (400.13 MHz, CDC1₃) 8.22 (s, 2H), 5.26 (m, 2H), 3.72 (m, 2H) 1.52 (m, 8H), 0.95 (m, 6Η;) ¹³C NMR (100.61 MHz, CDCI3) assigned to the dimer: 161.7 (CH), 89.8 (CH), 63.1 (CH), 30.8 (CH2), 18.4 (CH2), 13.7 (CH3); IR (neat): _Vmax (cm ): 3325 (s), 2959 (s), 1649 (s), 1530 (s), 1381 (m), 1123 (w); HRMS (ESI, 4500 V): m/z calc. for C₆Hi₂N0₂ ⁺ ([M + H]⁺) 130.0863, found 130.0858.

It was noted that the dimer exists as a mixture of diastereomers.

Example 27:

(35)-2-acetoxy- V-cyclopropyl-3-formamidohexanoyl amide (13). From 7: Aldehyde 7 (0.892 g, 6.91 mmol) was added to a solution of cyclopropyl isocyanide (0.410 g, 6.12 mmol) in CH₂C1₂ (110 ml) and stirred for 5 minutes at room temperature. Acetic acid (0.711 ml, 0.747 g, 12.44 mmol) was added and the yellow reaction mixture was stirred for 3 days at room temperature. The reaction mixture was washed twice with 100 ml saturated Na₂C03, followed by drying with Na₂S04 and concentration in vacuo. The crude was purified by silica gel flash chromatography (5% MeOH in CH₂C1₂, 1% triethylamine). (3S)-2-acetoxy-N-cyclopropyl-3- formamidohexanoyl amide (0.99 g, 3.87 mmol, 56%) was obtained as a white solid as a 78:22 mixture of diastereomers.

From 12: Dess Martin periodinane (5.66 g, 12.3 mmol) was added to a solution of (S)-N-(l hydroxypentan-2-yl)formamide (1.15 g, 8.8 mmol) in CH₂C1₂ (12 ml) at room temperature. The white suspension was stirred for 60 minutes and subsequently cyclopropyl isocyanide (0.74 g, 10.0 mmol) was added and stirred for 48 hours. The resulting suspension was filtrated and washed twice with 10 ml saturated Na₂C03, followed by drying with Na₂SC>4 and concentration in vacuo. The crude product was purified by silica gel flash chromatography (5% MeOH in CH₂CI₂, 1% triethylamine) to give 13 (1.34 g, 5.22 mmol, 60%) as a pale yellow solid as a 78:22 mixture of diastereomers.

*H NMR (130 °C, 400.13 MHz, DMSO-i¾: δ = 8.03 (s, 1H), 7.52 (m, 1H), 7.30 (m, 1H), 4.89 (d, J= 4.4, 1H), 4.28 (m, 1H), 2.65 (m, 1H), 2.17(s, 3H), 1.27-1.47 (m, 4H), 0.89 (t, J= 7.2, 3H), 0.63 (m, 2H), 0.48 (m, 2H); ¹³C NMR (125.78 MHz, DMSO-i ₆): δ = 169.8 (C), 168.5 (C), 160.6 (CH), 74.4 (CH), 47.5 (CH), 22.2 (CH), 18.4 (CH₃), 13.6 (CH₃), 5.7 (CH₂); IR (neat): v_max (cm ) 3283 (s), 2961 (w), 1744 (m), 1661 (s), 1530 (s), 1238 (s); HRMS (ESI, 4500 V): m/z calcd. for Ci₂H₂oN₂0₄Na⁺ ([M + Na]⁺) 279.1315, found 279.1325.

Example 28:

(35)-2-acetoxy-7V-cyclopropyl-3-isocyano-hexanoyl amide (4). N- methylmorpholine (0.57 ml, 0.562 g, 5.56 mmol) was added to a solution of (5)-l- (cyclopropylamino)-3-formamido-l-oxohexan-2-yl acetate (0.713 g, 2.78 mmol) in CH₂CI₂ (40 ml) at room temperature. The reaction mixture was cooled to -78 °C and triphosgene (0.289 g, 0.97 mmol) was quickly added and stirred for 5 minutes at this temperature. The resulting yellow solution was warmed up to -30 °C and was stirred for another 3 h. Subsequently, the reaction was quenched with water and extracted twice with CH₂CI₂ (40 ml). The organic layers were collected, dried with Na₂SC>4 and concentrated in vacuo. The crude product was purified by silica gel flash chromatography (2% MeOH in CH₂C1₂) to give 4 (0.578 g, 2.42 mmol, 87%) as a white solid. ^lU NMR (250.13 MHz, CDC1₃): δ = 6.28 (s, 1H), 5.25 (d, J = 2.5 Hz, 1H), 4.2 (m, 1H), 2.74 (m, 1H), 2.24 (s, 3H), 1.55 (m, 4H), 0.96 (m, 3H), 0.84 (m, 2H), 0.60 (m, 2H); ¹³C NMR (62.90 MHz, CDCI3): δ= 169.7 (C), 168.3 (C), 74.4 (CH), 47.5 (CH), 22.0 (CH), 20.6 (CH₃), 18.5 (CH₂), 13.5 (CH₃), 5.5 (CH₂); IR (neat): _Vmas(cm ): 3267 (s), 2959 (m), 1745 (m), 1643 (s), 1512 (m), 1221 (s); HRMS (ESI, 4500 V): m/z calcd. for Ci₂H₁₈N₂0₃Na⁺ ([M + Na]⁺) 261.1210, found 261.1214.

Example 29:

Compound 14. Isocyanide 4 (0.549 g, 2.3 mmol) was dropwise added to a solution of imine 3 (0.252 g, 2.3 mmol) and carboxylic acid 2 (0.602 g, 1.60 mmol) in CH₂C1₂ (5 ml) at room temperature. This yellow solution was stirred for 72 hours and afterwards diluted with 5 ml CH₂C1₂. The reaction mixture was washed twice with saturated Na₂C0₃ solution (10 ml) and twice with saturated NH₄CI. The organic layers were collected, dried with MgS04 and concentrated in vacuo. The crude product was purified by silica gel flash chromatography (5% MeOH in CH₂C1₂) to give 14 (0.876 g, 1.21 mmol, 76%) as a mixture of diastereomers.

lU NMR (500.23 MHz, CDC1₃): δ = 9.50 (s, 1H), 8.75 (d, J = 2.5, 1H), 8.59 (s, 1H), 8.35 (d, J = 9.0, 1H), 6.84 (d, J= 9.0, 1H), 6.44 (s, 1H), 5.20 (d, J= 3.0, 1H), 4.74 (d, J= 9.5, 1H), 4.58 (t, J = 7.5, 1H), 4.38 (m, 1H), 3.37 (d, J= 6.0, 1H), 2.82 (m, 1H), 2.69 (m, 1H), 2.11 (s, 3H), 1.26 (s, 2H), 0.97 (s, 9H), 0.86 (m, 3H), 0.84-2.00 (m, 21H), 0.76 (m, 2H), 0.51 (m, 2H);¹³C NMR (125.78 MHz, CDC1₃): δ = 170.5 (C), 169.3 (C), 162.9 (C), 147.4 (CH), 144.6 (CH), 144.2 (C), 142.8 (CH), 74.4 (CH), 66.6 (CH), 58.3 (CH), 56.6 (CH), 54.5 (CH₂), 44.9 (CH), 43.0 (CH), 41.3 (CH), 35.5 (C), 26.4 (CH₃), 20.8 (CH₃), 19.1 (CH₂), 13.8 (CH₃), 6.6 (CH₂); v_max (cm⁴): 3306 (m), 2928 (m), 2931 (m), 1743 (w), 1655 (s), 1520 (m), 1219 (m); HRMS (ESI, 4500 V): m/z calcd. for C₃8H₅7N₇0₇Na⁺ ([M + Na]⁺) 746.4212, found 746.4107.

Example 30:

Telaprevir (1). 250 μΐ of saturated K₂C0₃ was added to a solution of 14 (0.514 g, 0.75 mmol) in MeOH (20 ml) at room temperature. The reaction mixture was stirred for 30 minutes at room temperature resulting in a pale yellow suspension. After full conversion (as judged by TLC analysis), the reaction mixture was washed with 20 ml brine, the aqueous layer was washed again with 10 ml CH₂C1₂ (2x). The organic layers were collected, dried with MgS0₄ and concentrated in vacuo, to yield a pale yellow solid. The yellow solid was dissolved in CH₂CI₂ (10 ml) and Dess-Martin periodinane (0.650 g, 1.532 mmol) was added at room temperature. The reaction mixture was stirred overnight before adding saturated NaHC0₃ solution (10 ml) and saturated Na₂S₂0₃ solution (10 ml). This mixture was stirred for 10 minutes, separated and the aqueous layers were washed with EtOAc (2 x 10 ml). The organic layers were collected, dried with MgSC^ and concentrated in vacuo to give the crude product as an 83: 13:4 mixture of diastereomers. After silica gel flash chromatography (1% MeOH in CH₂C1₂), 1 (0.412 mg, 0.61 mmol, 80%) was obtained as a white solid. ^lU NMR (500.23 MHz, DMSO-i¾: 5 = 9.19 (d, J= 1.4 Hz, 1H), 8.91 (d, J= 24.5 Hz, 1H), 8.76 (dd, J = 1.5, 2.5 Hz, 1H), 8.71 (d, J= 5.3 Hz, 1H), 8.49 (d, J= 9.2 Hz, 1H), 8.25 (d, J = 6.8 Hz, 1H), 8.21 (d, J = 8.9 Hz, 1H), 4.94 (m, 1H), 4.68 (dd, J= 6.5, 9.0 Hz, 1H), 4.53 (d, J = 9.0 Hz, 1H), 4.27 (d, J = 3.5 Hz, 1H), 3.74 (dd, J = 8.0, 10 Hz, 1H), 2.74 (m, 1H), 3.64 (d, J = 3.5 Hz, 1H), 0.92 (s, 9H), 0.87 (t, 3H), 0.84-1.40 (m, 23H), 0.65 (m, 2H), 0.56 (m, 2H);

¹³C NMR (125.78 MHz, CDC1₃): δ = 197.0 (C), 171.8 (C), 170.4 (C), 169.0 (C), 162.1 (C), 161.9 (C), 147.9 (CH), 144.0 (C), 143.4 (CH), 56.4 (CH), 56.3 (CH), 54.2 (CH), 53.4 (CH), 42.3 (CH), 41.3 (CH), 32.1 (CH), 31.8 (CH), 31.6 (CH), 29.1 (CH), 28.0 (CH), 26.4 (CH₃); (cm⁴): 3302 (m), 2928 (m), 2858 (w), 1658 (s), 1620 (s), 1561 (s), 1442 (m);

HRMS (ESI, 4500 V): m/z calcd. for C₃6H₅3N₇0₆Na⁺ ([M + Na]⁺) 702.3950, found 702.3941.

………………

telaprevir according to Formula 1

http://www.google.im/patents/WO2013135870A1?cl=en

Telaprevir is a protease inhibitor that can be used as antiviral drug. By way of example, telaprevir inhibits the hepatitis C virus NS3-4A serine protease.

Although some processes for the synthesis of telaprevir or its pharmaceutical acceptable salts are available, it is an object of the present invention to provide an alternative process, in particular an enhanced process that overcomes at least one of the problems of the prior art processes.

Y. Yip et al. Bioorg. Med. Chem. Lett., 2004, 14, 5007 discloses the preparation of a 1 :1 mixture of isomers defined by Formula 5a (see Scheme 1 ) which isomers appear to have a stereochemical configuration other than that of telaprevir. WO 2007/022459 A2 discloses a process for preparing telaprevir, wherein in a first coupling step, a bicyclic pyrrolidine derivative is reacted with a protected amino acid, followed by a stepwise extension of the chain of the amino acid to provide a tripeptide as shown in Formula 2. Subsequently, a β-amino acid is added to the carbon chain-end opposite to said previously built chain. Finally, telaprevir is obtained in an oxidation step. Turner et al. (Chemical Communications 2010, 46(42), 7918) discloses a process for the preparation of teiaprevir by applying an Ugi reaction type process which reacts a compound of Formula 2

a chiral imine, namely (3aS,6aR)-1 ,3a,4,5,6,6a-hexahydrocyclopenta[c]pyrrole, which is obtained by enzyme technology and is thus difficult to prepare and is instabile and a relatively unstable isonitrile derivative of formula 4.

Summary of the invention The known processes for the preparation of teiaprevir are based on long linear sequences or require the use of labile, highly reactive agents and specific enzymes. The process described herein may for example allow to avoid the use of said labile, highly reactive reactants and specific enzymes. It was surprisingly found within the context of the present invention that teiaprevir may be prepared in a smaller number of process steps in a convergent manner by using stabile precursors (see an example process in Figure 1 ). The present invention may also contribute to preserving the desired stereochemical configuration during the process of preparing teiaprevir. In particular, it has been found that the desired stereochemical configuration may be preserved during the process of peptide bond formation in the compound according to Formula 5 when using the coupling agents described herein, in particular when using 2, 4,6-tripropyl-1 ,3,5,2,4,6- trioxatriphosphorinane-2,4,6-trioxide (T3P) or related compounds in dichloromethane. It is also possible to use a combination of a diimide coupling reagent, including but not being limited to dicyclohexylcarbodiimide (DCC), diispropylcarbodiimide (DIC) and 1-ethyl-3-(3- dimethy!aminopropyl)carbodiimide hydrochloride (EDC), with 1-hydroxy-benzotriazole (HOBt) or 1-hydroxy-7-aza-benzotriazole (HOAt) or related reagents for preparing teiaprevir. It has been found that the coupling agents are particularly effective when used in the presence of a lewis acid such as a copper salt. It was also unexpectedly found that the choice of solvent for carrying out the coupling reaction may further enhance the preservation of the stereochemical configuration during peptide bond formation in the compound according to Formula 5.

Furthermore, the expensive compound according to Formula 3

is used at a later stage of the process compared to the process of WO 2007/022459 A2, namely for coupling to the compound according to Formula 2 which already represents a dipeptide. Considering the yields of the single process steps, a smaller amount of the compound according to Formula 3 is required according to the invention, and, thus, the process may be less costly. Compared to the above method of Turner et al., it is not required to use a toxic and instable isonitrile compound. It has also been found that the process for preparing telaprevir may provide an advantage since fewer impurities such as epimeric forms and other byproducts may be formed.

Example 1b – (1S,3aR,6aS)-tert-butyl 2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3 dimethylbutanovDoctahydrocvclopentafclpyrrole-l- carboxylate (5b)

A round bottom flask is charged with 41 mg of 2 (0.11 mmol, 1 eq.) and 1 ml. of DCM is added. Then, 29 mg of 3b (0.16 mmol, 1.5 eq.) are added. After stirring for 5 min 190μΙ of T3P (50% in EtOAc, 0.32 mmol, 3 eq.) are added and the reaction mixture is stirred for 21 h at room temperature.

The reaction is then diluted with DCM and quenched with water. The aqueous layer is separated and re-extracted with DCM. The combined organic layers are washed with brine, dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography yielded 5b (26 mg, 43% yield), (d.r. = 7.5:1 ).

Example 1c – (1S,3aR.6aS)-tert-butyl 2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3 dimethylbutanoyl)octahvdrocvclopentarc1pyrrole-1- carboxylate (5b)

A round bottom flask is charged with 1g of 2 (2.66 mmol, 1 eq.) and 20 ml_ of DCM is added. Then, 0.73g of 3b (3.98 mmol, 1.5 eq.) are added. After stirring for 5 min 4.75mL of T3P (50% in EtOAc, 7.98 mmol, 3 eq.) are added and the reaction mixture is stirred for 21 h at room temperature. The reaction is then quenched with water. The aqueous layer is separated and re-extracted with DCM. The combined organic layers are washed with sat. NaHC0₃-solution, brine and then dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography yielded 5b (0.70g, 49% yield), (d.r. = 5.6:1 ).

Example 1d – (1S,3aR,6aS)-tert-butyl 2-gS)-2-((S)-2-cvclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3.3 dimethylbutanoyl)octahvdrocvclopentaMpyrrole-1- carboxylate (5b)

A round bottom flask is charged with 1.0g of 2 (2.66 mmol, 1 eq.) and 0.58g of 3b (3.19 mmol, 1.2 eq), then 8 mL of DMF is added and the mixture cooled to 0°C using an ice-bath. In a second flask, 0.36g CuCI₂ (2.66 mmol, 1 eq.) are dispersed in 5mL DMF, cooled to 0°C and the previously prepared solution is added to it. Now, 0.36g HOBt (2.66 mmol, 1 eq.) and 2.0g EDC HCI (10.43 mmol, 4 eq.) are added and the mixture is then stirred at r.t. for 16h.

The reaction is then quenched with 10ml_ 10% NH₃-solution and then extracted 4 times with a total of 60mL of EtOAc. The combined organic layers are then washed 3 times with dilute hydrochloric acid, once with sat. NaHC0₃-solution and brine, then dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography yielded 5b (0.78g, 54% yield), (d.r. = 53 : 1 ).

Example 1e – (1S.3aR.6aS)-tert-butyl 2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3 dimethylbutanoyl)octahvdrocvclopentarclpyrrole-1- carboxylate (5b)

A round bottom flask is charged with 1.25g PS-supported HOBt (1.07 mmol/mg) and 0.30g of 3b (1.65 mmol, 1.2 eq) and 0.36g CuCI₂ (2.66 mmol, 1 eq.). Then 15 ml_ of DMF are added and the mixture cooled to 0°C using an ice-bath, while mixing with a mechanical stirrer. In a second flask, 0.5g of 2 (1.3 mmol, 1 eq.) and 1.0g EDC HCI (5.21 mmol, 4 eq.) are dispersed in 12mL DMF, cooled to 0°C and added to the previously prepared solution. The mixture is then stirred at r.t. for 22h.

The reaction is then filtered and the filter washed with 15mL DMF. 50ml_ EtOAc are added to the filtrate, followed by 35ml_ 5% NH₃-solution. The aqueous layer is then separated and extracted 3 times with a total of 45ml_ of EtOAc. The combined organic layers are then washed once with 10% NH₃-solution, dilute hydrochloric acid, sat. NaHC0₃-solution and brine, then dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography yielded 5b (0.43g, 61 % yield), (d.r. = 18 : 1 ).

Example 1f – (1S.3aR,6aS)-tert-butyl 2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3 dimethylbutanovDoctahydrocvclopentafclpyrrole-l- carboxylate (5b)

A round bottom flask is charged with 2.0g of 2 (5.3 mmol, 1 eq.) and 1.17g of 3b (6.4 mmol, 1.2 eq), then 10 mL of DMF is added and the mixture cooled to 0°C using an ice-bath, then 0.72g CuCI₂ (5.3 mmol, 1 eq.) are added. In a second flask 0.72g HOBt (5.3 mmol, 1 eq.) and 1.34g DIC (10.6 mmol, 2 eq.) are dissolved in 5mL DMF, cooled to 0°C and added to the previously prepared solution. The mixture is then stirred at r.t. for 5h.

The reaction is then quenched with 30mL 2% NH₃-solution and then extracted 3 times with a total of 60ml_ of EtOAc. The combined organic layers are then washed 3 times with a total of 60mL of dilute hydrochloric acid, once with 20ml_ sat. NaHC0₃-solution and 20ml_ of brine, then dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash chromatography yielded 5b (2.59g, 90% yield), (d.r. = 340 : 1 )

Example 1g – Use of HOAT as anti-isomerisation reagent

To 4.4ml of a 0.6M HOAT solution in DMF (1.1eq, 2.63mmol) were added 0.6ml DMF.

Afterwards 1g of 2 (90% content, 1eq, 2.39mmol), 567mg of 3b (1.3eq, 3.11 mmol) and 391 mg DIC (1.3eq, 3.11 mmol) were added at room temperature. The reaction was stirred at room temperature for 23h. After 19h 86% conversion to 5b, with a d.r. 3.9/1 was observed. After 23h, with 87% conversion to 5b, and a d.r. 4.1/1 the conversion had stalled and the product was not isolated. Example 1 h – Use of CuCI? with in situ generation of AOC-Et from its HCI salt

353mg water free CuCI₂ (1.1 eq, 2.63mmol) was dissolved in 5ml DMF. To the solution 1 g 2 (90% content, 1 eq, 2.39mmol), 683mg 3b. HCI (1 .3eq, 3.1 1 mmol), 315mg NMM (1.3eq,

3.1 1 mmol) and 391 mg DIC (1.3eq, 3.1 1 mmol) were added at room temperature. The reaction mixture was stirred at room temperature. After two hours 2.6area% 2 was detected and yield was 96.5% (calculated via internal standard). After 5h less than 0.5area% 2 was detected and yield was 98.1 %. d.r. at both IPCs was 108/1.

Example 1 i – HOAT without CuCI? in DMF

To a solution of 0.5g 2 (90%, 1 eq, 1 .19mmol) and 179mg HOAT (1.1eq, 1.32mmol) in 2.5ml DMF 284mg 3b (1.3eq, 1 .55mmol) was added. Afterwards

241 μΙ DIC (1 .3eq, 1.55mmol) was added. Reaction was stirred at room temperature. After2.5h 91 % conversion and DR of 4.3/1 was observed. After 5h complete conversion

Was observed and DR of 4.1/1. No work was performed.

Example 1j – HOAT without CuCI, in THF/MED

To a suspension of 0.5g 2 (90, 1 eq. 1.19mmol) and 179mg HOAT (1 .1 eq, 1 ,32mmol) in 2.5ml of a 1/1 mixture of THF/MED (methylene chloride) 284mg 3b (1.3eq, 1.55mmol) was added. Afterwards, 241 μΙ DIC (1.3eq, 1 .55mmol) was added. Reaction was stirred at room

temperature. After2.5h 7.5% conversion and DR of 6.7/1 was observed. After 5h 80 conversion was observed and DR of 6.2/1 . After 19h 86% conversion and DR of 6.0/1 was found. No work was performed. Example 1 k – HOAT with CuCI? in DMF

177mg CuCI₂ (1 .1 eq, 1 .31 mmol) was dissolved in 2.5ml DMF. To the solution 0.5g 2 (90%, 1 eq, 1.19mmol), 179mg HOAT (1.1 eq, 1 .32mmol), 284mg 3b (1.3eq, 1.55mmol) and 241 μΙ DIC (1.3eq, 1.55mmol) was added. The reaction was stirred at room temperature for 13h. 95% conversion and DR of 48/1 was observed. No work up was performed.

Example 11 – Substochiometric amounts of CuCI? in DMF without HOAT

177mg CuCI₂ (0.55eq, 1.31 mmol) was dissolved in 5ml DMF. To the solution 1.0g 2 (90%, 1 eq, 2.39mmol), 683mg 3b.HCI (1.3eq, 3.11 mmol), 340μΙ NMM (1.3eq, 3.11 mmol) and 481 μΙ DIC (1.3eq, 3.11 mmol) was added. The reaction was stirred at room temperature for 4.5h, complete conversion and DR of 76/1 was observed. No separated work up was performed. Example 2a – Synthesis of the compound according to Formula 7

(1S.3aR.6aS)-2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3,3- dimethylbutanoyl)octahvdrocvclopentarclpyrrole-1 -carboxylic acid (7)

A round-bottom flask was charged with 6g of 5b (11.08 mmol, 1 eq.) and 85mL of THF and 26mL of H₂0 was added. Then 2.20g LiOH H₂0 (52.43 mmol, 4.7 eq.) were added and the mixture was stirred at r.t. for 18h.

Then 50ml_ EtOAc and 50mL H₂0 were added, and the aqueous layer separated. The organic layer was washed once more with 40ml_ H₂0. To the combined aqueous layers 50ml_ of EtOAc were added, and by slow addition of 2M HCI the pH was adjusted to 1.89. After separation of the aqueous layer, it was extracted once more with 50ml_ EtOAc, and the combined organic layers washed with brine, then dried over Na₂S0₄, filtered and concentrated in vacuo.

Purification by flash chromatography yielded 7 (4.83g, 85% yield).

Example 2b – Synthesis of the compound according to Formula 7

(1S,3aR,6aS)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3,3- dimethylbutanoyl)octahvdrocvclopenta[c1pyrrole-1 -carboxylic acid (7)

A round bottom flask is charged with 2.0g of 2 (5.3 mmol, 1 eq.) and 1.17g of 3b (6.4 mmol, 1.2 eq), then 10 mL of DMF is added and the mixture cooled to 0°C using an ice-bath, then 0.72g CuCI₂ (5.3 mmol, 1 eq.) are added. In a second flask 0.72g HOBt (5.3 mmol, 1 eq.) and 1 .34g DIC (10.6 mmol, 2 eq.) are dissolved in 3ml_ DMF, cooled to 0°C and added to the previously prepared solution. The mixture is then stirred at r.t. for 5h.

The reaction is then quenched with 30mL 2% NH₃-solution and then extracted 3 times with a total of 70ml_ of EtOAc. The combined organic layers are then washed with 15mL 2% NH₃– solution, 1 time with 20ml_ 1 M HCI, 3 times with a total of 60ml_ of dilute hydrochloric acid, once with 20ml_ sat. NaHC0₃-solution and 20ml_ of brine, then dried over Na₂S0₄, filtered and concentrated in vacuo. The residue (compound 5b – 2.59g, 4.78 mmol, 1 eq.) was dissolved in 27ml_ of a 1 :1 mixture THF/H₂0. Then 0.48g NaOH (1 1.95 mmol, 2.5 eq.) were added and the mixture was stirred at r.t. for 18h.

Then 20ml_ EtOAc and 10ml_ H₂0 were added, and the aqueous layer separated. The organic layer was washed once more with 20ml_ H₂0. To the combined aqueous layers 20ml_ of EtOAc were added, and by slow addition of 2M HCI the pH was adjusted to 1.27. After separation of the aqueous layer, it was extracted once more with 20ml_ EtOAc, and the combined organic layers washed with brine, then dried over Na₂S0₄, filtered and concentrated in vacuo to give 7 (2.82g, 93% yield). Trace metal analysis using ICP-OES showed residual copper < 1 ppm, wherein the following method was used:

Digestion: about 250mg of sample material was digested under pressure with a mixture of HNO₃+HCI in a closed quartz container which can be heated by microwave radiation.

Determination of Cu:

Measurement was performed with ICP-OES at 324,754nm, Axialplasm, simultaneous background correction; calibration with external standards, certified elemental standard of Merck, Device: Fabr. Thermo Electron, Type: IRIS Intrepid XSP II, Duo. Example 2c – Synthesis of the compound according to Formula 7

(1 S,3aR,6aSV2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3.3- dimethylbutanoyl)octahvdrocyclopenta[clpyrrole-1 -carboxylic acid (7)

7.07g water free CuCI₂ (1.1eq, 52.6mmol) was dissolved in 100ml DMF. To the solution 20g of 2 (90% content, 1eq, 47.82mmol), 10.51g of 3b (d.r. = 9:1) (1.2eq, 57.38mmol), 6.3ml NMM (1.2eq, 57.38mmol) and 9.6ml DIC (1.3eq, 62.16mmol) were added at 0°C. the reaction mixture was warmed to 40°C in 1.5h and stirred at that temperature until complete conversion was observed. To the reaction mixture isopropyl acetate was added followed by the addition of 10% HCI. The organic phase was separated and washed with 5% ammonia and 2% NaCI. The organic solvent was removed to dryness and 26.95g was isolated as a diasteromeric mixture of 9:1 detected via NMR.

6g of this material was dissolved in 15ml ethanol and 15ml water. To the mixture 1.15g sodium hydroxide was added. The reaction mixture was stirred at room temperature until no further conversion was observed. Ethanol was removed via distillation and water was added. The basic aqueous phase was washed with isopropyl acetate, the organic phase was re-extracted with water. To the combined aqueous phase Isopropyl acetate was added an pH was adjusted to 1.5 via addition of 10% HCI. The organic phase was separated and solvent was removed to dryness to yield 5.29g of compound 7 as a single diastereomer according to NMR analysis.

Example 2d – Use of CuCI? as anti isomerisation reagent (without any triazol reagent) 353mg water free CuCI₂ (1.1eq, 2.63mmol) was dissolved in 5ml DMF. To the solution 1g 2 (90% content, 1eq, 2.39mmol), 567mg 3b (1.3eq, 3.11 mmol) and 391 mg DIC (1.3eq,

3.1 1 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 19h full conversion to 5b with d.r. 116/1 was observed. To the reaction mixture 50ml of ethyl acetate was added and the occurring precipitation was removed via filtration. The organic phase was washed with 5% ammonia and the aqueous phase was reextracted with 50ml ethyl acetate. The combined organic phase was washed with 40ml 2M HCI and 40ml brine. After drying with sodium sulfate and filtration the organic solvent was removed via evaporation. The solid residue was dissolved in 20ml methylene chloride and again the solvent was removed to dryness. After drying (rt, 40mbar), 1.373g of a slightly yellow amorphous solid (NMR content 81.6%, yield 86.4%).

Example 3 – Synthesis of the compound according to Formula 6

(1S,3aR,6aSV2-((S)-2- SV2-cvclohexyl-2-(pyrazine-2-carboxamido)acetamidoV3,3- dimethylbutanoyl)-N-((S)-1-(cvclopropylamino)-2-hvdroxy-1-oxohexan-3- yl)octahvdrocvclopentarclpyrrole-1-carboxamide (6)

A round-bottom flask was charged with 11.87g of 7 (23.11 mmol, 1 eq.), 5.32g of EDC^*HCI (27.73mmol, 1.2 eq.), 3,74g of HOBt (27.73 mmol, 1.2 eq.) and 80 mL DCM were added. The mixture was cooled with an ice-bath and a suspension of 5.66g of 4 (25.42mmol, 1.1 eq.) in 50 mL of DCM containing 2.75g NEt₃ (27.73 mmol, 3.88mL, 1.2 eq.) was added. This mixture was then stirred at r.t. for 6h when conversion was complete.

The reaction was quenched by addition of 50ml_ H₂0, followed by dropwise addition of 6M HCI to adjust the pH to 1.45. The aqueous layer was separated and extracted once with 50ml_ DCM. The combined organic layers were washed with 50ml_ sat. NaHC0₃ solution and 50mL brine, dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by flash

chromatography yielded 6 (14.89g, 94% yield).

Example 4 – Synthesis of the compound according to Formula 1

(1S,3aR,6aS)-2-((S)-2-((S)-2-cvclohexyl-2-(pyrazine-2-carboxamido)acetamido)-3.3- dimethylbutanoyl)-N-((S)-1-(cvclopropylamino)-1 ,2-dioxohexan-3- yl)octahvdrocvclopentaFclpyrrole-1-carboxamide (Telaprevir) (1)

A round-bottom flask was charged with 2.00g of 6 (2.93 mmol, 1 eq.) and 20mL of DCM and then cooled with an ice-bath. 200μΙ of 15% KBr-solution and 800μΙ of sat. NaHC0₃-solution were added, followed by 1 1 mg of TEMPO (0.07mmol, 0.025 eq.) and 600μΙ 10% NaOCI- solution. After stirring at r.t. for 18h, another 1.2ml_ of 10% NaOCI-solution were added – after another 2h the reaction was complete.

The reaction mixture was then diluted with 10mL of H₂0. After separation of the aqueous layer it was extracted with 10ml_ of DCM. The combined organic layers were washed with 10ml_ of 1 % Na₂S0₃ and 10ml_ of H₂0, dried over Na₂S0₄, filtered and concentrated in vacuo.

The residue was then stirred in 40ml_ Et₂0, filtered, washed with 10ml_ of cold Et₂0 and then dried in vacuo to give crystalline 1 (1 .41g, 71 %).

Cited literature

WO 2007/022459 A2; Turner et al. (Chemical Communications 2010, 46(42), 7918); WO2010/126881 ; Y. Yip et al. Bioorg. Med. Chem. Lett., 2004, 14, 5007; Harbeson, S. L. et al. J. Med. Chem. 1994, 37, 2918-2929.

WO2007022459A2	18 Aug 2006	22 Feb 2007	Vertex Pharma	Processes and intermediates
WO2010126881A1	27 Apr 2010	4 Nov 2010	Vertex Pharmaceuticals Incorporated	Processes and intermediates

………………

PATENT

http://www.google.im/patents/WO2010126881A1?cl=en

5 6 7 8 (rac)

9 10 1

Scheme I

Scheme II

a cyclopropylamide of Formula 18 is prepared using the Passeπni reaction (see, e.g., A. Doemling et al., Angew. Chem., 2000, 1 12, 3300-3344). Scheme IV

Scheme V

The invention further relates to a process for piepaπng a compound of Formula 4

[0060] In some embodiments, the process for preparing compounds of Formula 4 includes the steps of i) providing an N-alkoxycarbonyl-S-azabicycloP 3 0]octane, ii) forming a 2-anion of the N-alkoxycarbonyl-3-azabicyclo[3 3 0]octane in the presence of a chelating agent, iii) treating the anion of step ii) with carbon dioxide to produce a cis /trans mixture of N-alkoxycarbonyl-octahydrocyclopenta[c]pyrrole- l-carboxyhc acids, iv) treating the mixture of step iii) with a strong base to produce an essentially pure trans- N-alkoxycarbonyl-octahydrocyclopenta[c]ρyrrole-l -carboxyhc acid, v) forming a salt of the carboxylic acid with an optically active amine, vi) crystallizing the salt, vii) esteπfying the salt provided in step vi), viii) removing the N-alkoxycarbonyl gioup to produce (lS,3aR,6aS)-f-butyl- octahydiocyclopenta[c)pyiτole-l -carboxylate, /-butyl ester, ix) reacting the bicyclic of step viii) with a protected amino acid of Formula 26,

26 wherein Z is an amine protecting group, in the piesence of a coupling reagent, to pioduce an amide-ester of Formula 27,

27 x) removing the protecting group Z from the amide-ester of step ix) to produce the amino compound of Formula 28,

28 xi) reacting the amino compound of Formula 28 with a protected amino acid of Formula 29

Z-HN^_/CO₂H

29 in the presence of a coupling reagent to produce a tripeptide of Formula 30;

30 xii) removing the protecting group Z in the tripeptide of Formula 30 to produce a free amino-tripeptide of Formula 31;

31 xiii) reacting the amino-tripeptide of Formula 31 with pyrazine-2-carboxylic acid in the presence of a coupling reagent to produce an amide-tripeptide ester of Formula 33;

33 xiv) hydrolyzing the ester of the amide-tripeptide ester of Formula 33 to produce an amide-tripeptide acid of Formula 34;

XV) reacting the amide-tπpeptide acid of Formula 34 with an aminohydroxy-amide of Formula 18

18 in the piesence of a coupling reagent to produce a hydroxy-tetrapeptide of Formula 35, and

35 xvi) oxidizing the hydroxy gioup of Formula 35 to produce the compound of Formula 4

Example 13: (lS,3aR,6aS)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3-dimethylbutanoyl)-N-((S)-l-(cyclopropylamino)-l,2- dioxohexan-3-yl)octahydrocyclopenta[c]pyrrole-l -carboxamide (4)

35 ₄

Method 1

[00256] A 500 inL 3-neck round bottomed flask equipped with an overhead stirrer, condenser, thermocouple, and nitrogen outlet was purged with nitrogen for several minutes A methylene chloride solution of the hydroxyamide peptide amide 35 (128 64 g, 16-17wt%, 20 6 g and 30 mmol of 35) in methylene chloride was added to the reaction flask, followed by the addition of 15% w/w aq NaBr (13 mL) and 7 5% w/w aq NaHCO₃ (52 mL) The solution was cooled to 5±3 ⁰C in an ice bath TEMPO (0 7 g) dissolved in methylene chloride (3 mL) was added to the reaction mixture In a separate Erlenmeyer flask, 10- 13% NaOCI solution (23 25 mL, titer = 108 mg/mL, 2 51 g, 33 7 mmol, 1 12 molar eq ) was diluted with water (70 mL) The NaOCI solution was charged to the reaction mixture via addition funnel at a rate that maintained the temperature below 8 ⁰C The reaction mixture was allowed to stir at 5±3 ⁰C for lhour The layers were separated and the organic layer was quenched with 10% (w/w) aq Na₂SOs (100 mL) and washed with water (100 mL) The organic phase was reduced to dryness at reduced pressure and the solid triturated with ethyl acetate (100 mL) and filtered on a Buchner funnel to give the title compound Method 2

[00257] TEMPO (1 09 g, 6 95 mmol) was added to the methylene chloπde solution of 35 from Example 12, Method 2, followed by a solution of sodium bicarbonate (21 89 g, 260 5 mmol) in water (400 mL) and the mixture cooled to 0-5 ⁰C A solution of sodium hypochlorite (122 17 g, 11 64 wt%, 191 04 mmol) was added over 2 hours while maintaining a temperature of 0-5 ⁰C The mixture was stirred for 1 hour at 0-5 ⁰C, then the phases separated The organic phase was washed with water (500 mL), 1 wt% aqueous sodium bisulfite (500 mL) and water (500 mL), then polish filtered The mixture was distilled at 38- 42 ⁰C, 710 mm Hg, to a volume of about 320 mL Ethyl acetate (44 mL) was added followed immediately by 1 5 g of seed ciystals of 4 and the mixture was stirred for 15 minutes at 38-42 ⁰C Ethyl acetate (800 mL) was added over 3 hours while maintaining a temperature of 38-42 ⁰C The mixture was then distilled at 38-42 ⁰C, 200-250 mm Hg, to a volume of about 400 mL Additional ethyl acetate (200 mL) was added over 0 5 hour The resultant slurry was cooled over 1 hour to 20-25 ⁰C and stirred an additional hour at the same temperature The mixture was filtered and the filter cake washed with ethyl acetate (twice, 300 mL each) and dned under vacuum with a nitrogen bleed at 45-55 ⁰C to give the title compound 4 as a white solid. Method 3

[00258] TEMPO (0 06 eq) was added to the CH₂CI₂ solution of 35 from Example 12, method 3, and the solution was stirred at 20-25 ⁰C until all TEMPO dissolved To this solution was added a solution of NaHCOi (1 5 eq ) in water (4 vol ) The resulting biphasic mixture was cooled to 0-5 ⁰C While maintaining the reaction temperature at 0-5 ⁰C, a 10-13 wt% NaOCl solution (1 10 eq ) was added over 2-3 hours and the mixture stirred for additional one hour The layers were separated and the organic layer was washed at 0-5 ⁰C with H₂O (5 vol ), 1 wt% Na₂SO₃ (5 vol ), and H₂O (5 vol ) Glacial acetic acid (0 12 eq ) was added to the solution of compound 4 in CH₂Cl₂ to stabilize compound 4

Example 14: Recrystallization of Compound of Formula 4.

[00259] The solution of Compound 4 from Example 13, Method 3, was filtered through Cehte, and the filtrate solution was reduced to 3 1 -3 3 volumes by vacuum distillation at lower than 20 ⁰C After distillation, the solution was brought to 38-42⁰C before EtOAc (0 80 vol ) was added, followed by the addition of Compound 4 seed (1 5 wt% relative to 34, Example 12) The resulting mixture was stirred for 15 minutes at 38-42 ⁰C EtOAc (8 vol ) was added over 3 hours to this mixture while maintaining a temperature of 38-42 ⁰C The total volume of the slurry was then reduced to 3 9-4 1 volumes by vacuum distillation at 38- 42 ⁰C To this mixture was added EtOAc (2 vol ) over 30 minutes while maintaining the batch temperature at 38-42 ⁰C The resulting slurry was then cooled to 20-25 ⁰C over 1 hour and stirred at 20-25 ⁰C for additional 1 hour. The slurry was filtered. The filter cake was washed with EtOAc (twice, 3 vol. each) and dried under vacuum with a nitrogen bleed at 45- 55 ⁰C for 6 hours.

[00260] To the dried filter cake was added 2.2-2.4 volumes of CH₂Ch to a total volume of 3.1-3.3 volumes. The mixture was broughl to 38-42 ⁰C to give a homogeneous solution. EtOAc (0.80 vol) was added, followed by the addition of Compound 4 seed ( 1.5 wt% relative to 34, Example 12). The resulting mixture was stirred for 15 minutes at 38-42 ⁰C. EtOAc (8 vol.) was added over 3 hours to this mixture while maintaining a temperature of 38-42 ⁰C. The total volume of the slurry was then reduced to 3.9-4.1 volumes by vacuum distillation at 38-42⁰C. EtOAc (2 vol.) was added over 30 minutes to this mixture while maintaining the batch temperature at 38-42°C. The resulting slurry was then cooled to 20-25 ⁰C over 1 hour and stirred at 20-25 ⁰C for additional one hour. The slurry was filtered and the filter cake was washed with EtOAc (twice, 3 vol. each) and dried under vacuum with a nitrogen bleed at 45-55 ⁰C for 12 hour to give purified Compound 4. ¹H NMR (500 MHz, CDCI3) 0.78 (m, 2H), 0.87 (m, 2H), 0.91 (s, 9H), 0.91 (t Lobscured], 3H), 0.98 (m, 4H), 1.08 (m, IH), 1.20 (m, 4H), 1.29 (m, IH), 1.40 (m, IH), 1..42 (m, 2H), 1.46 (m, IH), 1.48 (m, IH), 1.60 (m, IH), 1.70 (m, IH), 1.79 (m, IH), 1.83 (m, 2H), 1.88 (m, IH), 1.94 (m, IH), 2.67 (m, IH), 2.89 (bs, IH), 2.96 (bs, IH), 3.63 (d, IH), 3.99 (d, IH), 4.70 (s, IH), 4.82 (d, IH), 4.89 (t, IH), 5.65 (bs, IH), 7.74 (bs, IH), 8.00 (bs, I H), 8.06 (bs, IH), 8.29 (bs, IH), 8.60 (s, IH), 8.77 (s, I H), 9.42 (s, IH).

Example 15: (lS,3a#,6aS)-2-((,S>2-((S)-2-cyclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3-dimethylbutanoyl)-Λ’-((S)-l-(cyclopropylamino)-l,2-dioxo- hexan-3-yl)octahydrocyclopenta[c]pyrrole-l-carboxamide (4)

………………………

PATENT

http://www.google.im/patents/WO2007022459A2?cl=en

Example 1: N-/-butyloxycarbonyI-3-azabicycIo[3.3.0]octane (6)

Method 1

[00177] To a 2 L 3-necked round-bottom flask under nitrogen fitted with a mechanical stirrer, a 500 niL addition funnel, and a thermometer was charged 3-azabicyclo[3.3.0]nonane hydrochloride (100 g, 0.677 mol), potassium carbonate (187 g, 1.35 mol), /-butyl methyl ether (220 mL) and water (160 mL), with stirring. The mixture was cooled to 14-16 °C. In a separate 500 mL erylenmeyer flask was charged Boc₂O (di-t-butyl dicarbonate) (145 g, 0.644 mol) and t-butyl methyl ether (190 mL). The mixture was stirred until complete dissolution was obtained. The solution was poured into the addition funnel and added to the above reaction mixture, keeping the reaction temperature below 25 °C. Water (290 mL) was added to dissolve solids, and the mixture was stirred for 10-15 minutes. After separating the lower aqueous phase, the organic phase was washed with 5% aq. NaHSO₄ (twice, 145 mL each), then water (145 mL). The organic phase was concentrated and methyl t-butyl ether was added (1.3 L) to give a solution of the title compound in t-butyl methyl ether. See, e.g., R.Griot, HeIv. CMm. Acta., 42, 67 (1959).

Method 2

[00178] A solution of potassium carbonate (187 g, 1.35 mol) in water (160 mL) was added to a mixture of 3-azabicyclo[3.3.0]octane hydrochloride (100 g, 0.677 mol) and t-butyl methyl ether (220 mL), and the resultant mixture was cooled to 14-16 °C. A solution of Boc₂O (145 g, 0.644 mol) in t-butyl methyl ether (190 mL) was added while maintaining a temperature below 35 ⁰C. After the addition, the mixture was stirred for 1 hour then filtered. The solids were washed with MTBE (50 mL). The phases were separated and the organic phase washed with 5% aq. NaHSO₄ (twice, 145 mL each) and water (145 mL) and concentrated to 300 mL under vacuum. MTBE (300 mL) was added and the mixture concentrated to remove water to less than 550 ppm. The concentrate was diluted with MTBE (400 mL) to provide a solution of the title compound in MTBE.

Example 2: mc-2-(/-butoxycarbonyl)octahydrocyclopenta[c]pyrrole-l-carboxylic acid (7)

6 7

Method 1

[00179] The solution from Example 1, Method 1, was charged to a 5 L 4-necked flask fitted with a mechanical stirrer, an addition funnel, a ReactIR probe, and a thermometer. 3,7- Dipropyl-3,7-diazabicyclo[3.3.1]nonane (183 g, 0.88 mol) was charged to the flask. Data collection was started on the ReactIR instrument, and the solution was cooled to -72 to -75 ⁰C. sec-Butyllithium (600 mL, 1.6 M in cyclohexane) was slowly added to the reaction mixture, keeping the reaction temperature below -69 ⁰C. The addition was monitored with the ReactIR instrument, and the addition was stopped after the absorbance at 1698 cm^“1 had disappeared and the absorbance at 1654 cm^“1 ceased to increase for three consectutive scans (2-minute intervals). The solution was agitated for 3 hours at -75 to -72 °C. A 10% mixture of CO₂ in nitrogen was carefully sparged into the reaction mixture, keeping the reaction temperature below -70 °C. The sparge was stopped after the absorbance for CO₂ appeared in the ReactIR spectrum (2350 cm^“1). The mixture was warmed to 0-5 ⁰C, and a solution of 30 wt% NaHSO₄(1.4 L) was added. The mixture was warmed to 22-25 °C and stirred for 30 minutes. The aqueous phase was separated and the organic phase washed with water (700 mL). The aqueous phase was decanted and the organic phase concentrated to provide the title compound.

Method 2

[00180] A solution of 3,7-dipropyl-3,7-diazabicyclo[3.3.1]nonane (183 g, 0.87 mol) in

MTBE (300 mL) was added to the solution of N-t-butyloxycarbonyl-3- azabicyclo [3.3.0] octane from Example 1, Method 2 in a flask fitted with a mechanical stirrer, an addition funnel, a ReactIR probe, and a thermometer and the mixture was cooled to -75 to

-72 ⁰C. A solution of sec-butyllithium (510 mL, 1.6 M) was added, keeping the reaction temperature below -70 °C, until the absorbance at 1698 cm^“1 had disappeared and the absorbance at 1654 cm^“1 ceased to increase. The solution was stirred for 3 hours at -75 to -72

°C. The reaction mixture was sparged with 10% CO₂ in N₂ keeping the reaction temperature e ow – /υ “U. lne sparge was stoppe w en t e a sor ance or ₂ appears in the eact spectrum (2339 cm^“1). The mixture was warmed to 0-5 °C and a solution of 30 wt% NaHSO₄ (1.4 L) was added and the mixture was warmed to 22-25 °C then stirred 30 minutes. The phases were separated and the aqueous phase was checked to make sure the pH was lower than 3. The organic phase was washed with water (700 mL) then concentrated to 300 mJL Ethyl acetate (1.7 L) was added and the mixture concentrated to 300 mL twice to give a solution of the title compound in ethyl acetate.

Example 3: (S)-l,2,3,4-tetrahydronaphthalen-l-aminium (lS,3aR,6aS)-2-(f- butoxycarbonyl)octahydrocyclopenta[c]pyrrole-l-carboxylate (9a)

8 9a

Method 1

[00181] Ethyl acetate (2.3 L) was added to the residue of Example 2, method 1, and the mixture filtered through a pad of Celite^®. (S)-1 ,2,3,4-tetrahydro-l-naphthylamine (56.7 g, 0.385 mol) was added and the solution was stirred for 3-4 hours at 22-25 ⁰C. The mixture was filtered and the solids were rinsed with ethyl acetate (200 mL). The solids were dried at 20-30 ⁰C under vacuum for 4 hours to give 99.02 g of product (73% yield, 90% ee by chiral HPLC).

[00182] To a 3-necked RBF fitted with a temperature contoller, a mechanical stirrer, a reflux condenser, and a nitrogen bubbler, was charged the (S)-l,2,3,4-tetrahydro-l- naphthylammonium salt (88.98g, 0.22 mol), ethyl acetate (712 mL), and 2-propanol (666 mL). The mixture was warmed to 70-75 ⁰C with stirring. The mixture was stirred for 15-30 minutes, then cooled to -5 to -10 °C over 1 hour. The resultant slurry was filtered and the solids were rinsed with cold ethyl acetate (180 mL). The solids were dried in vacuo at 35-40 °C to give 7.37 g of a white solid (83% yield, 98% ee).

Method 2

[00183] The ethyl acetate solution of racemic N-t-butyloxycarbonyl-3- azabicyclo[3.3.0]octane-2-carboxylic acid from Example 2, Method 2, was added to a solution of (S)-l,2,3,4-tetrahydro-l-naphthylamine (56.7 g, 0.385 mol) in ethyl acetate (300 mL). The mixture was strirred for 3-4 hours at 22-25 °C, then filtered, and the solids washed with ethyl acetate (200 mL). The product was dried at 20-30 °C under vacuum for 4 hours to give the title compound (99.02 g, 36% yield) with a 95 to 5 diasteromer ratio. [00184] A mixture of the salt as prepared above (89.0 g), ethyl acetate, and 2-propanol was warmed to 70-75 ⁰C until complete dissolution. The mixture was cooled to -5 to -10 ⁰C over two hours and stirred for 3-4 hours. The mixture was filtered and the product dried at 35-40 ⁰C to give the title compound (73.7g, 83% yield, >99.5% ee).

Example 4: (R)-l-phenylethanaminium (lS,3aR,6aS)-2-(f- butoxycarbonyl)octahydrocyclopenta[c]pyrrole-l-carboxylate (9b)

8 9b

[00185] To a soution of racemic N-t-butyloxycarbonyl-3-azabicyclo[3.3.0]octane-2- carboxylic acid (4.66 g) in ethyl acetate (100 mL) was added (R)-α-methylbenzylamine (56.7 g) and the solution was stirred for 16 hr at 22-25 °C. The mixture was filtered and the solids were rinsed with ethyl acetate. The solids were dried at 20-30 °C under vacuum for 4 hours to give 1.47 g of product (43%, 82% ee, 92:8 ratio of exorendo diastereomers).

Example 5: (lS,3aR,6aS)-tf-butyl octahydrocyclopenta[c]pyrrole-l-carboxylate, t- butylester, oxalate

Method 1

[00186] A mixture of the (S)- 1 ,2,3 ,4-tetrahydro- 1 -naphthylammonium salt prepared as in

Example 3, Method 1 (81.7 g, 0.203 mol), /-butyl methyl ether (400 mL) and 5% NaHSO₄– H₂O (867 mL, 0.304 mol) was stirred for 30 minutes until all solids were dissolved. The organic phase was washed with water (334 mL) then concentrated to 259 mL. /-Butyl methyl ether (334 mL) was added and the solution was concentrated again to 259 mL. The addition- concentration process was repeated twice more. After the final concentration, t-BuOH (158 mL) and dimethylaminopyridine (5.04 g, 41.3 mmol) were added. A solution of BoC₂O (67.6 g, 0.31 mol) in t-butylmethyl ether (52.0 mL) was added. After stirring for 5 hours at ambient temperature, /-butyl methyl ether (158 mL) and 5% aqueous NaHSO₄-H₂O (260 mL) were added and the resultant mixture was stirred. The organic phase was washed with 5% aqueous NaCl (twice, 260 mL each). The organic phase was concentrated to 320 mL, and tetrahydrofuran (320 mL) was added. The organic phase was concentrated again to 320 mL, and tetrahydrofuran (320 mL) was added. After concentrating to 320 mL once more, methane sulfonic acid (80.1 g, 0.62 mol) was added and the solution was stirred at ambient temperature for 4.5 hours. The reaction mixture was added to a 30% aqueous solution of K₂CO₃ (571 mL) and stirred. The aqueous phase was extracted with isopropyl acetate (320 mL). The combined organic phases were concentrated to 320 mL, and isopropyl acetate (320 mL) was added. The organic solution was concentrated again to 320 mL. The organic phase was washed with water (320 mL). Isopropyl acetate (320 mL) was added to the organic phase and the solution was concentrated to 192 mL. Isopropyl acetate (320 mL) was added a second time, and the organic solution was concentrated to 192 mL. A solution of oxalic acid (24.1 g, 267 mmol) in isopropyl acetate (448 mL) was added to the orgam^‘c solution over 2 hours. The mixture was stirred for 2-4 hours, and the slurry was filtered. The white solids were rinsed with isopropyl acetate (100 mL) and dried at 35-40 ⁰C under vacuum to yield 52.6 g of the title compound (85% yield).

Method 2

[00187] A mixture of (S)- 1 ,2,3,4-tetrahydro- 1 -naphthylammonium salt as prepared by the method of Example 3, Method 2 (148 g, 0.609 mol), t-butyl methyl ether (726 mL) and 5% NaHSO₄-H₂O (1.58 L, 0.913 mol) was stirred until all of the solids had dissolved. The phases were separated and the organic phase washed with water (726 mL). The organic phase was concentrated to about 400 mL. t-Butyl methyl ether (726 mL) was added and the mixture concentrated to 590 mL. The addition of t-butyl methyl ether and concentration was repeated to give a final volume of 350 mL. Dimethylaminopyridine (8.42 g, 68.9 mmol) and t-butyl alcohol(260 mL) were added, followed by addition of a solution of BoC₂O (112 g, 0.52 mol) in MTBE (88 mL) over 0.5 hour. The mixture was stirred for 5 hours at 22-25 ⁰C.

A solution of 5% sodium bisulfate in water was added and the mixture stirred for 0.5 hour. The organic phase was washed with 5% sodium chloride (twice, 440 mL each) and concentrated to 270 mL. Tetrahydrofuran (540 mL) was added and the mixture concentrated to 270 mL; this procedure was repeated twice more to give a final volume of 270 mL. Methane sulfonic acid (67 mL) was added over 0.5 hour while maintaining a temperature of lower than 30 °C and the mixture stirred at 22-25 °C for 12 hours. The mixture was added to a 30% aqueous solution of pottassium carbonate (478 mL) while maintaining a temperature of 22-25 °C. The mixture was filtered, the phases separated and the aqueous phase extracted with isopropyl acetate (twice, 540 mL each). The organic phase was concentrated to 270 mL, then twice evaporated with isopropyl acetate (540 ml) to give a final volume of 540 mL. The organic phase was washed with water (twice, 540 mL), then twice evaporated with isopropyl acetate (320 mL) to give a final volume of 320 mL. Additional isopropyl acetate (429 mL) was added followed by addition of a solution of oxalic acid (40.4 g, 0.448 mol) in t- butylmethyl ether (321 mL) over 2 hours maintaining a temperature of 22-25 ⁰C. The mixture was stirred for 3 hours at 22-25 ⁰C then filtered. The filter cake was washed with isopropyl acetate (100 mL) and the prouduct dried at 35-40 °C under vacuum to give the title compound as a white solid (88.4g, 81%).

Example 6: (lS,3aR,6aS)-*-butyl 2-((S)-2-(benzyloxycarbonylamino)-3,3- dimethylbutanoyl)octahydrocyclopenta[c]pyrrole-l-carboxylate (27)

Method 1

[00188] A 3-L 3-neck round bottomed flask equipped with an overhead stirrer, condenser, thermocouple, and nitrogen outlet was purged with nitrogen for several minutes. In a separate flask, sulfuric acid (46.2 mL, 0.867 mol) was diluted with 442 mL of water. The solution was allowed to cool slightly. Cbz-L-tert-Leucine dicyclohexylamine salt (330.0 g, 0.739 mol) was charged to the reaction flask. t-Butyl methyl ether (1620 mL) was added to the reactor, and the mixture was stirred to suspend the salt. The acid solution prepared above was added to the reactor over about 10 minutes, keeping the temperature at 20±5 °C. The mixture was stirred at room temperature for approximately 1 hour, then diluted slowly with water (455 mL). Agitation was stopped, and the layers were allowed to settle. The lower (aqueous) phase was withdrawn to provide 1100 mL colorless solution of pH 1. To the organic phase remaining in the flask was charged additional water (200 mL). The mixture was stirred at room temperature for approximately lhour. Agitation was stopped, and the layers were allowed to settle. The lower (aqueous) phase was withdrawn to provide 50OmL colorless solution of pH 2. The organic phase was heated to about 35 °C, diluted with DMF (300 mL), and concentrated under reduced pressure to the point at which distillation slowed significantly, leaving a concentrate of about 500 mL. The concentrate was transferred without rinsing to a 1-L Schott bottle. The concentrate, a clear colorless solution, weighed 511.6 g. Based on solution assay analysis and the solution weight, the solution contained 187.2 g (0.706 mol) Cbz-L-tørt-Leucine.

[00189] To a 5-L 4-neck round bottomed flask equipped with an overhead stirrer, thermocouple, addition funnel and nitrogen inlet were charged HOBT^»H₂O (103.73 g, 0.678 mol, 1.20 molar eq.), EDC-HCl (129.48 g, 0.675 mol, 1.20 molar eq.) and DMF (480 mL). The slurry was cooled to 0-5 ⁰C. A 36.6 wt% solution of the acid of Cbz-L-tert-Leucine in DMF (491.3 g, 0.745 mol, 1.32 molar eq.) was added over 47 minutes to the reaction mixture while keeping the temperature at 0-5 °C. The reaction mixture was stirred for 1 hour and 27 minutes. A solution of 3-azabicyclo(3.3.0)octane-2-carboxylic acid /-butyl ester in isopropyl acetate (28.8 wt%, 414.3 g, 0.564 mol) was added over 53 minutes while keeping the reaction temperature at 0-5.1 ⁰C. The reaction mixture was warmed to 20±5 °C over about lhour. 4- Methylmorpholine (34.29 g, 0.339 mol, 0.60 molar eq.) was added over 5 minutes. The reaction mixture was agitated for 16 hours then isopropyl acetate (980 mL) was added to the reaction solution. A solution of histamine*2HCl (41.58 g, 0.226 mol, 0.40 molar eq.) in water (53.02 g) was added to the reaction mixture within 4 minutes, followed by 4- methylmorpholine (45.69 g, 0.45 mol, 0.80 molar eq.). The reaction mixture was sampled after 3.5 hours. Water (758 mL) was added, the mixture stirred for about 20 minutes, then allowed to settle for 11 minutes. The phases were separated. The aqueous phase was extracted with isopropyl acetate (716 mL) and the organic phases were combined. IN aq. HCl was prepared by adding 37 wt% hydrochloric acid (128.3 mL) to water (1435 ml). The organic phase was washed for about 20 minutes with the IN hydrochloric acid. A 10wt% aq. K₂CO₃ solution was prepared by dissolving K₂CO₃ (171 g, 1.23 mol, 2.19 molar eq.) in water (1540 mL). The organic phase was washed with the 10 wt% aq. K₂CO₃ solution for about 20minutes. The final clear, very slightly yellow organic solution, weighing 1862.1 g, was sampled and submitted for solution assay. Based on the solution assay and the weight of the solution, the solution contained 238.3 g (0.520 mol) of product of the title compound. ¹H NMR (DMSO-d₆, 500 MHz): δ 7.37 ppm (5 H, s), 7.25-7.33 ppm (1 H, m), 5.03 ppm (2 H₃ s), 4.17 ppm (1 H, d), 3.98 ppm (1 H, d), 3.67-3.75 ppm (2 H, m), 2.62-2.74 ppm (1 H, m), 2.48-2.56 ppm (1 H, m), 1.72-1.89 ppm (2 H, m), 1.60-1.69 ppm (1 H, m), 1.45-1.58 ppm (2 H, m), 1.38 ppm (9 H, s), 1.36-1.42 ppm (1 H, m), 0.97 ppm (9 H, s).

Method 2

[00190] A solution of potassium carbonate (73.3 g) in water (220 mL) was added to a suspension of (IS, 2S,5R) 3-azabicyclo[3.3.0]octane-2-carboxylic, t-butylester, oxalate (80.0 g,) in isopropyl acetate (400 mL) while maintaining a temperature of about 20 ⁰C. The mixture was stirred for 0.5 hour, tha phases separated and the organic phase washed with 25% w/w aqueous potassium carbonate (80 mL) to give a slution of the free base. In a separate flask, aqueous sulfuric acid (400 mL, 0.863 M) was added to a suspension of Cbz-t- leucine dicyclohexylamine salt (118.4g) in /-butylmethyl ether (640 mL) while maintaing a temperature of about 20 ⁰C. The mixture was stirred for 0.5 hour, the phases separated and the organic phase washed with water (200 mL). The phase were separated and N- methylmorpholine (80 mL) was added to the organic phase which was concentrated under reduced pressure at 40 ⁰C to 80 mL to give the free acid as a solution in N-methyl morpholine. This solution was added to a mixture of EDC* HCl (50.8 g) HOBt hydrate (40,6 g) in N-methylmorpholine (280 mL) at 0-10 ⁰C. The mixture was stirred for 1 hour at about 5 ⁰C. The solution of 3-azabicyclo[3.3.0]octane-2-carboxylic, t-butylester from above was added at 0-20 ⁰C followed by N-methylmorpholine (32 mL). The mixture was stired for 6 hour then diluted with isopropyl acetate (600 mL) followed by IN HCl (400 mL). After stirring 0.5 hour, the phases were separated and the organic phase washed with 25% w/w aqueous potassium carbonate (400 mL) and water (80 mL). The mixture was stirred for about 1 hour and the phases separated to give a solution of the title compound in isopropyl acetate.

Method 3

[00191] (IS, 2S,5R) 3-azabicyclo[3.3.0]octane-2-carboxylic, /-butylester, oxalate (1.0 eq.) was suspended in isopropyl acetate (6 vol.) and a solution of potassioum carbonate (3.0 eq.) in water (3.5 vol.) was added at 20-25⁰C. The mixture was stirred for 3 hours then the phases separated. The organic phase was washed with water (2 vol.). [00192] Cbz-Meucine dicyclohexylamine salt (1.05 eq.) was suspended in isopropyl acetate (6 vol.) and sulfuric acid (1.3 eq.) in water (5 vol.) was added at 20-25⁰C. The mixture was stirred for 30 minutes, the phases separated, and the organic phase washed twice with water (2.5 vol each).

[00193] The two solutions from above were combined and then cooled to 0-5⁰C. HOBt hydrate (1.1 eq.) and EDC (1.1 eq.) were suspended in the mixture and the mixture stirred for 6 hours. The mixture was washed with water (5 vol.) and the resulting organic phase treated with L-lysine (1 eq.) and-N-methylmorpholine (NMM) (2 eq.) at 20-25⁰C to destroy excess activated ester. The mixture was then washed with 5% potassium carbonate (5 vol.), IN hydrochloric acid (5 vol.), 5% potassium carbonate (5 vol.) and twice with water (5 vol. each) to give a solution of the title compound in isopropyl acetate.

Example 7: (lS,3aR,6aS)-*-butyI 2-((S)-2-amino-3,3-dimethyIbutanoyl)- octahydrocyclopenta[c]pyrrole-l-carboxylate (28)

Method 1

[00194] A 1 L Buchi hydrogenator was purged with nitrogen three times. A 307.8 g portion of a 12.8 wt% solution of (lS,3aR,6aS)-t-butyl 2-((S)-2-(benzyloxycarbonylamino)-3,3- dimethylbutanoyl)octahydrocyclopenta[c]pyrrole-l-carboxylate (as prepared by the method of Example 6, Method 1) in isopropyl acetate (39.39 g, 0.086 mol) was charged to the reactor. Isopropyl acetate (100 mL) was added to the reactor. A slurry of 50% water and wet 20% Pd(OH)₂/carbon (3.97 g) in isopropyl acetate (168 mL) was prepared and charged to the reactor and agitation was started. The reactor was pressurized to 30 psig with nitrogen gas and vented down to atmospheric pressure. This was repeated twice. The reactor was pressurized to 30 psig with hydrogen and vented down to atmospheric pressure. This was repeated twice. The reactor was pressurized to 30 psig with hydrogen and stirred at ambient temperature for 1 hour. The mixture was filtered using a Buchner funnel with a Whatman # 1 filter paper to remove catalyst. The filter cake was washed with isopropyl acetate (80 mL). The procedure was repeated twice more using 617 g and 290.6 g of the 12.8 wt% solution of the starting Cbz compound. The material from the three hydrogenations were combined and distilled under reduced pressure (28″ Hg). The resultant solution (468.68 g) was assayed for the title compound (23.2%, 98.9 % purity).

¹H NMR (DMSO-d₆, 500 MHz): δ 3.96 ppm (1 H, d), 3.67 ppm (1 H, dd), 3.53 ppm (1 H, dd), 3.19 ppm (1 H₅ s), 2.66-2.75 ppm (1 H, m), 2.49-2.53 ppm (1 H, m), 1.75-1.92 ppm (2 H, m), 1.66-1.74 ppm (1 H, m), 1.48-1.60 ppm (4 H, m), 1.38 ppm (9 H, s), 1.36-1.42 ppm (1 H₅ m), 0.91 ppm (9 H₅ s)

Method 2

[00195] The solution of the Cbz derivative 27 from Example 6, Method 2, was added to 20% Pd(OH)₂/water (50%, 12.2 g) in a hydrogenation apparatus. The apparatus was pressurized to 30 psi with hydrogen then stirred for 2 hr at about 20 ⁰C. The mixture was filtered to remove the catalyst, the filter cake washed with isopropyl acetate (160 mL). The combined filtrates were evaporated with about 4 volumes of heptane at 40 ⁰C 2 to 3 times to remove the isopropyl acetate. The resultant slurry was cooled to 0 ⁰C, filtered and the product dried under vacuum to give the title compound (78.8 g, 98.3% purity).

Method 3

[00196] Asolution of (1 S,3aR,6aS)-t-butyl 2-((S)-2-amino-3₅3-dimethylbutanoyl)- octahydrocyclopenta[c]pyrrole-l-carboxylate in isopropyl acetate from Example 6, Method 3₅ was added to 20% Pd(OH)₂ (2 wt% loading, 50% wet) and the mixture was hydrogenated at 2 bar and 20-25 ⁰C for 2 hours. The catalyst was removed by filtration and washed with isopropyl acetate (2 vol.). The filtrate was concentrated to 10 vol. under reduced pressure at 40 ⁰C to give a solution of the title compound in isopropyl acetate.

Example 8: (lS,3aR,6aS)-*-butyl 2-((S)-2-((S)-2-(benzyloxycarbonylamino)-2- cycIohexylacetamido)-3,3-dimethyIbutanoyl)octahydrocyclopenta[c]pyrrole-l- carboxylate (30)

Method 1 [00197] To a 3 L 3-neck round bottomed flask equipped with an overhead stirrer, thermocouple, addition funnel, nitrogen outlet and ice/water bath was charged HOBt*H₂O (51.74 g; 0.338 mol, 1.05 molar eq.), EDCΗC1 (64.8 g; 0.338 mol, 1.05 molar eq.) followed by DMF (197.1 g, 208.8 mL) and agitation was started. The slurry was cooled to 0-5 ⁰C, then a solution of the acid 29 (98.45 g; 0.338 mol, 1.05 molar eq.) in DMF (172.4 g; 182.9 mL) was prepared and charged to the addition funnel. This was added over about 30 minutes to the batch, maintaining the temperature at 0-5 ⁰C. Once addition was complete the reaction mixture was agitated at 0-5 °C for 2 hours. The solution of the amine 28 in isopropyl acetate (450 g solution; containing 104.4 g of acid 29, 0.322mol) was charged to an addition funnel and added drop wise over lhour maintaining the temperature at 0-5 ⁰C. Sample analysis indicated incomplete reaction and additional EDC hydrochloride (3.89 g) was added. After 3 hours, analysis of a sample showed 1.8% amine 28 remained. A slurry of HOBT»H₂O (2.59 g; 0.0169 mol), and EDC»HC1 (3.24 g; 0.0169 mol) was prepared in DMF (10.44 mL) and cooled to 0-5 ⁰C . A solution of acid 29 (4.92 g; 0.169 mol) in DMF (10.44 mL) was prepared and added to the slurry of EDC^»HC1 and HOBT in DMF over 30minutes, maintaining the reaction temperature at 0-5 ⁰C. The mixture was stirred for 1 hour at 0-5 ⁰C then added to the original mixture maintaining 0-5 ⁰C. The mixture was stirred for 14 hours at about 25 ⁰C. A solution of histamine^»2HCl (11.84 g; 0.064 mol) in water (8.9 mL) was prepared and added to the reaction mixture over 5-10 minutes. A charge of 4- methylmorpholine (13.01 g; 0.129 mol) was added to the batch over about 10 minutes, maintaining the batch temperature at 20±5 ⁰C. The reaction mixture was diluted with isopropyl acetate (443 mL), followed by water (585 mL). A solution of potassium carbonate (57.8 g) in water (585 mL) was added and the mixture was stirred for 0.5 hour. The layers were separated the aqueous layer was extracted twice with isopropyl acetate (twice, 235 mL each). The combined organic phases were washed with 18 % aqueous HCl in water (585 mL), then NaHCO₃ (43.25 g) in water (585 mL). The layers were separated to give a light yellow solution of product 30 in isopropyl acetate weighing 1159.3 g (1275 mL) containing 16.0 w/w % 30 in isopropyl acetate.

¹H NMR (DMSO-d₆, 500 MHz): δ 7.74 (IH, d), 7.36 (5H, m), 7.34-7.26 (IH, m), 5.01 (2H, s), 4.51 (IH, d), 4.02 (IH, t), 3.96 (IH, d), 3.73 (IH, m), 3.66 (IH, m), 3.68 (IH, m), 2.53 (IH, m), 1.86-1.76 (2H, m), 1.70-1.30 (1OH, m), 1.39 (9H, s), 1.15-0.85 (5H, m), 0.96 (9H, s).

Method 2

[00198] A solution of Cbz acid 29 (59.62 g) in N-methylpyrrolidone (126 mL) was added to a suspension of EDCHCL (39.23 g) HOBt hydrate (31.34 g) in N-methylpyrrolidone (221 mL) while maintaining a temperature of about 0 ⁰C. After the addition, the mixture was stirred for 1.5 hours at about 0 ⁰C. A solution of the amine 28 (63.24 g, as prepared in Example 7, Method 2) in isopropyl acetate (632 mL) was added to the mixture maintaining a temperature of about 0 ⁰C. After the addition the mixture was allowed to warm to room temperature and stirred for 5 hours. A solution of potassium carbonate (20.17g) in water (316 mL) was added while maintaining a temperature of about 20 ⁰C. The mixture was vigorously stirred for 0.5 hour. The phases were separated and the organic phase vigorously stirred with potassium carbonate (105.3 g) in water (316 mL). The organic phase was separated and washed with IN HCl (316 mL), and then water (158 mL) to give a 12.7% w/w solution of the title compound 30 in isopropyl acetate.

Method 3 I

[00199] To a solution of (1 S,3aR,6aS)-t-butyl 2-((S)-2-amino-3,3-dimethylbutanoyl)- octahydrocyclopenta[c]pyrrole-l-carboxylate (1 eq) in isopropyl acetate (10 vol) was added NMP (5 vol) followed by EDC (1.15 eq), HOBT hydrate (1.0 eq) and (S)-2- (benzyloxycarbonylamino)-2-cyclohexylacetic acid (29, 1.05 eq) and the suspension was stirred at 20-25⁰C for 4 hr. The mixture was washed with 5% potassium carbonate (5 vol). A mixture of glycine (1 eq), NMM (2 eq) and water (1 vol) was added and the mixture stirred for 4 hr. The mixture was then washed with 5% potassium carbonate (5 vol), IN hydrochloric acid (5 vol), 5% potassium carbonate (5 vol) and twice with water (5 vol each) to give a solution of the title compound in isopropyl acetate.

Example 9: (lS,3aR,6aS)-fert-butyl 2-((S)-2-((S)-2-amino-2-cyclohexylacetamido)-3,3- dimethylbutanoyl)octahydrocyclopenta[c]pyrrole-l-carboxylate (31)

Method 1

[00200] A 60-gallon hasteloy hydrogenating reactor was charged with a solution of the Cbz peptide 30 (15.1 kg) in isopropyl acetate (109 kg). This solution was reduced under vacuum at 50 ⁰C to 68 L. The mixture was then cooled to 25±5 °C and MeOH (15.4 kg) added. This mixture was drained into a container and the reactor was dried. To the dried reactor was charged Pd(OH)₂/C (20%, 1.51 kg). The solution containing the Cbz peptide 30 was added to W 2

the reactor and blanketed with hydrogen (30 psi). The reaction was stirred at 20±5 °C and at 150-220 rpm for 2 hours. After completion, a slurry of activated carbon (0.97 kg) in isopropyl acetate (6.8 kg) was added batch and the mixture stirred for 15 minutes. The mixture was filtered over Celite^® (2.0 kg) via Sparkler filter and through a 0.1 -um cartridge filter. The reactor was rinsed with isopropyl acetate (33.0 kg) and the rinse was combined with the reaction mixture. The system was rinsed additionally with a mixture of isopropyl acetate (25.6 kg) and MeOH (5.73 kg). The combined organics were reduced under vacuum at 65 °C to 30 L. The solution was cooled to 20-30 °C and heptane added (30.8 kg). Distillation was instituted again and the mixture reduced to 30 L. This procedure was repeated for a total of 4 heptane additions (as above) and solvent reductions (as above). The mixture was cooled to 0-5 ⁰C and the product filtered and washed with heptane (12.6 kg). The wet solid (14.0 kg) was dried under vacuum at 15-20 ⁰C to constant weight to give the title compound (10.17 kg).

¹H NMR (DMSOd₆, 500 MHz): δ 7.97 (IH, d), 4.49 (IH₅ d), 3.96 (IH, d), 3.76 (IH, m), 3.67 (IH, m), 3.05 (IH, d), 2.70 (IH, m), 2.53 (IH, m), 1.87-1.77 (2H, m), 1.7-1.3 (1OH, m), 1.39 (9H, s), 1.2-0.85 (5H, m), 0.96 (9H, s).

Method 2

[00201] The solution of compound 30 from Example 8, Method 1, was added to 50% wet 20 wt% Pd(OH)₂ on carbon (3.16 g) in a pressure reactor. The reactor was pressurized at 30 psi with hydrogen and the mixture stirred for about 1 hour. The catalyst was filtered, the filter washed with isopropyl acetate and the combined organics distilled to about 65 mL. The mixture was evaporated with heptane (316 mL) several times until analysis indicates <0.5% isopropyl acetate. The resultant slurry is diluted to about 320 mL then warmed to reflux. The solution was slowly cooled to about 5 ⁰C, the suspension stirred for 1 hour then filtered. The filter cake was washed with about 65 mL of heptane and the product dried under vacuum at 30⁰C to give the title compound (80.16 g) as a white solid.

Method 3

[00202] The solution of (1 S,3aR,6aS)-t-butyl 2-((S)-2-((S)-2-(benzyloxycarbonylamino)-2- cyclohexylacetamido)-3,3-dimethylbutanoyl)octahydrocyclopenta[c]pyrrole- 1 -carboxylate in isopropyl acetate from Example 9, Method 3, was added to 20% Pd(OH)₂ (2 wt% loading, 50% wet) and the mixture hydrogenated at 2 bar and 20-25 ⁰C for 2 hour. The catalyst was removed by filtration and washed with isopropyl acetate (1 vol.). The solvent was exchanged by distillation twice with heptane (8.6 vol.) at reflux. The mixture was cooled to 78 ⁰C over 1 hour, then to 22 ⁰C over 2 hours. After 1 hour at 22 ⁰C the suspension was filtered and the cake washed with heptane (3.2 vol.) and the product dried under vacuum at 30 ⁰C with a nitrogen purge to give the title compound.

Example 10: (lS,3aR,6aSH-butyl 2-((S)-2-((S)-2-cycIohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3-dimethylbutanoyl)octahydrocycIopenta[c]pyrrole-l- carboxylate (33)

Method 1

[00203] To a 10OmL round bottomed flask was added pyrazine-2-carboxylic acid 32 (1.6070 g, 12.95 mmol) and DMF (4 mL). The slurry was stirred at 20-25 ⁰C. Meanwhile, a solution of CDI was prepared by combining CDI (2.1012 g, 12.96 mmol, 1 molar eq.) and DMF (8.80 g, 9.3 mL) in a 25 mL flask. Mild heating (30 °C) aided in dissolution. The CDI solution was cooled to 20-25 ⁰C and added to the slurry of pyrazine-2-carboxylic acid. Stirring was continued for 1.5 hours to assure complete activation of the acid as carbon dioxide was produced as a byproduct. Meanwhile, the amine 31 (5.0002 g, 10.78 mmol) was dissolved in DMF (14.15 g, 15 mL) with mild warming to 30 °C aided in the dissolution of the material. This solution was cooled to 20-25 °C. The activated pyrazine solution was also cooled to about 15 ⁰C. The solution of compound 31 was added to the activated pyrazine carboxylic acid while maintaining the temperature at 30 ⁰C for about 1 hour. The solution was allowed to cool to 20-25 ⁰C then added to a solution of potassium carbonate (0.25 g) in water (100 mL) at 0 ⁰C. The mixture was filtered and washed with water (four times, 50 mL each). The filter cake was dried under vacuum beginning at 20-25 °C and warmed to 30 ⁰C after 24hours until the cake was constant weight to give the title compound (5.99 g). ¹H NMR (DMSO-d_6j 500 MHz): δ 9.19 ppm (1 H, d, J =1.3 Hz), 8.90 ppm (1 H, d, J = 2.5 Hz), 8.76 ppm (1 H, dd, J = 2.4 Hz, 1.5 Hz), 8.50 ppm (1 H, d, J = 9.2 Hz)₅ 8.22 ppm (1 H, d, J = 9.0 Hz), 4.68 ppm (1 H, dd, J = 9.1 Hz, 6.6 Hz)₅ 4.53 ppm (1 H, d, J = 9.0 Hz), 3.96 ppm (1 H₅ d, J = 4.2 Hz), 3.73 ppm (1 H₅ dd, J = 10.5 Hz, 7.5 Hz), 3.68 ppm (1 H, dd, J = 10.6 ppm, 3.4 ppm), 2.68-2.74 ppm (1 H, m), 2.52-2.58 ppm (1 H, m), 1.70-1.88 ppm (3 H, m), 1.51-1.69 ppm (7 H, m), 1.31-1.44 ppm (2 H, m), 1.39 ppm (9 H, s), 1.00-1.19 ppm (4 H₅ m), 0.97 ppm (9 H, s), 0.91-0.97 ppm (1 H, m).

Method!

[00204] Oxalyl chloride (11.29 mL) was added to a solution of pyrazine-2-carboxylic acid 32 and N-methylmorpholine (59.28 mL) in methylene chloride (150 mL) at about 30 ⁰C. The mixture was stirred for 0.5 hour, then a solution of the amine 31 (50.0 g) in methylene chloride (150 mL) was added at about 30 ⁰C. After 0.5 hour, the mixture was washed with water (250 mL). The aqueous phase was extracted with methylene chloride (100 mL) to give a solution of the title compound in methylene chloride which was used directly in the next step (Example 11, Method 2).

Example 11: (lS)-2-((S)-2-((S)-2-cyclohexyI-2-(pyrazine-2-carboxamido)acetamido)-3,3- dimethylbutanoyl)octahydrocyclopenta[c]pyrrole-l-carboxylic acid (34)

Method 1

[00205] Concentrated HCl (150 g, 0.015 mol, 1.2 molar eq.) was slowly added at 0 °C to a stirred solution of the pyrazinyl peptide 33 (50.0 g) in formic acid (100. Og). After 3.3 hours, the reaction mixture was diluted with 166.5 g of ice water. Methylene chloride (100 mL) was added and the reaction was stirred for 10 minutes to dissolve the product. The phases were separated and the aqueous layer extracted with methylene chloride (100 mL). The combined organic phases were washed with water (75 mL) then concentrated to about 1/3 volume at 50 ⁰C, 1 atm. Toluene (100 mL) was added at room temperature and the homogeneous solution was evaporated under vacuum at <56 ⁰C to about 1/3 volume. The mixture was cooled to 20- 25 ⁰C as a precipitate formed. Heptane (75 mL) was slowly added and the slurry stirred for 10-15 minutes. The slurry was filtered and the filter cake was washed with heptane (50 mL). The solids were dried under vacuum at 20-25 °C to give the title compound (15.19 g).

Method 2 [00206] The methylene chloride solution of the starting compound 33 from Example 10, Method 2, was cooled to 0-5 ⁰C then concentrated HCl (200 mL) was added while maintaining a temperature of <10 ⁰C. The mixture was stirred for 3 hours, then diluted with water (200 mL) while maintaining a temperature of < 10 ⁰C. The phases were separated and the aqueous phase extracted with methylene chloride (100 mL). The combined organic phases were washed with water (100 mL) and the aqueous wash phase extracted with methylene chloride. The combined organic extracts were refluxed under an inverse Dean- Stark trap to azeotrope water. The mixture was concentrated by distillation to a minimum volume then diluted with toluene (500 mL) then concentrated by distillation at atmospheric pressure to 250 mL. The mixture was slowly cooled to 20 ⁰C over about 6 hours. The resultant slurry was filtered, the filter cake washed with toluene (100 mL) then dried at about 45 ⁰C in a vacuum oven to provide the title compound (64.7 g) as a pale yellow powder containing about 17 % toluene.

Example 12: (lS,3aR,6aS)-2-((S)-2-((S)-2-cyclohexyl-2-(pyrazine-2- carboxamido)acetamido)-3,3-dimethylbutanoyl)-N-((3S)-l-(cyclopropylamino)-2- hydroxy-l-oxohexan-3-yl)octahydrocyclopenta[c]pyrrole-l-carboxamide (35)

Method 1

[00207] A 500 mL 3-neck round bottomed flask equipped with an overhead stirrer, condenser, thermocouple, and nitrogen outlet was purged with nitrogen for several minutes. The peptide-acid 34 (25.0 g, 0.049 mol), EDC-HCl (10.35 g, 0.054 mol, 1.1 molar eq.), and HOBt-H₂O (8.27 g, 0.054 mol, 1.1 molar eq.) were charged to the flask followed by 175 mL of methylene chloride. The mixture was stirred at room temperature for lhour then added over 20 minutes to a suspension of hydroxyamide-amine 18 (11.1 g, 0.054 mol, 1.1 molar eq.) in methylene chloride (75 mL) while maintaining a temperature below 10 °C. Upon W 2

complete addition, N-methylmorpholine (5.94 mL, 0.054 mol, 1.1 molar eq.) was added in 2 portions. The mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction was quenched by the addition OfNaHCO₃ (8.0 g) in 200 mL of water. The phases were separated and the organic layer washed with water (175 mL), 0.5 N aq. HCl (200 mL), water (three times, 200 mL each) and saturated NaCl (200 mL) to give a 16% by weight methylene chloride solution of the title compound 35 of 100 A% purity (molar yield 100%).

Method 2

[00208] N-methylmorpholine (38.19 mL, 347.3 mmol) was added to a mixture of the peptide-acid 34 (100.0 g, 89.2 wt%, 173.7 mmol), HOBt hydrate (26.79 g, 87.6 wt%, 173.7 mmol), EDCI (36.62 g, 191.04 mmol), and the hydroxyamide-amine 18 in methylene chloride over 30 minutes while maintaining a temperature of 0-5 ⁰C. After the addition, the mixture was warmed to 20 ⁰C and stirred for 5 hours. The mixture was then diluted with water (500 mL) and stirred for about 0.5 hour. The phases were separated and the organic phase washed with IN HCl (500 mL), 5 wt% aqueous sodium bicarbonate (500 mL) to give a solution of the title compound in methylene chloride, 98.5% AUC purity, 95% solution yield.

Method 3

[00209] Peptide acid 34 (1.00 eq.), EDCI (1.10 eq.), HOBt hydrate (1.00 eq.), and hydroxyamine 18^«HC1 (1.05 eq.) were suspended in CH₂Cl₂ (5 vol.) and the mixture was cooled to 0-5 ⁰C. NMM (2.0 eq) was added over 30-60 minutes while maintaining the reaction temperature below 5°C. The reaction mixture was warmed to 20-25 °C over 30 minutes and stirred for additional 5 hours. The reaction was washed with water (5 vol.), IN HCl (5 vol), and 5 wt% aqueous NaHCO₃ (5 vol.) to provide a solution of the title compound in CH₂Cl₂.

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SAXAGLIPTIN

diabetes, Uncategorized Comments Off

Mar 292015

SAXAGLIPTIN

Saxagliptin
CAS No.:	361442-04-8
Synonyms:	Saxagliptin 15ND2; Onglyza;
Formula:	C18H25N3O2
Exact Mass:	315.19500
Molecular Weight:	315.41000

SMILES:

C1[C@@H]2C[C@@H]2N([C@@H]1C#N)C(=O)[C@H](C34CC5CC(C3)CC(C5)(C4)O)N13c nmr predict

Saxagliptin, (1S,3S,5S)-2-(2S)-2-Amino-2-(3-hydroxyadamantan-1-yl)-acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile of the following chemical structure:

is a dipeptidyl peptidase IV (DPP4) inhibitor. Saxagliptin is marketed under the trade name ONGLYZA® by Bristol-Myers Squibb for the treatment of type 2 diabetes.

Saxagliptin and its hydrochloride and trifluoroacetic acid salts are disclosed in U.S. Pat. No. 6,395,767. In addition, U.S. Pat. No. 7,420,079 discloses Saxagliptin and its hydrochloride, trifluoroacetic acid and benzoate salts, as well as Saxagliptin monohydrate.

U.S. 2009/054303 and the corresponding WO 2008/131149 application disclose several crystalline forms of Saxagliptin and of Saxagliptin salts. The crystalline forms of Saxagliptin reported in that patent application are a monohydrate (denoted there as form H-1), a hemihydrate (denoted there as form H0.5-2), a dihydrate (denoted form H2-1) and an anhydrous form (denoted there as N-3).

WO 2005/117841 (the ‘841 application) describes the cyclization of Saxagliptin to form the therapeutically inactive cyclic amidine. The ‘841 application reports that such cyclization can occur both in solid state and solution state.

WO 2010/115974 discloses Forms: I-S, HT-S, IV-S, and HT-IV-S of Saxagliptin hydrochloride.

Org. Process Res. Dev., 2009, 13 (6), pp 1169–1176

DOI: 10.1021/op900226j

http://pubs.acs.org/doi/abs/10.1021/op900226j

The commercial-scale synthesis of the DPP-IV inhibitor, saxagliptin (1), is described from the two unnatural amino acid derivatives 2 and 3. After the deprotection of 3, the core of 1 is formed by the amide coupling of amino acid 2 and methanoprolinamide 4. Subsequent dehydration of the primary amide and deprotection of the amine affords saxagliptin, 1. While acid salts of saxagliptin have proven to be stable in solution, synthesis of the desired free base monohydrate was challenging due to the thermodynamically favorable conversion of the free amine to the six-membered cyclic amidine 9. Significant process modifications were made late in development to enhance process robustness in preparation for the transition to commercial manufacturing. The impetus and rationale for those changes are explained herein.

Monohydrate 1 was isolated as a white solid (58.2 kg, 88%).

1 H NMR (400 MHz, CD2Cl2- d6) δ 5.25 (dd, J1 ) J2 ) 1.0 Hz, 1H), 4.93 (dd, J1 ) 10.6 Hz, J2 ) 2.3 Hz, 1H), 3.55-3.50 (m, 1H), 3,35 (s, 1H), 2.45 (ddd, J1 ) 16.1 Hz, J2 ) 10.9 Hz, J3 ) 5.6 Hz, 1H), 2.25 (dd, J1 ) 13.6 Hz, J2 ) 2.5 Hz, 1H), 2.18-2.10 (m, 2H), 1.83-1.42 (m, 15H), 1.40-1.27 (m, 3H) 1.0-0.87 (m, 2H)

13C NMR (100 MHz, CD2Cl2) δ 173.43, 120.15, 68.83, 60.90, 46.57, 45.51, 45.08, 45.01, 41.62, 38.15, 37.92, 37.35, 35.88, 30.98, 30.93, 30.80, 18.00, 13.69.

MS (FAB) m/z 316 [M + H]+

1H NMR PREDICT

Saxagliptin NMR spectra analysis, Chemical CAS NO. 361442-04-8 NMR spectral analysis, Saxagliptin H-NMR spectrum

13C NMR PREDICT
Saxagliptin NMR spectra analysis, Chemical CAS NO. 361442-04-8 NMR spectral analysis, Saxagliptin C-NMR spectrum

………………

http://www.google.com/patents/WO2012162507A1?cl=en

two amino acid derivatives (A) and (B), described in further detail hereinbelow, coupled in the presence of a coupling reagent. The amide coupling of (S)-a[[(l,l-dimethyleethoxy)carbonyl]amino]-3- hydroxytricyclo [3.3.1.1]decane-l-acetic acid (A) and (lS,3S,5S)-2-azabicyclo[3.1.0]hexane-3- carboxamide (B), subsequent dehydration of the primary amide and deprotection of the amine affords saxagliptin (C).

synthetic route is disclosed as follows:

Scheme-IV

Scheme-V

………………

……………..

………….

Savage, Scott A., et al., “Preparation of Saxagliptin, a Novel DPP-IV Inhibitor“, Organic Process Research & Development, 2009, vol. 13, pp. 1169-1176.

REFERENCES

US6395767	16 Feb 2001	28 May 2002	Bristol-Myers Squibb Company	Cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl peptidase IV and method
US6995183	27 Jul 2004	7 Feb 2006	Bristol Myers Squibb Company	Adamantylglycine-based inhibitors of dipeptidyl peptidase IV and methods
US7186846	28 Mar 2005	6 Mar 2007	Bristol-Myers Squibb Company	Process for preparing a dipeptidyl peptidase IV inhibitor and intermediates employed therein
US7214702	23 May 2005	8 May 2007	Bristol-Myers Squibb Company	Reacting the amide compound with phosphorus oxychloride in an organic solvent; treating the reaction mixture with water to form (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxytricyclo[3.3.1.13,7]dec-1-yl)-1-oxoethyl]-2-azabicyclo[3.1.0]hexane-3-carbonitrile-hydrochloride
US7223573	2 May 2005	29 May 2007	Bristol-Myers Squibb Company	Enzymatic ammonolysis process for the preparation of intermediates for DPP IV inhibitors
US7420079	18 Nov 2003	2 Sep 2008	Bristol-Myers Squibb Company	Intermediates for making 1(alpha-amino-1-(cyclopropyl-fused pyrrolidinylcarbonyl)methyl)-3-hydroxyadamantanes, e.g., methyl 3-hydroxy-<a-oxotricyclo[3.3.1.13,7]decane-1-acetate
US7470810	11 Jan 2005	30 Dec 2008	Bristol-Myers Squibb Company	Such as 1-dodecane-thiotrifluoroacetate; alkyl/arylthiol is treated with trifluoroacetic anhydride in presence of pyridine, solvent (dichloromethane), and dimethylaminopyridine (DMAP) as catalyst; for protection of amino acids
US7741082	12 Apr 2005	22 Jun 2010	Bristol-Myers Squibb Company	Process for preparing dipeptidyl peptidase IV inhibitors and intermediates therefor
US7943656	18 Apr 2008	17 May 2011	Bristol-Myers Squibb Company	Crystal forms of saxagliptin and processes for preparing same
US20060035954	8 Aug 2005	16 Feb 2006	Sharma Padam N	Ammonolysis process for the preparation of intermediates for DPP IV inhibitors
WO2001068603A2	5 Mar 2001	20 Sep 2001	Bristol Myers Squibb Co	Cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl iv, processes for their preparation, and their use
WO2008131149A2	18 Apr 2008	30 Oct 2008	Squibb Bristol Myers Co	Crystal forms of saxagliptin and processes for preparing same
WO2010115974A1	9 Apr 2010	14 Oct 2010	Sandoz Ag	Crystal forms of saxagliptin
WO2011140328A1	5 May 2011	10 Nov 2011	Teva Pharmaceutical Industries Ltd.	Saxagliptin intermediates, saxagliptin polymorphs, and processes for preparation thereof

Citing Patent	Filing date	Publication date	Applicant	Title
US8748631 *	24 May 2012	10 Jun 2014	Apicore, Llc	Process for preparing saxagliptin and its novel intermediates useful in the synthesis thereof
US20130023671 *	24 May 2012	24 Jan 2013	Apicore, Llc	Process for preparing saxagliptin and its novel intermediates useful in the synthesis thereof

REFERENCES

1. Scott A. Savage, Gregory S. Jones, Sergei Kolotuchin, Shelly Ann Ramrattan, Truc Vu, and Rebert E. Waltermire (2009) Preparation of Saxagliptin, a Novel DPP-IV Inhibitor, Organic Process Research & Development., 13, 1169-1176.
2. Santosh K. Sing, Narendra Manne and Manojit Pal, (2008) Synthesis of (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile: A key intermediate for dipeptidyl peptidase IV inhibitors. Beilstein Journal of Organic Chemistry, 4, No. 20.
3. U.S. Pat. No. (2010) 0274025 A1.
4. U.S. Pat. No. (2006) 0035954 A1.
5. U.S. Pat. No. (2005) 0090539 A1.
6. Organic letters. (2001) Vol. 3, No.5, Page: 759-762
7. Tetrahedron 59 (2003) 2953-2989

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Uprosertib (GSK-2141795)

Uncategorized Comments Off

Mar 242015

Uprosertib (GSK-2141795)

GSK 2141795C

N-[(1S)-1-(aminomethyl)-2-(3,4-difluorophenyl)ethyl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide

N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)furan-2-carboxamide

2-Furancarboxamide, N-[(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl]-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-

Λ/-{(1 S)-2-amino-1-r(3,4-difluorophenyl)methyllethyl}-5-chloro-4-(4- chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxamide

N-{(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1-methyl-1Hpyrazol-5-yl)-2-furancarboxamide.

Cas 1047634-65-0 (GSK-2141795); BASE

CAS 1047635-80-2 (GSK-2141795 HCl salt)

Synonym: GSK-2141795; GSK2141795; GSK 2141795; GSK795; GSK-795; GSK 795. Uprosertib. UNII ZXM835LQ5E

IUPAC/Chemical name:

N-((S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl)-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-carboxamide

C18H16Cl2F2N4O2
Exact Mass: 428.06184
Molecular Weight: 429.25

Elemental Analysis: C, 50.37; H, 3.76; Cl, 16.52; F, 8.85; N, 13.05; O, 7.45

Mechanims of Action:Akt inhibitor
Indication:Cancer Treatment
Drug Company:GlaxoSmithKline

PHASE 2… CANCER

Uprosertib, also known as GSK2141795 and GSK795, is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity.

The National Cancer Institute (NCI) is evaluating the compound in phase II clinical studies for the treatment of endometrial carcinoma and multiple myeloma in combination with trametinib.

GSK-2141795, an oral AKT inhibitor, is in early clinical trials at GlaxoSmithKline for the treatment of solid tumors and lymphoma. The company is conducting phase II clinical trials for the treatment of patients with BRAF wild-type mutation melanoma and for the treatment of recurrent or persistent cervical cancer in combination with trametinib.

Akt inhibitor GSK2141795 binds to and inhibits the activity of Akt, which may result in inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.

QC data:

View NMR, View HPLC, View MS …… MEDKOO

PATENT

PATENT	SUBMITTED	GRANTED
Inhibitors of AKT Activity [US2011071182]	2011-03-24
INHIBITORS OF Akt ACTIVITY [US2010267759]	2010-10-21
INHIBITORS OF AKT ACTIVITY [US2009209607]	2009-08-20
INHIBITORS OF Akt ACTIVITY [US2010041726]	2010-02-18

More information about this drug

The chemical structures of Afuresertib (GSK-2110183) and GSK-2141795 are very similar as shown below:

Fig 1. chemical structures of Afuresertib (GSK-2110183) and GSK-2141795

PATENT

WO 2008098104 OR EP2117523

http://www.google.com/patents/EP2117523A1?cl=en

Scheme 2

11-1 I-2

II-3 II-4

Reagents: (a) PyBrop, (i-Pr)2NEt, 1 ,1-dimethylethyl (2-amino-3- phenylpropyl)carbamate, DCM, RT; (b) 5-(5,5-dimethyl-1 ,3,2-dioxaborinan-2-yl)-1- methyl-1 H-pyrazole, K2CO3, Pd(PPh3)4, dioxane/H2O; (c) TFA / DCM, RT.

Preparation 7

Preparation of 5-(5,5-dimethyl-1 ,3,2-dioxaborinan-2-yl)-1 -methyl-1 H-pyrazole

To a solution of 1 -methyl pyrazole (4.1 g, 50 mmole) in THF (100 ml.) at 00C was added n-BuLi (2.2M in THF, 55 mmole). The reaction solution was stirred for 1 hour at RT and then cooled to -78°C [J. Heterocyclic Chem. 41 , 931 (2004)]. To the reaction solution was added 2-isopropoxy-4,4,5,5-tetramethyl-1 ,3,2-dioxaborolane (12.3 ml_, 60 mmole). After 15 min at -78°C, the reaction was allowed to warm to 00C over 1 hour. The reaction was diluted with saturated NH4CI solution and extracted with DCM. The organic fractions were washed with H2O (2 x 100 ml_), dried over Na2SO4 and concentrated under vacuum to afford a tan solid (8.0 g, 77%) which was used without further purification. LCMS (ES) m/z 127 (M+H)+ for [RB(OH)2]; 1H NMR (CDCI3, 400 MHz) δ 7.57 (s, 1 H), 6.75 (s, 1 H), 4.16 (s, 3H), and 1.41 (s, 12H).

Example . .24

UPROSERTIB

Preparation Λ/-{(1 S)-2-amino-1-r(3,4-difluorophenyl)methyllethyl}-5-chloro-4-(4- chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxamide

a) methyl 4-(1-methyl-1H-pyrazol-5-yl)-2-furancarboxylate

A solution of methyl 4-bromo-2-furancarboxylate (470 mg, 2.29 mmol), potassium carbonate (1584 mg, 11.46 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)-1 H-pyrazole (525 mg, 2.52 mmol)[prepared according to Preparation 7] and bis-(tri-t-butylphosphine)Palladium (0) (58.6 mg, 0.12 mmol) in 1 ,4-dioxane (9.55 ml) and water (1.9 ml) was stirred at 80 0C. After 1 hr, the solution was partitioned between H2O-DCM and the aqueous phase was washed several times with DCM. The combined organic fractions were dried over I^^SOφ concentrated and purified via column chromatography (30% EtOAc in hexanes) affording the title compound (124 mg, 0.60 mmol, 26 % yield) as a white powder: LCMS (ES) m/e 206 (M+H)+.

b) methyl 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylate

A solution of methyl 4-(1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylate (412 mg, 2.0 mmol) and N-chlorosuccinimide (267 mg, 2.0 mmol) in DMF (10 ml.) was heated at 75 0C for 30 minutes. Another batch of N-chlorosuccinimide (267 mg, 2.0 mmol) was added. After 1 hr, the mixture was concentrated and purified using silica gel and eluting with 0-55% ethyl acetate / hexane to afford the title compound as a white solid (225 mg, 0.82 mmol, 71 % yield) : LCMS (ES) m/e 276 (M+H)+.

c) 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylic acid

A solution of methyl 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-2- furancarboxylate (224 mg, 0.82 mmol) in 6N sodium hydroxide (1.36 ml, 8.2 mmol) and tetrahydrofuran (5 ml) was stirred at 70 0C in a sealed tube for 1 h. The resulting solution was cooled and then partitioned between H2O-DCM. The aqueous phase was adjusted to pH ~4 and then washed several times with DCM. The combined organic fractions were dried over Na2SO4 and concentrated affording the title compound (201 mg, 0.77 mmol, 94 % yield) as a yellow oil: LCMS (ES) m/e 262 (M+H)+.

d) 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-N-{(1S)-2-(3,4-difluorophenyl)-1- [(1 ,3-dioxo-1 ,3-dihydro-2H-isoindol-2-yl)methyl]ethyl}-2-furancarboxamide

To a solution of 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-2- furancarboxylic acid (200 mg, 0.77 mmol)[prepared according to the procedure of Preparation 6], 2-[(2S)-2-amino-3-(2,4-difluorophenyl)propyl]-1 H-isoindole-1 ,3(2H)- dione (254 mg, 0.80 mmol) and N,N-diisopropylethylamine (0.40 ml, 2.30 mmol) in DCM (10 ml) was added bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (536 mg, 1.15 mmol). After stirring at ambient temperature for 20 hrs, the mixture was concentrated and purified with silica gel column eluting with gradient (0-50% ethyl acetate/hexanes) to afford the title compounds as an off-white foamy solid (304 mg, 0.54 mmol, 71 % yield): LCMS (ES) m/e 560(M+H)+.

e) Λ/-{(1 S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1- methyl-1 /-/-pyrazol-5-yl)-2-furancarboxamide

To a solution of 5-chloro-4-(4-chloro-1-methyl-1 H-pyrazol-5-yl)-N-{(1S)-2- (3,4-difluorophenyl)-1 -[(1 ,3-dioxo-1 ,3-dihydro-2H-isoindol-2-yl)methyl]ethyl}-2- furancarboxamide (304 mg, 0.54 mmol) in methanol (5 ml) at 25 0C was added hydrazine (0.08 ml, 2.7 mmol) dropwise. After 12h, the solution was concentrated, dry loaded onto silica and purified by column chromatography (5% MeOH in DCM (1 % NH4OH)). The free base was converted to the HCI salt by addition of excess 4M HCI in dioxane (1 ml) to the residue in MeOH (2 ml) affording the HCI salt of the title compound as a yellow solid:

LC-MS (ES) m/z 430(M+H)+,

1H NMR (400 MHz, MeOD) δ ppm 2.91 – 3.05 (m, 2 H) 3.17 – 3.28 (m, 2 H) 3.81 (s, 3 H) 4.57 (d, J=9.60 Hz, 1 H) 7.12 (br. s., 1 H) 7.18-7.28 (m., 2 H) 7.36-7.39 (m, 1 H) 7.58 (s, 1 H).

SYNTHESIS ELABORATED

STEP A

4,5-dibromo-2-furancarboxylic acid in methanol , sulfuric acid methyl 4,5-dibromo-2-furancarboxylate LCMS (ES) m/e 283 (M+H)+

STEP B

methyl 4,5-dibromo-2-furancarboxylate and isopropylmagnesium chloride ,to give methyl 4-bromo-2-furancarboxylate

LCMS (ES) m/e 204,206 (M, M+2)+

STEP C

methyl 4-bromo-2-furancarboxylate and NCS in N,N-dimethylformamide methyl 4-bromo-5-chloro-2-furancarboxylate LCMS (ES) m/e 238,240,242 (M, M+2, M+4)+

STEP D

methyl 4-bromo-5-chloro-2-furancarboxylate , 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole prepared according toPreparation 7], potassium carbonate and bis(tri-t-butylphosphine)paliadium(0) in 1,4-dioxane (19.14 ml) and water ……methyl 5-chloro-4-(1-methyl-1H-pyrazol-5-yl)-2-furancarboxylate obtained. LCMS m/e ES 240, 242 (M, M+2)+

STEP E

a) 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxylic acid.

A solution of methyl 5-chloro-4-(1-methyl-1H-pyrazol-5-yl)-2-furancarboxylate [prepared according to Example

127] and n-chlorosuccinimide (166 mg, 1.25 mmol) yielding 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxylic acid. LCMS (ES) m/e 261,263 (M, M+2)+

STEP F

Reacting 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxylic acid [prepared according to the procedure of Preparation 6], 2-[(2S)-2-amino-3-(2,4-difluorophenyl)propyl]-1H-isoindole-1,3(2H)-dione and N,N-diisopropylethylamine in DCM was added bromo-tris-pyrrolidino-phosphonium hexafluorophosphate …….obtd

5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-N-{(1S)-2-(3,4-difluorophenyl)-1-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]ethyl}-2-furancarboxamide. the uproserib precursor

LCMS (ES) m/e 560(M+H)+

NOTE STRUCTURE OF 2-[(2S)-2-amino-3-(2,4-difluoro phenyl)propyl]-1H-isoindole-1,3(2H)-dione

SEE http://www.google.com/patents/WO2010093885A1?cl=en

Preparation 1

Preparation of 2-[(2S)-2-amino-3-(3,4-difluorophenyl)propyl1-1 /-/-isoindole-1 ,3(2H)-dione a) 1 ,1-dimethylethyl [(1 S)-2-(3,4-difluorophenyl)-1-(hydroxymethyl)ethyl]carbamate

To a solution of Λ/-{[(1 ,1-dimethylethyl)oxy]carbonyl}-3,4-difluoro-L-phenylalanine (2.0 g, 6.7 mmol) in THF (35 ml.) at 0 0C stirred was added BH3-THF (30 ml_, 30 mmol- 1 M in THF). After 12h, the reaction was quenched with AcOH:MeOH (1 :4, 20 ml.) and partitioned between saturated aqueous NaHCO3 and CHCI3. The aqueous phase was then extracted several times with CHCI3. The combined organic fractions were concentrated and the resulting white solid (7.0 g, 74%) used without further purification: LCMS (ES) m/e 288 (M+H)+.

b) 1 ,1-dimethylethyl {(1 S)-2-(3,4-difluorophenyl)-1-[(1 ,3-dioxo-1 ,3-dihydro-2/-/-isoindol-2- yl)methyl]ethyl}carbamate

To a solution of 1 ,1-dimethylethyl [(1 S)-2-(3,4-difluorophenyl)-1-

(hydroxymethyl)ethyl]carbamate (2.65 g, 9.22 mmol), polymer bound triphenylphosphine (5.33 g, 1 1.5 mmol, 2.15 mmol/g) and phthalimide (1.63 g, 10.9 mmol) in THF (50 ml.) at 25 0C was added diisopropyl azodicarboxylate (1.85 ml_, 11.3 mmol). After stirring at RT for 1 h, the reaction solution was filtered and concentrated. The residue was adsorbed onto silica and purified via column chromatography to yield product (0.33 g) as a white solid: LCMS (ES) m/z 417 (M+H)+.

c) 2-[(2S)-2-amino-3-(3,4-difluorophenyl)propyl]-1 H-isoindole-1 ,3(2H)-dione

To a solution of 1 ,1-dimethylethyl {(1S)-2-(3,4-difluorophenyl)-1-[(1 ,3-dioxo-1 ,3- dihydro-2H-isoindol-2-yl)methyl]ethyl}carbamate (0.33 g, 0.79 mmol) in CHCI3:MeOH (10:3, 13 mL) at RT was added 4M HCI in dioxane (5 mL, 20 mmol). After 12h, the solvents were removed and affording the title compound (0.29 g, quant.) as a white HCI salt which was used without further purification: LCMS (ES) m/z 317 (M+H)+.

FINAL STEP

conversion of precursor to uprosertb

UPROSERTIB PRECURSOR GIVES

UPROSERTIB

N-{(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1-methyl-1Hpyrazol-5-yl)-2-furancarboxamide.

5-chloro-4-(4-chloro-1-methyl-1Hpyrazol-5-yl)-N-{(1S)-2-(3,4-difluorophenyl)-1-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)methyl]ethyl}-2-furancarboxamide in methanol (5 ml) AND hydrazine …..N-{(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1-methyl-1Hpyrazol-5-yl)-2-furancarboxamide.

SYNTHESIS OF INTERMEDIATES

Example 127

a) methyl 4,5-dibromo-2-furancarboxylate

To a solution of 4,5-dibromo-2-furancarboxylic acid (25 g, 93 mmol) in methanol (185 ml) was added sulfuric acid (24.7 ml, 463 mmol). The resulting solution stirred at 50 0C over 12h. The solution was partitioned between H2O-DCM and the aqueous phase was washed several times with DCM. The combined organic fractions were dried over I^^SOφ concentrated and used directly without further purification providing methyl 4,5-dibromo-2-furancarboxylate (23.67 g, 83 mmol, 90 % yield), LCMS (ES) m/e 283, 285, 287 (M, M+2, M+4)+.b) methyl 4-bromo-2-furancarboxylate Br

To a solution of methyl 4,5-dibromo-2-furancarboxylate (3.3 g, 1 1.62 mmol) in tetrahydrofuran (46 ml) at -40 0C was added isopropylmagnesium chloride (6.97 ml, 13.95 mmol). After 1 h, Water (11 ml) was added and the solution warmed to 25 0C. The reaction mixture was then partitioned between H2O-DCM and the aqueous phase was washed several times with DCM. The combined organic fractions were dried over Na2SOφ concentrated and purified by column chromatography (3% EtOAc in hexanes) affording methyl 4-bromo-2-furancarboxylate (1.4 g, 6.49 mmol, 56 % yield) as a yellow solid: LCMS (ES) m/e 205, 207 (M, M+2)+.

c) methyl 4-bromo-5-chloro-2-furancarboxylate

A solution of methyl 4-bromo-2-furancarboxylate (1.4 g, 6.83 mmol) and NCS (0.912 g, 6.83 mmol) in N,N-dimethylformamide (13.7 ml) was stirred in a sealed tube for 1 h at 100 0C. After 1 h, the solution was partitioned between DCM- H2O and the aqueous phase was washed several times with DCM. The combined organic fractions were dried over I^^SOφ concentrated and purified via column chromatography (2-10% EtOAc in hexanes) affording methyl 4-bromo-5-chloro-2- furancarboxylate (1.348 g, 5.12 mmol, 75 % yield) as a white solid: LCMS (ES) m/e 238, 240, 242 (M, M+2, M+4)+.

d) methyl 5-chloro-4-(1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylate

A solution of methyl 4-bromo-5-chloro-2-furancarboxylate (1.1 g, 4.59 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl)-1 H-pyrazole (1.05 g, 5.05 mmol)[prepared according to Preparation 7], potassium carbonate (3.17 g, 22.97 mmol) and bis(tri-t-butylphosphine)palladium(0) (0.117 g, 0.23 mmol) in 1 ,4- dioxane (19.14 ml) and water (3.83 ml) was stirred at 80 0C in a sealed tube for 1 h. The reaction mixture was partitioned between H2O-DCM and the aqueous phase was washed several times with DCM. The combined organic fractions were dried over Na2SOφ concentrated and purified via column chromatography (silica, 4-25% EtOAc in hexanes) yielding methyl 5-chloro-4-(1-methyl-1 H-pyrazol-5-yl)-2- furancarboxylate (800 mg, 2.53 mmol, 55 % yield) as a yellow oil: LCMS m/e ES 240, 242 (M, M+2)+.

e) 5-chloro-4-(1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylic acid

A solution of methyl 5-chloro-4-(1-methyl-1 H-pyrazol-5-yl)-2- furancarboxylate (300 mg, 1.25 mmol) in 6N sodium hydroxide (4.16 ml, 24.93 mmol) and tetrahydrofuran (5.4 ml) was stirred at 70 0C in a sealed tube for 1 h. The resulting solution was cooled and then partitioned between H2O-DCM. The aqueous phase was adjusted to pH ~4 and then washed several times with DCM. The combined organic fractions were dried over Na2SO4 and concentrated affording 5-chloro-4-(1-methyl-1 H-pyrazol-5-yl)-2-furancarboxylic acid (267 mg, 0.59 mmol, 47 % yield) as a white foam: LCMS (ES) m/e 265 (M+H)+.

References

1: Dumble M, Crouthamel MC, Zhang SY, Schaber M, Levy D, Robell K, Liu Q, Figueroa DJ, Minthorn EA, Seefeld MA, Rouse MB, Rabindran SK, Heerding DA, Kumar R. Discovery of Novel AKT Inhibitors with Enhanced Anti-Tumor Effects in Combination with the MEK Inhibitor. PLoS One. 2014 Jun 30;9(6):e100880. doi: 10.1371/journal.pone.0100880. eCollection 2014. PubMed PMID: 24978597; PubMed Central PMCID: PMC4076210.

2: Pachl F, Plattner P, Ruprecht B, Médard G, Sewald N, Kuster B. Characterization of a chemical affinity probe targeting Akt kinases. J Proteome Res. 2013 Aug 2;12(8):3792-800. doi: 10.1021/pr400455j. Epub 2013 Jul 3. PubMed PMID: 23795919.

3: Pal SK, Reckamp K, Yu H, Figlin RA. Akt inhibitors in clinical development for the treatment of cancer. Expert Opin Investig Drugs. 2010 Nov;19(11):1355-66. doi: 10.1517/13543784.2010.520701. Epub 2010 Sep 16. Review. PubMed PMID: 20846000; PubMed Central PMCID: PMC3244346.

Tadalafil Analytical/Spectral Visit

Uncategorized Comments Off

Mar 112015

Tadalafil

INTRODUCTION Tadalafil is a potent and selective phosphodiesterase-5 (PDE-5) inhibitor, asecondary messenger for the smoothmuscle relaxing effects of nitric oxide,which plays an important role in thevasodilation of erectile tissues.1-3 OralPDE-5 inhibitors have become the preferredfirst-line treatment for erectile dysfunction worldwide.4

PREPARATION

Diastereoselective synthesis of (+)-tadalafil (1)describes a process for the synthesis of tadalafil (1) and itsintermediate of formula5which involves reactingD-tryptophan methylester 2 with a piperonal 3 in the presence of methanol and conc. HCl to

give compound 4 . The later compound is then reacted with chloroacetyl chloride in the presence of NaHCO 3

to afford the intermediate5, which is reacted with methylamine in chloroform to give tadalafil in 88% yield

Stereoselective synthesis of (+)-tadalafil (1) and(+)-6-epi-tadalafil (8)[20]The target isomeric tadalafil molecule is shown . Thus,D-tryptophan methyl ester reacted with piperonal3under Pictet–Spen-gler reaction condition (TFA/CH2Cl2/MeOH) to furnish two diastereo-mers4and6in 25% and 24% yields, respectively. Condensation of4or6with chloroacetyl chloride provided acylated intermediate 5or7in almostquantitative yield. Subsequent cyclization of5withN-methyl amine inmethanol at 50C for 16 h provided diastereomers tadalafil (1) in 54%yield. Compound1is in full accordance with the literature data {[a]D20¼+71.4 (c 1.00, CHCl3); lit. [a]D20¼+71.2 (c 1.00, CHCl3)}[17,18]. Thus,under the elongated reaction time, 48 h, compound8was obtained fromprecursor7with decreased yield of 21%

depicts an efficient and stereospecific synthesis of tadalafil (1)as well as 12a-epi-tadalafil (11). Pictet–Spengler reaction ofD-trypto-phan methyl ester hydrochloride9with equal molar piperonal byrefluxing for 4 h in nitromethane affordedcis-10-HCl in 98% ee and94% yield. The hydrochloride salt ofcistetrahydro-b-carboline deriva-tivecis-10-HCl was directly treated with 1.5 equiv of chloroacetyl chlo-ride in dichloromethane at 0o

C in the presence of 3 equiv oftriethylamine to formN-chloroacetyl tetrahydro-b-carboline derivative5

in 92% yield. Then compound5reacted with 5 equiv of methylamineovernight in DMF at room temperature to furnish tadalafil1in95% yields.

US PATENT

D. Ben-Zion, D. Dov, United States Patent, US 2006/0276652 A1, 2006.

B.D. Pandurang, B.B. Bharat, S.S. Sachin, P.S. Pranay, United States Patent, US 7, 223,

863 B2, 2007.

FROM L TRYPTOPHAN

X. Sen, S. Xiao-Xin, X. Jing, Y. Jing-Jing, L. Shi-Ling, L. Wei-Dong, Tetrahedron

Asymmetr. 20 (2009) 2090.

S. Xiao-Xin, L. Shi-Ling, X. Wei, X. Yu-Lan, Tetrahedron Asymmetr. 19 (2008) 435

S. Xiao, X. Lu, X.-X. Shi, Y. Sun, L.-L. Liang, X.-H. Yu, J. Dong, Tetrahedron Asymmetr.

20 (2009) 430.

IR OF TADALAFIL

1H NMR OF TADALAFIL

13 C NMR OF TADALAFIL

COSY NMR OF TADALAFIL

DEPT NMR OF TADALAFIL

HSQC NMR OF TADALAFIL

HMBC NMR OF TADALAFIL

MASS SPECTRUM OF TADALAFIL

UV OF TADALAFIL

RAMAN SPEC OF TADALAFIL

SECTION 1 SECTION 2 .. SECTION 3 Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 103–113 Analysis of illegally manufactured formulations of tadalafil (Cialis®) by 1H NMR, 2D DOSY 1H NMR and Raman spectroscopy Saleh Trefia, Corinne Routaboul b, Saleh Hamieh a, Veronique Gilard ´ a, Myriam Malet-Martino a,∗, Robert Martino a a Groupe de RMN Biom´edicale, Laboratoire SPCMIB (UMR CNRS 5068), France b Service commun de spectroscopie IR et Raman, Universit´e Paul Sa LC-DAD apparatus and chromatographic conditions HPLC was carried out using a Waters 2695 Alliance model with a Waters 2996 diode array detector. The analytical column was a reversed-phase column Luna C18 (100 mm × 3 mm i.d.; 3m particle size; Phenomenex, UK). The column temperature was 30 ◦C. The mobile phase consisted of a mixture (35:65, v/v) of acetonitrile and phosphate buffer (10 mmol L−1, pH 3). The flow rate was 0.6 mL min−1 and the volume injected 10 L. A detection wavelength of 225 nm was chosen as it allows the detection of all tadalafil or sildenafil analogues. For quantitative analysis, a calibration curve was constructed from the analysis of four solutions containing pure tadalafil in a concentration range of 0.01–0.1 mg mL−1. Each standard solution was injected in triplicate in the chromatographic system. The linearity (R2 > 0.999) was evaluated by least-squares linear regression analysis. LC–MS analysis The HPLC system used consisted of an Agilent 1100 series apparatus. An Applied System QTRAP triple quadrupole mass spectrometer, equipped with a turbo ion spray (TIS) interface, was used for detection. Both were controlled by an Agilent Analyst software (version 1.4). HPLC conditions were as follows. The column temperature was 30 ◦C. The mobile phase consisted of a mixture (50:50, v/v) of acetonitrile and a buffer solution (ammonium acetate 10 mmol L−1, pH 7). The flow rate was 0.6 mL min−1 and the volume injected 5 L. The mass spectrometer was operated in positive ionisation mode with TIS heater set at 450 ◦C. Nitrogen served both as auxiliary, collision gas and nebuliser gas. The operating conditions for TIS interface were—(i) in MS mode: mass range 200–550m (1 s), step size 0.1m; Q1 TIS MS spectra were recorded in profile mode, IS 5000 V, DP 85 V; (ii) in MS–MS mode: precursor mass 489 m; mass range 10–500 m (0.35 s); step size 0.15m; LC–MS–MS spectra were rec d in profile mode, IS 5000 V, DP 85 V and CE 40 V Fig. 3. DOSY NMR spectra in CD3CN:D2O (80:20) of genuine Eli Lilly Cialis® (A), formulation 6 Fig. 2. Raman spectra of pure tadalafil (A) and genuine Eli Lilly Cialis®: whole tablet (B), uncoated tablet from 200 to 1800 cm−1 (C), from 2500 to 3200 cm−1 (D). TiO2; talc (as shoulders of TiO2 bands); () lactose; () sodium lauryl sulfate; () magnesium stearate; (T) tadalafil. …………… Instrumentation The HPLC system consisted of a 1100 series quaternary pump, degasser, automatic injector, thermostatted column compartment, and diode array detector (Agilent Technologies, Palo Alto, CA);Vortex TecnoKartell TK3; shaker BIOSAN Multi Bio RS-24, and innovative mixing cycle (VWR international, USA).The data were collected using the system software (Chemstation 1990- 2002, Agilent Technologies). Chromatographic Conditions The separation was achieved on an Agilent LiChrospher 100, C18 column, 5-μm particle size, 250 x 4 mm I.D., with a 2-μm precolumn filter.The mobile phase consisted of 65% water acidified with glacial acetic acid (0.1 mM, pH 2.5- 2.7) and 35% acetonitrile. The flow rate was 0.8 mL/min, and UV detection was performed at 280 nm. All analyses were made at room temperature. The injection volume was 25 μL, and a small volume of air was bubbled through each sample before injection. pg 171-175

Lydia Rabbaa

Saint Joseph University, Lebanon · faculty of pharmacy

…………………………………… Research In Pharmaceutical Biotechnology Vol. 2(1), pp. 001-006, February, 2010 Available online at http://www.academicjournals.org/RPB Validation and stability indicating RP-HPLC method for the determination of tadalafil API in pharmaceutical formulations B. Prasanna Reddy1*, K. Amarnadh Reddy2 and M. S. Reddy3 1Department of Quality control, Nosch Labs Pvt Ltd, Hyderabad-500072, A.P, India. 2 Department of AR and D, Aurigene Discovery Technologies Ltd, Bangalore, India. 3Department of Plant Pathology and Entomology, Auburn University, USA.

Battu.Prasanna Reddy Ph.D

The present study describes the development and subsequent of a stability indicating RP-HPLC method for the analysis of tadalafil. The samples separated on an Inertsil C18, (5 m , 150 mm x 4.6 mm i.d) by isocratic run using acetonitrile and phosphate buffer as mobile phase), with a flow rate of 0.8 ml/min, and the determination wavelength was 260 nm for analysis of tadalafil. The described method was linear within range of 70 – 130 μg/ml (r2 = 0.999). The precision, ruggedness and robustness values were also within the prescribed limits (< 1% for system precision and < 2% for other parameters). Tadalafil was exposed to acidic, basic, oxidative and thermal stress conditions and the stressed samples were analyzed by the proposed method. Chromatographic peak purity results indicated the absence of coeluting peaks with the main peak of tadalafil, which demonstrated the specificity of assay method for estimation of tadalafil in presence of degradation products. The proposed method can be used for routine analysis of tadalafil in quality control laboratories. Tadalafil hydro-2-methyl-6-[3,4-(methylenedioxy)phenyl]pyrazino-[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione (Figure1), is a phosphodiesterase type 5 inhibitor used in the management of erectile dysfunction. It is not officially included in any of the pharmacopoeias. It is listed in the Merck Index (Budavari et al., 2001) and Martindle and complete drug reference (Sean et al., 2002). There are several (Cheng et al., 2005) methods for determination of tadalafil such as HPLC-EIMS (Zhu et al., 2005) and capillary electrophoresis methods (Aboul-Enein, 2005) and by HPLC (Aboul, 1994). The present work was designed to develop a simple, precise and rapid analytical LC procedure, which would serve as stability indicating assay method for analysis of tadalafil active pharmaceutical ingredient. *Corresponding author. E-mail: drbpkreddy@gmail.com. Tel: +91-9848392677. Prasanna Reddy. Manager, Quality Control, Nosch Labs Pvt Ltd. Hyderabad, INDIA http://bloggerbattu.blogspot.in/ REFERENCES 1. Pomerol JM, Rabasseda X.Tadalafil, a furtherinnovation in the treatment of sexual dysfunction. Drugs Today (Barc). 2003;39:103-113. 2. Francis SH, Corbin JD. Molecular mechanismsand pharmacokinetics of phosphodiesterase-5 antagonists. Curr Urol Rep. 2003;4:457-465. 3. Seftel AD. Phosphodiesterase type 5 inhibitordifferentiation based on selectivity, pharmacokinetic,and efficacy profile. Clin Cardiol.2004;27(4 suppl 1):I14-I19. 4 Bella AJ, Brock GB.Tadalafil in the treatment of erectile dysfunction. Curr Urol Rep. 2003;4:472-478. 7A. Daugan, P. Grondin, C. Ruault, A.-C. Le Monnier de Gouville, H. Coste, J. Kirilovsky,F. Hyafil, R. Labaudinie

re, J. Med. Chem. 46 (2003) 4525.

[8] A. Daugan, P. Grondin, C. Ruault, A.-C. Le Monnier de Gouville, H. Coste, J.M. Linget,

J. Kirilovsky, F. Hyafil, R. Labaudinie`

re, J. Med. Chem. 46 (2003) 4533.

[9] M.W. Orme, J.C. Sawyer, L.M. Schultze, World Patent WO 02/036593 17 S. Xiao-Xin, L. Shi-Ling, X. Wei, X. Yu-Lan, Tetrahedron Asymmetr. 19 (2008) 435.

[18] Merck index 2006, 14th edition pages 1550–1551.

[19] N.M. Graham, M.N.A. Charlotte, G. Eugene, A.M. William, Bioorg. Med. Chem. Lett. 13

(2003) 1425.

[20] Y. Zhang, Q. He, H. Ding, X. Wu, Y. Xie, Org. Prep. Proced. Int. 37 (2005) 99.

Tadalafil
Systematic (IUPAC) name


(6R–trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1′, 2′:1,6] pyrido[3,4-b]indole-1,4-dione
Clinical data
Trade names	Cialis
AHFS/Drugs.com	monograph
MedlinePlus	a604008
Pregnancy category	B
Legal status	℞ Prescription only
Routes	Oral
Pharmacokinetic data
Bioavailability	varies
Protein binding	94%
Metabolism	CYP3A4 (liver)
Half-life	17.5 hours
Excretion	feces (> 60%), urine (> 30%)
Identifiers
CAS number	171596-29-5
ATC code	G04 BE08
PubChem	CID 110635
DrugBank	DB00820
ChemSpider	99301
UNII	742SXX0ICT
KEGG	D02008
ChEBI	CHEBI:71940
ChEMBL	CHEMBL779
PDB ligand ID	CIA (PDBe, RCSB PDB)
Chemical data
Formula	C₂₂H₁₉N₃O₄
Molecular mass	389.404 g/mol

Some new cancer drugs are available in other countries, but not in India. BY E. Kumar Sharma, Dec 22, 2013

cancer Comments Off

Mar 102015

E. Kumar Sharma Follow @EKumarSharma Edition:Dec 22, 2013

http://businesstoday.intoday.in/story/some-new-cancer-drugs-still-not-available-in-india/1/201095.html

Burixafor 布利沙福

phase 2 Comments Off

Mar 102015

Burixafor is a potent and selective chemokine CXCR4 antagonist developed by TaiGen Biotechnology (www.taigenbiotech.com.tw).

The SDF1/CXCR4 pathway plays key roles in homing and mobilization of hematopoietic stem cells and endothelial progenitor cells. In a mouse model, burixafor efficiently mobilizes stem cells (CD34+) and endothelial progenitor cells (CD133+) from bone marrow into peripheral circulation. It can be used in hematopoietic stem cell transplantation, chemotherapy sensitization and other ischemic diseases.

Because TaiGen has filed an IND (CXHL1200371) for burixafor as a chemotherapy sensitizer in October 2012, the new application (CXHL1400844) may supplement a new indication. Phase II clinical trials (NCT02104427) are currently underway in the US, with Phase IIa (NCT01018979, NCT01458288) already completed.

TaiGen plans to initiate clinical trials of burixafor as a chemotherapy sensitizer in China shortly. Burixafor’s annual sales are estimated at $1.1 billion by consultancy company JSB. This compound is protected by patent WO2009131598.

SEE……….http://newdrugapprovals.org/2014/06/09/scinopharm-to-provide-active-pharmaceutical-ingredient-%E8%8B%B1%E6%96%87%E5%90%8D%E7%A7%B0-burixafor-to-ftaigen-for-novel-stem-cell-drug/

英文名称Burixafor

TG-0054

(2-{4-[6-amino-2-({[(1r,4r)-4-({[3-(cyclohexylamino)propyl]amino}methyl)cyclohexyl]methyl}amino)pyrimidin-4-yl]piperazin-1-yl}ethyl)phosphonic acid

[2-[4-[6-Amino-2-[[[trans-4-[[[3-(cyclohexylamino)propyl]amino]methyl]cyclohexyl]methyl]amino]pyrimidin-4-yl]piperazin-1-yl]ethyl]phosphonic acid

1191448-17-5

C27H51N8O3P, 566.7194

chemokine CXCR 4 receptor antagonist;

Taigen Biotechnology Co., Ltd.

ScinoPharm to Provide Active Pharmaceutical Ingredient to F*TaiGen for Novel Stem Cell Drug
MarketWatch
The drug has received a Clinical Trial Application from China’s FDA for the initiation of … In addition, six products have entered Phase III clinical trials.

read at

http://www.marketwatch.com/story/scinopharm-to-provide-active-pharmaceutical-ingredient-to-ftaigen-for-novel-stem-cell-drug-2014-06-08

2D chemical structure of 1191448-17-5

TAINAN, June 8, 2014 — ScinoPharm Taiwan, Ltd. (twse:1789) specializing in the development and manufacture of active pharmaceutical ingredients, and TaiGen Biotechnology (4157.TW; F*TaiGen) jointly announced today the signing of a manufacturing contract for the clinical supply of the API of Burixafor, a new chemical entity discovered and developed by TaiGen. The API will be manufactured in ScinoPharm’s plant in Changshu, China. This cooperation not only demonstrates Taiwan’s international competitive strength in new drug development, but also sees the beginning of a domestic pharmaceutical specialization and cooperation mechanisms, thus establishing a groundbreaking milestone for Taiwan’s pharmaceutical industry.

Dr. Jo Shen, President and CEO of ScinoPharm said, “This cooperation with TaiGen is of representative significance in the domestic pharmaceutical companies’ upstream and downstream cooperation and self-development of new drugs, and indicates the Taiwanese pharmaceutical industry’s cumulative research and development momentum is paving the way forward.” Dr. Jo Shen emphasized, “ScinoPharm’s Changshu Plant provides high-quality API R&D and manufacturing services through its fast, flexible, reliable competitive advantages, effectively assisting clients of new drugs in gaining entry into China, Europe, the United States, and other international markets.”

ScinoPharm President, CEO and Co-Founder Dr. Jo Shen

According to Dr. Ming-Chu Hsu, Chairman and CEO of TaiGen, “R&D is the foundation of the pharmaceutical industry. Once a drug is successfully developed, players at all levels of the value chain could reap the benefit. Burixafor is a 100% in-house developed product that can be used in the treatment of various intractable diseases. The cooperation between TaiGen and ScinoPharm will not only be a win-win for both sides, but will also provide high-quality novel dug for patients from around the world.”

Burixafor is a novel stem cell mobilizer that can efficiently mobilize bone marrow stem cells and tissue precursor cells to the peripheral blood. It can be used in hematopoietic stem cell transplantation, chemotherapy sensitization and other ischemic diseases. The results of the ongoing Phase II clinical trial in the United States are very impressive. The drug has received a Clinical Trial Application from China’s FDA for the initiation of a Phase II clinical trial in chemotherapy sensitization under the 1.1 category. According to the pharmaceutical consultancy company JSB, with only stem cell transplant and chemotherapy sensitizer as the indicator, Burixafor’s annual sales are estimated at USD1.1 billion.

ScinoPharm currently has accepted over 80 new drug API process research and development plans, of which five new drugs have been launched in the market. In addition, six products have entered Phase III clinical trials. Through the Changshu Plant’s operation in line with the latest international cGMP plant equipment and quality management standards, the company provides customers with one stop shopping services in professional R&D, manufacturing, and outsourcing, thereby shortening the customer development cycle of customers’ products and accelerating the launch of new products to the market.

TaiGen’s focus is on the research and development of novel drugs. Besides Burixafor, the products also include anti-infective, Taigexyn®, and an anti-hepatitis C drug, TG-2349. Taigexyn® is the first in-house developed novel drug that received new drug application approval from Taiwan’s FDA. TG-2349 is intended for the 160 million global patients with hepatitis C with huge market potential. TaiGen hopes to file one IND with the US FDA every 3-4 years to expand TaiGen’s product line.

About ScinoPharm

ScinoPharm Taiwan, Ltd. is a leading process R&D and API manufacturing service provider to the global pharmaceutical industry. With research and manufacturing facilities in both Taiwan and China, ScinoPharm offers a wide portfolio of services ranging from custom synthesis for early phase pharmaceutical activities to contract services for brand companies as well as APIs for the generic industry. For more information, please visit the Company’s website at http://www.scinopharm.com

About TaiGen Biotechnology

TaiGen Biotechnology is a leading research-based and product-driven biotechnology company in Taiwan with a wholly-owned subsidiary in Beijing, China. The company’s first product, Taigexyn®, have already received NDA approval from Taiwan’s FDA. In addition to Taigexyn®, TaiGen has two other in-house discovered NCEs in clinical development under IND with US FDA: TG-0054, a chemokine receptor antagonist for stem cell transplantation and chemosensitization, in Phase 2 and TG-2349, a HCV protease inhibitor for treatment of chronic hepatitis infection, in Phase 2. Both TG-0054 and TG-2349 are currently in clinical trials in patients in the US.

SOURCE ScinoPharm Taiwan Ltd.

TG-0054 is a potent and selective chemokine CXCR4 (SDF-1) antagonist in phase II clinical studies at TaiGen Biotechnology for use in stem cell transplantation in cancer patients. Specifically, the compound is being developed for the treatment of stem cell transplantation in multiple myeloma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma and myocardial ischemia.

Preclinical studies had also been undertaken for the treatment of diabetic retinopathy, critical limb ischemia (CLI) and age-related macular degeneration. In a mouse model, TG-0054 efficiently mobilizes stem cells (CD34+) and endothelial progenitor cells (CD133+) from bone marrow into peripheral circulation.

BACKGROUND

Chemokines are a family of cytokines that regulate the adhesion and transendothelial migration of leukocytes during an immune or inflammatory reaction (Mackay C.R., Nat. Immunol, 2001, 2:95; Olson et al, Am. J. Physiol. Regul. Integr. Comp. Physiol, 2002, 283 :R7). Chemokines also regulate T cells and B cells trafficking and homing, and contribute to the development of lymphopoietic and hematopoietic systems (Ajuebor et al, Biochem. Pharmacol, 2002, 63:1191). Approximately 50 chemokines have been identified in humans. They can be classified into 4 subfamilies, i.e., CXC, CX3C, CC, and C chemokines, based on the positions of the conserved cysteine residues at the N-terminal (Onuffer et al, Trends Pharmacol ScI, 2002, 23:459). The biological functions of chemokines are mediated by their binding and activation of G protein-coupled receptors (GPCRs) on the cell surface.

Stromal-derived factor- 1 (SDF-I) is a member of CXC chemokines. It is originally cloned from bone marrow stromal cell lines and found to act as a growth factor for progenitor B cells (Nishikawa et al, Eur. J. Immunol, 1988, 18:1767). SDF-I plays key roles in homing and mobilization of hematopoietic stem cells and endothelial progenitor cells (Bleul et al, J. Exp. Med., 1996, 184:1101; and Gazzit et al, Stem Cells, 2004, 22:65-73). The physiological function of SDF-I is mediated by CXCR4 receptor. Mice lacking SDF-I or CXCR4 receptor show lethal abnormality in bone marrow myelopoiesis, B cell lymphopoiesis, and cerebellar development (Nagasawa et al, Nature, 1996, 382:635; Ma et al, Proc. Natl. Acad. ScI, 1998, 95:9448; Zou et al, Nature, 1998, 393:595; Lu et al, Proc. Natl. Acad. ScI, 2002, 99:7090). CXCR4 receptor is expressed broadly in a variety of tissues, particularly in immune and central nervous systems, and has been described as the major co-receptor for HIV- 1/2 on T lymphocytes. Although initial interest in CXCR4 antagonism focused on its potential application to AIDS treatment (Bleul et al, Nature, 1996, 382:829), it is now becoming clear that CXCR4 receptor and SDF-I are also involved in other pathological conditions such as rheumatoid arthritis, asthma, and tumor metastases (Buckley et al., J. Immunol., 2000, 165:3423). Recently, it has been reported that a CXCR4 antagonist and an anticancer drug act synergistically in inhibiting cancer such as acute promuelocutic leukemia (Liesveld et al., Leukemia

Research 2007, 31 : 1553). Further, the CXCR4/SDF-1 pathway has been shown to be critically involved in the regeneration of several tissue injury models. Specifically, it has been found that the SDF-I level is elevated at an injured site and CXCR4-positive cells actively participate in the tissue regenerating process.

………………………………………………………………………..

http://www.google.com/patents/WO2009131598A1?cl=en

Compound 52

Example 1 : Preparation of Compounds 1

1-1 1-Ii 1-m

^ ^–\\ Λ xCUNN H ‘ ‘22.. P rdu/’C^ ^. , Λ>\V>v

Et3N, TFAA , H_, r [ Y I RRaanneeyy–NNiicckkeell u H f [ Y | NH2

CH2CI2, -10 0C Boc^ ‘NNA/ 11,,44–ddιιooxxaannee B Boocer”1^”–^^ LiOH, H2O, 50 0C

1-IV 1-V

Water (10.0 L) and (BoC)2O (3.33 kgg, 15.3 mol) were added to a solution of trans-4-aminomethyl-cyclohexanecarboxylic acid (compound 1-1, 2.0 kg, 12.7 mol) and sodium bicarbonate (2.67 kg, 31.8 mol). The reaction mixture was stirred at ambient temperature for 18 hours. The aqueous layer was acidified with concentrated hydrochloric acid (2.95 L, pH = 2) and then filtered. The resultant solid was collected, washed three times with water (15 L), and dried in a hot box (60 0C) to give trα/?5-4-(tert-butoxycarbonylamino-methyl)-cyclo-hexanecarboxylic acid (Compound l-II, 3.17 kg, 97%) as a white solid. Rf = 0.58 (EtOAc). LC-MS m/e 280 (M+Na+). 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, IH), 2.98 (t, J= 6.3 Hz, 2H), 2.25 (td, J = 12, 3.3 Hz, IH), 2.04 (d, J= 11.1 Hz, 2H), 1.83 (d, J= 11.1 Hz, 2H), 1.44 (s, 9H), 1.35-1.50 (m, 3H), 0.89-1.03 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 181.31, 156.08, 79.12, 46.41, 42.99, 37.57, 29.47, 28.29, 27.96. M.p. 134.8-135.0 0C. A suspension of compound l-II (1.0 kg, 3.89 mol) in THF (5 L) was cooled at

-10 0C and triethyl amine (1.076 L, 7.78 mol) and ethyl chloroformate (0.441 L, 4.47 mol) were added below -10 0C. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was then cooled at -100C again and NH4OH (3.6 L, 23.34 mol) was added below -10 0C. The reaction mixture was stirred at ambient temperature for 18 hours and filtered. The solid was collected and washed three times with water (10 L) and dried in a hot box (6O0C) to give trans-4- (tert-butoxycarbonyl-amino-methyl)-cyclohexanecarboxylic acid amide (Compound l-III, 0.8 kg, 80%) as a white solid. Rf= 0.23 (EtOAc). LC-MS m/e 279, M+Na+. 1H NMR (300 MHz, CD3OD) δ 6.63 (brs, IH), 2.89 (t, J= 6.3 Hz, 2H), 2.16 (td, J = 12.2, 3.3 Hz, IH), 1.80-1.89 (m, 4H), 1.43 (s, 9H), 1.37-1.51 (m, 3H), 0.90-1.05 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 182.26, 158.85, 79.97, 47.65, 46.02, 39.28, 31.11, 30.41, 28.93. M.p. 221.6-222.0 0C.

A suspension of compound l-III (1.2 kg, 4.68 mol) in CH2Cl2 (8 L) was cooled at -1O0C and triethyl amine (1.3 L, 9.36 mol) and trifluoroacetic anhydride (0.717 L, 5.16 mol) were added below -10 0C. The reaction mixture was stirred for 3 hours. After water (2.0 L) was added, the organic layer was separated and washed with water (3.0 L) twice. The organic layer was then passed through silica gel and concentrated. The resultant oil was crystallized by methylene chloride. The crystals were washed with hexane to give £rαns-(4-cyano-cyclohexylmethyl)-carbamic acid tert-butyl ester (Compound 1-IV, 0.95 kg, 85%) as a white crystal. Rf = 0.78 (EtOAc). LC-MS m/e 261, M+Na+. 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, IH), 2.96 (t, J = 6.3 Hz, 2H), 2.36 (td, J= 12, 3.3 Hz, IH), 2.12 (dd, J= 13.3, 3.3 Hz, 2H), 1.83 (dd, J = 13.8, 2.7 Hz, 2H), 1.42 (s, 9H), 1.47-1.63 (m, 3H), 0.88-1.02 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 155.96, 122.41, 79.09, 45.89, 36.92, 29.06, 28.80, 28.25, 28.00. M.p. 100.4~100.6°C.

Compound 1-IV (1.0 kg, 4.196 mol) was dissolved in a mixture of 1 ,4-dioxane (8.0 L) and water (2.0 L). To the reaction mixture were added lithium hydroxide monohydrate (0.314 kg, 4.191), Raney-nickel (0.4 kg, 2.334 mol), and 10% palladium on carbon (0.46 kg, 0.216 mol) as a 50% suspension in water. The reaction mixture was stirred under hydrogen atmosphere at 5O0C for 20 hours. After the catalysts were removed by filtration and the solvents were removed in vacuum, a mixture of water (1.0 L) and CH2Cl2 (0.3 L) was added. After phase separation, the organic phase was washed with water (1.0 L) and concentrated to give £rα/?s-(4-aminomethyl- cyclohexylmethyl)-carbamic acid tert- butyl ester (compound 1-V, 0.97 kg, 95%) as pale yellow thick oil. Rf = 0.20 (MeOH/EtOAc = 9/1). LC-MS m/e 243, M+H+. 1H NMR (300 MHz, CDCl3) δ 4.67 (brs, IH), 2.93 (t, J= 6.3 Hz, 2H), 2.48 (d, J= 6.3 Hz, 2H), 1.73-1.78 (m, 4H), 1.40 (s, 9H), 1.35 (brs, 3H), 1.19-1.21 (m, IH), 0.77-0.97 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 155.85, 78.33, 48.27, 46.38, 40.80, 38.19, 29.87, 29.76, 28.07. A solution of compound 1-V (806 g) and Et3N (1010 g, 3 eq) in 1-pentanol

(2.7 L) was treated with compound 1-VI, 540 g, 1 eq) at 900C for 15 hours. TLC showed that the reaction was completed. Ethyl acetate (1.5 L) was added to the reaction mixture at 25°C. The solution was stirred for 1 hour. The Et3NHCl salt was filtered. The filtrate was then concentrated to 1.5 L (1/6 of original volume) by vacuum at 500C. Then, diethyl ether (2.5 L) was added to the concentrated solution to afford the desired product 1-VII (841 g, 68% yield) after filtration at 250C .

A solution of intermediate 1-VII (841 g) was treated with 4 N HCl/dioxane (2.7 L) in MeOH (8.1 L) and stirred at 25°C for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated to 1.5 L (1/7 of original volume) by vacuum at 500C. Then, diethyl ether (5 L) was added to the solution slowly, and HCl salt of 1-VIII (774 g) was formed, filtered, and dried under vacuum (<10 torr). For neutralization, K2CO3 (2.5 kg, 8 eq) was added to the solution of HCl salt of 1-VIII in MeOH (17 L) at 25°C. The mixture was stirred at the same temperature for 3 hours (pH > 12) and filtered (estimated amount of 1-VIII in the filtrate is 504 g). Aldehyde 1-IX (581 g, 1.0 eq based on mole of 1-VII) was added to the filtrate of 1-VIII at 0-100C. The reaction was stirred at 0-100C for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (81 g, 1.0 eq based on mole of 1-VII) was added at less than 100C and the solution was stirred at 10-150C for Ih. The solution was concentrated to get a residue, which then treated with CH2Cl2 (15 L). The mixture was washed with saturated aq. NH4Cl solution (300 mL) diluted with H2O (1.2 L). The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (short column, EtOAc as mobile phase for removing other components; MeOH/28% NH4OH = 97/3 as mobile phase for collecting 1-X) afforded crude 1-X (841 g). Then Et3N (167 g, leq) and BoC2O (360 g, leq) were added to the solution of

1-X (841 g) in CH2Cl2 (8.4 L) at 25°C. The mixture was stirred at 25°C for 15 hours. After the reaction was completed as evidenced by TLC, the solution was concentrated and EtOAc (5 L) was added to the resultant residue. The solution was concentrated to 3L (1/2 of the original volume) under low pressure at 500C. Then, n-hexane (3 L) was added to the concentrated solution. The solid product formed at 500C by seeding to afford the desired crude product 1-XI (600 g, 60% yield) after filtration and evaporation. To compound 1-XI (120.0 g) and piperazine (1-XII, 50.0 g, 3 eq) in 1- pentanol (360 niL) was added Et3N (60.0 g, 3.0 eq) at 25°C. The mixture was stirred at 1200C for 8 hours. Ethyl acetate (480 mL) was added to the reaction mixture at 25°C. The solution was stirred for Ih. The Et3NHCl salt was filtered and the solution was concentrated and purified by silica gel (EtOAc/MeOH = 2:8) to afforded 1-XIII (96 g) in a 74% yield.

A solution of intermediate 1-XIII (100 mg) was treated with 4 N HCl/dioxane (2 mL) in CH2Cl2 (1 mL) and stirred at 25°C for 15 hours. The mixture was concentrated to give hydrochloride salt of compound 1 (51 mg). CI-MS (M+ + 1): 459.4

Example 2: Preparation of Compound 2

Compound 2 Intermediate 1-XIII was prepared as described in Example 1.

To a solution of 1-XIII (120 g) in MeOH (2.4 L) were added diethyl vinyl phosphonate (2-1, 45 g, 1.5 eq) at 25°C. The mixture was stirred under 65°C for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2 = 8/92) to get 87 g of 2-11 (53% yield, purity > 98%, each single impurity <1%) after analyzing the purity of the product by HPLC.

A solution of 20% TFA/CH2C12 (36 mL) was added to a solution of intermediate 2-11 (1.8 g) in CH2Cl2 (5 mL). The reaction mixture was stirred for 15 hours at room temperature and concentrated by removing the solvent to afford trifluoracetic acid salt of compound 2 (1.3 g). CI-MS (M+ + 1): 623.1

Example 3 : Preparation of Compound 3

TMSBr H H

s U

Intermediate 2-11 was prepared as described in Example 2. To a solution of 2-11 (300 g) in CH2Cl2 (1800 mL) was added TMSBr (450 g, 8 eq) at 10-150C for 1 hour. The mixture was stirred at 25°C for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 400C.

CH2Cl2 was added to the mixture to dissolve the residue. TMSBr and solvent were removed under vacuum again to obtain 36O g crude solid after drying under vacuum (<1 torr) for 3 hours. Then, the crude solid was washed with 7.5 L IPA/MeOH (9/1) to afford compound 3 (280 g) after filtration and drying at 25°C under vacuum (<1 torr) for 3 hours. Crystallization by EtOH gave hydrobromide salt of compound 3 (19Og). CI-MS (M+ + 1): 567.0.

The hydrobromide salt of compound 3 (5.27 g) was dissolved in 20 mL water and treated with concentrated aqueous ammonia (pH=9-10), and the mixture was evaporated in vacuo. The residue in water (30 mL) was applied onto a column (100 mL, 4.5×8 cm) of Dowex 50WX8 (H+ form, 100-200 mesh) and eluted (elution rate, 6 mL/min). Elution was performed with water (2000 mL) and then with 0.2 M aqueous ammonia. The UV-absorbing ammonia eluate was evaporated to dryness to afford ammonia salt of compound 3 (2.41 g). CI-MS (M+ + 1): 567.3.

The ammonia salt of compound 3 (1.5 g) was dissolved in water (8 mL) and alkalified with concentrated aqueous ammonia (pH=l 1), and the mixture solution was applied onto a column (75 mL, 3×14 cm) of Dowex 1X2 (acetate form, 100-200 mesh) and eluted (elution rate, 3 mL/min). Elution was performed with water (900 mL) and then with 0.1 M acetic acid. The UV-absorbing acetic acid eluate was evaporated, and the residue was codistilled with water (5×50 mL) to afford compound 3 (1.44 g). CI-MS (M+ + 1): 567.4. Example 4: Preparation of Compound 4

Compound 4

Intermediate 1-XIII was obtained during the preparation of compound 1. To a solution of diethyl vinyl phosphonate (4-1, 4 g) in CH2Cl2 (120 mL) was added oxalyl chloride (15.5 g, 5 eq) and the mixture was stirred at 300C for 36 hours. The mixture were concentrated under vacuum on a rotatory evaporated to give quantitatively the corresponding phosphochloridate, which was added to a mixture of cyclohexyl amine (4-II, 5.3 g, 2.2 eq), CH2Cl2 (40 mL), and Et3N (6.2 g, 2.5 eq). The mixture was stirred at 35°C for 36 hours, and then was washed with water. The organic layer was dried (MgSO4), filtered, and evaporated to afford 4-III (4.7 g, 85% yield) as brown oil.

Compound 4-III (505 mg) was added to a solution of intermediate 1-XIII (500 mg) in MeOH (4 mL). The solution was stirred at 45°C for 24 hours. The solution was concentrated and the residue was purified by column chromatography on silica gel (EtOAc/ MeOH = 4: 1) to afford intermediate 4-IV (420 mg) in a 63% yield.

A solution of HCl in ether (5 mL) was added to a solution of intermediate 4- IV (420 mg) in CH2Cl2 (1.0 mL). The reaction mixture was stirred for 12 hours at room temperature and concentrated by removing the solvent. The resultant residue was washed with ether to afford hydrochloride salt of compound 4 (214 mg). CI-MS (M+ + 1): 595.1

Preparation of compound 51

TMSBr

Intermediate l-II was prepared as described in Example 1. To a suspension of the intermediate l-II (31.9 g) in toluene (150 mL) were added phosphorazidic acid diphenyl ester (51-1, 32.4 g) and Et3N (11.9 g) at 25°C for 1 hour. The reaction mixture was stirred at 800C for 3 hours and then cooled to 25°C. After benzyl alcohol (51-11, 20 g) was added, the reaction mixture was stirred at 800C for additional 3 hours and then warmed to 1200C overnight. It was then concentrated and dissolved again in EtOAc and H2O. The organic layer was collected. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with 2.5 N HCl, saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (EtOAc/Hexane = 1 :2) to give Intermediate 51-111 (35 g) in a 79% yield. A solution of intermediate 51-111 (35 g) treated with 4 N HCl/dioxane (210 rnL) in MeOH (350 mL) was stirred at room temperature overnight. After ether (700 mL) was added, the solution was filtered. The solid was dried under vacuum. K2CO3 was added to a suspension of this solid in CH3CN and ώo-propanol at room temperature for 10 minutes. After water was added, the reaction mixture was stirred at room temperature for 2 hours, filtered, dried over anhydrous MgSO4, and concentrated. The resultant residue was purified by column chromatography on silica gel (using CH2Cl2 and MeOH as an eluant) to give intermediate 51-IV (19 g) in a 76% yield. Intermediate 1-IX (21 g) was added to a solution of intermediate 51-IV (19 g) in CH2Cl2 (570 mL). The mixture was stirred at 25°C for 2 hours. NaBH(OAc)3 (23 g) was then added at 25°C overnight. After the solution was concentrated, a saturated aqueous NaHCO3solution was added to the resultant residue. The mixture was then extracted with CH2Cl2. The solution was concentrated and the residue was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 51-V (23.9 g) in a 66% yield.

A solution of intermediate 51-V (23.9 g) and BoC2O (11.4 g) in CH2Cl2 (200 mL) was added to Et3N (5.8 mL) at 25°C for overnight. The solution was then concentrated and the resultant residue was purified by column chromatography on silica gel (using EtOAc and Hexane as an eluant) to give intermediate 51-VI (22 g) in a 77% yield.

10% Pd/C (2.2 g) was added to a suspension of intermediate 51-VI (22 g) in MeOH (44 mL). The mixture was stirred at ambient temperature under hydrogen atmosphere overnight, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 51-VII (16.5 g) in a 97% yield.

Intermediate 51-VII (16.5 g) and Et3N (4.4 mL) in 1-pentanol (75 mL) was allowed to react with 2,4-dichloro-6-aminopyrimidine (1-VI, 21 g) at 1200C overnight. The solvent was then removed and the residue was purified by column chromatography on silica gel (using EtOAc and hexane as an eluant) to afford intermediate 51-VIII (16.2 g) in a 77% yield.

A solution of intermediate 51-VIII (16.2 g) and piperazine (1-XII, 11.7 g) in 1-pentanol (32 mL) was added to Et3N (3.3 mL) at 1200C overnight. After the solution was concentrated, the residue was treated with water and extracted with CH2Cl2. The organic layer was collected and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc/ MeOH to 28% NH40H/Me0H as an eluant) to afford Intermediate 51-IX (13.2 g) in a 75% yield. Diethyl vinyl phosphonate (2-1) was treated with 51-IX as described in

Example 3 to afford hydrobromide salt of compound 51. CI-MS (M+ + 1): 553.3

………………………………….

Preparation of Compound 1

Water (10.0 L) and (Boc)2O (3.33 kgg, 15.3 mol) were added to a solution of trans-4-aminomethyl-cyclohexanecarboxylic acid (compound 1-I, 2.0 kg, 12.7 mol) and sodium bicarbonate (2.67 kg, 31.8 mol). The reaction mixture was stirred at ambient temperature for 18 hours. The aqueous layer was acidified with concentrated hydrochloric acid (2.95 L, pH=2) and then filtered. The resultant solid was collected, washed three times with water (15 L), and dried in a hot box (60° C.) to give trans-4-(tert-butoxycarbonylamino-methyl)-cyclo-hexanecarboxylic acid (Compound 1-II, 3.17 kg, 97%) as a white solid. Rf=0.58 (EtOAc). LC-MS m/e 280 (M+Na+). 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, 1H), 2.98 (t, J=6.3 Hz, 2H), 2.25 (td, J=12, 3.3 Hz, 1H), 2.04 (d, J=11.1 Hz, 2H), 1.83 (d, J=11.1 Hz, 2H), 1.44 (s, 9H), 1.35˜1.50 (m, 3H), 0.89˜1.03 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 181.31, 156.08, 79.12, 46.41, 42.99, 37.57, 29.47, 28.29, 27.96. M.p. 134.8˜135.0° C.

A suspension of compound 1-II (1.0 kg, 3.89 mol) in THF (5 L) was cooled at 10° C. and triethyl amine (1.076 L, 7.78 mol) and ethyl chloroformate (0.441 L, 4.47 mol) were added below 10° C. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was then cooled at 10° C. again and NH4OH (3.6 L, 23.34 mol) was added below 10° C. The reaction mixture was stirred at ambient temperature for 18 hours and filtered. The solid was collected and washed three times with water (10 L) and dried in a hot box (60° C.) to give trans-4-(tert-butoxycarbonyl-amino-methyl)-cyclohexanecarboxylic acid amide (Compound 1-III, 0.8 kg, 80%) as a white solid. Rf=0.23 (EtOAc). LC-MS m/e 279, M+Na+. 1H NMR (300 MHz, CD3OD) δ 6.63 (brs, 1H), 2.89 (t, J=6.3 Hz, 2H), 2.16 (td, J=12.2, 3.3 Hz, 1H), 1.80˜1.89 (m, 4H), 1.43 (s, 9H), 1.37˜1.51 (m, 3H), 0.90˜1.05 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 182.26, 158.85, 79.97, 47.65, 46.02, 39.28, 31.11, 30.41, 28.93. M.p. 221.6˜222.0° C.

A suspension of compound 1-III (1.2 kg, 4.68 mol) in CH2Cl2 (8 L) was cooled at 10° C. and triethyl amine (1.3 L, 9.36 mol) and trifluoroacetic anhydride (0.717 L, 5.16 mol) were added below 10° C. The reaction mixture was stirred for 3 hours. After water (2.0 L) was added, the organic layer was separated and washed with water (3.0 L) twice. The organic layer was then passed through silica gel and concentrated. The resultant oil was crystallized by methylene chloride. The crystals were washed with hexane to give trans-(4-cyano-cyclohexylmethyl)-carbamic acid tent-butyl ester (Compound 1-IV, 0.95 kg, 85%) as a white crystal. Rf=0.78 (EtOAc). LC-MS m/e 261, M+Na+. 1H NMR (300 MHz, CDCl3) δ 4.58 (brs, 1H), 2.96 (t, J=6.3 Hz, 2H), 2.36 (td, J=12, 3.3 Hz, 1H), 2.12 (dd, J=13.3, 3.3 Hz, 2H), 1.83 (dd, J=13.8, 2.7 Hz, 2H), 1.42 (s, 9H), 1.47˜1.63 (m, 3H), 0.88˜1.02 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 155.96, 122.41, 79.09, 45.89, 36.92, 29.06, 28.80, 28.25, 28.00. M.p. 100.4˜100.6° C.

Compound 1-IV (1.0 kg, 4.196 mol) was dissolved in a mixture of 1,4-dioxane (8.0 L) and water (2.0 L). To the reaction mixture were added lithium hydroxide monohydrate (0.314 kg, 4.191), Raney-nickel (0.4 kg, 2.334 mol), and 10% palladium on carbon (0.46 kg, 0.216 mol) as a 50% suspension in water. The reaction mixture was stirred under hydrogen atmosphere at 50° C. for 20 hours. After the catalysts were removed by filtration and the solvents were removed in vacuum, a mixture of water (1.0 L) and CH2Cl2 (0.3 L) was added. After phase separation, the organic phase was washed with water (1.0 L) and concentrated to give trans-(4-aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester (compound 1-V, 0.97 kg, 95%) as pale yellow thick oil. Rf=0.20 (MeOH/EtOAc=9/1). LC-MS m/e 243, M+H+. 1H NMR (300 MHz, CDCl3) δ 4.67 (brs, 1H), 2.93 (t, J=6.3 Hz, 2H), 2.48 (d, J=6.3 Hz, 2H), 1.73˜1.78 (m, 4H), 1.40 (s, 9H), 1.35 (brs, 3H), 1.19˜1.21 (m, 1H), 0.77˜0.97 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 155.85, 78.33, 48.27, 46.38, 40.80, 38.19, 29.87, 29.76, 28.07.

A solution of compound 1-V (806 g) and Et3N (1010 g, 3 eq) in 1-pentanol (2.7 L) was treated with compound 1-VI, 540 g, 1 eq) at 90° C. for 15 hours. TLC showed that the reaction was completed.

Ethyl acetate (1.5 L) was added to the reaction mixture at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was filtered. The filtrate was then concentrated to 1.5 L (1/6 of original volume) by vacuum at 50° C. Then, diethyl ether (2.5 L) was added to the concentrated solution to afford the desired product 1-VII (841 g, 68% yield) after filtration at 25° C.

A solution of intermediate 1-VII (841 g) was treated with 4 N HCl/dioxane (2.7 L) in MeOH (8.1 L) and stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated to 1.5 L (1/7 of original volume) by vacuum at 50° C. Then, diethyl ether (5 L) was added to the solution slowly, and HCl salt of 1-VIII (774 g) was formed, filtered, and dried under vacuum (<10 ton). For neutralization, K2CO3 (2.5 kg, 8 eq) was added to the solution of HCl salt of 1-VIII in MeOH (17 L) at 25° C. The mixture was stirred at the same temperature for 3 hours (pH>12) and filtered (estimated amount of 1-VIII in the filtrate is 504 g).

Aldehyde 1-IX (581 g, 1.0 eq based on mole of 1-VII) was added to the filtrate of 1-VIII at 0-10° C. The reaction was stirred at 0-10° C. for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (81 g, 1.0 eq based on mole of 1-VII) was added at less than 10° C. and the solution was stirred at 10-15° C. for 1 h. The solution was concentrated to get a residue, which then treated with CH2Cl2 (15 L). The mixture was washed with saturated aq. NH4Cl solution (300 mL) diluted with H2O (1.2 L). The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (short column, EtOAc as mobile phase for removing other components; MeOH/28% NH4OH=97/3 as mobile phase for collecting 1-X) afforded crude 1-X (841 g).

Then Et3N (167 g, 1 eq) and Boc2O (360 g, 1 eq) were added to the solution of 1-X (841 g) in CH2Cl2 (8.4 L) at 25° C. The mixture was stirred at 25° C. for 15 hours. After the reaction was completed as evidenced by TLC, the solution was concentrated and EtOAc (5 L) was added to the resultant residue. The solution was concentrated to 3 L (1/2 of the original volume) under low pressure at 50° C. Then, n-hexane (3 L) was added to the concentrated solution. The solid product formed at 50° C. by seeding to afford the desired crude product 1-XI (600 g, 60% yield) after filtration and evaporation.

To compound 1-XI (120.0 g) and piperazine (1-XII, 50.0 g, 3 eq) in 1-pentanol (360 mL) was added Et3N (60.0 g, 3.0 eq) at 25° C. The mixture was stirred at 120° C. for 8 hours. Ethyl acetate (480 mL) was added to the reaction mixture at 25° C. The solution was stirred for 1 h. The Et3NHCl salt was filtered and the solution was concentrated and purified by silica gel (EtOAc/MeOH=2:8) to afforded 1-XIII (96 g) in a 74% yield.

To a solution of 1-XIII (120 g) in MeOH (2.4 L) were added diethyl vinyl phosphonate (1-XIV, 45 g, 1.5 eq) at 25° C. The mixture was stirred under 65° C. for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2=8/92) to get 87 g of 1-XV (53% yield, purity>98%, each single impurity<1%) after analyzing the purity of the product by HPLC.

A solution of 20% TFA/CH2Cl2 (36 mL) was added to a solution of intermediate 1-XV (1.8 g) in CH2Cl2 (5 mL). The reaction mixture was stirred for 15 hours at room temperature and concentrated by removing the solvent to afford trifluoracetic acid salt of compound 1 (1.3 g).

CI-MS (M++1): 623.1.

(2) Preparation of Compound 2

Intermediate 1-XV was prepared as described in Example 1.

To a solution of 1-XV (300 g) in CH2Cl2 (1800 mL) was added TMSBr (450 g, 8 eq) at 10-15° C. for 1 hour. The mixture was stirred at 25° C. for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 40° C. CH2Cl2 was added to the mixture to dissolve the residue. TMSBr and solvent were removed under vacuum again to obtain 360 g crude solid after drying under vacuum (<1 torr) for 3 hours. Then, the crude solid was washed with 7.5 L IPA/MeOH (9/1) to afford compound 2 (280 g) after filtration and drying at 25° C. under vacuum (<1 ton) for 3 hours. Crystallization by EtOH gave hydrobromide salt of compound 2 (190 g). CI-MS (M++1): 567.0.

The hydrobromide salt of compound 2 (5.27 g) was dissolved in 20 mL water and treated with concentrated aqueous ammonia (pH=9-10), and the mixture was evaporated in vacuo. The residue in water (30 mL) was applied onto a column (100 mL, 4.5×8 cm) of Dowex 50WX8 (H+ form, 100-200 mesh) and eluted (elution rate, 6 mL/min). Elution was performed with water (2000 mL) and then with 0.2 M aqueous ammonia. The UV-absorbing ammonia eluate was evaporated to dryness to afford ammonia salt of compound 2 (2.41 g). CI-MS (M++1): 567.3.

The ammonia salt of compound 2 (1.5 g) was dissolved in water (8 mL) and alkalified with concentrated aqueous ammonia (pH=11), and the mixture solution was applied onto a column (75 mL, 3×14 cm) of Dowex 1×2 (acetate form, 100-200 mesh) and eluted (elution rate, 3 mL/min). Elution was performed with water (900 mL) and then with 0.1 M acetic acid. The UV-absorbing acetic acid eluate was evaporated, and the residue was codistilled with water (5×50 mL) to afford compound 2 (1.44 g). CI-MS (M++1): 567.4.

(3) Preparation of Compound 3

Intermediate 1-XIII was obtained during the preparation of compound 1.

To a solution of diethyl vinyl phosphonate (3-I, 4 g) in CH2Cl2 (120 mL) was added oxalyl chloride (15.5 g, 5 eq) and the mixture was stirred at 30° C. for 36 hours. The mixture were concentrated under vacuum on a rotatory evaporated to give quantitatively the corresponding phosphochloridate, which was added to a mixture of cyclohexyl amine (3-II, 5.3 g, 2.2 eq), CH2Cl2 (40 mL), and Et3N (6.2 g, 2.5 eq). The mixture was stirred at 35° C. for 36 hours, and then was washed with water. The organic layer was dried (MgSO4), filtered, and evaporated to afford 3-III (4.7 g, 85% yield) as brown oil.

Compound 3-III (505 mg) was added to a solution of intermediate 1-XIII (500 mg) in MeOH (4 mL). The solution was stirred at 45° C. for 24 hours. The solution was concentrated and the residue was purified by column chromatography on silica gel (EtOAc/MeOH=4:1) to afford intermediate 3-IV (420 mg) in a 63% yield.

A solution of HCl in ether (5 mL) was added to a solution of intermediate 3-IV (420 mg) in CH2Cl2 (1.0 mL). The reaction mixture was stirred for 12 hours at room temperature and concentrated by removing the solvent. The resultant residue was washed with ether to afford hydrochloride salt of compound 3 (214 mg).

CI-MS (M++1): 595.1.

(4) Preparation of Compound 4

Compound 4 was prepared in the same manner as that described in Example 2 except that sodium 2-bromoethanesulfonate in the presence of Et3N in DMF at 45° C. was used instead of diethyl vinyl phosphonate. Deportations of amino-protecting group by hydrochloride to afford hydrochloride salt of compound 4.

CI-MS (M++1): 567.3

(5) Preparation of Compound 5

Compound 5 was prepared in the same manner as that described in Example 2 except that diethyl-1-bromopropylphosphonate in the presence of K2CO3 in CH3CN was used instead of diethyl vinyl phosphonate.

CI-MS (M++1): 581.4

(6) Preparation of Compound 6

Compound 6 was prepared in the same manner as that described in Example 5 except that 1,4-diaza-spiro[5.5]undecane dihydrochloride was used instead of piperazine.

CI-MS (M++1): 649.5

(7) Preparation of Compound 7

Intermediate 1-II was prepared as described in Example 1.

To a suspension of the intermediate 1-II (31.9 g) in toluene (150 mL) were added phosphorazidic acid diphenyl ester (7-I, 32.4 g) and Et3N (11.9 g) at 25° C. for 1 hour. The reaction mixture was stirred at 80° C. for 3 hours and then cooled to 25° C. After benzyl alcohol (7-II, 20 g) was added, the reaction mixture was stirred at 80° C. for additional 3 hours and then warmed to 120° C. overnight. It was then concentrated and dissolved again in EtOAc and H2O. The organic layer was collected. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with 2.5 N HCl, saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (EtOAc/Hexane=1:2) to give Intermediate 7-III (35 g) in a 79% yield.

A solution of intermediate 7-III (35 g) treated with 4 N HCl/dioxane (210 mL) in MeOH (350 mL) was stirred at room temperature overnight. After ether (700 mL) was added, the solution was filtered. The solid was dried under vacuum. K2CO3 was added to a suspension of this solid in CH3CN and iso-propanol at room temperature for 10 minutes. After water was added, the reaction mixture was stirred at room temperature for 2 hours, filtered, dried over anhydrous MgSO4, and concentrated. The resultant residue was purified by column chromatography on silica gel (using CH2Cl2 and MeOH as an eluant) to give intermediate 7-IV (19 g) in a 76% yield.

Intermediate 1-IX (21 g) was added to a solution of intermediate 7-IV (19 g) in CH2Cl2 (570 mL). The mixture was stirred at 25° C. for 2 hours. NaBH(OAc)3(23 g) was then added at 25° C. overnight. After the solution was concentrated, a saturated aqueous NaHCO3 solution was added to the resultant residue. The mixture was then extracted with CH2Cl2. The solution was concentrated and the residue was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 7-V (23.9 g) in a 66% yield.

A solution of intermediate 7-V (23.9 g) and Boc2O (11.4 g) in CH2Cl2 (200 mL) was added to Et3N (5.8 mL) at 25° C. for overnight. The solution was then concentrated and the resultant residue was purified by column chromatography on silica gel (using EtOAc and Hexane as an eluant) to give intermediate 7-VI (22 g) in a 77% yield. 10% Pd/C (2.2 g) was added to a suspension of intermediate 7-VI (22 g) in MeOH (44 mL). The mixture was stirred at ambient temperature under hydrogen atmosphere overnight, filtered, and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc and MeOH as an eluant) to afford intermediate 7-VII (16.5 g) in a 97% yield.

Intermediate 7-VII (16.5 g) and Et3N (4.4 mL) in 1-pentanol (75 mL) was allowed to react with 2,4-dichloro-6-aminopyrimidine (1-VI, 21 g) at 120° C. overnight. The solvent was then removed and the residue was purified by column chromatography on silica gel (using EtOAc and hexane as an eluant) to afford intermediate 7-VIII (16.2 g) in a 77% yield.

A solution of intermediate 7-VIII (16.2 g) and piperazine (1-XII, 11.7 g) in 1-pentanol (32 mL) was added to Et3N (3.3 mL) at 120° C. overnight. After the solution was concentrated, the residue was treated with water and extracted with CH2Cl2. The organic layer was collected and concentrated. The residue thus obtained was purified by column chromatography on silica gel (using EtOAc/MeOH to 28% NH4OH/MeOH as an eluant) to afford Intermediate 7-IX (13.2 g) in a 75% yield.

Diethyl vinyl phosphonate (2-I) was treated with 7-IX as described in Example 3 to afford hydrobromide salt of compound 7.

CI-MS (M++1): 553.3

(8) Preparation of Compound 8

Cis-1,4-cyclohexanedicarboxylic acid (8-I, 10 g) in THF (100 ml) was added oxalyl chloride (8-II, 15.5 g) at 0° C. and then DMF (few drops). The mixture was stirred at room temperature for 15 hours. The solution was concentrated and the residue was dissolved in THF (100 ml). The mixture solution was added to ammonium hydroxide (80 ml) and stirred for 1 hour. The solution was concentrated and filtration to afford crude product 8-III (7.7 g).

Compound 8-III (7.7 g) in THF (200 ml) was slowly added to LiAlH4 (8.6 g) in THF (200 ml) solution at 0° C. The mixture solution was stirred at 65° C. for 15 hours. NaSO4.10H2O was added at room temperature and stirred for 1 hours. The resultant mixture was filtered to get filtrate and concentrated. The residue was dissolved in CH2Cl2 (100 ml). Et3N (27 g) and (Boc)2O (10 g) were added at room temperature. The solution was stirred for 15 h, and then concentrated to get resultant residue. Ether was added to the resultant residue. Filtration and drying under vacuum afforded solid crude product 8-IV (8.8 g).

A solution of compound 8-IV (1.1 g) and Et3N (1.7 g) in 1-pentanol (10 ml) was reacted with 2,4-dichloro-6-aminopyrimidine (1-VI, 910 mg) at 90° C. for 15 hours. TLC showed that the reaction was completed. Ethyl acetate (10 mL) was added to the reaction mixture at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was removed. The filtrate was concentrated and purified by silica gel (EtOAc/Hex=1:2) to afford the desired product 8-V (1.1 g, 65% yield).

A solution of intermediate 8-V (1.1 g) was treated with 4 N HCl/dioxane (10 ml) in MeOH (10 ml) and stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The mixture was concentrated, filtered, and dried under vacuum (<10 ton). For neutralization, K2CO3 (3.2 g) was added to the solution of HCl salt in MeOH (20 ml) at 25° C. The mixture was stirred at the same temperature for 3 hours (pH>12) and filtered. Aldehyde 1-IX (759 mg) was added to the filtrate at 0-10° C. The reaction was stirred at 0-10° C. for 3 hours. TLC showed that the reaction was completed. Then, NaBH4 (112 mg) was added at less than 10° C. and the solution was stirred at 10-15° C. for 1 hour. The solution was concentrated to get a residue, which was then treated with CH2Cl2 (10 mL). The mixture was washed with saturated NH4Cl (aq) solution. The CH2Cl2 layer was concentrated and the residue was purified by chromatography on silica gel (MeOH/28% NH4OH=97/3) to afford intermediate 8-VI (1.0 g, 66% yield).

Et3N (600 mg) and Boc2O (428 mg) were added to the solution of 8-VI (1.0 g) in CH2Cl2 (10 ml) at 25° C. The mixture was stirred at 25° C. for 15 hours. TLC showed that the reaction was completed. The solution was concentrated and purified by chromatography on silica gel (EtOAc/Hex=1:1) to afford intermediate 8-VII (720 mg, 60% yield).

To a solution compound 8-VII (720 mg) and piperazine (1-XII, 1.22 g) in 1-pentanol (10 mL) was added Et3N (1.43 g) at 25° C. The mixture was stirred at 120° C. for 24 hours. TLC showed that the reaction was completed. Ethyl acetate (20 mL) was added at 25° C. The solution was stirred for 1 hour. The Et3NHCl salt was removed and the solution was concentrated and purified by silica gel (EtOAc/MeOH=2:8) to afford 8-VIII (537 mg) in 69% yield.

To a solution of 8-VIII (537 mg) in MeOH (11 ml) was added diethyl vinyl phosphonate (2-I, 201 mg) at 25° C. The mixture was stirred under 65° C. for 24 hours. TLC and HPLC showed that the reaction was completed. The solution was concentrated and purified by silica gel (MeOH/CH2Cl2=1:9) to get 8-IX (380 mg) in a 57% yield.

To a solution of 8-IX (210 mg) in CH2Cl2 (5 ml) was added TMSBr (312 mg) at 10-15° C. for 1 hour. The mixture was stirred at 25° C. for 15 hours. The solution was concentrated to remove TMSBr and solvent under vacuum at 40° C., then, CH2Cl2 was added to dissolve the residue. Then TMSBr and solvent were further removed under vacuum and CH2Cl2 was added for four times repeatedly. The solution was concentrated to get hydrobromide salt of compound 8 (190 mg).

CI-MS (M++1): 566.9

To do a job well is one thing, but to consistently deliver a product that is nearly flawless is quite a different challenge. For its new molecule burixafor, the Taiwanese drug discovery firm TaiGen Biotechnology instructed its contract manufacturing partners to achieve 99.8% purity in the production of the active pharmaceutical ingredient (API).

Discovered in TaiGen’s labs in 2006, burixafor is in Phase II clinical trials in both the U.S. and China for use in stem cell transplants and cancer chemotherapy. Avecia, a unit of Japan’s Nitto Denko, manufactures the drug substance in the U.S., where burixafor was tested for the first time on human patients. When TaiGen later initiated clinical trials in China, it chose the Taiwanese firm ScinoPharm to produce the drug at its plant in Changshu, near Shanghai. Under Chinese law, only drugs made domestically can be tested in China.

NITTO DENKO Avecia Inc.

It is rare for a drug discovery firm to select two companies to scale up the production of a new molecule. TaiGen went one step further by paying both contract manufacturers to reach an extremely high level of purity.

“We are trying to avoid any unwanted side effects during the trials,” says C. Richard King, TaiGen’s senior vice president of research. Drug regulators in the U.S. and China “need very tight specifications these days for new drugs,” he adds.

TaiGen registered burixafor with the U.S. Food & Drug Administration in 2007. When it contracted Girindus America (bought by Avecia in 2013) to manufacture it that year, TaiGen specified purification by column chromatography, a cumbersome and relatively expensive procedure when carried out on a large scale. “Our process development efforts were racing against the clinical trials launch schedule,” King recalls. Column chromatography, he points out, is a “tedious approach, but it works.”

By the time ScinoPharm was hired last year, TaiGen’s process development team had come up with a simpler and more elegant process. But its purity demands hadn’t changed.

“Usually, clients are satisfied with a purity level of 98% to 99%,” says Koksuan Tang, head of operations at ScinoPharm’s Changshu plant. “To go from 99% to 99.8% is very different.” The manufacturing of burixafor, he adds, involves five chemical steps and two purification steps. Upstream of the API, ScinoPharm also produces burixafor’s starting material.

Purity level aside, burixafor is not a particularly difficult compound to make, Tang says. Nonetheless, the process supplied by TaiGen had to be adjusted for larger-scale production. “If you heat up 10 g in the lab, it takes two minutes, but in a plant, it could take as long as two hours,” he says.

Although, while hydrogen chloride gas can be controlled effectively when making minute quantities of a compound in the lab, it’s another challenge to handle large volumes of the toxic substance at the plant level. To safely execute one reaction step, ScinoPharm dissolved HCl in a special solvent that does not affect the purity profile of burixafor.

TaiGen selected ScinoPharm as its China contractor after a careful process that involved two visits to Changshu by TaiGen’s senior managers, Tang recalls. ScinoPharm’s track record of meeting regulatory requirements in different countries, including China, was a plus, Tang believes. Its ability to produce both for clinical trials and in larger quantities after commercial launch was also decisive.

Operational since 2012, ScinoPharm’s Changshu site can deliver products under Good Manufacturing Practices in quantities ranging from grams to kilograms. It employs 220 people.

“Moving from the single-kilogram quantities we make now to hundreds of kilograms will require some adjustment to the process, but we believe we can deliver,” says Tang’s colleague Sing Ping Lee, senior director of product technical support in Changshu. One thing to keep in mind, he notes, is that Chinese regulatory standards for drug production are actually more restrictive than those in the U.S. or Europe, going so far as specifying what equipment manufacturers need to use.

Other than complying with Chinese regulators, one reason TaiGen needed to carefully select its China contractor is that the two companies could well be long-term partners, since TaiGen believes it has the ability to market the drug on its own in China, Taiwan, and Southeast Asia. In the event of approvals elsewhere, TaiGen plans to license the compound to a large drug company, which may or may not stick with ScinoPharm or Avecia.

Relatively unknown outside Taiwan, TaiGen was formed in 2001 by Ming-Chu Hsu, the founder of the Division of Biotechnology & Pharmaceutical Research at Taiwan’s National Health Research Institutes. The holder of a Ph.D. in biochemistry from the University of Illinois, Urbana-Champaign, she headed oncology and virology research at Roche for more than 10 years before returning to Taiwan in 1998.

Ming-Chu Hsu, Chairman & CEO, TaiGen Biotechnology, Taiwan

TaiGen employs about 80 people, three-quarters of whom are in R&D. The company develops its own drugs in-house and also in-licenses molecules that are in early stages of development. The company licenses out the molecules for the European Union and U.S. markets but seeks to retain Asian marketing rights. Burixafor was discovered in TaiGen’s own labs in Taipei. To come up with it, researchers used a high-throughput screening approach that involved 130,000 compounds, including the design and synthesis of 1,500 new compounds. “It went back and forth between chemistry and biology many times,” recalls King, TaiGen’s research head.

A so-called CXCR4 chemokine receptor antagonist, burixafor mobilizes hematopoietic stem cells and endothelial progenitor cells in human bone marrow and channels them into the peripheral blood within three hours of ingestion, according to results of Phase I and Phase II trials.

In the U.S., burixafor is undergoing clinical trials for use during stem cell transplantation in patients with multiple myeloma, non-Hodgkin’s lymphoma, or Hodgkin’s disease. In China, TaiGen is testing it as a chemotherapy sensitizer in relapsed or refractory adult acute myeloid leukemia.

Owing to its activity on CXCR4 chemokine receptors, the drug could also fight age-related macular degeneration and diabetic retinopathy diseases, as well as find use in tissue repair, King says. For clinical trials in the U.S., TaiGen has partnered with Michael W. Schuster, a medical doctor who conducts research at Stony Brook University Hospital in New York.

Dr. Michael Schuster is Gift of Life’s Medical Director, as well as the Director of the Hematopoietic Stem Cell Transplantation Program and Hematologic Malignancy Program of Stony Brook University Hospital in New York

Typical structure of a chemokine receptor

TaiGen sees particular potential for burixafor in stem cell applications. For example, patients undergoing hematopoietic stem cell transplantation often must take a granulocyte colony-stimulating factor plus a Sanofi drug called Mozobil to stimulate stem cell production. TaiGen says burixafor could accomplish this goal on its own in multiple myeloma patients. It cites one consulting firm forecast that puts eventual sales at more than $1 billion per year.

Sanofi drug called Mozobil to stimulate stem cell production

With that kind of potential, the company is counting on significant interest among licensors, any one of which might want to engage its own contract producer of burixafor. If that happens, a third manufacturer will have to learn to reach 99.8% purity.

TaiGen Biotechnology Co., Ltd.

7F,138 Shin Ming Rd. Neihu Dist., Taipei, Taiwan 114 R.O.C

Tel: 886-2-81777072　|　886-2-27901861

Fax: 886-2-27963606

Taipei Railway Station front

Taipei Songshan Airport

Scinopharm

ScinoPharm (Changshu) Pharmaceuticals, Ltd.

ScinoPharm is currently expanding its manufacturing and process development capabilities by adding significant production and technical capacity in Mainland China at its new Changshu site.

ScinoPharm Changshu is located in the Changshu Economic Development Zone (CEDZ), near Suzhou City, Jingsu Province, China on a 6.6-hectare site.

The facilities will include a R&D centre and production plants fully compliant with U.S. and international GMP standards. The Changshu plant, slated to be fully completed by 2012, will be used for the production of GMP grade pharmaceutical intermediates initially, and later be equipped to handle API production. China’s market for better quality APIs has grown considerably, and local formulation companies are encouraged to utilize APIs from companies having DMFs filed in advanced countries. ScinoPharm had closed its site in Kunshan and relocated the production and R&D groups to Changshu in the 4th quarter of 2011. These groups will continue to be expanded to meet growing demand for ScinoPharm products by both multinational and local formulation companies.

The small and medium-sized production units had been operational in the 4th quarter of 2011. The large production Bays plus a peptide purification unit, a high potency unit and a physical property processing facility will be operational by the end of 2012. Using advanced engineering designs, this site will also have the capability to process high potency, injectable grade products.

ScinoPharm Changshu will adopt the same quality systems as ScinoPharm Taiwan, and will therefore comply with ICH guidelines and FDA 21 CFR Parts 210 & 211.

TAIPEI