RG 6080

phase 1, Uncategorized Comments Off

Jul 152016

RG-6080

Sulfuric acid, mono[(1R,2S,5R)-2-[[(2-aminoethoxy)amino]carbonyl]-7-oxo-1,6-diazabicyclo[3.2.1]oct-6-yl] ester

A β-lactamase inhibitor potentially for the treatment of bacterial infections.

RG-6080; FPI-1459; OP-0595

CAS No. 1452458-86-4

Molecular Formula	C₉ H₁₆ N₄ O₇ S
Formula Weight	324.31

Originator Fedora Pharmaceuticals
Developer Meiji Seika Pharma
Class Antibacterials; Azabicyclo compounds
Mechanism of Action Beta lactamase inhibitors

Phase IBacterial infections

Most Recent Events

13 Jan 2015 OP 0595 licensed to Roche worldwide, except Japan ,
30 Nov 2014 Meiji Seika Pharma completes a phase I trial in Healthy volunteers in Australia (NCT02134834)
01 May 2014 Phase-I clinical trials in Bacterial infections (in volunteers) in Australia (IV)

SYNTHESIS

WO 2015046207,

CONTD…………………..

CONTD………………………………..

Patent

WO 2015053297

The novel heterocyclic compound in Japanese Patent 4515704 (Patent Document 1), preparation and shown for their pharmaceutical use, sodium trans-7-oxo-6- (sulfooxy) as a representative compound 1,6-diazabicyclo [3 .2.1] discloses an octane-2-carboxamide (NXL104). Preparation in regard to certain piperidine derivatives which are intermediates Patent 2010-138206 (Patent Document 2) and JP-T 2010-539147 (Patent Document 3) are shown at further WO2011 / 042560 (Patent Document 4) NXL104 to disclose a method for producing the crystals.

　In Patent 5038509 (Patent Document 5) (2S, 5R) -7- oxo -N- (piperidin-4-yl) -6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane – 2- carboxamide (MK7655) is shown, discloses the preparation of certain piperidine derivatives with MK7655 at Patent 2011-207900 (Patent Document 6) and WO2010 / 126820 (Patent Document 7).

　The present inventors also disclose the novel diazabicyclooctane derivative represented by the following formula (VII) in Japanese Patent Application 2012-122603 (Patent Document 8).

Patent Document 1: Japanese Patent No. 4515704 Pat
Patent Document 2: Japanese Patent Publication 2010-138206 Pat
Patent Document 3: Japanese patent publication 2010-539147 Pat
Patent Document 4: International Publication No. WO2011 / 042560 Patent
Patent Document 5: Japanese Patent No. 5038509 Pat
Patent Document 6: Japanese Patent Publication 2011-207900 Pat
Patent Document 7: International Publication No. WO2010 / 126820 Patent
Patent Document 8: Japanese Patent application 2012-122603 Pat.

[Chemical formula 1] (In the formula, R ³ are the same as those described below)

Reference Example
5 of 5 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)
Formula 43]

step 1 tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate 　(IV-1)(2S, 5R)-6-(benzyloxy) -7-oxo-1,6-diazabicyclo [3.2.1] octane-2-carboxylic acid (4 .30g, dehydrated ethyl acetate (47mL) solution of 15.56mmol) was cooled to -30 ℃, isobutyl chloroformate (2.17g, washing included dehydration ethyl acetate 1mL), triethylamine (1.61g, washing included dehydration ethyl acetate 1 mL), successively added dropwise, and the mixture was stirred 1 hour at -30 ° C.. To the reaction solution tert- butyl 2-dehydration of ethyl acetate (amino-oxy) ethyl carbamate (3.21g) (4mL) was added (washing included dehydration ethyl acetate 1mL), raising the temperature over a period of 1.5 hours to 0 ℃, It was further stirred overnight. The mixture of 8% aqueous citric acid (56 mL), saturated aqueous sodium bicarbonate solution (40 mL), sequentially washed with saturated brine (40 mL), dried over anhydrous magnesium sulfate, filtered, concentrated to 5 mL, up to 6mL further with ethanol (10 mL) It was replaced concentrated. Ethanol to the resulting solution (3mL), hexane the (8mL) in addition to ice-cooling, and the mixture was stirred inoculated for 15 minutes. The mixture was stirred overnight dropwise over 2 hours hexane (75 mL) to. Collected by filtration the precipitated crystals, washing with hexane to give the title compound 5.49g and dried in vacuo (net 4.98 g, 74% yield). HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 50/50, 1.0 mL / min, UV 210 nm, Retweeted 4.4 min; ¹ H NMR (400 MHz, CDCl ₃ ) [delta] 1.44 (s, 9H), 1.56-1.70 (m, 1H), 1.90-2.09 (m, 2H), 2.25-2.38 (m, 1H), 2.76 (d, J = 11.6 Hz, 1H), 3.03 (br.d., J = 11.6 Hz , 1H), 3.24-3.47 (m, 3H), 3.84-4.01 (m, 3H), 4.90 (d, J = 11.6 Hz, 1H), 5.05 (d, J = 11.6 Hz, 1H), 5.44 (br. . s, 1H), 7.34-7.48 (yd, 5H), 9.37 (Br.S., 1H); MS yd / z 435 [M + H] ⁺ .

Step 2

tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate
(V-1) tert-butyl {2 – [({[( 2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl ] carbonyl} amino) oxy] ethyl} carbamate (3.91 g, to a methanol solution (80 mL) of 9.01mmol), 10% palladium on carbon catalyst (50% water, 803 mg) was added, under hydrogen atmosphere and stirred for 45 minutes . The reaction mixture was filtered through Celite, after concentrated under reduced pressure to give 3.11g of the title compound (quantitative).
HPLC: COSMOSIL 5C18 MS-II 4.6 × 150 mm, 33.3 mM phosphate buffer / MeCN = 75/25, 1.0 mL / min, UV 210 nm, Retweeted 3.9 from min; ¹ H NMR (400 MHz, CD ₃ OD) [delta] 1.44 (s, 9H) , 1.73-1.83 (m, 1H), 1.86-1.99 (m, 1H), 2.01-2.12 (m, 1H), 2.22 (br.dd., J = 15.0, 7.0 Hz, 1H), 3.03 (d, J= 12.0 Hz, 1H), 3.12 (br.d., J = 12.0 Hz, 1H), 3.25-3.35 (m, 2H), 3.68-3.71 (m, 1H), 3.82-3.91 (m, 3H); MS M / Z 345 [M Tasu H] ^Tasu .

Step 3
Tetrabutylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl } amino) oxy] ethyl} carbamate
(VI-1) tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct 2-yl] carbonyl} amino) oxy] ethyl} carbamate (3.09g, in dichloromethane (80mL) solution of 8.97mmol), 2,6- lutidine (3.20mL), sulfur trioxide – pyridine complex (3 .58g) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was poured into half-saturated aqueous sodium bicarbonate solution, washed the aqueous layer with chloroform, tetrabutylammonium hydrogen sulfate to the aqueous layer and (3.47 g) chloroform (30 mL) was added and stirred for 10 minutes. The aqueous layer was extracted with chloroform, drying the obtained organic layer with anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the title compound 5.46g (91% yield).
HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 80/20, 1.0ML / Min, UV210nm, RT 2.0 Min; ¹ H NMR (400 MHz, CDCl ₃ ) Deruta 1.01 (T, J = 7.4 Hz, 12H), 1.37-1.54 (m , 8H), 1.45 (s, 9H), 1.57-1.80 (m, 9H), 1.85-1.98 (m, 1H), 2.14-2.24 (m, 1H), 2.30- 2.39 (m, 1H), 2.83 (d, J = 11.6 Hz, 1H), 3.20-3.50 (m, 11H), 3.85-3.99 (m, 3H), 4.33-4.38 (m, 1H), 5.51 (br s , 1H), 9.44 (Br.S., 1H); MS yd / z 425 [M-Bu ₄ N + 2H] ⁺ .

Step 4 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VII-1)
tetra butylammonium tert- butyl {2 – [({[( 2S, 5R) -7- oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (5.20g, 7.82mmol) in dichloromethane (25mL) solution of ice-cold under trifluoroacetic acid (25mL), and the mixture was stirred for 1 hour at 0 ℃. The reaction mixture was concentrated under reduced pressure, washed the resulting residue with diethyl ether, adjusted to pH7 with aqueous sodium bicarbonate, subjected to an octadecyl silica gel column chromatography (water), after freeze drying, 1.44 g of the title compound obtained (57% yield).
HPLC: COSMOSIL 5C18 MS-II 4.6X150mm, 33.3MM Phosphate Buffer / MeCN = 99/1, 1.0ML / Min, UV210nm, RT 3.1 Min; ¹ H NMR (400 MHz, D ₂O) Deruta 1.66-1.76 (M, 1H), 1.76-1.88 (m, 1H ), 1.91-2.00 (m, 1H), 2.00-2.08 (m, 1H), 3.02 (d, J = 12.0 Hz, 1H), 3.15 (t, J = 5.0 Hz , 2H), 3.18 (br d , J = 12.0 Hz, 1H), 3.95 (dd, J = 7.8, 2.2 Hz, 1H), 4.04 (t, J = 5.0 Hz, 2H), 4.07 (dd, J = 6.4 3.2 Hz &, 1H); MS yd / z 325 [M + H] ⁺ .

PATENT

WO 2015046207

Example
64 tert-butyl {2 – [({[( 2S, 5R) -6- hydroxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy ] ethyl} carbamate (V-1)
[of 124]

tert- butyl {2 – [({[(2S, 5R) -6- benzyloxy-7-oxo-1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl } carbamate (example 63q, net 156.42g, 360mmol) in methanol solution (2.4L) of 10% palladium carbon catalyst (50% water, 15.64g) was added, under an atmosphere of hydrogen, stirred for 1.5 hours did. The catalyst was filtered through celite, filtrate was concentrated under reduced pressure until 450mL, concentrated to 450mL by adding acetonitrile (1.5 L), the mixture was stirred ice-cooled for 30 minutes, collected by filtration the precipitated crystals, washing with acetonitrile, and vacuum dried to obtain 118.26g of the title compound (net 117.90g, 95% yield). Equipment data of the crystals were the same as those of the step 2 of Reference Example 3.

Example
65 (2S, 5R)-N- (2-aminoethoxy) -7-oxo-6- (sulfooxy) 1,6-diazabicyclo [3.2.1] octane-2-carboxamide (VI-1)
[of 125]

　tert- butyl {2 – [({[(2S, 5R) -1,6- -6- hydroxy-7-oxo-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate (example 64,537.61g, 1.561mol) in acetonitrile (7.8L) solution of 2,6-lutidine (512.08g), sulfur trioxide – pyridine complex (810.3g) was added, at room temperature in the mixture was stirred overnight. Remove insolubles and the mixture was filtered, the filtrate concentrated to 2.5 L, diluted with ethyl acetate (15.1L). The mixture was extracted with 20% phosphoric acid 2 hydrogencarbonate aqueous solution (7.8L), the resulting aqueous layer into ethyl acetate (15.1L), added tetrabutylammonium hydrogen sulfate (567.87g), was stirred for 20 min. The organic layer was separated layers, dried over anhydrous magnesium sulfate (425 g), after filtration, concentration under reduced pressure, substituted concentrated tetrabutylammonium tert- butyl with dichloromethane (3.1L) {2 – [({[(2S, 5R ) -7-oxo-6 (sulfooxy) 1,6-diazabicyclo [3.2.1] oct-2-yl] carbonyl} amino) oxy] ethyl} carbamate was obtained 758g (net 586.27g, Osamu rate 84%).

　The tetra-butyl ammonium salt 719g (net 437.1g, 0.656mol) in dichloromethane (874mL) solution was cooled to -20 ℃, dropping trifluoroacetic acid (874mL) at 15 minutes, 1 the temperature was raised to 0 ℃ It was stirred time. The reaction was cooled to -20 ° C. was added dropwise diisopropyl ether (3.25L), and the mixture was stirred for 1 hour the temperature was raised to 0 ° C.. The precipitate is filtered, washed with diisopropyl ether to give the title compound 335.36g of crude and vacuum dried (net 222.35g, 99% yield).

　The title compound of crude were obtained (212.99g, net 133.33g) and ice-cold 0.2M phosphate buffer solution of pH5.3 mix a little at a time, alternating between the (pH6.5,4.8L). The solution was concentrated under reduced pressure to 3.6L, it was adjusted to pH5.5 at again 0.2M phosphate buffer (pH6.5,910mL). The solution resin purification (Mitsubishi Kasei, SP207, water ~ 10% IPA solution) is subjected to, and concentrated to collect active fractions, after lyophilization, to give the title compound 128.3 g (96% yield). Equipment data of the crystals were the same as those of step 3 of Reference Example 3.

PATENT

US 20140288051

WO 2014091268

WO 2013180197

US 20130225554

///////////RG-6080, 1452458-86-4, FPI-1459, OP-0595, , β-lactamase inhibitor, bacterial infections, Fedora parmaceuticals, Meiji Seika Pharma

MK-8876

phase 1, Uncategorized Comments Off

Jul 022016

MK 8876
CAS 1426960-33-9

2-(4-Fluorophenyl)-5-(11-fluoro-6H-pyrido[2′,3′:5,6][1,3]oxazino[3,4-a]indol-2-yl)-N-methyl-6-(N-methylmethanesulfonamido)-1-benzofuran-3-carboxamide

2-(4-Fluorophenyl)-5-(11-fluoro-6H-pyrido[2′,3′:5,6][1,3]oxazino[3,4-a]indol-2-yl)-N-methyl-6-[methyl(methylsulfonyl)amino]-3-benzofurancarboxamide

Molecular Formula		C₃₂H₂₄F₂N₄O₅S
Molecular Weight		614.62

Originator Merck & Co
Class Antivirals

Phase I Hepatitis C

Most Recent Events

11 Oct 2013 Phase-I clinical trials in Hepatitis C in Germany (PO)
11 Oct 2013 Phase-I clinical trials in Hepatitis C in Moldova (PO)
23 Aug 2013 Preclinical trials in Hepatitis C in USA (PO)

DATA

2-(4-Fluorophenyl)-5-(11-fluoro-6H-pyrido[2′,3′:5,6][1,3]oxazino[3,4-a]indol-2-yl)-N-methyl-6-(N-methylmethanesulfonamido)-1-benzofuran-3-carboxamide

MK-8876 off-white solid

¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (q, J = 4.7 Hz, 1H), 8.06–8.01 (m, 2H), 8.05 (s, 1H), 7.86 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.46–7.40 (m, 2H), 7.29–7.22 (m, 1H), 7.11 (s, 1H), 6.94 (dd, J = 10.6, 7.9 Hz, 1H), 6.27 (s, 2H), 3.31 (s, 3H), 2.96 (s, 3H), 2.85 (d, J = 4.7 Hz, 3H);

¹³C NMR (125.7 MHz, DMSO-d₆) δ 162.86, 162.82 (d, J_C–F = 248.5 Hz), 155.74 (d, J_C–F = 246.1 Hz), 153.80, 152.43, 152.28, 147.20, 137.08, 137.00 (d, J_C–F = 10.8 Hz), 136.36, 136.20, 132.37, 129.50 (d, J_C–F = 8.6 Hz), 127.17, 125.45 (d, J_C–F = 3.1 Hz), 125.08, 125.02, 123.70 (d, J_C–F = 7.7 Hz), 122.28, 117.23 (d, J_C–F = 22.4 Hz), 116.01 (d, J_C–F = 21.9 Hz), 113.65, 111.76, 106.90 (d,J_C–F = 3.5 Hz), 105.32 (d, J_C–F = 18.5 Hz), 94.16, 73.57, 39.39, 37.24, 26.16;

HR-ESI-MS m/zcalcd for C₃₂H₂₅N₄O₅SF₂⁺ [M + H]⁺ 615.1514, found 615.1500.

. HPLC Method and Retention Time Data

HPLC Method
column	Ascentis Express C₁₈ 2.7 μm (fused core), 100 mm × 4.6 mm
detection	UV at 210 nm
column temperature	40 °C
flow rate	1.8 mL/min
injection volume	5.0 μL
gradient	90% A to 5% A over 11 min, hold at 5% A for 2 min, 5% A back to 90% A over the next 0.1 min, and then hold at 90% A for 2.9 min
run time	16 min
data collection	acquisition for the first 13 min
mobile phases	solvent A: water with 0.1% H₃PO₄
	solvent B: acetonitrile

Retention Time Data
identity	t_R (min)
boronic acid 27	4.24
desbromoarene 28	5.33
MK-8876 (1)	7.89
chloropyridine starting material 2	8.03
BHT	10.22

SYNTHESIS

Figure imgf000211_0002

Figure imgf000212_0002

Figure imgf000213_0001

CONTD……………

MK 8876

Figure imgf000207_0002

Figure imgf000211_0001

Figure imgf000211_0002

Figure imgf000212_0002

Figure imgf000213_0001

Figure imgf000213_0002

Figure imgf000214_0001

Figure imgf000207_0001

MK 8876

Patent

WO 2013033900

Scheme 1

Scheme 2

Scheme 3

Scheme 4

EXAMPLES

Example 1

Preparation of Compound 1

THIS COMPD HAS ONE FLUORO MISSING, APPLY TO YOUR MK 8876

Step 1 – Synthesis of 2,6-dichloropyridin-3-ol

Η₂0₂ (1.60 g, 47.12 mmol) was added slowly to the solution of compound 2,6- dichloropyridin-3-ylboronic acid (3 g, 15.71 mmol) in CH₂CI₂ (30 mL) at 0 °C. After stirred at room temperature for about 15 hours, the mixture was quenched with sat. Na₂S203 aqueous (50 mL) and adjusted to pH < 7 with IN HC1. The mixture was extracted with EtOAc (40 mL x 3). The organic layer was washed with brine (100 mL), dried over Na₂S0₄, filtered and the solvent was evaporated to provide2,6-dichloropyridin-3-ol (2.34 g, yield: 91.4%). 1H-NMR (CDC1₃, 400 MHz) δ 7.30 (d, / = 8.4 Hz, 1H), 7.19 (d, / = 8.4 Hz, 1H), 5.70 (br, 1H).

– Synthesis of 2,6-dichloro- -methoxypyridine

To a solution of 2,6-dichloropyridin-3-ol (16.3 g, 0.1 mol) and K₂C0₃ (41.4 g, 0.3 mol) in DMF (200 mL) were added Mel (21.3 g, 0.15 mol). The mixture was allowed to stir at 80 °C for 2 hours. The mixture was then diluted with water (200 mL) and extracted with EtOAc (200 mL x 3). The organic layer was washed with brine (200 mL x 3), dried over Na₂S0₄, filtered and the solvent was evaporated to provide 2,6-dichloro-3-methoxypyridine (17.0 g, yield: 96.0%). 1H-NMR (CDC1₃, 400 MHz) δ 7.12-7.18 (m, 2H), 3.86 (s, 3H). Step 3 – Synthesis of2-(6-chloro-3-methoxypyridin-2-yl)-lH-indole

To a degassed solution of compound 2,6-dichloro-3-methoxypyridine (8.9 g, 0.05 mol), (l-(tert-butoxycarbonyl)-lH-indol-2-yl)boronic acid (13 g, 0.05 mol) and K₃PO₄ (31.8 g, 3.0 mol) in DMF (100 mL) was added Pd(dppf)Cl₂ (3.65 g, 0.005 mol) under N₂. The mixture was heated at 60 °C for about 15 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and filtered. The filtrate was washed with H₂0, brine, dried over Na₂S0₄. After being concentrated in vacuo, the resulting residue was purified using prep-HPLC to provide the desired product of 2-(6-chloro-3-methoxypyridin-2-yl)-lH-indole (9.0 g, yield:

69.8%). 1H-NMR (CDC1₃, 400 MHz) δ 9.52 (s, 1H), 7.65 (d, / = 7.6 Hz, 1H), 7.38-7.43 (m, 2H), 7.07-7.26 (m, 4H), 4.03 (s, 3H).

Step 4 – Synthesis of6-chlor -2-(lH-indol-2-yl)pyridin-3-ol

BBr₃ (0.4 mL, 0.39 mmol) was added to the solution of 2-(6-chloro-3- methoxypyridin-2-yl)-lH-indole (50 mg, 0.194 mmol) in CH₂C1₂ (0.5 mL) at -78 °C under N₂. The mixture was allowed to stir at room temperature for 3 hours. The mixture was then quenched with CH₃OH (10 mL) at -78 °C. After being concentrated in vacuo, the resulting residue was purified using prep-TLC (PE : EtOAc = 2.5 : 1) to afford the desired product of 6- chloro-2-(lH-indol-2-yl)pyridin-3-ol (40 mg, yield: 85.1%). 1H-NMR (CDC1₃, 400 MHz) δ 10.09 (s, 1H), 9.72 (s, 1H), 7.50 (d, / = 7.9 Hz, 1H), 7.17-7.32 (m, 3H), 7.08-7.14 (m, 1H), 6.87-6.96 (m, 2H).

Step 5 – Synthesis of 2-chlo -6H-pyrido[2′ ,3′ : 5 ,6] [ 1 ,3]oxazino[3 ,4-a]indole

To a solution of chloroiodomethane (3.51 g, 20.0 mmol) and K₂CO₃ (1.38 g, 10.0 mmol) in DMF (50 mL) was allowed to stir at 100 °C, 6-chloro-2-(lH-indol-2-yl)pyridin-3-ol (480 mg, 2.0 mmol) in DMF (50 mL) was added dropwise. After addition, the mixture was allowed to stir for another 0.5 hours. The mixture was then diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The organic layer was washed with brine (100 mL x 3), dried over Na₂S0₄ and concentrated. The residue was purified using prep-TLC (PE : EtOAc = 3 1) to afford the desired product of 2-chloro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (260 mg, yield: 50.7%). 1H-NMR (CDC1₃, 400 MHz) δ 7.63 (d, / = 8.0 Hz, 1H), 7.22-7.27 (m, 3H), 7.19 (d, / = 2.4 Hz, 1H), 7.08-7.12 (m, 2H), 5.86 (s, 2H).

Step 6 – Synthesis of2-(4-fluowphenyl)-N-methyl-6-(N-methylmethylsulfonamido)-5-(6H- pyridol 2 ‘,3’:5,6][ l, mpound 1 )

THIS COMPD HAS ONE FLUORO MISSING, APPLY TO YOUR MK 8876

To a degassed solution of 2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzofuran-3- carboxamide (502 mg, 1.0 mmol), 2-chloro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (256 mg, 1.0 mmol) and K₃PO₄ (636 mg, 3.0 mmol) in dioxane : H₂0 (1.5 mL : 0.4 mL) was added Pd₂(dba)₃ (91 mg, 0.1 mmol) and X-phos (91 mg, 0.2 mmol) under N₂. The mixture was heated to 110 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and filtered. The filtrate was washed with H₂0, brine, dried over Na₂S0₄. After being concentrated in vacuo, the resulting residue was purified using prep-HPLC to provide the desired product of Compound 1 (275 mg, yield: 46.1%). 1H-NMR (CDC1₃, 400 MHz) δ 7.88-7.94 (m, 3H), 7.61-7.63 (m, 2H), 7.40 (s, 2H), 7.09-7.28 (m, 6H), 5.94 (s, 2H), 5.86 (d, / = 4.4 Hz, 1H), 3.29 (s, 3H), 2.92 (d, / = 5.2 Hz, 3H), 2.65 (s, 3H). MS (M+H)⁺: 596.

Compounds 2-15, depicted in the table below, were prepared using the method described above.

COMPD 2 IS MK 8876

Figure imgf000031_0001

PATENT

WO 2013033971

Example 81

Preparation of Compound 2

Synthesis of ethyl 3- 4-fluorophenyl)-3-oxopropanoate

Diethyl carbonate (130 g, 1.1 mol) was dissolved in a suspension ofNaH (60% in oil, 50.2 g, 1.3 mol) in anhydrous tetrahydrofuran (1.5 L), and then l-(4-fluorophenyl)ethanone (150 g, 1.09 mol) was added dropwise at 70 °C. The resulting mixture was stirred at 70 °C for 3 hours. After the reaction mixture was cooled to room temperature and poured into HCl (1 N). The mixture was extracted with EtOAc, the organic phase was dried with anhydrous NaS0₄ and concentrated in vacuo. The resulting residue was purified using column chromatography (eluted with petroleum ether / EtOAc = 50 / 1) to provide ethyl 3-(4-fluorophenyl)-3-oxopropanoate (217 g, yield: 95%). 1H-NMR (CDC1₃, 400 MHz) δ 7.92-7.97 (m, 2H), 7.07-7.13 (m, 2H), 4.14-4.20 (m, 2H), 3.93 (s, 2H), 1.22 (d, J= 7.2 Hz, 3H). MS (M+H)⁺: 211. Step 2 – Synthesis of ethyl 5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate

A solution of ethyl 3-(4-fluorophenyl)-3-oxopropanoate (130 g, 0.6 mol), 4- bromophenol (311 g, 1.8 mol) and FeCl₃-6H₂0 (19.5 g, 0.09 mol) in DCE (700 mL) was heated to reflux, and then 2-(tert-butylperoxy)-2-methylpropane (193 g, 1.32 mol) was added dropwise under nitrogen. After 6 hours of refluxing, the mixture was cooled to RT, quenched with saturated NaHS0₃ and extracted with dichloromethane. The organic phases were washed with water, brine and dried over Na₂S0₄, filtered and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether / dichloromethane = 15 / 1) to provide the crude product, which was crystallized from cold MeOH to provde ethyl 5-bromo-2- (4-fluorophenyl)benzofuran-3-carboxylate (37 g, yield: 14.3%) as solid. 1H- MR (CDC1₃, 400 MHz) δ 8.12 (s, 1H), 7.97-8.01 (m, 2H), 7.37 (d, J= 4.0 Hz, 1H), 7.32 (d, J= 8.0 Hz, 1H), 7.11 (t, J= 8.0 Hz, 2H), 4.32-4.38 (m, 2H), 1.36 (t, J= 8.0 Hz, 3H). MS (M+H)⁺: 363 / 365.

Step 3 – Synthesis of eth l 5-bromo-2-(4-fluorophen -6-nitrobenzofuran-3-carboxylate

To a solution of ethyl 5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate (50 g,

137.6 mmol) in CHC1₃ (500 mL), fuming HN0₃ (50 mL) was added dropwise at -15 °C and the mixture was stirred for 0.5 hour. The reaction mixture was poured into ice water and extracted with CH₂C1₂. The organic layer was washed with a.q. sat. NaHC0₃ and brine, after removed the most of solvent, the resulting residue was crystallized with petroleum ether / dichloromethane = 20 / 1 to provide product of ethyl 5-bromo-2-(4-fluorophenyl)-6-nitrobenzofuran-3-carboxylate (35 g, yield: 66%). 1H- MR (CDC1₃, 400 MHz) δ 8.36 (s, 1H), 8.02-8.04 (m, 3H), 7.13-7.18 (m, 2H), 4.36-4.41 (m, 2H), 1.37 (t, J= 4.0 Hz, 3H). MS (M+H)⁺: 408 / 410.

Step 4 – Synthesis of ethyl 6-amino-5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate

A mixture of ethyl 5-bromo-2-(4-fluorophenyl)-6-nitrobenzofuran-3-carboxylate (52 g, 127 mmol), iron filings (21.3 g, 382.2 mmol) and H₄C1 (41 g, 764.4 mmol) in MeOH / THF / H₂0 (2 / 2 / 1, 500 mL) was stirred at reflux for 3 hour. After filtered and concentrated, the resulting residue was purified using column chromatography (petroleum ether / EtOAc / dichloromethane = 20 : 1 : 20) to provide ethyl 6-amino-5-bromo-2-(4-fluorophenyl) benzofuran-3-carboxylate (40 g, yield: 82%). 1H- MR (CDC1₃, 400 MHz) δ 8.01 (s, 1H), 7.94-7.98 (m, 2H), 7.08 (t, J= 8.0 Hz, 2H), 6.83 (s, 1H), 4.32-4.36 (m, 2H), 4.18 (s, 2H), 1.35 (t, J= 8.0 Hz, 3H). MS (M+H)⁺: 378 / 380.

Step 5 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid eth l ester

MsCI (31.7 g, 277.5 mmol) was added to a solution of ethyl 6-amino-5-bromo-2- (4-fluorophenyl)benzofuran-3-carboxylate (35 g, 92.5 mmol) and pyridine (60 mL) in

dichloromethane (300 mL) at 0 °C. After stirred overnight at room temperature, the mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over Na₂S0₄, filtered and concentrated in vacuo, the resulting residue was purified using crystallized with EtOAc to provde the pure product of ethyl 5-bromo-2-(4-fluorophenyl)-6- (methylsulfonamido)benzofuran-3-carboxylate (35 g, yield: 82%). 1H- MR (CDC1₃, 400 MHz) δ 8.27 (s, 1H), 8.01-8.05 (m, 2H), 7.87 (s, 1H), 7.15-7.19 (m, 2H), 6.87 (s, 1H), 4.38-4.43 (m, 2H), 3.00 (s, 3H), 1.40 (t, J= 40 Hz, 3H). MS (M+H)⁺: 456 / 458.

Step 6 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid

To a solution of ethyl 5-bromo-2-(4-fluorophenyl)-6-(methylsulfonamido) benzofuran-3-carboxylate (53 g, 0.23 mol) in dioxane / H₂0 (5 / 1, 600 mL) was added

LiOH-H₂0 (25 g, 1.17 mol), and the mixture was stirred at 100 °C for 3 hours. After

concentrated, the resulting residue was dissolved in H₂0, 1 N HCl was added until pH reached 3, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na₂S0₄ and filtered. The solvent was removed to provide the product of 5-bromo-2-(4- fluorophenyl)-6-(methylsulfonamido)benzofuran-3-carboxylic acid (48 g, yield: 96%).¹H- MR (DMSO- e, 400 MHz) δ 13.49 (s, 1H), 9.67 (s, 1H), 8.30 (s, 1H), 8.12-8.17 (m, 2H), 7.87 (s, 1H), 7.45-7.50 (m, 2H), 3.16 (s, 3H). MS (M+H)⁺: 428 / 430. Step 7 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid methylamide

A solution of 5-bromo-2-(4-fluorophenyl)-6-(methylsulfonamido) benzofuran-3- carboxylic acid (33 g, 77 mmol), HOBT (15.6 g, 115.5 mmol) and EDCI (22.2 g, 115.5 mmol) in DMF (250 mL) was stirred at room temperature. After 2 hours, Et₃N (50 mL) and CH₃ H₂ (HC1 salt, 17.7 g, 231 mmol) was added to the mixture, and the mixture was stirred overnight. After the solvent was removed, H₂0 was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with H₂0, brine and concentrated in vacuo. The resulting residue was washed with EtOAc to provide the product of 5-bromo-2-(4-fluorophenyl)-N- methyl-6-(methylsulfonamido)benzofuran-3-carboxamide (32 g, yield: 94%). 1H- MR (DMSO- ck, 400 MHz) δ 9.55 (br s, 1H), 8.46-8.48 (m, 1H), 8.12-8.17 (m, 2H), 7.96 (s, 1H), 7.87 (s, 1H), 7.45-7.50 (m, 2H), 3.16 (s, 3H), 2.93 (d, J= 8.4 Hz, 3H). MS (M+H)⁺: 441 / 443.

Step 8 – Synthesis of 5-bromo-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido benzofuran-3-carboxamide

CH₃I (31.6 g, 223 mmol) was added to a mixture of 5-bromo-2-(4-fluorophenyl)- N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide (32 g, 74 mmol), K₂C0₃ (25.6 g, 186 mmol) and KI (246 mg, 1.5 mmol) in DMF (150 mL) under N₂ protection. The mixture was stirred at 80-90 °C overnight. After concentrated in vacuo, the resulting residue was washed with water (200 mL) and EtOAc (200 mL) to provide the product of 5-bromo-2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)benzofuran-3-carboxamide (31.5 g, 94%). 1H- MR (CDCI₃, 400 MHz) δ 8.16 (s, 1H), 7.88-7.92 (m, 2H), 7.70 (s, 1H), 7.18-7.23 (m, 2H), 5.78 (br s, 1H), 3.34 (s, 3H), 3.09 (s, 3H), 3.00 (d, J= 4.8 Hz, 3H). MS (M+H)⁺: 455 / 457. Step 9 – Synthesis of 2-(4-fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)-5-(4, 4, 5, 5- tetramethyl-1 -dioxaborolan-2-yl)benzofuran-3-carboxamide

a degassed solution of 5-bromo-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)benzofuran-3-carboxamide (1.0 g, 2.2 mmol) and pinacol diborane (2.79 g, 11.0 mmol) in 1,4-Dioxane (25 mL) was added KOAc (647 mg, 6.6 mmol) under N₂ and stirred for 4 hours at room temperature. Then Pd(dppf)Cl₂ (60 mg) was added, and the mixture was stirred for another 30 minutes. Then the mixture was put into a pre-heated oil-bath at 130 °C and stirred for another 1 hour under N₂. The reaction mixture was cooled to room

temperatureand concentrated and extracted with EtOAc. The organic layers were washed with brine, dried over Na₂S0₄. After concentrated, the crude product of the boronic ester was purified using column chromatography (petroleum ether / EtOAc = 5 / 1 to 2 / 1) to obtain 2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzofuran-3-carboxamide as white solid (700 mg, yield: 64%). ¹H- MR (CDCI₃, 400 ΜΗζ) δ 8.17 (s, 1H), 7.87-7.91 (m, 2H), 7.52 (s, 1H), 7.11 (t, 7= 7.6 Hz, 2H), 5.81 (d, 7= 2.8 Hz, 1H), 3.30 (s, 3H), 2.97 (d, 7= 5.2 Hz, 3H), 2.90 (s, 3H), 1.31 (s, 12H). MS (M+H)⁺: 503.

Step 10 – Synthesis of tert-butyl 4-fluoro-lH-indole-l -car boxy late

To a solution of 4-fluoro-lH-indole (5 g, 0.11 mol) and DMAP (150 mg, 3%Wt) in THF (50 mL) was added (Boc)₂0 (8.5 g, 0.04 mol) dropwise. The mixture was stirred at room temperature for 2 hours. The organic solvent was removed in vacuo, and the resulting residue was purified using column chromatography (pure petroleum ether) to provide tert-butyl 4-fluoro- lH-indole-l-carboxylate (8.3 g, yield: 96%). 1H- MR (CDC1₃, 400 MHz) δ 7.92 (d, J= 8.4 Hz, 1H), 7.55 (d, J= 3.6 Hz, 1H), 7.23 (m, 1H), 6.90 (m, 1H), 6.66 (d, J= 3.6 Hz, 1H), 1.67 (s, 9H). MS (M+H)⁺: 236.

Step 11 – Synthesis of (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid

To a solution of diisopropylamine (7.5 mL, 0.11 mol) in THF (35 mL) at 0 °C was added «-BuLi (21 mL, 0.055 mol) dropwise. The mixture was stirred at 0 °C for 40 minutes. Then the mixture was cooled to -78 °C. Tert-butyl 4-fluoro-lH-indole-l-carboxylate (5 g, 0.02 mol) in THF (13 mL) was added dropwise slowly. After addition, the mixture was stirred at -78 °C for 2 hours. Then triisopropyl borate (3.29 g, 0.03 mol) was added. The mixture was stirred at -78 °C for another 40 minutes. The reaction was monitored using TLC. When the reaction was completed, the mixture was adjusted to pH = 6 with 1 N HC1. After extracted with EtOAc (25 mL x 3), the combined organic layers were washed with brine (50 mL), dried over Na₂S0₄, filtered and concentrated in vacuo. The obtained solid was recrystallized with EtOAc and petroleum ether to provide (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid (4.5 g, yield: 76.7%, which might be unstable at high temp, work up, store in fridge). ¹H- MR (CDC1₃, 400 MHz) δ 7.77 (d, J= 8.4 Hz, 1H), 7.57 (s, 1H), 7.44 (s, 2H), 7.24 (m, 1H), 6.90 (m, 1H), 1.66 (s, 9H). MS (M+H)⁺: 280.

Step 12 – Synthesis of 6-chloro-2-iodopyridin-3-ol

6-chloropyridin-3-ol (5.0 g, 38.6 mmol) was dissolved in water (50 mL) and placed under an N₂ atmosphere. Na₂C0₃ (8.2 g, 77.4 mmol) was added followed by iodine (9.8 g, 38.8 mmol). The reaction mixture was stirred at room temperature for 2 hours. The mixture was poured into 1M Na₂S₂0₃ and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na₂S0₄ and concentrated to provide the product of 6-chloro-2- iodopyridin-3-ol (7.0 g, yield: 70.9%). 1H- MR (CDC1₃, 400 MHz) δ 7.17 (d, J= 8.4 Hz, 1H), 7.06 (d, J= 8.4 Hz, 1H). MS (M+H)⁺: 256 / 258.

Step 13 – Synthesis of 6-chloro-2-(4-fluoro-lH-indol-2-yl)pyridin-3-ol

A mixture of (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid (5 g, 18.0 mmol), 6-chloro-2-iodopyridin-3-ol (3.82 g, 15.0 mol) and NaHC0₃ (3.78 g, 45.0 mol) in 1, 4-dioxane (76 mL) and water (7 mL) was stirred at room temperature for 15 minutes. Then Pd(PPh₃)₂Cl₂ (527 mg, 0.75 mmol) was added under nitrogen atmosphere, and the mixture was heated at 100 °C under N₂ for 16 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), filtered and concentrated in vacuo. The resulting residue was diluted with H₂0 (60 mL) and EtOAc (30 mL), and the layer was separated, the aqueous layer was extracted with EtOAc (3*30 mL). The combined organic layers were washed with brine (50 mL), dried over Na₂S0₄, filtered and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether / EtOAc = 20 / 1 ~ 3 / 1) to provide 6-chloro-2- (4-fluoro-lH-indol-2-yl)pyridin-3-ol (3 g, yield: 76.5%). 1H- MR (MeOD, 400 MHz) δ 7.36 (s, 1H), 7.23-7.27 (m, 2H), 7.03-7.11 (m, 2H), 6.63-6.68 (m, 1H). MS (M+H)⁺: 263 / 265.

Ste 14 – Synthesis of 2-chloro-ll-fluoro-6H-pyrido[2′,3′:5, 6][l,3]oxazino[3,4-a]indole

A solution of 6-chloro-2-(4-fluoro-lH-indol-2-yl)pyridin-3-ol (2 g, 7.6 mmol) and Cs₂C0₃ (7.46 g, 22.89 mmol) in DMF (100 mL) was stirred at 100 °C (internal temperature) for 15 min, and then chloroiodomethane (2.85 g, 15.3 mmol) in DMF (2 mL) was added dropwise. After the reaction was completed, the mixture was filtered and concentrated in vacuo. The resulting residue was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic layer was washed with brine, dried over Na₂S0₄ and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether:EA=10: l) to provde 2-chloro-l l-fluoro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (1.8 g, yield: 86.1%). 1H- MR (DMSO-i¾, 400 MHz) δ 7.64 (d, J= 8.8 Hz, 1H), 7.39-7.46 (m, 2H), 7.21-7.25 (m, 1H), 7.06 (s, 1H), 6.88-6.92 (m, 1H), 6.18 (s, 2H). MS (M+H)⁺: 275 / 277. Step 15 – Synthesis of5-(ll-fluoro-6H-pyrido[2 3′:5, 6][l,3]oxazino[3,4-a]indol-2-yl)-2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)benzofuran-3-carboxam

To a degassed solution of 2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzofuran-3- carboxamide (100 mg, 0.199 mmol), 2-chloro-l l-fluoro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4- a]indole (56 mg, 0.199 mmol) and Κ₃Ρ0₄·3Η₂0 (159 mg, 0.597 mmol) in dioxane / H₂0 (0.8 mL / 0.2 mL) was added Pd₂(dba)₃ (9 mg, 0.01 mmol) and X-Phos (9 mg, 0.02 mmol) under N₂. The mixture was heated at 80 °C for 1 hour. The mixture was then diluted with water (30 mL) and extracted with EtOAc (15 mL x 3). The organic layer was washed with brine (20 mL), dried over Na₂S0₄ and concentrated in vacuo. The resulting residue was purified using prep-TLC (petroleum ether / EtOAc = 1 : 1.5) to provde the pure product of 5-(l l-fluoro-6H- pyrido [2′, 3 ‘ : 5 , 6] [ 1 , 3 ]oxazino [3 ,4-a]indol-2-yl)-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)benzofuran-3-carboxamide (60 mg, 48.8%). 1H- MR (CDC1₃, 400 MHz) δ: 7.99 (s, 1H), 7.93-7.96 (m, 2H), 7.65 (s, 1H), 7.45-7.50 (m, 2H), 7.17-7.21 (m, 4H), 7.10 (d, J= 8.0 Hz, 1H), 6.81-6.85 (m, 1H), 5.98 (s, 3H), 3.35 (s, 3H), 2.98 (d, J= 4.8 Hz, 3H), 2.72 (s, 3H). MS (M+H)⁺: 615.

Paper

We describe the route development and multikilogram-scale synthesis of an HCV NS5B site D inhibitor, MK-8876. The key topics covered are (1) process improvement of the two main fragments; (2) optimization of the initially troublesome penultimate step, a key bis(boronic acid) (BBA)-based borylation; (3) process development of the final Suzuki–Miyaura coupling; and (4) control of the drug substance form. These efforts culminated in a 28 kg delivery of the desired active pharmaceutical ingredient.

Process Development of the HCV NS5B Site D Inhibitor MK-8876

Michael J. Williams^†, Qinghao Chen^*^†, Lorenzo Codan^§, Renee K. Dermenjian^†, Spencer Dreher^†, Andrew W. Gibson^‡, Xianliang He^∥, Yan Jin^†, Stephen P. Keen^‡, Alfred Y. Lee^†, David R. Lieberman^‡, Wei Lin^∥, Guiquan Liu^∥, Mark McLaughlin^†, Mikhail Reibarkh^†, Jeremy P. Scott^‡, Sophie Strickfuss^‡, Lushi Tan^†, Richard J. Varsolona^†, and Feng Wen^∥

^† Department of Process Research and Development, Merck Research Laboratories, Rahway, New Jersey 07065, United States

^‡ Department of Process Chemistry, Merck Sharp & Dohme Ltd., Hertford Road, Hoddesdon, Hertfordshire EN11 9BU, United Kingdom

^§ Werthenstein BioPharma GmbH (MSD Switzerland), Industrie Nord 1, CH-6105 Schachen, Switzerland

^∥ WuXi AppTec Co., Ltd., No. 1 Building, #288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai 200131, China

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00405

*E-mail: qinghao.chen@merck.com

http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00405

PAPER

Using the Teasdale method, purge factor estimates for six impurities identified as mutagenic alerts in the synthesis of MK-8876 are compared to actual measured amounts of these impurities determined via appropriate analytical methods. The results from this comparison illustrate the conservative nature of purge factor estimates, meaning that overprediction of mutagenic impurity purging is unlikely when using this method. Industry and regulatory acceptance of the purge factor estimation method may help minimize analytical burden in pharmaceutical development projects.

Evaluation and Control of Mutagenic Impurities in a Development Compound: Purge Factor Estimates vs Measured Amounts

Mark McLaughlin^*^†, Renee K. Dermenjian^†, Yan Jin^‡, Artis Klapars^†, M. Vijayaraj Reddy^†, and Michael J. Williams^†

^† Merck and Co., Rahway, New Jersey 07065, United States

^‡ Advanced Polymer Technology, The Dow Chemical Company, 400 Arcola Road, Collegeville, Pennsylvania 19426, United States

Org. Process Res. Dev., 2015, 19 (11), pp 1531–1535

DOI: 10.1021/acs.oprd.5b00263

*E-mail: mark_mclaughlin@merck.com.

This article is part of the Genotoxic Impurities 2015 special issue.

http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00263?journalCode=oprdfk

WO2004041201A2 *	Oct 31, 2003	May 21, 2004	Viropharma Incorporated	Benzofuran compounds, compositions and methods for treatment and prophylaxis of hepatitis c viral infections and associated diseases
WO2011106992A1 *	Mar 2, 2011	Sep 9, 2011	Merck Sharp & Dohme Corp.	Inhibitors of hepatitis c virus ns5b polymerase

WO2004041201A2 *	Oct 31, 2003	May 21, 2004	Viropharma Incorporated	Benzofuran compounds, compositions and methods for treatment and prophylaxis of hepatitis c viral infections and associated diseases
WO2010030592A1 *	Sep 8, 2009	Mar 18, 2010	Bristol-Myers Squibb Company	Compounds for the treatment of hepatitis c
WO2011106992A1 *	Mar 2, 2011	Sep 9, 2011	Merck Sharp & Dohme Corp.	Inhibitors of hepatitis c virus ns5b polymerase

Citing Patent	Filing date	Publication date	Applicant	Title
WO2014123794A1 *	Feb 3, 2014	Aug 14, 2014	Merck Sharp & Dohme Corp.	Tetracyclic heterocycle compounds and methods of use thereof for the treatment of hepatitis c
WO2014123795A2 *	Feb 3, 2014	Aug 14, 2014	Merck Sharp & Dohme Corp.	Tetracyclic heterocycle compounds and methods of use thereof for the treatment of hepatitis c
WO2014123795A3 *	Feb 3, 2014	Oct 30, 2014	Merck Sharp & Dohme Corp.	Tetracyclic heterocycle compounds and methods of use thereof for the treatment of hepatitis c
US9242998	Feb 3, 2014	Jan 26, 2016	Merck Sharp & Dohme Corp.	Tetracyclic heterocycle compounds and methods of use thereof for the treatment of hepatitis C

//////MK-8876, 1426960-33-9, Merck & Co, Antivirals, Phase I, Hepatitis C

Fc7cccc6c7cc2n6COc1ccc(nc12)c3cc4c(cc3N(C)S(C)(=O)=O)oc(c4C(=O)NC)c5ccc(F)cc5

Recilisib Sodium, EX-RAD

phase 1, Uncategorized Comments Off

Jun 162016

Recilisib Sodium

C₁₆H₁₂ClNaO₄S
Molecular Weight:	358.771849 g/mol

Recilisib sodium.png

A protein kinase inhibitor potentially for the treatment of acute radiation syndrome.

sodium;4-[(E)-2-[(4-chlorophenyl)methylsulfonyl]ethenyl]benzoate

Onc-01210; ON-01210.Na, Ex-RAD; ON 01210.Na; ON-01210; ON-01210-Na; Recilisib

CAS No. 334969-03-8(free)

CAS 922139-31-9(Recilisib sodium)

Benzoic acid, 4-[(1E)-2-[[(4-chlorophenyl)methyl]sulfonyl]ethenyl]-, sodium salt (1:1)

Onconova Therapeutics Inc, Univ Temple INNOVATOR

Stephen C Cosenza, Lawrence Helson,Premkumar E Reddy, Ramana M V Reddy INVENTORS

Company	Onconova Therapeutics Inc.
Description	Synthetic, low molecular weight radioprotectant that modulates DNA repair pathways
Molecular Target	DNA
Mechanism of Action	Radioprotectant
Therapeutic Modality	Small molecule

Latest Stage of Development	Phase I
Standard Indication	Poisoning
Indication Details	Prevent radiation poisoning; Provide radation protection; Treat and prevent acute radiation syndrome (ARS)

Originator Onconova Therapeutics
Class Radioprotectives; Small molecules; Sulfonamides
Mechanism of Action Apoptosis inhibitors; Protein kinase inhibitors

Orphan Drug Status Yes – Acute radiation syndrome

Phase I Acute radiation syndrome

Most Recent Events

22 Apr 2016 Phase I development is ongoing in the US (PO & SC)
20 Mar 2014 Recilisib receives Orphan Drug status for Acute radiation syndrome in USA
03 Oct 2012 Phase-I clinical trials in Acute radiation syndrome in USA (PO)

Ex-Rad (or Ex-RAD), also known by the code name ON 01210.Na, or recilisib sodium (INN, USAN) is a drug developed by Onconova Therapeutics and the U.S. Department of Defense.^[1]^[2] This newly developed compound is said to be a potent radiation protection agent. Chemically, it is the sodium salt of 4-carboxystyryl-4-chlorobenzylsulfone.^[3]

Clinical trials

The results of two Phase I clinical studies in healthy human volunteers indicate that subcutaneously injected Ex-Rad is safe and well tolerated, with “no evidence of systemic side effects”.^[4] A study in mice demonstrated the efficacy of Ex-Rad by increasing the survival rate of mice exposed to typically lethal whole-body irradiation. The study tested oral and parenteral administration of Ex-Rad for both pre- and post-exposure radiomitigation.^[1]

Research on Ex-Rad has involved collaboration with the Armed Forces Radiobiology Research Institute (AFRRI), the Department of Biochemistry and Molecular & Cellular Biology at Georgetown University, Long Island University‘s Arnold & Marie Schwartz College of Pharmacy, and the Department of Oncological Sciences at the Mt. Sinai School of Medicine.^[1]

Mechanism of action

Onconova suggests that Ex-Rad protects cells exposed to radiation against DNA damage, and that the drug’s mechanism of action does not involve scavenging free radicals or arresting the cell cycle. Instead, they claim it employs a “novel mechanism” involving “intracellular signaling, damage sensing, and DNA repair pathways”.^[4] Ex-RAD is a chlorobenzylsulfone derivative that works after free radicals have damaged DNA. Onconova CEO Ramesh Kumar believes this is a better approach than trying to scavenge free radicals. “Free radicals are very short-lived, and so the window of opportunity to give a drug is very narrow,” he says. In cell and animal models, Ex-RAD protects hematopoieticand gastrointestinal tissues from radiation injury when given either before or after exposure.^[5]

While anti-radiation suits or other protective gear may be effective at reducing radiation exposure, such gear is expensive, unwieldy, and generally not available to public. Moreover, radioprotective gear will not protect normal tissue adjacent to a tumor from stray radiation exposure during radiotherapy. Pharmaceutical radioprotectants offer a cost-efficient, effective and easily available alternative to radioprotective gear. However, previous attempts at radioprotection of normal cells with pharmaceutical compositions have not been entirely successful. For example, cytokines directed at mobilizing the peripheral blood progenitor cells confer a myeloprotective effect when given prior to radiation (Neta et al., Semin. Radiat. Oncol. 6:306-320, 1996), but do not confer systemic protection. Other chemical radioprotectors administered alone or in combination with biologic response modifiers have shown minor protective effects in mice, but application of these compounds to large mammals was less successful, and it was questioned whether chemical radioprotection was of any value (Maisin, J. R., Bacq and Alexander Award Lecture. “Chemical radioprotection: past, present, and future prospects”, Int J. Radiat Biol. 73:443-50, 1998). Pharmaceutical radiation sensitizers, which are known to preferentially enhance the effects of radiation in cancerous tissues, are clearly unsuited for the general systemic protection of normal tissues from exposure to ionizing radiation.

The major biological effects of radiation exposure are the destruction of bone marrow cells, gastrointestinal (GI) damage, lung pneumonitis, and central nervous system (CNS) damage. The long-term effects of radiation exposure include an increase in cancer rates. It has been estimated that the exposure of 100 rems (roentgen equivalent man: a measurement used to quantify the amount of radiation that would produce harmful biological effects) would produce ARS symptoms. Exposure levels above 300 rems would result in the death of approximately 50% of the exposed population.

The α,β-unsaturated aryl sulfones, in particular benzyl styryl sulfones, provide significant and selective systemic protection of normal cells from radiation-induced damage in animals. When used in radiotherapy techniques, these compounds also exhibit independent toxicity to cancer cells. These α,β-unsaturated aryl sulfones, in particular benzyl styryl sulfones, are described in U.S. Pat. Nos. 6,656,973 and 6,667,346, which are particularly incorporated herein by reference in their entirety. Although these compounds are stable in solid state their aqueous formulations for parenteral administration are pH sensitive and pose challenging hurdles to overcome physical stability. The most likely causative factor may be attributed to the reactive styryl sulfone conjugated double bond, which is prone to Michael addition by nucleophiles and eventual fallout of the conjugated addition product.

U.S. Patent No. 6,656,973, describes in vitro pharmacological effects of DMSO solubilization of a benzyl styryl sulfone (e.g. ON 01210.NA) but fails to disclose a composition comprising ON 01210. NA formulation and specifically, a shelf stable formulation which is suitable for administration to humans.

PCT Application WO 2007/016201 describes pharmaceutical solution compositions for parenteral administration for reducing toxic effects of ionizing radiation in a subject, comprising an effective amount of at least one radioprotective α,β-Unsaturated aryl sulfone, and at least one component selected from the group consisting of a) a water soluble polymer in an amount between about 0.5% and about 90% w/v, b) at least one chemically modified cyclodextrin in an amount between about 20% and about 60% w/v, and c) DMA in an amount between 10% and about 50% w/v.

U.S. Patent Application 20090247624, and corresponding PCT Application WO 2008/105808, are directed to aqueous solutions, which comprise between about 20 mg/ml to about 100 mg/ml of at least one α,β-unsaturated aryl sulfone (e.g., the compound ON 01210. Na ((E)-4-Carboxystyryl-4-chlorobenzylsulfone sodium salt, a cosolvent in an amount between about 25% and about 90% w/v (e.g., about 50% PEG 400), wherein the composition is buffered and exists within the range of about pH 7.0 to about pHIO (e.g., 0.2M Tris-EDTA, pH about 8.5). The aforementioned solution formulations have exhibited a sub-optimal shelf life and lack a preferred degree of solubility and/or stability. These formulations evolved progressively as a result of addressing the most challenging aspects in the formulation and drug development field, namely, solubility and stability parameters that defined the long term viability of these formulations. There seems to be a delicate balance between pH, solubility and stability of the active moiety in aqueous milieu, wherein achieving such balance and development of a shelf stable aqueous formulation has presented a formidable challenge. Therefore, a shelf stable effective solution formulation that prevents the breakdown of the therapeutically active entity and keeps the drug in the solution at the desired pH was most desired and significant effort was directed towards this goal.

What is needed therefore, is a shelf stable effective solution formulation of radioprotective α,β-unsaturated aryl sulfones that prevents the breakdown of the therapeutically active entity and keeps the drug in the solution at the desired pH. This invention solves these and other long felt needs by providing improved solution formulation of radioprotective α,β- unsaturated aryl sulfones having improved physical and chemical stability and enhanced shelf life.

SYNTHESIS BY WORLDDRUGTRACKER

PATENT

WO 2011119863

An exemplary species of a radioprotective α,β-unsaturated aryl sulfone is ON 01210.Na. ON 01210.Na is a derivative of chlorobenzylsulfone. This compound is described in U.S. Pat. Nos. 6,656,973 and 6,667,346 as exhibiting valuable prophylactic properties which mitigate the effects of accidental and intentional exposure to life-threatening levels of irradiation. Hence, a systematic development of this compound is described with the objective of developing a shelf stable formulation.

Table 1 describes the general physical properties of ON. 1210. Na. The exemplary compound is a sodium salt of (E)-4-Carboxystyryl-4-chlorobenzylsulfone.

TABLE 1

Physical Properties of ON.1210.Na

Chemical Structure

Chemical Name (E)-4-Carboxystyryl-4-chlorobenzylsulfone,

Sodium Salt

Empirical Formula C₁₆H₁₂ClNa0₄S

Molecular Weight 358.79

Physical Nature White crystalline flakes

Melting Point 354-356° C.

Solubility Soluble in water at 8-10 mg/ml

The compound ON 01210. Na appears to form at least one polymorph. X-ray diffraction pattern, for example, of precipitated ON 01210. Na is different from that of the originally synthesized compound. Polymorphs of ON 01210.Na are intended to be within the scope of the claims appended hereto.

EXAMPLE 1

Preparation of ON 01210. Na

4-Chlorobenzyl-4-carboxystyryl sulfone (ON 01210) (49 g; 0.145 mol) was taken in a one-liter conical flask and 500 ml of distilled water was added. Sodium hydroxide solution (16 ml: 10 M stock) (0.150 mol.) was added to the conical flask. The contents of the flask were then boiled with stirring till ON 01210 was completely dissolved. The solution was then cooled to room temperature and shining crystals separated were filtered through a fluted filter paper. The crystalline material was dried under vacuum to yield (48 g) (92% yield) of pure ON 1210. Na.

EXAMPLE II

Preparation of ON 01210. Na Formulation A (Without Vitamin E TPGS)

TRIS (968.0 mg), EDTA (233.8 mg), and deionized (DI) water (24 ml) were combined in a beaker equipped with a Teflon coated stirring bar. The mixture was stirred until complete dissolution occurred, and the resulting solution was covered with aluminum foil and allowed to stir gently overnight at room temperature. The following morning, PEG 400 NF (40.0 ml) was added to the TRIS/EDTA aqueous solution with continued stirring. The vessel containing PEG 400 NF was rinsed with DI water (2 x 3.2 ml), and the rinsate added to the formulation mixture. After stirring the mixture to homogeneity (approx. 10 minutes), the pH was measured to be 9.46 using a calibrated electronic pH meter. The pH was adjusted to 8.37 (target pH = 8.40) by the careful addition of 98 pipet drops of 1.0 M HCl (aq) with stirring and allowed to fully equilibrate over a 10-15 minute period. Once the pH steadied at 8.37, ON 01210. Na (4.0 g) was added to the stirring formulation mixture. Complete dissolution required vigorous stirring and brief periodic sonication to break up ON 01210.Na clumps over a two hour period. After complete dissolution of ON 01210. Na, DI water (approx. 5 ml) was added to bring the final volume to approximately 80 milliliters. The pH of the resulting solution was determined to be 8.31, and thus 20 pipet drops of 1.0N NaOH(aq) were added to adjust the final formulation batch (defined as ON 01210.Na Formulation A) pH to 8.41-8.42. Formulation A was 0.22 micron filtered using a 100 ml Gastight Syringe equipped with a Millex^®GP filter unit (Millipore Express^® PES Membrane; Lot No R8KN13888).

PATENT

WO 2008105808

PATENT

WO 2007016201

PATENT

WO 2002069892

The α,β unsaturated aryl sulfones are characterized by cis-trans isomerism resulting from the presence of one or more double bonds. The compounds are named according to the Cahn-Ingold-Prelog system, the IUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc., New York, NY, 4^th ed., 1992, p.

127-138. Stearic relations around a double bond are designated as “Z” or “E”.

(E)-α,β unsaturated aryl sulfones may be prepared by Knoevenagel condensation of aromatic aldehydes with benzylsulfonyl acetic acids or arylsulfonyl acetic acids. The procedure is described by Reddy et al, Ada. Chim. Hung. 115:269-71 (1984); Reddy et al, Sulfur Letters 13:83-90 (1991); Reddy et al, Synthesis No. 4, 322-23 (1984); and Reddy et al, Sulfur Letters 7:43-48 (1987), the entire disclosures of which are incorporated herein by reference.
According to the Scheme 1 below, R_a and R_b each represent from zero to five substituents on the depicted aromatic nucleus. For purposes of illustration, and not limitation, the aryl groups are represented as phenyl groups, that is, the synthesis is exemplified by the preparation of styryl benzylsulfones. Accordingly, the benzyl thioacetic acid B is formed by the reaction of sodium thioglycollate and a benzyl chloride A. The benzyl thioacetic acid B is then oxidized with 30% hydrogen peroxide to give a corresponding benzylsulfonyl acetic acid C. Condensation of the benzylsulfonyl acetic acid C with an aromatic aldehyde D via a Knoevenagel reaction in the presence of benzylamine and glacial acetic acid yields the desired (E)-styryl benzylsulfone E.

Scheme 1

The following is a more detailed two-part synthesis procedure for preparing (E)-styryl benzylsulfones according to the above scheme.

General Procedure 1: Synthesis (E)-Styryl Benzylsulfones
Part A. To a solution of (8g, 0.2 mol) sodium hydroxide in methanol (200 ml), thioglycollic acid (0.1 mol) is added slowly and the precipitate formed is dissolved by stirring the contents of the flask. Then an appropriately substituted benzyl chloride (0.1 mol) is added stepwise and the reaction mixture is refluxed for 2-3 hours. The cooled contents are poured onto crushed ice and neutralized with dilute hydrochloric acid (200 ml). The resulting corresponding benzylthioacetic acid (0.1 mol) is subjected to oxidation with 30% hydrogen peroxide (0.12 mol) in glacial acetic acid (125 ml) by refluxing for 1 hour. The contents are cooled and poured onto crushed ice. The separated solid is recrystalized from hot water to give the corresponding pure benzylsulfonylacetic acid.
Part B. A mixture of the benzylsulfonyl acetic acid (10 mmol), an appropriately substituted aromatic aldehyde (10 mmol), and benzylamine (0.2 ml) in glacial acetic acid (12 ml) is refluxed for 2-3 hours. The contents are cooled and treated with cold ether (50 ml). Any product precipitated out is separated by filtration. The filtrate is diluted with more ether and washed successively with a saturated solution of sodium bicarbonate (20 ml), sodium bisulfite (20 ml), dilute hydrochloric acid (20 ml) and finally with water (35 ml). Evaporation of the dried ethereal layer yields styryl benzylsulfones as a solid material.

According to an alternative to Part A, the appropriate benzylsulfonylacetic acids may be generated by substituting a thioglycollate

HSCH₂COOR for thioglycollic acid, where R is an alkyl group, typically C1-C6 alkyl. This leads to the formation of the alkylbenzylthioacetate intermediate (F),

which is then converted to the corresponding benzyl thioacetic acid B by alkaline or acid hydrolysis.

(E)-styryl phenyl sulfones (formula I: n=zero; Q_ls Q₂ = substituted or unsubstituted phenyl) are prepared according to the method of General Procedure 1, replacing the benzylsulfonyl acetic acid in Part B with the appropriate substituted or unsubstituted phenylsulfonyl acetic acid.

(Z)-Styryl benzylsulfones are prepared by the nucleophilic addition of the appropriate thiols to substituted phenylacetylene with subsequent oxidation of the resulting sulfide by hydrogen peroxide to yield the (Z)-styryl benzylsulfone. The procedure is generally described by Reddy et al., Sulfur Letters 13:83-90 (1991), the entire disclosure of which is incorporated herein as a reference.
In the first step of the (Z)-styryl benzylsulfones synthesis, the sodium salt of benzyl mercaptan or the appropriate substituted benzyl mercaptan is allowed to react with phenylacetylene or the appropriate substituted phenylacetylene forming the pure (Z)-isomer of the corresponding styryl benzylsulfide in good yield.
In the second step of the synthesis, the (Z)-styryl benzylsulfide intermediate is oxidized to the corresponding sulfone in the pure (Z)-isomeric form by treatment with hydrogen peroxide.
The following is a more detailed two-part synthesis procedure for preparing (Z)-styryl benzylsulfones:

Procedure 2: Synthesis of (Z)-Styryl Benzylsulfones
Part A. To a refluxing methanolic solution of substituted or unsubstituted sodium benzylthiolate prepared from 460 mg (0.02g atom) of (i) sodium, (ii) substituted or unsubstituted benzyl mercaptan (0.02 mol) and (iii) 80 ml of absolute methanol, is added freshly distilled substituted or unsubstituted phenylacetylene. The mixture is refluxed for 20 hours, cooled and then poured on crushed ice. The crude product is filtered, dried and recrystalized from methanol or aqueous methanol to yield a pure (Z)- styryl benzylsulfide.
Part B. An ice cold solution of the (Z)- styryl benzylsulfide (3.0g) in 30 ml of glacial acetic acid is treated with 7.5 ml of 30% hydrogen peroxide. The reaction mixture is refluxed for 1 hour and then poured on crushed ice. The separated solid is filtered, dried, and recrystalized from 2-propanol to yield the pure (Z)-styryl benzylsulfone. The purity of the compounds is ascertained by thin layer chromatography and geometrical configuration is assigned by analysis of infrared and nuclear magnetic resonance spectral data.

The bis(styryl) sulfones of formula IN are prepared according to Procedure 3:
Procedure 3
Synthesis of (E)(E)- and (E)(Z)-bis(Styryl) Sulfones
To freshly distilled phenyl acetylene (51.07 g, 0.5 mol) is added sodium thioglycollate prepared from thioglycollic acid (46 g, 0.5 mol) and sodium hydroxide (40 g, 1 mol) in methanol (250 ml). The mixture is refluxed for 24 hours and poured onto crushed ice (500 ml) after cooling. The styrylthioacetic acid, formed after neutralization with dilute hydrochloric acid (250 ml), is filtered and dried; yield 88 g (90%); m.p. 84-86°C.
The styrylthioacetic acid is then oxidized to styrylsulfonylacetic acid as follows. A mixture of styrylthioacetic acid (5 g, 25 mmol) in glacial acetic acid (35 ml) and 30% hydrogen peroxide (15 ml) is heated under reflux for 60 minutes and the mixture is poured onto crushed ice (200 ml) after cooling. The compound separated is filtered and recrystalized from hot water to give white crystalline flakes of (Z)-styrylsulfonylacetic acid; yield 2.4 g (41%); m.p. 150-51°C.
A solution of (Z)-styrylsulfonylacetic acid (2.263 g, 10 m mol) in glacial acetic acid (6 ml) is mixed with an aromatic aldehyde (10 mmol) and benzylamine (0.2 ml) and refluxed for 3 hours. The reaction mixture is cooled, treated with dry ether (50 ml), and any product separated is collected by filtration. The filtrate is diluted with more ether and washed successively with a saturated solution of sodium hydrogen carbonate (15 ml), sodium bisulfite (15 ml), dilute hydrochloric acid (20 ml) and finally with water (30 ml). Evaporation of the dried ethereal layer yields (E)(Z)-bis(styryl)sulfones.
(E),(E)-bis(styryl)sulfones are prepared following the same procedure as described above with exception that sulfonyldiacetic acid is used in place of (Z)-styrylsulfonylacetic acid, and twice the amount of aromatic aldehyde (20 mmol) is used.

The styryl sulfones of formula N, which are systematically identified as 2-(phenylsulfonyl)-l-phenyl-3-phenyl-2-propen-l-ones, may be prepared according to either Method A or Method B of Procedure 4:

Procedure 4
Synthesis of 2-(Phenylsulfonyl)-l-phenyl-3-phenyl-2-propen-l-ones
These compounds are synthesized by two methods which employ different reaction conditions, solvents and catalysts.
Method A: Phenacyl aryl sulfones are made by refluxing α-bromoacetophenones (0.05 mol) and sodium arylsulfinates (0.05 mol) in absolute ethanol (200 ml) for 6-8 hours. The product which separates on cooling is filtered and washed several times with water to remove sodium bromide. The product is then recrystalized from ethanol: phenacyl-phenyl sulfone, m.p. 90-91°C; phenacyl-p-fluorophenyl sulfone, m.p. 148-149°C; phenacyl-p-bromophenyl sulfone, m.p. 121-122°C; phenacyl-p-methoxyphenyl sulfone, m.p. 104-105°C; p-nitrophenacyl-phenyl sulfone, m.p. 136-137°C.
A solution of phenacyl aryl sulfone (0.01 mol) in acetic acid (10 ml) is mixed with an araldehyde (0.01 mol) and benzylamine (0.02 ml) and refluxed for 3 hours. The solution is cooled and dry ether (50 ml) is added. The ethereal solution is washed successively with dilute hydrochloric acid, aqueous 10% NaOH, saturated NaHSO3 solution and water. Evaporation of the dried ethereal layer gives a solid product which is purified by recrystallization.

Method B: Dry tetrahydrofuran (200 ml) is taken in a 500 ml conical flask flushed with nitrogen. To this, a solution of titanium (IN) chloride (11 ml, 0.01 mol) in absolute carbon tetrachloride is added dropwise with continuous stirring. The contents of the flask are maintained at -20°C throughout the course of the addition. A mixture of phenacyl aryl sulfone (0.01 mol) and aromatic aldehyde (0.01 mol) is added to the reaction mixture and pyridine (4 ml, 0.04 mol) in tetrahydrofuran (8 ml) is added slowly over a period of 1 hour. The contents are stirred for 10-12 hours, treated with water (50 ml) and then ether (50 ml) is added. The ethereal layer is separated and washed with 15 ml of saturated solutions of 10% sodium hydroxide, sodium bisulfite and brine. The evaporation of the dried ethereal layer yields 2-(phenylsulfonyl)-l-phenyl-3-phenyl-2 propen-l-ones.

PATENT

https://www.google.com/patents/CN104817488A?cl=en

The structure of this medicine formula (I) shown below,

Wherein, R1 is absent or is halogen, C1-3 alkyl, alkoxy and -CF3; R2 is absent or is halogen, C1-3 alkyl, alkoxy and -cf3; structural formula (I) The method for the preparation of compounds as follows:

WO2007016201A2	Jul 28, 2006	Feb 8, 2007	Onconova Therapeutics, Inc.	FORMULATION OF RADIOPROTECTIVE α, β UNSATURATED ARYL SULFONES
WO2008105808A2	Jul 27, 2007	Sep 4, 2008	Onconova Therapeutics, Inc.	FORMULATIONS OF RADIOPROTECTIVE α, β UNSATURATED ARYL SULFONES
US6656973	Nov 27, 2002	Dec 2, 2003	Temple University – Of The Commonwealth System Of Higher Education	(E)-4-carboxystyrl-4-chlorobenzyl sulfone and pharmaceutical compositions thereof
US6667346	Feb 28, 2002	Dec 23, 2003	Temple University – Of The Commonwealth System Of Higher Education	Method for protecting cells and tissues from ionizing radiation toxicity with α, β unsaturated aryl sulfones
US6982282 *	May 17, 2002	Jan 3, 2006	Sonus Pharmaceuticals, Inc.	Emulsion vehicle for poorly soluble drugs
US20090247624	Jul 27, 2007	Oct 1, 2009	Onconova Therapeutics Inc.	Formulations of radioprotective alpha beta unsaturated aryl sulfones

References

“Onconova Therapeutics presents new data demonstrating radioprotection by Ex-RAD at RRS annual meeting” (Press release). EurekAlert. 2010-09-27. Archived from the originalon 2011-03-22. Retrieved 2011-03-22.
Hipp, Van (2011-03-16). “Ex-Rad, the U.S. Military’s Radiation Wonder Drug”. FoxNews.com (FOX News Network). Archived from the original on 2011-03-26. Retrieved 2011-03-26.
Ghosh, Sanchita P.; Perkins, Michael W.; Hieber, Kevin; Kulkarni, Shilpa; Kao, Tzu-Cheg; Reddy, E. Premkumar; Reddy, M. V Ramana; Maniar, Manoj; Seed, Thomas; Kumar, K. Sree (2009). “Radiation Protection by a New Chemical Entity, Ex-Rad™: Efficacy and Mechanisms”. Radiation Research 171 (2): 173–9. doi:10.1667/RR1367.1. PMID 19267542.
“Ex-RAD® for Protection from Radiation Injury”. Onconova Therapeutics. 2009. Archived from the original on 2011-03-22. Retrieved 2011-03-22.
http://cen.acs.org/articles/90/i26/Drugs-Never-Used.html^{[full citation needed]}
Kouvaris, J. R.; Kouloulias, V. E.; Vlahos, L. J. (2007). “Amifostine: The First Selective-Target and Broad-Spectrum Radioprotector”. The Oncologist 12 (6): 738–47.doi:10.1634/theoncologist.12-6-738. PMID 17602063.
http://www.news-medical.net/news/20110323/Cellerant-commences-CLT-008-Phase-III-trial-in-patients-with-leukemia.aspx
Reliene, Ramune; Pollard, Julianne M.; Sobol, Zhanna; Trouiller, Benedicte; Gatti, Richard A.; Schiestl, Robert H. (2009). “N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals”. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 665: 37. doi:10.1016/j.mrfmmm.2009.02.016.
Mansour, Heba H.; Hafez, Hafez F.; Fahmy, Nadia M.; Hanafi, Nemat (2008). “Protective effect of N-acetylcysteine against radiation induced DNA damage and hepatic toxicity in rats”.Biochemical Pharmacology 75 (3): 773–80. doi:10.1016/j.bcp.2007.09.018. PMID 18028880.
Demirel, C; Kilçiksiz, S; Ay, OI; Gürgül, S; Ay, ME; Erdal, N (2009). “Effect of N-acetylcysteine on radiation-induced genotoxicity and cytotoxicity in rat bone marrow”. Journal of radiation research 50 (1): 43–50. doi:10.1269/jrr.08066. PMID 19218780.
Demirel, C; Kilciksiz, S; Evirgen-Ayhan, S; Gurgul, S; Erdal, N (2010). “The preventive effect of N-acetylcysteine on radiation-induced dermatitis in a rat model”. Journal of the Balkan Union of Oncology 15 (3): 577–82. PMID 20941831.
Geiger, Hartmut; Pawar, Snehalata A; Kerschen, Edward J; Nattamai, Kalpana J; Hernandez, Irene; Liang, Hai Po H; Fernández, Jose Á; Cancelas, Jose A; Ryan, Marnie A; Kustikova, Olga; Schambach, Axel; Fu, Qiang; Wang, Junru; Fink, Louis M; Petersen, Karl-Uwe; Zhou, Daohong; Griffin, John H; Baum, Christopher; Weiler, Hartmut; Hauer-Jensen, Martin (2012).“Pharmacological targeting of the thrombomodulin–activated protein C pathway mitigates radiation toxicity”. Nature Medicine 18 (7): 1123–9. doi:10.1038/nm.2813. PMC 3491776.PMID 22729286.

External links

Onconova Therapeutics

Patent ID	Date	Patent Title
US2015265549	2015-09-24	STABLE AQUEOUS FORMULATION OF (E)-4-CARBOXYSTYRYL-4-CHLOROBENZYL SULFONE
US2015238448	2015-08-27	FORMULATION OF RADIOPROTECTIVE ALPHA, BETA UNSATURATED ARYL SULFONES
US2013012588	2013-01-10	COMPOSITIONS AND METHODS FOR PREVENTION AND TREATEMENT OF WOUNDS
US2013012589	2013-01-10	STABLE AQUEOUS FORMULATION OF (E)-4-CARBOXYSTYRYL-4-CHLOROBENZYL SULFONE
US2011250184	2011-10-13	METHODS FOR DETERMINING EFFICACY OF A THERAPEUTIC REGIMEN AGAINST DELETERIOUS EFFECTS OF CYTOTOXIC AGENTS IN HUMAN
US2011028504	2011-02-03	Formulation of radioprotective alpha beta unsaturated aryl sulfones
US2009247624	2009-10-01	FORMULATIONS OF RADIOPROTECTIVE ALPHA BETA UNSATURATED ARYL SULFONES

Ex-Rad
Identifiers

CAS Number	922139-31-9
PubChem	23668369
Properties
Chemical formula	C₁₆H₁₂ClNaO₄S
Molar mass	358.77 g·mol⁻¹

//////////Onc-01210, ON-01210.Na, 334969-03-8, 922139-31-9, Recilisib Sodium, , A protein kinase inhibitor, treatment of acute radiation syndrome, Orphan Drug Status, Ex-RAD

C1=CC(=CC=C1CS(=O)(=O)C=CC2=CC=C(C=C2)C(=O)[O-])Cl.[Na+]

GSK-2879552

phase 1, Uncategorized Comments Off

Jun 142016

GSK-2879552

CAS 1401966-69-5 (ABS), 1401966-63-9(REL)

C₂₃ H₂₈ N₂ O_2, 364.48

Benzoic acid, 4-[[4-[[[(1R,2S)-2-phenylcyclopropyl]amino]methyl]-1-piperidinyl]methyl]-

4-((4-((((lR,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoic acid

4-[[4-[[[(1R,2S)-2-Phenylcyclopropyl]amino]methyl]-1-piperidinyl]methyl]benzoic acid
4-[[4-[[((1R,2S)-2-Phenylcyclopropyl)amino]methyl]piperidin-1-yl]methyl]benzoic acid

4-((4-((((1R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-1-yl)methyl)benzoic acid

Glaxosmithkline Llc INNOVATOR

Neil W. Johnson, Jiri Kasparec, William Henry Miller, Meagan B. Rouse, Dominic Suarez, Xinrong Tian,

A LSD1 inhibitor potentially for the treatment of small cell lung cancer and acute myeloid leukemia.

GSK2879552 is an orally available, irreversible, inhibitor of lysine specific demethylase 1 (LSD1), with potential antineoplastic activity. Upon administration, GSK2879552 binds to and inhibits LSD1, a demethylase that suppresses the expression of target genes by converting the dimethylated form of lysine at position 4 of histone H3 (H3K4) to mono- and unmethylated H3K4. LSD1 inhibition enhances H3K4 methylation and increases the expression of tumor-suppressor genes. This may lead to an inhibition of cell growth in LSD1-overexpressing tumor cells. LSD1, overexpressed in certain tumor cells, plays a key role in tumor cell growth and survival. Check for active clinical trials or closed clinical trials using this agent.

GSK-2879552 chemical structure

Formula: C₂₃H₂₉ClN₂O₂
M.Wt: 400.94

GSK2879552, LSD1 Inhibitor

CAS 1902123-72-1

Molecular Weight:	437.41
Formula:	C₂₃H₂₈N₂O₂.2HCl

Chromatin modification plays an essential role in transcriptional regulation (T. Kouzarides, 2007, Cell 128: 693-705). These modifications, which include DNA methylation, histone acetylation and hsitone methylation, are disregulated in tumors. This epigenetic disregulation plays an important role in the silencing of tumor suppressors and overexpression of oncogenes in cancer (M. Esteller, 2008, N Engl J Med 358: 1148-59. P. Chi et al, 2010, Nat Rev Cane 10:457-469.). The enzymes that regulate histone methylation are the histone methyl transferases and the histone demethylases.

Lysine-specific demethylase 1 (LSDl; also known as BHC110) is a histone lysine demethylase reported to demethylate H3K4mel/2 (Y. Shi et al, 2004, Cell 119: 941-953) and H3K9mel/2 (R. Schule et al.,2005, Nature 437: 436-439). LSDl is overexpressed in multiple human cancers, including prostate where it is associated with more frequent relapse (P. Kahl et al, 2006, Cane. Res. 66: 11341-11347), breast (J. Kirfel et al, 2010, Carcinogenesis 31: 512-520) neuroblastoma (J. Kirfel et al, 2009, Cane. Res. 69: 2065-2071. G. Sun et al, 2010, Mol. Cell. Biol. 28: 1997-2000). LSDl is essential for transcriptional regulation mediated by a number of nuclear hormone receptors, including androgen receptor in prostate cancer (R. Schuele et al, 2005, Nature 437: 436-439. R. Schuele et al, 2007, Nat. Cell Biol. 9: 347-353. R. Schuele et al, 2010, Nature 464: 792-796), estrogen receptor in breast carcinomas (M.G. Rosenfeld et al, 2007, Cell 128: 505-518), and TLX receptor in neuorblastoma (S. Kato et al, 2008, Mol. Cell. Biol. 28: 3995-4003). These studies have shown that knockdown of LSDl expression results in decreased cancer cell proliferation. Additionally, LSDl is overexpressed in multiple cancer types that are nuclear hormone receptor-independent. Those tumors include ER-negative breast (J. Kirfel et al, 2010, Carcinogenesis 31: 512-520), small-cell lung, bladder, head & neck, colon, serous ovary, and kidney Wilm’s tumor. Therefore, potent selective small molecule inhibitors of LSDl may be useful for treatment of cancers that are nuclear hormone receptor-dependent and/or nuclear hormone receptor-independent.

The compositions and methods provided herein can potentially be useful for the treatment of cancer including tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to tumor types such as astrocytic, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral, ovarian, prostate and thyroid carcinomas and sarcomas. More specifically, these compounds can potentially be used to treat: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi’s sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm’s tumor

(nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing’s sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, meduUoblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre -tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes

(carcinoma); Hematologic: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplasia syndrome), Hodgkin’s disease, non-Hodgkin’s lymphoma (malignant lymphoma); Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi’s sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” as provided herein, includes a cell afflicted by any one of or related to the above identified conditions.

SYNTHESIS

GSK-2879552

PATENT

WO 2012135113

https://www.google.co.in/patents/WO2012135113A2?cl=en

Example 2

1 , 1 -Dimethylethyl 4-( { \( 1 R,2S)-2-phenylcyclopropyl] amino I methyl)- 1 -piperidinecarboxylate

Following a procedure analogous to the procedure described in Example 1 using [(1R,2S)-2-phenylcyclopropyl]amine ((-) isomer) (94 mg, 0.703 mmol) afforded 1,1 -dimethylethyl 4-({[(lR,2S)-2-phenylcyclopropyl]amino}methyl)-l-piperidinecarboxylate (92 mg, 0.264 mmol, 56.4 % yield) as white solid. 1H NMR (400 MHz, METHANOL-d₄) δ 7.29 – 7.37 (m, 2H), 7.23 – 7.28 (m, 1H), 7.17 – 7.22 (m, 2H), 4.14 (d, J= 12.63 Hz, 2H), 3.14 (d, J = 7.07 Hz, 2H), 3.01 (dt, J= 4.14, 7.64 Hz, 1H), 2.81 (br. s., 2H), 2.53 (ddd, J= 3.54, 6.63, 10.29 Hz, 1H), 1.97 (ddd, 1H), 1.80 (d, J= 12.13 Hz, 2H), 1.55 (ddd, J= 4.29, 6.63, 10.55 Hz, 1H), 1.47 (s, 9H), 1.36 – 1.45 (m, 1H), 1.23 (qd, J= 4.29, 12.38 Hz, 2H); LC-MS Rt = 0.78 min; MS (ESI): 331.3 [M+H]⁺.

Example 6

[(lR,2S)-2-Phenylcyclopropyll(4-piperidinylmethyl)amine

Following a procedure analogous to the procedure described in Example 4 using 1,1-dimethylethyl 4-({[(lR,2S)-2-phenylcyclopropyl]amino}methyl)-l-piperidinecarboxylate (Example 2, 60 mg, 0.182 mmol) afforded [(lR,2S)-2-phenylcyclopropyl](4-piperidinylmethyl)amine (41 mg, 0.146 mmol, 80 % yield)as white solid. 1H NMR (400 MHz, METHANOLS) δ 7.29 – 7.38 (m, 2H), 7.23 – 7.29 (m, 1H), 7.18 – 7.23 (m, 2H), 3.47 (d, J= 13.39 Hz, 2H), 3.21 (d, 2H), 2.89 – 3.13 (m, 3H), 2.60 (ddd, J= 3.79, 6.57, 10.36 Hz, 1H), 2.13 – 2.28 (m, J= 3.85, 3.85, 7.61, 11.21 Hz, 1H), 1.99 – 2.13 (m, 2H), 1.49 – 1.71 (m, 3H), 1.35 – 1.48 (m, 1H); LC-MS Rt = 0.44 min; MS (ESI): 231.2

Example 26

4-((4-(((trans-2-phenylcyclopropyl)amino)methyl)piperidin- 1 -yl)methyl)benzoic acid

To the solution of 2,2,2-trifluoro-N-(trans-2-phenylcyclopropyl)-N-(piperidin-4-ylmethyl)acetamide (200 mg, 0.613 mmol, Example l ib) and 4-(bromomethyl)benzoic acid (198 mg, 0.919 mmol) in acetonitrile (6 mL) was added potasium carbonate (254 mg, 1.838 mmol). The reaction mixture was stirred for 3 hours at the 90 °C. The reaction mixture was then filtered and evaporated. The crude oil was mixed with 10 mL of 10 % acetic acid and 10 mL of ethyl acetate. Layers were separated, and the organic layer was discharged. Aqueous layer was neutralized with 1 M Na₂C0₃, and the product was extracted into 10 mL of ethyl acetate. The organic layer was washed with brine, dried over MgS0₄, filtered and evaporated. The oil was dissolved in 6 ml of EtOH and 3 ml of 1 M NaOH. The reaction mixture was stirred for 20 min, and then it was concentrated. The solution was then partioned between 2 ml of water and 5 mL of ethyl acetate. The organic layer was separated and evaporated. The oil was purified on preparatory HPLC (2 to 10 % AcCN: H₂0 with 0.1 % formic acid modifier). The fractions were collected. To each

fraction was added 1 ml of 1 M HCl, and the fractions were evaporated to dryness. 4-((4-(((trans-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoic acid (50 mg, 0.118 mmol, 19.33 % yield) was isolated as a white solid. 1H NMR (400 MHz,

METHANOLS) δ 8.16 (d, J= 8.34 Hz, 2H), 7.70 (d, J= 8.34 Hz, 2H), 7.30 – 7.37 (m, 2H), 7.23 – 7.29 (m, 1H), 7.20 (d, J= 7.33 Hz, 2H), 4.44 (br. s., 2H), 3.57 (d, J= 11.62 Hz, 2H), 3.07 – 3.27 (m, 4H), 3.04 (dt, J= 3.95, 7.52 Hz, 1H), 2.59 (ddd, J= 3.54, 6.57, 10.11 Hz, lH), 2.12 (d, J= 13.89 Hz, 3H), 1.54 – 1.81 (m, 3H), 1.42 (q, 1H); LC-MS Rt = 0.47 min; MS (ESI): 365.3 [M+H]⁺.

[M+H]⁺.

Example 29

4-((4-((((lR,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoic acid

Step 1.

tert-Butyl 4-((4-(hydroxymethyl)piperidin-l-yl)methyl)benzoate

tert-Butyl 4-(bromomethyl)benzoate (1 g, 3.13 mmol) and piperidin-4-ylmethanol (0.361 g, 3.13 mmol) were dissolved in acetonitrile (25 mL). K₂CO₃ (1.300 g, 9.40 mmol) was added and the reaction mixture was heated to reflux for 20 min. The reaction mixture was cooled down to room temperature, filtered and evaporated. The resulting solid was partitioned between ethyl acetate (50mL) and 1 M HC1 (50 mL). The layers were separated and the aqueous layer was washed with ethyl acetate and the organic layers were discarded. The aqueous layer was basified with 8 M NaOH to pH -10 and extracted 2 times with 50 mL of ethyl acetate. The organic layers were combined, washed with brine and dried over MgSC^, filtered and evaporated. tert-Butyl 4-((4- (hydroxymethyl)piperidin-l-yl)methyl)benzoate (0.95 g, 2.99 mmol, 95 % yield) was isolated as yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 7.95 (d, J= 8.34 Hz, 2H), 7.39 (d, J = 8.08 Hz, 2H), 3.56 (s, 2H), 3.51 (d, J = 6.57 Hz, 2H), 2.90 (d, J= 11.37 Hz, 2H), 1.94 – 2.04 (m, 2H), 1.73 (d, J= 14.15 Hz, 2H), 1.61 (s, 9H), 1.40 – 1.56 (m, 2H), 1.30 – 1.37 (m, 2H); LC-MS Rt = 0.67 min; MS (ESI): 306.2 [M+H]⁺.

Step 2.

tert-Butyl 4-((4-formylpiperidin- 1 -yl)methyl)benzoate

To a solution of oxalyl chloride (0.408 mL, 4.67 mmol) in dichloromethane (5 mL) at -60 °C was added a solution of DMSO (0.508 mL, 7.15 mmol) in 15 mL of dichloromethane over 30 minutes. The reaction was stirred for 30 minutes at -60 °C A solution of tert-butyl 4-((4-(hydroxymethyl)piperidin-l-yl)methyl)benzoate (950 mg, 3.11 mmol) in 5 mL of dichloromethane was added over 10 minutes at -60 °C. The reaction mixture was stirred for 3 hours at – 60 °C, then triethylamine (2.168 mL, 15.55 mmol) was added and after 10 minutes 10 mL of water was added. The reaction mixture was allowed to warm up to the room temperature. The layers were separated. The pH of the water layer was adjusted to ~7 with 1 M HC1 and then extracted with 20 mL of dichloromethane. The combined organic layers were washed with water and brine, then dried over MgSO, filtered and evaporated. The resulting oil was purified on a silica column eluting with EtOAc to yield tert-butyl 4-((4-formylpiperidin-l-yl)methyl)benzoate (550 mg, 1.722 mmol, 55.4 % yield) as a yellow oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 9.67 (d, J= 1.26 Hz, 1H), 7.96 (d, J= 8.34 Hz, 2H), 7.38 (d, J= 8.34 Hz, 2H), 3.56 (s, 2H), 2.75 – 2.92 (m, 2H), 2.21 – 2.35 (m, 1H), 2.14 (t, J= 10.48 Hz, 2H), 1.91 (dd, J= 2.78, 13.14 Hz, 2H), 1.65 – 1.81 (m, 2H), 1.58 – 1.64 (m, 9H); LC-MS Rt = 0.69 min; MS (ESI): 304.2

[M+H]⁺, 322.2 [M+H₂0]⁺, 336.6 [M+Na]⁺

Step 3.

tert-Butyl 4-((4-(((( 1 R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin- 1 -yl)methyl)benzoate

To a solution of tert-butyl 4-((4-formylpiperidin-l-yl)methyl)benzoate (6.7 g, 22.08 mmol) in methanol (50 mL) was added (lR,2S)-2-phenylcyclopropanamine (3.53 g, 26.5 mmol). The reaction mixture was refluxed for 5 minutes then cooled down to the room temperature. Sodium cyanotrihydroborate (2.082 g, 33.1 mmol) was added. The reaction mixture was stirred 1 hour at room temperature. Water (50 mL) was added. The reaction was concentrated and 50 mL of dichloromethane was added. The layers were separated. The organics were washed with 10 % acetic acid (50 mL). The layers were separated and 50 mL of brine was added slowly as a solid crashed out. The solid was filtered and suspended in isopropanol. The suspension was sonicated and filtered. tert-Butyl 4-((4-(((( 1 R,2S)-2-phenylcyclopropyl)amino)methyl)piperidin- 1 -yl)methyl)benzoate (5.8 g, 13.65 mmol, 61.8 % yield) was isolated as a white solid. 1H NMR (400 MHz,

METHANOLS) δ 8.07 (d, J= 8.34 Hz, 2H), 7.70 (d, J= 8.08 Hz, 2H), 7.28 – 7.37 (m, 2H), 7.10 – 7.28 (m, 3H), 4.43 (br. s., 2H), 3.54 (d, J= 10.86 Hz, 2H), 3.08 – 3.26 (m, 4H), 3.03 (dt, J= 3.76, 7.39 Hz, 1H), 2.54 – 2.71 (m, 1H), 2.03 – 2.29 (m, 3H), 1.67 – 1.84 (m, 2H), 1.58 – 1.67 (m, 10H), 1.40 (q, J = 6.82 Hz, lH); LC-MS Rt = 0.76 min; MS (ESI): 421.4 [M+H]⁺.

Step 4.

4-((4-((((lR,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoic acid

A suspension of tert-butyl 4-((4-((((lR,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoate (5.8 g, 13.79 mmol) in HCL – 1 M (80 ml, 80 mmol) was heated to 89 °C (internal temperature) for 2 hr. The solution was cooled down to the room temperature and held in an ice -bath for 1 hour and then filtered. 4-((4-((((lR,2S)-2-phenylcyclopropyl)amino)methyl)piperidin-l-yl)methyl)benzoic acid (3.8 g, 8.25 mmol, 59.8 % yield) was isolated as white solid. 1H NMR (400 MHz, METHANOL-d₄) 5 8.15 (d, J= 8.34 Hz, 2H), 7.72 (d, J= 8.59 Hz, 2H), 7.29 – 7.37 (m, 2H), 7.14 – 7.28 (m, 3H), 4.45 (br. s., 2H), 3.55 (d, J= 10.36 Hz, 2H), 3.07 – 3.29 (m, 4H), 3.04 (dt, J= 3.98, 7.71 Hz, 1H), 2.61 (ddd, J= 3.66, 6.57, 10.23 Hz, 1H), 1.98 – 2.31 (m, 3H), 1.72 (br. s., 2H), 1.62 (ddd, J= 4.42, 6.51, 10.55 Hz, 1H), 1.41 (q, J= 6.82 Hz, lH); LC-MS Rt = 0.49 min; MS (ESI): 365.3 [M+H]⁺.

Neil Johnson

US Lead of Chemistry Talent Development, External Engagement and Recruitment at GSK

https://www.linkedin.com/in/neil-johnson-6628894

Experience

US Lead of Chemistry Talent Development, External Engagement and Recruitment

GSK

March 2016 – Present (4 months)Greater Philadelphia Area

Manager

GSK

July 1999 – Present (17 years)

Investgator

GlaxoSmithKline

1999 – Present (17 years)

Senior Scientist

Cephalon

September 1994 – June 1999 (4 years 10 months)

Education

The Johns Hopkins University

Doctor of Philosophy (PhD), Organic Chemistry

1988 – 1994

Fort Lewis College

BS, Chemistry

1984 – 1988

///////////GSK-2879552, 1401966-63-9, , A LSD1 inhibitor, small cell lung cancer, acute myeloid leukemia, 1401966-69-5, 1902123-72-1

O=C(O)C1=CC=C(CN2CCC(CN[C@H]3[C@H](C4=CC=CC=C4)C3)CC2)C=C1

O=C(O)c1ccc(cc1)CN2CCC(CC2)CN[C@@H]4C[C@H]4c3ccccc3

Zamicastat

Uncategorized Comments Off

Apr 122016

2D chemical structure of 1080028-80-3

CAS 1080028-80-3 BASE

1383828-47-4 OF HCL SALT

C₂₁ H₂₁ F₂ N₃ O S BASE

2H-Imidazole-2-thione, 1-[(3R)-6,8-difluoro-3,4-dihydro-2H-1-benzopyran-3-yl]-1,3-dihydro-5-[2-[(phenylmethyl)amino]ethyl]-(R)-5-(2-(Benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione

(R)-5-(2-(Benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione

Molecular Weight, 401.47 BASE


BIAL – PORTELA & CA., S.A. [PT/PT]; À Avenida da Siderurgia Nacional P-4745-457 S. Mamede do Coronado (PT)

Zamicastat is a dopamine beta-monooxygenase inhibitor in phase I clinical studies at BIAL for the treatment of hypertension and heart failure.
Zamicastat is a potent and selective dopamine β-mono-oxygenase inhibitor. Zamicastat Prevents the Deterioration of Cardiometabolic and Inflammatory Biomarkers in a Genetic Model of Salt-sensitive Hypertension. Chronic high salt intake deteriorates several cardiometabolic and inflammatory biomarkers in Dahl/SS rats, which can be prevented by dopamine β-hydroxylase inhibition with zamicastat.
crystalline forms of l-[(3R)-6,8-difluoro- 3,4-dihydro-2H-l-benzopyran-3-yl]-l,3-dihydro-5-[2-[(phenylmethyl)amino]ethyl]-2H- imidazole-2-thione, i.e. the Renantiomer of and processes for preparing the same. Background and prior art:Interest in the development of inhibitors of dopamines-hydroxylase (ϋβΗ) has centred on the hypothesis that inhibition of this enzyme may provide significant clinical improvements in patients suffering from cardiovascular disorders such as hypertension or chronic heart failure. The rationale for the use of ϋβΗ inhibitors is based on their capacity to inhibit the biosynthesis of noradrenaline, which is achieved via enzymatic hydroxylation of dopamine. Activation of neurohumoral systems, chiefly the sympathetic nervous system, is the principal clinical manifestation of congestive heart failure (Parmley, W.W., Clinical Cardiology, 18: 440-445, 1995). Congestive heart failure patients have elevated concentrations of plasma noradrenaline (Levine, T.B. et al., Am. J. Cardiol., 49: 1659-1666, 1982), increased central sympathetic outflow (Leimbach, W.N. et al., Circulation, 73: 913- 919, 1986) and augmented cardiorenal noradrenaline spillover (Hasking, G.J. et al., Circulation, 73:615-621, 1966). Prolonged and excessive exposure of the myocardium to noradrenaline may lead to down-regulation of cardiac β] -adrenoceptors, remodelling of the left ventricle, arrhythmias and necrosis, all of which can diminish the functional integrity of the heart. Congestive heart failure patients who have high plasma concentrations of noradrenaline also have the most unfavourable long-term prognosis (Cohn, J.N. et al., N. Engl. J. Med., 311 :819-823, 1984). Of greater significance is the observation that plasma noradrenaline concentrations are already elevated in asymptomatic patients with no overt heart failure and can predict ensuing mortality and morbidity (Benedict, C.R. et al., Circulation, 94:690-697, 1996). An activated sympathetic drive is not therefore merely a clinical marker of congestive heart failure, but may contribute to progressive worsening of the disease. Potent dopamines-hydroxylase inhibitors having high potency and significantly reduced brain access are disclosed in WO 2008/136695. WO 2008/136695 describes compounds of formula I: I where R_ls R₂ and R₃ are the same or different and signify hydrogens, halogens, alkyl, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R4 signifies -alkylaryl or – alkylheteroaryl; X signifies CH₂, oxygen atom or sulphur atom; n is 2 or 3; including the individual (R)- and (S)-enantiomers or mixtures of enantiomers thereof; and including pharmaceutically acceptable salts and esters thereof, wherein the term alkyl means hydrocarbon chains, straight or branched, containing from one to six carbon atoms, optionally substituted by aryl, alkoxy, halogen, alkoxycarbonyl or hydroxycarbonyl groups; the term aryl means a phenyl or naphthyl group, optionally substituted by alkyl, alkyloxy, halogen or nitro group; the term halogen means fluorine, chlorine, bromine or iodine; the term heteroaryl means heteroaromatic group. In particular, WO 2008/136695 describes l-[(3R)-6,8-difluoro-3,4-dihydro-2H-l-benzopyran-3-yl]-l,3-dihydro-5-[2- [(phenylmethyl)amino]ethyl]-2H-Imidazole-2-thione. Processes for the preparation of compounds of formula I, and in particular l-[(3R)-6,8- difluoro-3,4-dihydro-2H-l-benzopyran-3-yl]-l,3-dihydro-5-[2-[(phenylmethyl)amino] ethyl] -2H-Imidazole-2-thione, are described in WO 2008/136695 and are incorporated by reference herein. It is known that polymorphic forms of the same drug may have substantially different pharmaceutically important properties such as dissolution characteristics and bioavailability as well as stability of the drug. Furthermore, different forms may have different particle size, hardness and glass transition temperature. Thus, one form may provide significant advantages over other forms of the same drug in solid dosage form manufacture processes, such as accurate measurement of the active ingredients, easier filtration, or improved stability during granulation or storage. Furthermore, a particular process suitable for one form may also provide drug manufacturers several advantages such as economically or environmentally suitable solvents or processes, or higher purity or yield of the desired product.

PATENT

http://www.google.com/patents/WO2012087174A2?cl=en

Preparation of compound 2

[0090] Six lots of compound 2 (designated as lots 1, 2, 3, 4, 5 and 6) were prepared. The starting materials were prepared according to the following experimental protocols.

Lot 1 (Form A)

To a suspension of (R)-5-(2-aminoethyl)-l-(6,8-difluorochroman-3-yl)-lH- imidazole-2(3H)-thione (6.23 g, 20 mmol) in a mixture of Dichloromethane (DCM – 40 ml) and Methanol (40.0 ml) was added BENZALDEHYDE (2.230 ml, 22.00 mmol). To the resulting clear solution SODIUM CYANOBOROHYDRIDE (1.9 g, 28.7 mmol) was added in portions at 20-25°C to avoid intensive foaming and the solution was stirred at 20- 25°C for 40 h. The solution was quenched at 20-25°C with IN HC1 (35 ml), neutralised with 3N NaOH (35 ml), the mixture was extracted with DCM (200 ml). The organic phase was washed with brine, dried (MgS04), evaporated to dryness. The oily residue crystallised from 2-propanol (40 ml) at 20-25°C over a week-end. The crystals were collected, washed with 2-propanol, dried to give 5.2 g of the crude product. Re- crystallisation from 2-propanol-DCM hasn’t removed all impurities. Everything collected, evaporated with silica, applied on a column, eluted with Ethyl Acetate (EA)->EA-MeOH 9:1->4: 1, fractions 8-25 collected to give 3.8 g. Re-crystallised from 2-propanol (45 ml) and DCM (120 ml, removed on a rotavap) to give 2.77 g => initial lot (a) (HPLC 98.3% area) and 0.3 g of undissolved filtered off, by TLC right product. Initial lot (a) re- crystallised from 2-propanol (35 ml) and DCM (95 ml, removed on a rotavap) to give 2.51 g => initial lot (b) (HPLC 98.3% area). Combined with the above undissolved, re- crystallised from acetonitrile (200 ml, reflux to ice bath) to give 2.57 g => initial lot (c) (HPLC 98.8% area). Re-crystallised from acetonitrile (180 ml, reflux to 15°C) to give 2.25 g => Lot 1 (HPLC 99.2% area), mp 190-92°C. Lot 2 (Form A)

[0092] (R)-5-(2-(benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole- 2(3H)-thione (12 g, 29.9 mmol) was dissolved with heating to reflux in Tetrahydrofuran (300 ml), the solution was cooled to 5-10°C, Water (510 ml) was added slowly (approx 10 min) with stirring. The mixture was stirred for 1 h, solid was collected, washed with water, dried to give 11.73 g of product, by HPLC 1% of (R)-5-(2-Aminoethyl)-l-(6,8- difluorochroman-3-yl)-l,3-dihydroimidazole-2-thione hydrochloride and 1% of less polar impurity. The product was dissolved in Tetrahydrofuran (300 ml) with heating to reflux, 2- Propanol (150 ml) was added, the solution was concentrated to approx 100 ml (crystallisation occured), stirred in ice for 1.5 h. Solid was collected, washed with 2- propanol, dried to give 11.2 g of product, by HPLC 0.8% of (R)-5-(2-aminoethyl)-l-(6,8- difluorochroman-3-yl)-lH-imidazole-2(3H)-thione hydrochloride and 0.5% of less polar impurity. The product was dissolved in Tetrahydrofuran (300 ml) with heating to reflux, 2- Propanol (150 ml) was added, the solution was concentrated to approx 100 ml (crystallisation occured), stirred at 20-25°C for 1 h. Solid was collected, washed with 2- propanol, dried to give (R)-5-(2-(benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH- imidazole-2(3H)-thione (10.22 g, 25.5 mmol, 85 % yield).,

Lot 3 (form B)

To (R)-5-(2-aminoethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole-2(3H)- thione (2.36 g, 7.58 mmol) in a mixture of Methanol (15.00 ml) and Dichloromethane (15 ml) was added BENZALDEHYDE (0.845 ml, 8.34 mmol). To the resulting clear solution SODIUM CYANOBOROHYDRIDE (0.702 g, 10.61 mmol) was added in portions at 20- 25°C to avoid intensive foaming and the solution was stirred at 20-25°C for 40 h. The solution was quenched at 20-25°C with IN HC1 (12 ml), neutralised with 3N NaOH (12 ml), the mixture was extracted with DCM (100 ml). The organic phase was washed with brine, dried (MgS04), evaporated to dryness. The residue was purified on a column with EA-MeOH 9: 1 as eluent, fractions collected, concentrated to approx 20 ml, cooled in ice. The precipitate collected, washed with Ethyl Acetate-Petroleum Ether 1 : 1, dried on air to give (R)-5-(2-(benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole-2(3H)- thione (1.55 g, 3.86 mmol, 50.9 % yield). Lot 4 (Form A)

To a 500 mL flask set up for atmospheric distillation was added (R)-5-(2- (benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole-2(3H)-thione (20 g, 49,8 mmol) and Tetrahydrofuran (400 ml) to afford a suspension. The suspension was heated until full dissolution was achieved (61°C) whereupon it was filtered. The resulting solution was then heated to 66°C in order to commence the distillation. A mixture of Water (125 ml) & 2-Propanol (125 ml) was added at the same rate as the distillate was collected. The distillation was continued until 400 mL of distillate was collected. Crystallisation commenced after ~320 mL of distillate was collected. The suspension was cooled to 20°C and aged for 45 min. before filtering and washing with additional 2- propanol (80 mL) and then dried under vacuum at 50°C overnight to give (R)-5-(2- (benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole-2(3H)-thione (18.79 g, 94%). Lot 5 (Form A)

To a mixture of Methanol (66 L) and Water (10 L) at 20°C was added purified (R)-5-(2-(benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH-imidazole-2(3H)-thione hydrochloride (4.37 kg, 9.98 mol) to afford a suspension. The reaction mixture was then heated to 67°C to affect complete dissolution, whereupon IN Sodium hydroxide (10.48 L_s 10.48 mol, 1.05 eq) was added in a single portion. The reaction mixture was adjusted back to 67°C and held at 67°C for 30 min. The reaction mixture was then cooled to 20°C and aged at 20°C for at least 30 min. The reaction was then filtered and the filter cake washed with aqueous Methanol (1 : 1 v/v, 20 L), sucked down for 15 min. and then dried at 45°C under vacuum, to afford (R)-5-(2-(benzylamino)ethyl)-l-(6,8-difluorochroman-3-yl)-lH- imidazole-2(3H)-thione (3.855 kg, 96%) as a pale tan crystalline solid.

PATENT

WO 2015038022

http://www.google.com/patents/WO2015038022A1?cl=en

processes .

(J?) -5- (2-Aminoethyl) -1- (6, 8-difluorochroman-3-yl) -1, 3-dihydroimidazole-2 -thione hydrochloride (the compound of formula 1, below) is a potent, non-toxic and peripherally selective inhibitor of ϋβΗ, which can be used for treatment of certain cardiovascular disorders. Compound 1 is disclosed in WO2004/033447 , along with processes for its preparation.

The process disclosed in WO2004/033447 involves the reaction of ( R) – 6 , 8 -difluorochroman-3 -ylamine hydrochloride (the structure of ( R) -6, 8-difluorochroman-3 -ylamine is shown below as compound QA) , [4 – ( tert-butyldimethylsilanyloxy) -3 -oxobutyl] carbamic acid tert-butyl ester and potassium thiocyanate .

(R) -6 , 8-difluorochroman- 3 -ylamine (compound QA) is a key intermediate in the synthesis of compound 1. The stereochemistry at the carbon atom to which the amine is attached gives rise to the stereochemistry of compound 1, so it is advantageous that compound QA is present in as pure enantiomeric form as possible. In other words, the (R) -enantiomer of compound QA should be in predominance, with little or no (S) enantiomer present. Thus, the process for preparing compound QA will advantageously produce compound QA with as high enantiomeric excess (ee) as possible.

Advantageous processes for preparing, for example, the compound of formula QA have now been found. In one aspect, the processes involve a biotransformation step. In another aspect, the processes involve chemical transformation. The processes may also be employed in the preparation of similar precursors useful in the production of other peripherally-selective inhibitors of dopamine -β -hydroxylase .

WO2008/136695 discloses a compound of formula YA, its (R) or (S) enantiomer, a mixture of its (R) and (S) enantiomers, or pharmaceutically acceptable salts thereof.

The (R) -enantiomer of the compound of formula YA has been found to be a potent dopamines-hydroxylase inhibitor having high potency and significantly reduced brain access.

As disclosed in WO2008/136695 , the compound of formula YA may be prepared by reacting the compound of formula 1 with benzaldehyde under reductive alkylation conditions. In particular, (R) -5- (2 -aminoethyl ) -1- (6 , 8-difluorochroman-3 -yl) – 1 , 3 -dihydroimidazole-2 -thione and benzaldehyde may be reacted in the presence of a solvent or mixture of solvents, and a reducing agent such as sodium cyanoborohydride or sodium triacetoxyborohydride .

process comprises the following steps:

The route from 2 , 4-difluorophenol may be as described 9/064210.

Preferably, the reagents and conditions are:

(i) H₂S0₄, acetic acid

(ii) NaOCl, MeOH/water

(iii) Ru-based catalyst, H₂, 30 bars, MeOH

(iv) aqueous KOH, MeOH, L-tartaric acid

(v) KSCN, AcOH/lPA

(vi) NaBH₄, BF₃.THF complex, THF then IPA

n one aspect, the process comprises the following steps

i. KOH, Thioglycolic acid or cysteine

ii. MEK

According to an aspect of the present invention, there is provided the following 2 -part synthetic route from the starting material 2 , 4 -difluorophenol to (R) -5- (2 -aminoethyl ) -1- (6 , 8-difluorochroman-3 -yl) -1 , 3 -dihydroimidazole-2 – thione

hydrochloride :

Part (1)

Preferred reagents and conditions:

a) HMTA, CF₃COOH, 115°C, 18 hours

b) CH₂CHCN, DABCO, DMF, water, 70°C, 16 hours

c) H₂S0₄, AcOH, 100°C, 1 hour

d) NaClO, NaOH, MeOH, 25°C, 24 hours

e) (R) -C3 -TunePhosRu (acac) 2 S/C 3000, 30 bar H₂, MeOH, 80°C, 20 hours

f) Water, 2-propanol, reflux to 20°C

g) 40% KOH, MeOH, reflux, 24 hours

h) L-tartaric acid, ethanol, water, RT, 1 hour

Part (2)

Preferred reagents and conditions

a’) methyl vinyl ketone, t-BuONa, EtOAc, EtOH, 40-50°C, 2-3 hours

Br₂, MeOH, 20-25°C, 5 hours

water, reflux, 1 hour

KOH, AcOH, reflux, 1 hour

HCl, water, 2-propanol, 75 °C, 4 hours

KSCN, AcOH, 100°C, 2-4 hours

NaHC0₃, water, EtOH

NaBH₄, 2-propanol, THF, water, 20-25°C, 16 hours

HCl, 2-propanol, water, reflux, 1-2 hours

The ( R ) -5- (2-Aminoethyl) -1- (6, 8-difluorochroman-3 -yl) -1,3-dihydroimidazole-2 – thione hydrochloride may then be used to

prepare (R) -5- (2- (benzylamino) ethyl) -1- (6, 8-difluorochroman-3 -yl) -lH-imidazole-2 (3H) -thione as follows.

Preferred reaction conditions/reagents:

q) NaBH(OAc)₃, PhCHO, IPA;

t) NaOH, MeOH , H₂0

Either r) and s) :

r) HCI aq;

s) MeOH/Toluene;

Or n) , o) and p) :

n) HCI aq;

o) MeOH, toluene;

p) IPA.

EXAMPLES

Example 1

Nitro chromene synthesis

To 3 , 5-difluoro-2-hydroxybenzaldehyde (lOg, 63mmol, leq) , di-n-butylamine (4.1g, 32mmol, 0.5eq) , phtalic anhydride (18.7g, 126mmol, 2eq) in toluene (500mL) was added nitroethanol (5.75g, 63mmol, leq) . The round bottomed flask fitted with a dean stark apparatus was refluxed for 18h. The mixture was cooled and nitroethanol (5.75g, 63mmol, leq) was added. The resulting reaction mixture was then reflux for 12h. After cooling, the solution was evaporated down to approximately 150mL and purified over silica gel (eluent ethyl acetate : hexane 1:1) this gave several fractions that contained only the product by TLC, these was evaporated under reduced pressure to yield 1.8g which was 100% pure by HPLC aera. Several more fractions were collected containing a mixture of product and starting material. These were combined and washed with 2% NaOH solution (2x50mL) to remove starting material. The organic layer was washed with water (50mL) , dried over sodium sulfate and evaporated under reduced pressure to give 2.49g of brown solid ( 100% pure by HPLC aera) . More fractions were collected. These were combined, washed with 2% NaOH solution (3xl00mL) , water (lOOmL) and dried over sodium sulfate. This was then filtered and evaporated down in vacuum to yield 6.14g of a brown solid which was 91.3% pure by HPLC aera. 6 , 8 -difluoro-3 -nitro-2H-chromene (9.90g, 73.4%) was obtained as a brown solid.

Example 2

Nitro chromene synthesis with column purification

To a solution of isobenzofuran-1 , 3 -dione (4,68 g, 31,6 mmol) , 3 , 5-difluoro-2 -hydroxybenzaldehyde (2,5 g, 15,81 mmol) in Toluene (25 ml) was added 2 -nitroethanol (2,88 g, 31,6 mmol). The resulting mixture was heated to reflux overnight (Dean stark) .

The reaction conversion was checked by TLC (eluent PE/EtOAc 9:1) . A yellow spot was observed and corresponds to the expected product .

Reaction was cooled to room temperature and a plug of silica gel was performed. A pale brown solid (3.9g) was obtained. “””H-NMR showed presence of product and starting material. The solid was dissolved in diethylether and the organic layer was washed with aqueous sodium carbonate, dried over Na₂S0₄, filtered and concentrated under reduced pressure. A pale brown solid (1.7g,) was obtained. The ¹H-NMR was indicated no starting material but still polymer from nitroethanol and residue of phtalic anhydride. A second silica plug (eluent: PE/EtOAc 95:5) was done. A pale yellow solid (1.5g) was obtained. ¹H-NMR of solid showed only product and polymer. The solid was recrystallized from methanol/water . A pale yellow solid (1.05g, 31.2%) was obtained.

Example 3

Nitro chromene synthesis without column purification

To a solution of isobenzofuran- 1 , 3 -dione (18,74 g, 127 mmol) , 3 , 5-difluoro-2 -hydroxybenzaldehyde (10 g, 63,3 mmol) in Toluene (100 ml) was added 2 -nitroethanol (6,86 ml, 95 mmol) . The resulting mixture was heated to reflux for 24h (Dean stark) .

The reaction conversion was checked by HPLC and by ¹H-NMR. Only 50% conversion was obtained.

The reaction mixture was cooled to room temperature and diluted with DCM (lOOmL) and 1M NaOH solution (200mL) .

The biphasic system was stirred for 30 minutes and then separated (very difficult to see phase separation) . The aqueous layer was washed with DCM (50mL) and the combined organic layers were washed twice with water (2x50ml) , dried over sodium sulfate. The filtered organic layer was concentrated under reduced pressure. To the residue was added methanol (50mL) . The methanol was then removed by distillation under reduced pressure. A brown solution precipitated when most of the methanol was removed. More methanol was added and more solid crushed out then few drops of water was added to increase the product precipitation. The brown slurry was stirred for 30 minutes and filtered. The brown solid was washed with methanol/water (1:9, 5mL) and dried in a vacuum oven at 40°C for 12h.6, 8-difluoro-3 -nitro-2H-chroraene (4,9 g, 22,99 mmol,) was obtained as brown solid in 36.3% yield.

HPLC showed a purity of 98% and ¹H-NMR confirmed the structure and purity around 95%

Example 4

Reduction of nitro chromene to nitro-alkane (racemic mixture)

To a suspension of 6 , 8 -difluoro-3 -nitro-2H-chromene (213mg, 0,999 mmol) and silica (0,8 g, 0,999 mmol) in a mixture of CHC1₃ (10 ml) and IPA (3,4 ml) at 0°C was added portion wise sodium borohydride (95 mg, 2,498 mmol). The resulting mixture was stirred at 0°C for 45 minutes. Reaction conversion was checked by HPLC. 1 mL of acetic acid was added at 0°C and the resulting mixture was stirred for 30 minutes at room temperature. The slurry was filtered and the silica was washed with DCM. The filtrate was diluted with ethyl acetate and water and the biphasic system was separated. The aqueous layer was back extracted with ethyl acetate. The combined organic layers were washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure.

6 , 8-difluoro-3 -nitrochroman (196mg, 0,911 mmol, 91 % yield) was obtained as a pale yellow oil.

Example 5

Preparation of 6 , 8 -difluorochroman-3 -one from nitro chromene

A solution of 6, 8-difluoro-3 -nitro-2H-chromene (lOOmg, 0,469 mmol) in acetic acid (0.5 ml) is added slowly to a stirred slurry of iron (262 mg, 4,69 mmol) in acetic acid (1 ml) at 60.deg. C. The reaction mixture is stirred at 60. °C for 2 hour then allowed to cool to room temperature and stirred overnight. The reaction mixture is poured onto ice-water (30 ml) and filtered through Celite. The solid was wash with dichloromethane (DCM) (50 ml) . The organic portion is separated and washed with water (2 x 30 ml) and brine (30 ml) , dried over MgS0₄, filtered and concentrated in vacuo to give a brown oil. 6,8-difluorochroman-3 -one (75 mg, 0,407 mmol, 87 % yield) was obtained as a brown oil.

Example 6

Preparation of 6 , 8-difluorochroman-3 -one from methyl 6,8-difluoro-2H-chromen-3 -yl-carbamate

Methanol (1000m ml) was added to a slurry of methyl fluoro-2H-chromen-3 -yl -carbamate (250 g, 1.037 mol) hydrogen chloride 6N (2000 ml, 12 mol) at room temperature. The resulting mixture was reflux and stirred for 2 hours. Reaction monitored by HPLC.

Reaction was not complete but was stopped in order to avoid degradation of the product. The yellow solution was cooled to room temperature. A slurry (two type of solid) was observed and diluted with diethyl ether (300mL) . The resulting slurry was stirred at 5°C for 1 hour then filtered. The yellow solid was washed with water. The resulting wet yellow solid was suspended in diethylether (400mL) and petroleum ether (PE) (400mL) was added. Slight yellow solid was stirred at room temperature overnight, filtered and washed with PE (300mL) , dried in a vacuum oven at 30 °C for 4h. The wet sample was checked by NMR. No starting material was detected. A pale yellow solid (72.5g, solid 1) was obtained. The mother liquors were concentrated to dryness. A yellow solid was obtained, suspended in diethyl ether and PE. The slurry was then stirred for 4 hours, filtered, washed with PE . A dark yellow solid (4.5g, solid 2) was obtained. Solid 1 (2g) was diluted in DCM and washed with water (pH =6). The organic layer was then dried over Na₂S0₄, filtered, concentrated to dryness. A crystalline pale yellow solid (1.9g, solid 3) was obtained. NMR showed the same purity for solid 3 as for solid 1. The remaining part of solid 1 was then diluted in DCM. The resulting organic layer was washed with water, dried over Na₂S0₄, filtered and then concentrated to dryness. Slight yellow crystalline solid (68.5g, solid 4) was obtained. NMR confirmed high quality material.

Loss on Drying (LOD) : 1.03% .

Example 7

Biotransformation: Transaminases

Codexis transaminases ATA-025, ATA-251 and ATA-P2-A07 recognized 6 , 8 -difluorochroman-3 -one as the substrate and produced the corresponding 6 , 8 -difluorochroman-3 -amine .

PATENT

WO 2014077715

WO 2013002660

WO 2008136695

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International Journal of Pharmaceutics (Amsterdam, Netherlands) (2016), 501(1-2), 102-111.

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