Home » Uncategorized (Page 73)

Category Archives: Uncategorized

New Website ECA Validation Group: Version 02 of ECA´s Good Practice Guide on Validation online available

February 4, 2016 3:26 am / Leave a comment

The ECA Validation Group was founded in autumn 2011 by representatives of the pharmaceutical industry after ECA´s 4th European GMP Conference. The mission of the group is to assemble knowledge on Validation, for example by continuously developing ECA´s Process Validation Good Practice Guide. Now the Validation Group launched a new website.

Since the ECA Foundation was established back in 1999 its mission has been to provide support to the Pharmaceutical Industry and Regulators to promote the move towards a harmonised set of GMP and regulatory guidelines by providing information and interpretation of new or updated guidances. For that purpose the ECA has initiated and established various working and interest groups concentrating on different topics.

The ECA Validation Group was founded in autumn 2011 by representatives of the pharmaceutical industry after ECA´s 4th European GMP Conference. This group’s mission is to assemble knowledge on Validation, for example by continuously developing ECA´s Process Validation Good Practice Guide.

Now the group launched its new website to provide members and those interested with information and practical tools. Here’s what you can find on the new website:

Current News
A news archive
Training Courses and Validation Conferences
ECA´s Process Validation Good Practice Guide
Discussion Forum
Presentations
Useful links
Q&A section
Membership information

Members of the group have now the opportunity to download the version 2 of ECA´s Good Practice Guide on Validation free of charge. On 174 pages the revised Good Practice Guide comprises the main elements of the new validation approach (“what to do”). On the other hand, it also serves as a supporting guide for the implementation (“how to do”).

To find out more we invite you to visit the ECA´s Validation Group new website.

//////

PF 04995274, a 5-HT4Partial Agonist

February 3, 2016 5:19 am / Leave a comment

PF-04995274,

(R)-4-((4-(((4-(Tetrahydrofuran-3-yloxy)-1,2-benzisoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol

4-(4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidin-1-ylmethyl)-tetrahydro-pyran-4-ol

CAS 1331782-27-4
UNII: XI179PG9LV

MF C23-H32-N2-O6

MW 432.5138

a 5-HT4Partial Agonist

PHASE 1 Alzheimer’s type dementia.

Pfizer Inc. INNOVATOR

5-HT₄ agonists have attracted attention for therapeutic value in the treatment of Alzheimer’s Disease (AD) and cognitive impairment.Acting to increase levels of acetylcholine and soluble APP alpha, 5-HT₄ agonists have the potential to demonstrate both ameliorative and disease modifying effects

(R)-4-((4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2/-/-pyran-4-ol and pharmaceutically acceptable salts thereof. This invention also is directed, in part, to a method for treating a 5-HT₄ mediated disorder in a mammal. Such disorders include acute neurological and psychiatric disorders, stroke, cerebral ischemia, spinal cord trauma, head trauma, perinatal hypoxia, cardiac arrest, hypoglycemic neuronal damage, dementia, Alzheimer’s disease, Huntington’s Chorea, amyotrophic lateral sclerosis, ocular damage, retinopathy, cognitive disorders, idiopathic and drug- induced Parkinson’s disease, muscular spasms and disorders associated with muscular spasticity including tremors, depression, epilepsy, convulsions, migraine, urinary incontinence, substance tolerance, substance withdrawal, psychosis, schizophrenia, anxiety, mood disorders, trigeminal neuralgia, hearing loss, tinnitus, macular degeneration of the eye, gastroesophageal reflux disease, gastrointestinal disease, gastric motility disorder, non-ulcer dyspepsia, functional dyspepsia, irritable bowel syndrome, constipation, dyspepsia, esophagitis, gastroesophageral disease, nausea, emesis, brain edema, pain, tardive dyskinesia, sleep disorders, attention deficit/hyperactivity disorder, attention deficit disorder, disorders that comprise as a symptom a deficiency in attention and/or cognition, and conduct disorder

^a(a) SOCl₂, DMAP, acetone, DME, RT, 81%;

(b) DEAD, PPh₃, THF, RT, 65%;

(d) K₂CO₃, water, MeOH, 50 °C, 76%;

(e) CDI, THF, 50 °C, 43%;

(f) DEAD, PPh₃, THF, reflux, 51%;

(g) HCl, Et₂O, RT, 81%;

(h) TEA, MeOH, reflux, 50%.

PAPER

Journal of Medicinal Chemistry (2012), 55(21), 9240-9254

http://pubs.acs.org/doi/abs/10.1021/jm300953p

The cognitive impairments observed in Alzheimer’s disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT₄) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT₄ agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure–response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT₄ partial agonist candidates 2d and 3 were chosen for clinical development.

PATENT

https://www.google.co.in/patents/WO2011101774A1?cl=en

(R)-4-((4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol , hereinafter referred to as “Compound X,” and having the following structure:

Compound X

Example 1 : Synthesis of iR)-4-ii4-i(4-itetrahvdrofuran-3-yloxy)benzord1isoxazol-3-yloxy)methyl)piperidin-1 -yl)methyl)tetrahvdro- 2 -pyran-4-ol

Methyl 2-fluoro-6-hydroxybenzoate (2): To a 20L jacketed reactor were charged 2-fluoro-6-hydroxybenzoic acid (Oakwood Products; 0.972 kg, 6.31 mol), methanol (7.60 L) and sulfuric acid (0.710 kg, 7.24 mol, 1 .15 eq). The jacket temperature was heated to 60°C and the reaction mixture was stirred for 45 h. The reaction mixture was concentrated under vacuum and approximately 7.5 L of methanol distillates were collected. The resulting thin oil was cooled to 20°C. Water (7.60 L) and ethyl acetate (7.60 L) were charged to the reactor, and the product extracted into the organic layer. The EtOAc solution was washed with a solution of sodium bicarbonate (1.52 Kg) in water (6.92 L) followed by a brine solution of sodium chloride (1.74 kg) in water (4.08 L). The resulting EtOAc solution was concentrated to dryness. A light orange oil was isolated; the oil slowly crystallized upon standing to give the title compound (2) (0.952 Kg, 5.60 mol, 89% yield). ¹ H NMR (400 MHz, CDCI₃) δ ppm 3.97 (s, 3H), 6.59 (ddd, J=10.9, 8.2,1 .2, 1 H), 6.76 (dt, J=8.2, 1 .1 , 1 H), 7.35 (td, J=8.6, 6.3, 1 H), 1 1.24 (s, 1 H); ¹³C NMR (400 MHz, CDCI₃) δ ppm 52.65, 102.56 (d, J=13), 106.90 (d, J=23), 1 13.31 (d, J=3.1 ), 135.34 (d, J=1 1 .5), 161 .02, 163.31 (d, J=62.2), 169.87 (d, 3.8); MS 171.045 (m+1 ). 2-Fluoro-N,6-dihydroxybenzamide (3): To a 50L reactor was charged water (4.47 L) and hydroxylamine sulfate (6.430 kg, 39.17 mol), the mixture was stirred at 25°C. A solution of potassium carbonate (3.87 Kg, 27.98 mol) in water (5.05 L) was slowly added to the reaction mixture to form a thick white mixture that was stirred at 20°C. A solution of methyl 2-fluoro-6-hydroxybenzoate (2) (0.952 Kg, 5.60 mol) in methanol (9.52 L) was slowly added to the reactor resulting in mild off gassing. The reaction mixture was then heated to 35°C and stirred for 20 h. The reaction mixture was cooled to 15°C and stirred for 1 h. The mixture was filtered to remove inorganic material. The reactor was rinsed with methanol (2.86 L) and the tank rinse was used to wash the inorganic cake.

Analysis of the cake indicated that it contained product. To a 20L reactor was charged methanol (10 L) and the inorganic cake and the mixture was stirred at 25°C for 30 min. The mixture was filtered and the cake washed with methanol (3 L).

The combined filtrates were charged back into the reactor and concentrated under vacuum with the jacket temperature set at 40°C until approximately 10 L remained. The mixture was held at 25°C and cone. HCI (5.51 L) was added. The reactor was cooled to 15°C and stirred for 2 h. The white slurry was filtered and the resulting product cake was washed with water (4.76L), blown dry with nitrogen and then dried in a vacuum oven at 40°C for 12 h. The desired product (3) (747 g, 4.36 mol), was isolated in 78% yield. ¹ H NMR (400 MHz, CD₃OD) δ ppm 4.91 (s, 3H), 6.63 (ddd, J=10.9, 8.5, 0.8, 1 H), 6.72 (dt, J=8.2, 0.8, 1 H), 7.31 (td, J=8.2, 6.6, 1 H); MS 172.040 (m+1 ).

4-Fluorobenzo[d]isoxazol-3-ol (4): To a 20L jacketed reactor were charged tetrahydrofuran (2.23 L) and 1 ,1 ‘-carbonyldiimidazole (0.910 Kg, 5.64 mol). The resulting mixture was stirred at 20°C. Then a solution of 2-fluoro-N,6-dihydroxybenzamide (3) (744 g, 4.34 mol) in tetrahydrofuran (4.45 L) was slowly charged to the reactor maintaining the temperature below 30°C and stirred at 25°C for 30 min during which some off gassing was observed. The reaction mixture was heated to 60°C over 30 min and stirred for 6 h. The reactor was cooled to 20°C followed by the addition of 1 N aqueous hydrogen chloride (7.48L) over 15 min to adjust the pH to 1. The jacket temperature was set to 35°C and the reaction mixture concentrated under vacuum to remove approximately 6.68L of THF. The reactor was cooled to 15°C and stirred for 1 h. The resulting white slurry was filtered, the cake was washed with water (3.71 L) and dried in a vacuum oven at 40°C for 12 h. The desired product, (4) (597 g, 3.90 mol), was isolated in 90% yield. ¹ H NMR (400 MHz, CD₃OD) δ ppm 4.93 (b, 1 H), 6.95 (dd, J=10.1 , 8.6, 1 H), (d, J=8.6, 1 H), 7.52-7.57 (m, 1 H); LRMS 154.029 (m+1 ).

Tert-butyl 4-(tosyloxymethyl)piperidine-1-carboxylate (5): To a 20L jacketed reactor were charged dichloromethane (8 L), N-boc-4-piperdine methanol (0.982 Kg, 4.56 mol) and p-toluenesulfonyl chloride (0.970 Kg, 5.09 mol) and the resulting mixture was stirred at 20°C for 5 min. Triethylamine (0.94 Kg, 9.29 mol) was added to the reactor via an addition funnel and the resulting deep red solution was stirred at 25°C for 16 h. A solution of sodium carbonate (0.96 Kg, 9.06 mol) in water (7.04 L) was charged to the reaction mixture and stirred for 1 h at 20°C. The phases were split and the organic layer washed with brine (6 L) and concentrated at 40°C to a low stir volume. Dimethylacetamide (2 L) was charged to the reactor and concentration continued under full vacuum at 40°C for 1 h. The solution of tert-butyl 4-(tosyloxymethyl)piperidine-l -carboxylate (5) in dimethyl acetamide was held for further processing. Yield was assumed to be 100% with approximately

90% potency. A sample was pulled and concentrated to dryness for purity analysis. ¹ H NMR (400 MHz, CDCI₃) δ ppm 1 .02-1 .12 (m, 2H), 1.14 (s, 9H), 1 .59-1.64 (m, 2H), 1.75-1.87 (m, 1 H), 2.43 (s, 3H), 2.55-2.75 (m, 2H), 3.83 (d, J=6.7, 2H), 3.95-4.20 (b, 2H), 7.33 (d, 8.6, 2H), 7.76 (d, 8.2, 2H); ¹³C NMR (400 MHz, CDCI₃) δ ppm 21 .64, 28.15, 28.39, 35.74, 73.97, 79.50, 126.99, 127.84, 129.86, 132.84, 144.84, 154.63; LRMS 739.329 (2m+1 ).

Tert-butyl 4-((4-fluorobenzo[d]isoxazol-3-yloxy)methyl)piperidine-1-carboxylate (6): To a 20L jacketed reactor were charged dimethylacetamide (4.28 L), tert-butyl 4-(tosyloxymethyl)piperidine-1 -carboxylate (5) (1.68 Kg, 4.56 mol), 4-fluorobenzo[d]isoxazol-3-ol (4) (540 g, 3.51 mol), and potassium carbonate (960 g, 6.98 mol) resulting in a thick beige slurry. The reaction mixture was heated to 50°C and stirred for 20 h and then cooled to 20°C, followed by the addition of water (7.5 L) and ethyl acetate (5.37 L). After mixing for 15 min, the phases were settled and split. The organic layer was washed with water (5.37 L), sending the aqueous wash to waste. The organic mixture was distilled under vacuum with a maximum jacket temperature of 40°C until approximately 5 L remained in the reactor. Methanol (2.68 L) was added and the resulting solution concentrated under vacuum to about 3 L of a yellow oil. Methanol (2.68 L) was charged to the reactor and the resulting solution was stirred at 25°C for 15 min. Water (0.54 L) was added over 15 min resulting in a white slurry. The mixture was cooled to 15°C, stirred for 1 h and then filtered. The filter cake was washed with a solution of water (0.54 L) in methanol (2.14 L), then air dried for 30 min, transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (6) (746 g, 2.13 mol), was isolated in 61 % yield. ¹ H NMR (400 MHz, CDCI₃) δ ppm 1.23-1 .37 (m, 2H), 1 .45 (s, 9H), 1 .78-1 .88 (m, 2H), 2.04-2.17 (m, 1 H), 2.67-2.83 (m, 2H), 4.02-4.26 (m, 2H), 4.28 (d, 6.6, 2H), 6.89 (dd, J=8.6, 7.5, 1 H), 7.21 (d, J=9, 1 H), (td, 8.6, 4.9); LRMS 351.171 (m+1 ).

(R)-Tert-butyl 4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidine-1-carboxylate (8): To a 20 L glass reactor with the jacket set to 20°C were charged (R)-tetrahydrofuran-3-ol (7) (297 g, 3.37 mol) and dimethylacetamide (5.1 L). 2.0 M sodium bis(trimethylsilyl)amide in THF (1.37 L, 2.74 mol) was slowly added via an addition funnel while maintaining a pot temperature less than 30°C. The resulting orange/red solution was stirred at 25°C for 30 min. Then, tert-butyl 4-((4-fluorobenzo[d]isoxazol-3-yloxy)methyl)piperidine-1 -carboxylate (6) (640.15 g, 1.83 mol) was charged and the reaction mixture was stirred at 25°C for 16 h. The reaction mixture was cooled to 20°C and water (6.4 L) was slowly added over 45 min maintaining a pot temperature of less than 35°C. Ethyl acetate (6 L) was added and the biphasic mixture was stirred for 15 min and then separated. The aqueous layer was back extracted with additional ethyl acetate (4 L). The combined organics were then washed with water (5 L) and a 20% brine solution (5 L). The organic mixture was concentrated under vacuum with the jacket temperature set to 40°C to approximately 3 L and held for further processing. Quantitative yield of the desired product, (8) (0.76 Kg, 1 .82 mol), in ethyl acetate was assumed. A sample was pulled and concentrated to dryness for purity analysis. ¹ H NMR (400 MHz, CDCI₃) δ ppm 1 .25-1.38 (m, 2H), 1 .44 (s, 9H), 1.76-1 .84 (m, 2H), 1 .89-1.97 (b, 1 H), 1 .99-2.12 (m, 1 H), 2.14-2.28 (m, 2H), 2.63-2.84 (m, 2H), 3.90-4.21 (m, 6H), 4.24 (d, J=6.3, 2H), 5.00-5.05 (m, 1 H), 6.48 (d, J=8.2, 1 H), 6.98 (d, J=8.6, 1 H), 7.37 (t, J=8.2, 1 H); LRMS 419.216 (m+1 ).

(R)-3-(Piperidin-4-ylmethoxy)-4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazole 4-methylbenzenesulfonate (9): To a 20L jacketed reactor charged ethyl acetate (6.1 L), (R)-tert-butyl 4-((4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidine-1 -carboxylate (8) (0.76 kg, 1 .82 mol) and p-toluenesulfonic acid monohydrate (0.413 kg, 2.17 mol) and stirred at 20°C for 30 min. The reactor jacket was heated from 20 to 65°C over

1 h and then held at 65°C for 16 h. The reactor was cooled to 15°C over 1 h and granulated for 2 h. The resulting slurry was filtered, the cake was washed with EtOAc (3 L) and then air dried on the filter for 30 min. The cake was transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (9) (854 g, 1.74 mol), was isolated in 96% yield (two steps). ¹ H NMR (400

MHz, CD₃OD) δ ppm 1.54-1 .67 (m, 2H), 2.04-2.18 (m, 3H), 2.19-2.36 (m, 2H), 2.33 (s, 3H), 3.01 -3.12 (m, 2H), 3.41-3.50 (m, 2H), 3.86-4.01 (m, 4H), 4.26 (d, J=6.3, 2H), 4.90 (s, 2H), 5.14-5.19 (m, 1 H), 6.72 (d, J=8.2, 1 H), 7.02 (d, J=8.6, 1 H), 7.21 (d, J=7.8, 2H), 7.48 (t, J=8.6, 1 H), 7.70 (d, J=8.2, 2H); LRMS 319.165 (m+1 ).

(R)-4-((4-((4-(Tetrahydrofuran-3-yloxy)benzo[d]isoxazol-3-yloxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (11): To a

20L jacketed reactor were charged water (7.5 L) and sodium carbonate (0.98 kg); the mixture was stirred at 20°C until all solids had dissolved. Then (R)-3-(piperidin-4-ylmethoxy)-4-(tetrahydrofuran-3-yloxy)benzo[d]isoxazole 4-methylbenzenesulfonate (9) (750 g, 1 .53 mol) and ethyl acetate (6.0 L) were added to the reactor and stirred at 20°C for 30 min. The phases were split and the lower aqueous layer was back extracted twice with ethyl acetate (6.0 L and then 3.75 L). The organic layers were combined in the 20L reactor and washed twice with brine (3.0 L). The ethyl acetate solution was concentrated to under vacuum at 45°C to a low stir volume. Isopropyl alcohol (3.75 L) was added and concentration continued until 2 L remained in the reactor.

Additional isopropyl alcohol (2.75 L) was added and the mixture cooled to 25°C. To the reactor was charged 1 ,6-dioxaspiro[2.5]octane (10) (260 g, 2.29 mol) and the resulting solution heated to 50°C and stirred for 16 h. The reaction mixture was cooled to 30°C and water (15 L) was added over 60 min. Product crystallized from solution and the resulting slurry was cooled to 15°C over 1 h and then granulated for 4 h. The product was filtered and washed with water (3.75 L). The cake was blown dry with nitrogen for 30 min and then transferred to a vacuum oven and dried at 40°C for 12 h. The desired product, (11 ) (588 g, 1 .36 mol), was isolated in 89% yield.

¹ H NMR (400 MHz, CDCI₃) δ ppm 1 .41-1 .63 (m, 6H), 1.71 -1.81 (m, 2H), 1.81 -1.94 (m, 1 H), 2.17-2.26 (m, 2H), 2.33 (s, 2H), 2.4 (td, J=1 1.7, 2.3, 2H), 2.92 (d, J=1 1 .8, 2H), 3.46 (s, 1 H), 3.71-3.84 (m, 4H), 3.91 -4.10 (m, 4H), 4.24 (d, J=5.9, 2H), 5.03-5.08 (m, 1 H), 6.50 (d, J=8.2, 1 H), 7.00 (d, J=8.2, 1 H), 7.38 (t, J=8.2, 1 H);

¹³C NMR (400 MHz, CDCI₃) δ ppm 29.1 1 , 33.10, 35.20, 36.92, 36.96, 56.15, 63.93, 67.14, 67.46, 68.27, 72.94, 74.06, 78.37, 103.17, 105.15, 131.71 , 152.71 , 166.02, 166.28;

LRMS 433.232 (m+1 ).

Example 2: Synthesis of iR)-4-ii4-i(4-itetrahvdrofuran-3-yloxy)benzord1isoxazol-3-yloxy)methyl)piperidin-1 -yl)methyl)tetrahvdro- 2H-pyran-4-ol

5-Hydroxy-2,2-dimethyl-benzo[1,3]dioxin-4-one: Thionyl chloride (83.8 g, 0.71 mol) was slowly added to a solution of 2,6-dihydroxy-benzoic acid (77 g, 0.5 mol), acetone (37.7 g, 0.65 mol) and DMAP (3.1 g, 0.025 mol) in dimethoxyethane (375 mL). The mixture was stirred at RT for 7 h. The residue obtained after concentration under reduced pressure was dissolved in ethyl

acetate and washed with water and aqueous saturated sodium bicarbonate solution. The organic layer was dried (Na₂S0₄) and concentrated to afford 79 g desired product as a red solid (81 % yield). ¹ H NMR (400 MHz, CDCI₃) δ ppm 1 .68 (s, 6H), 6.37 (dd, J=8, 0.8, 11-1) 6.56 (dd, J=8, 0.8, 1 H), 7.34 (t, J=8, 1 H), 10.27( brs, 1 H).

2,2-Dimethyl-5-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[1,3]dioxin-4-one:

Diethyl azodicarboxylate (130.5 g, 0.75 mol) was added in a dropwise fashion to a mixture of 5-hydroxy-2,2-dimethyl-benzo[1 ,3]dioxin-4-one (100 g, 0.51 mol), triphenylphosphine (196.5 g, 0.75 mol), and (S)-tetrahydro-furan-3-ol (44 g, 0.5 mol) in 600 ml. of anhydrous THF. The resulting mixture was stirred at RT for 18 h. The solvent was removed under reduced pressure and the crude material was purified on a silica gel flash column, eluting with petroleum ether/ ethyl acetate (15:1 -> 3:1 ). 86 g (65% yield) of product was isolated as a colorless oil. ¹ H NMR (400 MHz, CDCI₃) δ ppm 1.67 (s, 6H), 2.30 (m, 2H), 4.2 (m, 4H) 4.97 (m, 1 H), 6.49 (d, J=8.4, 1 H) 6.51 (d, J=8.4, 1 H), 7.39 (t,

J=8.4, 1 H).

2-Hydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzoic acid methyl ester: Potassium carbonate (134.8 g, 0.98 mol) was added to a solution of 2,2-dimethyl-5-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[1 ,3]dioxin-4-one (86 g, 0.33 mol) in 1 L methanol. The mixture was stirred at RT for 2 h, then concentrated in vacuo. The residue was dissolved in ethyl acetate and washed with aqueous ammonium chloride solution. The organic layer was dried (Na₂S0₄) and concentrated to afford 72 g of the product as a yellow solid (92% yield). ¹ H NMR (400 MHz, CDCI₃) δ ppm 2.20 (m, 2H), 3.99 (s, 3H), 4.80(m, 4H). 4.94 (m, 1 H), 6.31 (dd, J=8.4, 0.8, 1 H), 6.59 (dd, J=8.4, 0.8, 1 H), 7.30 (t, J=8.4, 1 H).

2,N-Dihydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzamide: Potassium carbonate (121 g. 0.867mmol) was added portionwise to a solution of hydroxylamine sulfate (120 g, 0.732 mol) in 360 ml. of water at 0°C. After stirring for 30 min, sodium sulfite (3.74 g, 0.029 mol) and a solution of 2-hydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzoic acid methyl ester (35 g, 0.146 mol) in 360 ml. of methanol were added and the mixture was stirred at 50°C for 30 h. Methanol was removed from the cooled reaction mixture under reduced pressure and the resulting aqueous layer was acidified with 2N HCI. The aqueous layer was extracted with ethyl acetate and the organic layer was dried (Na₂S0₄) and concentrated to afford 25 g (76% yield ) of the product as a yellow solid. ¹ H NMR (400 MHz, CDCI₃) δ ppm 2.00 (m, 1 H), 2.15 (m, 1 H), 3.80 (m, 4H), 5.05 (m, 1 H), 6.48 (d, J=8, 1 H), 6.49 (d, J=8, 1 H), 7.19 (t, J=8, 1 H), 10.41 (brs, 1 H), 1 1.49 (brs, 1 H); LRMS m/z 239 (m+1 ).

4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-ol: A solution of 2, N-dihydroxy-6-[(R)-(tetrahydro-furan-3-yl)oxy]-benzamide (25 g, 0.105 mol) in 250 ml. of THF was heated to 50°C. Carbonyl diimidazole was added portionwise and the resulting mixture was stirred at 50°C for 14 h. After cooling to RT, 100 ml. of 2N HCI was added and the aqueous layer was extracted with ethyl acetate. The combined organic layers were then extracted three times with 10% aqueous potassium carbonate. The potassium carbonate aqueous extracts were washed with ethyl acetate and then acidified to pH 2 – 3 with 2N HCI. The acidified aqueous layer was extracted with ethyl acetate. The ethyl acetate extracts were washed with brine, dried (Na₂S0₄) and concentrated to afford 20 g of product as a yellow solid (43% yield). ¹ H NMR (400 MHz, CDCI₃) δ ppm 2.20 (m, 2H), 3.89 (m, 1 H), 4.01 (m, 3H), 5.05 (m, 1 H), 6.48 (d, J=7.6, 1 H). 6.92 (d, J=7.6, 1 H), 7.37 (t, J=7.6, 1 H); LRMS m/z 222 (m+1 ).

4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidine-1-carboxylic acid tert-butyl ester: Diethyl azodicarboxylate (15.6 g, 0.09 mol) was added to a mixture of 4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-ol (10 g, 0.045 mol), 4-hydroxymethyl-piperidine-1 -carboxylic acid tert-butyl ester (1 1.6 g, 0.054 mol) and triphenylphosphine (23.5 g, 0.09 mol) in 300 mL THF. After the addition was complete the mixture was heated at reflux for 18 h. After concentration in vacuo, the crude product was purified on a silica gel flash column, eluting with petroleum ether/ ethyl acetate (15:1 -» 5:1 ) to afford 22 g of the product as an oil (51 % yield). ¹ H NMR (400 MHz, CDCI₃) δ ppm 1.25 (m, 2H), 1.39 (s, 9H), 1.76 (m, 2H), 1.99 (m, 1 H). 2.15 (m, 2H), 2.70 (bt, J=1 1.6, 2H), 3.95 (m, 4H). 4.13 (m, 2H). 4.34 (d J=6.4, 2H), 4.98 (m, 1 H), 6.43 (d, J=8, 1 H), 6.93 (d, J=8, 1 H), 7.31 (t, J=8, 1 H).

3-(Piperidin-4-ylmethoxy)-4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazole: A 0°C solution of 4-{4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidine-1 -carboxylic acid tert-butyl ester in 500 mL ether was treated with a saturated solution of HCI (g) in 200 mL ether. After addition was complete, the mixture was warmed to RT and stirred for 16 h. The reaction mixture was filtered. The white solid was washed with ethyl acetate followed by ether and dried to yield 15 g (81 % yield) of the desired product as a white solid. ¹ H NMR (400 MHz, CD₃OD) 5 ppm 1 .51 – 1.69 (m, 2 H) 2.04 – 2.19 (m, 3 H) 2.22 – 2.37 (m, 2 H) 2.99 – 3.14 (m, 2 H) 3.40 – 3.51 (m, 2 H) 3.85 – 4.02 (m, 4 H) 4.25 – 4.31 (m, 2 H) 5.17 (td, J= >1^ , 1 .56 Hz, 1 H) 6.72 (d, J=8.00 Hz, 1 H) 7.01 (d, J=8.59 Hz, 1 H) 7.47 (t, J=8.20 Hz, 1 H); LRMS m/z 319 (m+1 ).

4-(4-{4-[(R)-(Tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazol-3-yloxymethyl}-piperidin-1-ylmethyl)-tetrahydro-pyran-4-ol: 1 ,6-Dioxa-spiro[2.5]octane (Focus Synthesis; 9.7 g, 0.084 mol) and triethylamine (8.6 g, 0.084 mol) were added to a solution of 3-(piperidin-4-ylmethoxy)-4-[(R)-(tetrahydro-furan-3-yl)oxy]-benzo[d]isoxazole (15 g, 0.042 mol) in 200 mL methanol. The resulting solution was heated at reflux for 18 h. The cooled mixture was concentrated and ethyl acetate and water were added to the residue. The layers were separated and the organic extracts were washed with brine, dried (Na₂S0₄) and concentrated to provide 17 g crude product as a yellow oil. The crude material was purified by prep HPLC to afford 10 g of the desired product as a white solid. (50% yield).

¹ H NMR (400 MHz, CDCI₃) δ ppm 1.41 -1.63 (m, 6H), 1.71-1.81 (m, 2H), 1 .81 -1 .94 (m, 1 H), 2.17-2.26 (m, 2H), 2.33 (s, 2H), 2.4 (td, J=1 1 .7, 2.3, 2H), 2.92 (d, J=1 1.8, 2H), 3.46 (s, 1 H), 3.71-3.84 (m, 4H), 3.91-4.10 (m, 4H), 4.24 (d, J=5.9, 2H), 5.03-5.08 (m, 1 H), 6.50 (d, J=8.2, 1 H), 7.00 (d, J=8.2, 1 H), 7.38 (t, J=8.2, 1 H);

¹³C NMR (101 MHz, CDCI₃) δ ppm 29.1 1 , 33.10, 35.20, 36.92, 36.96, 56.15, 63.93, 67.14, 67.46, 68.27, 72.94, 74.06, 78.37, 103.17, 105.15, 131.71 , 152.71 , 166.02, 166.28.

PAPER

Two Routes to 4-Fluorobenzisoxazol-3-one in the Synthesis of a 5-HT₄Partial Agonist

Daniel W. Widlicka^*^†, John C. Murray^†, Karen J. Coffman^†, Chunguang Xiao^‡, Michael A. Brodney^†, Joseph P. Rainville^†, and Brian Samas^†

^† Groton Laboratories, Worldwide Research & Development, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340,United States

^‡ Porton Fine Chemical, 1 Fine Chemical Zone, Chongqing Chemical Industrial Park, Changshou, Chongqing 401221China

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00389

Publication Date (Web): February 2, 2016

*E-mail: daniel.w.widlicka@pfizer.com.

http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00389

A potent 5-HT₄ partial agonist, 1 (PF-04995274), targeted for the treatment of Alzheimer’s disease and cognitive impairment, has been prepared on a multi-kilogram scale. The initial synthetic route, that proceeded through a 4-substituted 3-hydroxybenzisoxazole core, gave an undesired benzoxazolinone through a Lossen-type rearrangement. Route scouting led to two new robust routes to the desired 4-substituted core. Process development led to the efficient assembly of the API on a pilot plant scale under process-friendly conditions with enhanced throughput. In addition, crystallization of a hemicitrate salt of the API with pharmaceutically beneficial properties was developed to enable progression of clinical studies.

REFERNCES

Noguchi, H.; Waizumi, N. Preparation of benzisoxazole derivatives for treatment of 5-HT4 mediated disorders. PCT Int. Appl. WO/2011/101774 A1, 20110825

////////PF-04995274, PF 04995274, PFIZER, Alzheimer’s type dementia, PHASE 1

c1cc2c(c(c1)O[C@@H]3CCOC3)c(no2)OCC4CCN(CC4)CC5(CCOCC5)O

New TRPV1 Antagonist From Neurogen Corporation

February 1, 2016 3:50 am / Leave a comment

MK ? NGD?

MK 2295; NGD 8243 may be???????

CAS 878811-00-8 FREE FORM

Molecular Formula:	C₂₇H₃₁FN₆O₂
Molecular Weight:	490.572443 g/mol

6-[(3R)-4-[6-(4-fluorophenyl)-2-[(2R)-2-methylpyrrolidin-1-yl]pyrimidin-4-yl]-3-methylpiperazin-1-yl]-5-methylpyridine-3-carboxylic acid

6-{4-[6-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidin-4-yl]-3-(R)-methyl-piperazin-1-yl}-5-methyl-nicotinic acid

3-Pyridinecarboxylic acid, 6-[(3R)-4-[6-(4-fluorophenyl)-2-[(2R)-2-methyl-1-pyrrolidinyl]-4-pyrimidinyl]-3-methyl-1-piperazinyl]-5-methyl-

Neurogen Corp INNOVATOR

MESYLATE

CAS 1855897-95-8

6-((R)-4-(6-(4-Fluorophenyl)-2-((R)-2-methylpyrrolidin-1-yl)pyrimidin-4-yl)-3-methylpiperazin-1-yl)-5-methylnicotinic acid methanesulfonic acid salt

white solid. ¹H NMR (CD₃OD, 400 MHz) δ 1.37 (d, 3H, J= 6.4 Hz), 1.48 (d, 3H, J = 6.7 Hz), 1.84 (m, 1H), 2.09 (m, 1H), 2.17–2.25 (m, 2H), 2.42 (s, 3H), 2.66 (s, 3H), 3.10 (dt, 1H, J = 12.3 and 3.3 Hz), 3.28 (dd, 1H, J = 13.1 and 3.7 Hz), 3.65–3.72 (m, 3H), 3.78 (m, 1H), 3.87 (m, 1H), 4.49 (m, 1H), 4.63 (m, 3H), 4.96 (br m, 1H), 6.61 (s, 1H), 7.32 (m, 2H), 7.82 (m, 2H), 8.05 (m, 1H), 8.69 (d, 1H, J = 1.9 Hz);

¹³C NMR (CD₃OD, 125 MHz) δ 19.4, 24.5, 33.5, 39.6, 41.5, 48.6, 50.0, 50.9, 54.1, 56.9, 94.8, 117.3 (d, J = 22.5 Hz), 122.1, 125.0, 130.1 (d, J = 3.3 Hz), 131.8 (d, J = 8.9 Hz), 142.1, 148.7, 153.1, 153.3, 162.4, 165.4, 166.4, (d, J = 251.3 Hz), 168.8;

¹⁹F NMR (CD₃OD, 470 MHz) δ −108.6.

Anal. Calcd For C₂₈H₃₅FN₆O₅S: C, 57.32; H, 6.01; N, 14.32. Found: C, 57.34; H, 6.13; N, 14.29.

Activated by a wide range of stimuli such as capsaicin, acid, or heat, the transient receptor potential vanilloid-1 (TRPV1) has been identified as a potential treatment for chronic pain.TRPV1 is a highly characterized member of the TRP cation channel family believed to be involved in a number of important biological roles and plays a role in the transmission of pain.TRPV1 activation inhibits the transition of pain signals from the periphery to the central nervous system (CNS), leading to the possible development of analgesic and anti-inflammatory agents. TRPV1 antagonists have also been evaluated in multiple clinical trials where hyperthermic effects seen preclinically are also observed in humans

PATENT

http://www.google.com.na/patents/US20110003813

6-{4-[6-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidin-4-yl]-3-(R)-methyl-piperazin-1-yl}-5-methyl-nicotinic acid 1. 1-(5-Bromo-3-methyl-pyridin-2-yl)-3-(R)-methyl-piperazine

Heat a solution of 2,5-dibromo-3-methyl-pyridine (Chontech Inc., Waterford, Conn.) (2.0 g, 7.97 mmol), (R)-2-methyl-piperazine (ChemPacific Corp., Baltimore, Md.; 3.2 g, 31.9 mmol) in DMA at 130° C. for 16 h. Partition the reaction mixture between water and EtOAc. Wash the EtOAc layer with water (1×) and brine (1×), dry (Na₂SO₄) and concentrate under reduced pressure to give 1-(5-bromo-3-methyl-pyridin-2-yl)-3-(R)-methyl-piperazine as a solid.

2. 2,4-dichloro-6-(4-fluorophenyl)pyrimidine

Dissolve 4-fluorobromobenzene (8.75 g, 0.05 moles) in anhydrous ether (80 mL) under nitrogen atmosphere and cool to −78° C. Add dropwise 1.6 M n-BuLi (34 mL, 0.055 moles) and stir at −78° C. for 45 min. Dissolve 2,4-dichloropyrimidine (7.45 g, 0.05 moles) in Et₂O (100 mL) and add dropwise to the reaction mixture. Warm the reaction mixture to −30° C. and stir at this temperature for 30 min followed by 0° C. for 30 min. Quench the reaction mixture with AcOH (3.15 mL, 0.055 moles) and water (0.5 mL, 0.027 moles) dissolved in THF (5.0 mL). Add dropwise a THF (40 mL) solution of DDQ (11.9 g, 0.053 moles) to the reaction mixture. Bring the reaction mixture to room temperature and stir at room temperature for 30 min. Cool the reaction mixture to 0° C., add 3.0 N aq. NaOH (35 mL) and stir for 30 min. Decant the organic layer from the reaction mixture and wash the brown solid with Et₂O (3×100 mL). Combine the organic layers, wash several times with saturated NaCl solution and dry with MgSO₄. Filter and evaporate under vacuum to afford a brown colored solid. Purify by flash column chromatography using 5% EtOAc/hexane to afford the title product as a white solid.

3. 4-[4-(5-Bromo-3-methyl-pyridin-2-yl)-2-(R)-methyl-piperazin-1-yl]-2-chloro-6-(4-fluoro-phenyl)-pyrimidine

Heat a mixture of 2,4-dichloro-6-(4-fluoro-phenyl)-pyrimidine (6.0 g, 24.7 mmol), 1-(5-bromo-3-methyl-pyridin-2-yl)-3-(R)-methyl-piperazine (7.0 g, 25.9 mmol) and K₂CO₃(6.8 g, 49.4 mmol) in DMA at 60° C. for 16 h. Partition the mixture between EtOAc and water, dry (Na₂SO₄) the organic layer and concentrate under reduced pressure. Purify with flash silica gel column eluting with 15% EtOAc/hexanes. Concentrate under reduced pressure to give the title compound.

4. 4-[4-(5-Bromo-3-methyl-pyridin-2-yl)-2-(R)-methyl-piperazin-1-yl]-6-(4-fluoro-phenyl)-2-(2-(R)-methyl-pyrrolidin-1-yl)-pyrimidine

Heat a mixture of 4-[4-(5-bromo-3-methyl-pyridin-2-yl)-2-(R)-methyl-piperazin-1-yl]-2-chloro-6-(4-fluoro-phenyl)-pyrimidine (7.7 g, 16.2 mmol), (R)-2-methylpyrrolidine hydrobromide [prepared essentially as described by Nijhuis et. al. (1989) J. Org. Chem. 54(1):209] (3.5 g, 21.1 mmol) and K₂CO₃(5.1 g, 37.3 mmol) in DMA at 110° C. for 16 h. Partition the mixture between EtOAc and water, dry (Na₂SO₄) the organic layer and concentrate under reduced pressure. Purify with flash silica gel column eluting with 10% EtOAc/hexanes. Concentrate under reduced pressure to give the title compound.
5. 6-{4-[6-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidin-4-yl]-3-(R)-methyl-piperazin-1-yl}-5-methyl-nicotinonitrile
To a mixture of 4-[4-(5-bromo-3-methyl-pyridin-2-yl)-2-(R)-methyl-piperazin-1-yl]-6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidine (700 mg, 1.33 mmol) and Zn(CN)₂(94 mg, 0.799 mmol) in DMF, add Pd(PPh₃)₄(77 mg, 0.067 mmol). Purge the reaction mixture for 10 min with dry N₂. Heat the stirring reaction mixture overnight at 80° C., cool to room temperature and partition between water and EtOAc. Dry the solution (Na₂SO₄), concentrate under reduced pressure. Purify the residue by flash column eluting with EtOAc-Hexanes (1:1) to afford the title compound as a white solid.
6. 6-{4-[6-(4-Fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidin-4-yl]-3-(R)-methyl-piperazin-1-yl}-5-methyl-nicotinic acid
Heat a solution of 6-{4-[6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-1-yl)-pyrimidin-4-yl]-3-(R)-methyl-piperazin-1-yl}-5-methyl-nicotinonitrile (100 mg, 0.212 mmol) in 12 M HCl for 3 hours at 90° C. Concentrate the mixture under reduced pressure. Add a small amount of water, adjust the pH to 6-7, and collect the resulting white precipitate to afford the title compound as a off-white solid. ¹H NMR (300 MHz, DMSO-d₆): δ 1.24 (m, 6H, 2×CH₃))_;1.61 (m, 1H,); 1.84 (m, 1H); 1.98 (m, 2H); 2.34 (s, 3H, Ar—CH₃); 2.91 (m, 1H); 3.08 (m, 1H); 3.26 (m, 2H); 3.56 (m, 2H); 3.74 (m, 1H); 4.21 (m, 1H); 4.35 (m, 1H); 4.74 (m, 1H); 6.57 (s, 1H); 7.26 (m, 2H); 7.91 (d, 1H, J=3 Hz); 8.15 (m, 2H); 8.60 (d, 1H, J=3 Hz).

END…………………

MESYLATE NMR

¹H NMR (CD₃OD, 400 MHz) δ 1.37 (d, 3H, J= 6.4 Hz), 1.48 (d, 3H, J = 6.7 Hz), 1.84 (m, 1H), 2.09 (m, 1H), 2.17–2.25 (m, 2H), 2.42 (s, 3H), 2.66 (s, 3H), 3.10 (dt, 1H, J = 12.3 and 3.3 Hz), 3.28 (dd, 1H, J = 13.1 and 3.7 Hz), 3.65–3.72 (m, 3H), 3.78 (m, 1H), 3.87 (m, 1H), 4.49 (m, 1H), 4.63 (m, 3H), 4.96 (br m, 1H), 6.61 (s, 1H), 7.32 (m, 2H), 7.82 (m, 2H), 8.05 (m, 1H), 8.69 (d, 1H, J = 1.9 Hz);

¹⁹F NMR (CD₃OD, 470 MHz) δ −108.6.

PATENT

http://www.google.ga/patents/WO2006026135

Scheme 1

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8

Scheme 9

Scheme 10

Scheme 14

Scheme 15

Scheme 16

Scheme 17

Scheme 18

Scheme 19

Scheme 20

6-{4-[6~(4-Fluoro-phenyl)-2-(2~methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]-3-(R)-met}τyl- piperazin-l-yl}-5-methyl-nicotinic acid

Heat a solution of 6-{4-[6-(4-fluoro-phenyl)-2-(2-methyl-pyrrolidin-l-yl)-pyrimidin-4-yl]- 3-(R)-methyl-piperazin-l-yl}-5-methyl-nicotinonitrile (100 mg, 0.212 mmol) in 12 M HCl for 3 hours at 9O⁰C. Concentrate the mixture under reduced pressure. Add a small amount of water, adjust the pH to 6-7, and collect the resulting white precipitate to afford the title compound as a off-white solid. ¹H NMR (300 MHz, DMSO-d₆): δ 1.24 (m, 6H, 2xCH₃)); 1.61 (m, 1Η,); 1.84 (m, 1Η); 1.98 (m, 2Η); 2.34 (s, 3H, Ar-CH₃); 2.91 (m, 1Η); 3.08 (m, 1Η); 3.26 (m, 2Η); 3.56 (m, 2H); 3.74 (m, IH); 4.21 (m, IH); 4.35 (m, IH); 4.74 (m, IH); 6.57 (s, IH); 7.26 (m, 2H); 7.91 (d, IH, J = 3Hz); 8.15 (m, 2H); 8.60 (d, IH, J = 3Hz).

PAPER

Development of a Multikilogram Scale Synthesis of a TRPV1 Antagonist

Jeffrey T. Kuethe^*, Michel Journet, Zhihui Peng, Dalian Zhao, Arlene McKeown, and Guy R. Humphrey

Department of Process Chemistry, Merck & Co., Inc., Rahway, New Jersey 07065, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00388

Publication Date (Web): January 13, 2016

*E-mail: Jeffrey_Kuethe@merck.com.

Abstract

A highly efficient, regioselective five-step synthesis of the TRPV1 antagonist 1 is described. The coupling of piperazine 7 with dichloropyrimidine 8 proceeded via a regioselective Pd-mediated amination affording product 11 in excellent yield. Conversion of the penultimate product 14 afforded 1 through formation of a magnesium ate complex and trapping with CO₂.

http://pubs.acs.org/doi/suppl/10.1021/acs.oprd.5b00388

http://pubs.acs.org/doi/suppl/10.1021/acs.oprd.5b00388/suppl_file/op5b00388_si_001.pdf

Patent	Submitted	Granted
Substituted biaryl piperazinyl-pyridine analogues [US7662830]	2006-06-08	2010-02-16
SUBSTITUTED BIARYL PIPERAZINYL-PYRIDINE ANALOGUES [US2011003813]	2011-01-06

Blum, C. A.; Brielmann, H.; Chenard, B. L.; Zheng, X. Preparation of substituted biaryl piperazinyl-pyridine analogues as capsaicin modulators. PCT Int. Appl. WO 2006026135 A2 20060309, 2006.

Neurogen Corporation, a Subsidiary of Ligand Pharmaceuticals Inc., 11119 North Torrey Pines Road, Suite 200, La Jolla, CA 92037, U.S.A.

Neurogen and Merck Agreement for Next-Generation Pain Drugs Consummated

Source	Press Release
Company	Neurogen, Merck & Co
Tags	Central Nervous System, Research Collaboration
Date	January 16, 2004

Branford, CT — January 16, 2004 — Neurogen Corporation (Nasdaq: NRGN) today announced that it has consummated its previously announced alliance with Merck & Co ., Inc. (NYSE: MRK) to discover and develop next-generation drugs for the treatment of pain. The deal received clearance from the Federal Trade Commission under the Hart-Scott-Rodino Act and the companies have now commenced the collaboration. The alliance, announced December 1, 2003, enables Merck , through a subsidiary, and Neurogen to pool drug candidates targeting the vanilloid receptor (VR1 ), a key integrator of pain signals in the nervous system, and combine their ongoing VR1 programs to form a global research and development collaboration.

With consummation of the deal, Neurogen has received $30 million from Merck , including a $15 million up-front license fee payment and a $15 million equity investment in Neurogen common stock. Under the agreement, Merck has purchased 1,783,252 shares of newly issued Neurogen common stock at $8.41 per share, the average market price per share for the 25 trading days preceding regulatory clearance. Merck ‘s new shareholder position represents approximately 9% of Neurogen ‘s 19,873,464 total shares outstanding.

About Neurogen

Neurogen Corporation targets new small molecule drugs to improve the lives of patients suffering from disorders with significant unmet medical need. Neurogen has generated a portfolio of compelling new drug candidates through its Accelerated Intelligent Drug Discovery (AIDD(TM)) system, its expertise in cellular functional assays, and its depth in medicinal chemistry. Neurogen conducts its research and development independently and, when advantageous, collaborates with world-class pharmaceutical companies to obtain additional resources and to access complementary expertise.

////////

n1c(nc(cc1c2ccc(cc2)F)N3CCN(C[C@H]3C)c4ncc(cc4C)C(=O)O)N5CCC[C@H]5C

5-Bromo-1-methyl-1H-imidazole-4-carbonitrile

January 30, 2016 2:51 pm / Leave a comment

Source: 5-Bromo-1-methyl-1H-imidazole-4-carbonitrile

Fresolimumab

January 30, 2016 4:23 am / 1 Comment on Fresolimumab

Fresolimumab
GC 1008, GC1008
UNII-375142VBIA

cas 948564-73-6

Structure

immunoglobulin G4, anti-(human transforming growth factors beta-1, beta-2 (G-TSF or cetermin) and beta-3), human monoclonal GC-1008 γ4 heavy chain (134-215′)-disulfide with human monoclonal GC-1008 κ light chain, dimer (226-226”:229-229”)-bisdisulfide
immunoglobulin G4, anti-(transforming growth factor β) (human monoclonal GC-1008 heavy chain), disulfide with human monoclonal GC-1008 light chain, dimer

For Idiopathic Pulmonary Fibrosis, Focal Segmental Glomerulosclerosis,and Cancer

An anti-TGF-beta antibody in phase I clinical trials (2011) for treatment-resistant primary focal segmental glomerulosclerosis.

A pan-specific, recombinant, fully human monoclonal antibody directed against human transforming growth factor (TGF) -beta 1, 2 and 3 with potential antineoplastic activity. Fresolimumab binds to and inhibits the activity of all isoforms of TGF-beta, which may result in the inhibition of tumor cell growth, angiogenesis, and migration. TGF-beta, a cytokine often over-expressed in various malignancies, may play an important role in promoting the growth, progression, and migration of tumor cells.

Fresolimumab (GC1008) is a human monoclonal antibody^[1] and an immunomodulator. It is intended for the treatment of idiopathic pulmonary fibrosis (IPF), focal segmental glomerulosclerosis, and cancer^[2]^[3] (kidney cancer and melanoma).

It binds to and inhibits all isoforms of the protein transforming growth factor beta (TGF-β).^[2]

History

Fresolimumab was discovered by Cambridge Antibody Technology (CAT) scientists^[4] and was one of a pair of candidate drugs that were identified for the treatment of the fatal condition scleroderma. CAT chose to co-develop the two drugs metelimumab (CAT-192) and fresolimumab with Genzyme. During early development, around 2004, CAT decided to drop development of metelimumab in favour of fresolimumab.^[5]

In February 2011 Sanofi-Aventis agreed to buy Genzyme for US$ 20.1 billion.^[6]

As of June 2011 the drug was being tested in humans (clinical trials) against IPF, renal disease, and cancer.^[7]^[8] On 13 August 2012, Genzyme applied to begin a Phase 2 clinical trial in primary focal segmental glomerulosclerosis^[9] comparing fresolimumab versus placebo.

As of July 2014, Sanofi-Aventis continue to list fresolimumab in their research and development portfolio under Phase II development.^[10]

References

1 WHO Drug Information

2 National Cancer Institute: Fresolimumab

3 Statement On A Nonproprietary Name Adopted By The USAN Council – Fresolimumab
4 Grütter, Christian; Wilkinson, Trevor; Turner, Richard; Podichetty, Sadhana; Finch, Donna; McCourt, Matthew; Loning, Scott; Jermutus, Lutz; Grütter, Markus G. (2008-12-23). “A cytokine-neutralizing antibody as a structural mimetic of 2 receptor interactions”. Proceedings of the National Academy of Sciences 105 (51): 20251–20256. doi:10.1073/pnas.0807200106. ISSN 0027-8424. PMC 2600578. PMID 19073914.
5 http://www.independent.co.uk/news/business/news/cat-may-abandon-skin-drug-after-trial-results-disappoint-569445.html
6 http://www.bbc.co.uk/news/business-12477750
7 http://www.genengnews.com/gen-news-highlights/scientists-trigger-white-fat-to-become-brown-fat-like-to-treat-obesty-and-type-2-diabetes/81245389/
8 Clinicaltrials.gov for Fresolimumab
9 http://clinicaltrials.gov/show/NCT01665391
10 http://en.sanofi.com/rd/rd_portfolio/rd_portfolio.aspx

Fresolimumab
Monoclonal antibody
Type	Whole antibody
Source	Human
Target	TGF beta 1, 2 and 3
Clinical data
Legal status	Investigational
Identifiers
CAS Number	948564-73-6
ATC code	None
ChemSpider	none
KEGG	D09620
Chemical data
Formula	C₆₃₉₂H₉₉₂₆N₁₆₉₈O₂₀₂₆S₄₄
Molar mass	144.4 kDa

////////////

Monoclonal antibodies for the immune system

Immune system (“-l(i[m])-“)

Human (“-limu-“)	immunosuppression: Abrilumab Adalimumab Anifrolumab Atorolimumab Belimumab Brodalumab Carlumab Dupilumab Durvalumab Eldelumab Fresolimumab Golimumab Lerdelimumab Lirilumab Mavrilimumab Metelimumab Morolimumab Namilumab Oxelumab§ Placulumab Sarilumab Sifalimumab Tabalumab Ulocuplumab Varlilumab immune activation: Ipilimumab Nivolumab Pembrolizumab Tremelimumab Urelumab other: Bertilimumab Zanolimumab

Mouse (“-limo-“)	Afelimomab Elsilimomab Faralimomab Gavilimomab Inolimomab Maslimomab Nerelimomab Odulimomab Telimomab aritox Vepalimomab Zolimomab aritox

Chimeric (“-lixi-“)	Basiliximab Clenoliximab Galiximab Gomiliximab Infliximab Keliximab Lumiliximab Priliximab Teneliximab Vapaliximab

Humanized (“-lizu-“)	immunosuppressive: Aselizumab Apolizumab Benralizumab^† Cedelizumab Certolizumab pegol Daclizumab Eculizumab Efalizumab Epratuzumab Erlizumab Etrolizumab Fontolizumab Itolizumab Lampalizumab Ligelizumab Lulizumab pegol Mepolizumab Mogamulizumab Natalizumab Ocrelizumab Omalizumab Ozoralizumab Pascolizumab Pateclizumab Pembrolizumab Pexelizumab Pidilizumab PRO 140^† Reslizumab Rontalizumab Rovelizumab Ruplizumab Quilizumab Samalizumab Siplizumab Talizumab Teplizumab Tocilizumab Toralizumab Tregalizumab Vatelizumab Vedolizumab Visilizumab TGN1412^§ non-immunosuppressive: Ibalizumab^†

Chimeric + humanized (“-lixizu-“)	Otelixizumab

Interleukin (“-k(i[n])-“)

Human (“-kinu-“)	Briakinumab Canakinumab Fezakinumab Fletikumab Guselkumab Secukinumab Sirukumab Tralokinumab Ustekinumab

Humanized (“-kizu-“, “-kinzu-“)	Anrukinzumab Clazakizumab Enokizumab Gevokizumab Ixekizumab Lebrikizumab Olokizumab Perakizumab Tildrakizumab

Inflammatory lesions (“-les-“)

Mouse (“-leso-“)	Besilesomab Fanolesomab^‡ Lemalesomab Sulesomab

^#WHO-EM
^‡Withdrawn from market
Clinical trials:
- ^†Phase III
- ^§Never to phase III

v t e Index of the immune system

Description	Physiology cells autoantigens autoantibodies complement surface antigens IG receptors

Disease	Allergies Immunodeficiency Immunoproliferative immunoglobulin disorders Hypersensitivity and autoimmune disorders Neoplasms and cancer

Treatment	Procedures Drugs antihistamines immunostimulants immunosuppressants monoclonal antibodies

What was the drug in Clinical Trial Tragedy In France Jan 2016

January 23, 2016 12:22 pm / 2 Comments on What was the drug in Clinical Trial Tragedy In France Jan 2016

3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.png

BIA 10-2474

cas 1233855-46-3

3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide

1H-Imidazole-1-carboxamide, N-cyclohexyl-N-methyl-4-(1-oxido-3-pyridinyl)-

C₁₆ H₂₀ N₄ O_2, 300.36

Bial-Portela & Ca. S.A.

BIA 10-2474 is an experimental fatty acid amide hydrolase inhibitor^[1] developed by the Portuguese pharmaceutical company Bial-Portela & Ca. SA. The drug was developed to relieve pain,^[2]^[3] to ease mood and anxiety problems, and to improve movement coordination linked to neurodegenerative illnesses.^[4] It interacts with the human endocannabinoid system.^[5]^[6] It has been linked to severe adverse events affecting 5 patients in a drug trial in Rennes, France, and at least one death, in January 2016.^[7]

Synthesis

WO 2014017938

BIAL – PORTELA & Cª, S.A.

Example 5. 3-(l-(cyclohexyl(methyl)carbamoyl-lfl-imidazol-4-yl)pyridine l-oxide (compound A)

C₁₆H₂₀N₄O C16H20N4O2

MW 284,36 MW 300,36

To a solution of N-cyclohexyl-N-methyl-4-(pyridm-3-yl)-lH-imidazole-l-carboxamide in dichioromethane at 25°C was added peracetic acid (38%; the concentration is not critical, and may be varied) in a single portion. The reaction mixture was then maintained at 25°C for at least 20 h, whereupon the reaction was washed four times with water (in some embodiments, the water for the extraction step may be supplemented with a small amount (e.g. 1%) of acetic acid, which helps to promote product solubility in the DCM). The dichioromethane solution was then filtered prior to diluting with 2-propanol. Dichioromethane (50%) was then distilled off under atmospheric pressure, whereupon, 2-propanol was charged at the same rate as the distillate was collected. The distillation was continued until >90% of the dichioromethane was collected. The resulting suspension was then cooled to 20°C and aged for at least 30 min. prior to cooling to 0°C and aging for a further 60 min. The reaction mixture was then filtered and the product washed with additional 2-propanol, before drying at 50°C under vacuum to afford the title compound as an off-white crystalline solid.

The purity of the product was ascertained by HPLC, with identity confirmable by NMR. The yield was consistently >80% in several production runs.

PATENT

WO 2012015324

Example 1. Preparation of N-cyclohexyl-N-methyl-4-(pyridin-3yl)-lH-imidazole-l-carboxamide

C₈H₇N₃ C₁₅H_{1 1}N₃0₂ C₁₆H₂₀N₄O

MW 145,16 MW 265,27 MW 284,36

To a suspension of 3-(l/ -imidazol-4-yl)pyridine in tetrahydrofuran (THF) containing pyridine at 25°C was slowly added a solution of phenyl chloroformate in THF over 60 to 90 min. The resulting fine white suspension was then maintained at 25°C for at least 60 min. before the addition of N-methyl- -cyclohexylamine in a single portion, causing the suspension to thin and become yellow in colour. The reaction mixture was then stirred for 90 min. before filtering and washing the filter cake with additional THF. The mother liquors were then maintained at 25°C for at least 18 h, whereupon 65% of the volume of THF was distilled off under atmospheric pressure. The resulting solution was then diluted with 2-propanol and maintained at > 50°C for 10 min. prior to cooling down to 20°C. The resulting suspension was aged at 20°C for 15 min. prior to cooling to 0°C and aging for a further 60 min. The reaction mixture was then filtered and the product was washed with additional 2-propanol, before drying at 50°C under vacuum to afford the title compound as an off-white crystalline solid.

The purity of the product was ascertained by HPLC, with identity confirmable by NMR. The yield was consistently around 50% in several production runs.

Example 2. 3-(l-(cyclohexyl(methyl)carbamoyl-l//-imidazol-4-yl)pyridine 1 -oxide (compound A)

C₁₆H₂₀N₄O Ci6H2oN₄0₂

MW 284,36 MW 300,36

To a solution of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-lH-imidazole-l-carboxamide in dichloromethane at 25°C was added peracetic acid (38%; the concentration is not critical, and may be varied) in a single portion. The reaction mixture was then maintained at 25°C for at least 20 h, whereupon the reaction was washed four times with water. The dichloromethane solution was then filtered prior to diluting with 2-propanol. Dichloromethane (50%) was then distilled off under atmospheric pressure, whereupon, 2-propanol was charged at the same rate as the distillate was collected. The distillation was continued until >90% of the dichloromethane was collected. The resulting suspension was then cooled to 20°C and aged for at least 30 min. prior to cooling to 0°C and aging for a further 60 min. The reaction mixture was then filtered and the product washed with additional 2-propanol, before drying at 50°C under vacuum to afford the title compound as an off-white crystalline solid.

The purity of the product was ascertained by HPLC, with identity confirmable by NMR. The yield was consistently >80% in several production runs. It will be appreciated that this gives an overall yield of compound A many times greater than that achieved in the prior art.

In a further run of this synthesis, in a 2L reactor to a mixture of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-l H- imidazole-l-carboxamide (90 g, 317 mmol) and dichloromethane (1350 ml) was added peracetic acid (84 ml, 475 mmol). The reaction mixture was stirred at 25°C. Completion of the reaction was monitored by HPLC for the disappearance of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-lH- imidazole- 1-carboxamide. After reaction completion a solution of sodium metabisulfite (60.2 g, 317 mmol) in water (270ml) was added to the reaction mixture maintaining the temperature below 30°C. After phase separation the organic phase was washed with water. After phase separation the organic phase was concentrated at atmospheric pressure until 5 vol. Then solvent was swapped to isopropanol (1350 ml) and the suspension was cooled to 0°C during 4 hours and stirred at that temperature for 1 hour. The resulting solid was collected by filtration and was rinsed with water (270 ml) and isopropanol (270 ml) to afford a white crystalline solid in 84.8g (89%).

PATENT

WO 2010074588

Preparation of compound 362 a) N-cyclohexyl-N-methyl-4-(pyridin-3-yl)- 1 H-imidazole- 1 -carboxamide

To a stirred suspension of 3-( 1 H-imidazol-4-yl)pyridine dihydrochloride (1.745 g, 8 mmol) in a mixture of tetrahydrofuran (29 mL) and DMF (2.90 mL) was added potassium 2-methylpropan-2-olate (1.795 g, 16.0 mmol) and the mixture was refluxed for 30 minutes. The resulting brown suspension was cooled to room temperature and treated with pyridine (0.979 mL, 12 mmol) and N,N-dimethylpyridin-4-amine (0.098 g, 0.8 mmol), followed by the addition of cyclohexyl(methyl)carbamic chloride (1.476 g, 8.4 mmol). The reaction was heated to 90 ⁰C overnight, whereupon the mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried (MgSO^) and filtered. After evaporation, the crude product was chromatographed over silica gel using a dichloromethane/methanol (9:1) mixture. Homogenous fractions were pooled and evaporated to leave a white powder, (160 mg, 7 %).

b) 3-( 1 -(cyclohexyl(methyl)carbamoyl)- 1 H-imidazol-4-yl)pyridine 1 -oxide

To a stirred solution of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-l H-imidazole- 1 -carboxamide (90 mg, 0.317 mmol) in chloroform (5 mL) was added 3-chlorobenzoρeroxoic acid (149 mg, 0.475 mmol) in one portion. The reaction was allowed to stir at room temperature for 20 h. TLC showed the reaction to be complete and the mixture was evaporated to dryness. The residue was triturated with ether and the resulting white crystals were filtered off and dried in air. Recrystallisation from hot isopropanol gave a white powder (46 mg, 46 %).

Structure and action

French newspaper Le Figaro has obtained Bial study protocol documents listing the the chemical name of BIA-10-2474 as 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.^[8] A Bial news release described BIA-10-2474 as “a long-acting inhibitor of FAAH”.^[9]

Fatty acid amide hydrolase (FAAH) is an enzyme which degrades endocannabinoid neurotransmitters like anandamide,^[10] which relieves pain and can affect eating and sleep patterns.^[11]^[12] FAAH inhibitors have been proposed for a range of nervous-system disorders including anxiety, alcoholism, pain and nausea.

The Portuguese pharmaceutical company Bial holds several patents on FAAH enzyme inhibitors.^[12]^[13]^[14]^[15]

No details of the preclinical testing of this molecule have been made public by the manufacturer Bial. However, the French newspaper Le Figaro has obtained and published an apparently legitimate copy of the full clinical trial protocol (BIA-102474-101).^[8] The protocol presents a summary of what appears to be a full package of pharmacodynamic, pharmacokinetic and toxicological studies that might be expected to support a first-in-man study, including safety pharmacology studies in two species (rat, dog) and repeated dose toxicity studies in four species (13 week sub-chronic studies in mouse, rat, dog and monkey). The summary presented however includes no assessment of the relevance of the animal species selected for study (that is, in terms of physiological and genetic similarities with humans and the mechanism of action of the study drug).

Of note, few adverse events were observed in any of the studies, with the 13-week oral No Observed Adverse Effect Level (NOAEL) varying between 10 mg/kg/day in mice to 75 mg/kg/day in monkeys. The authors suggest that these were the maximum doses tested in these studies, though it is not clear. The authors also report no effects of significance in the animal models used for the CNS safety pharmacology studies, which studied a dose of up to 300 mg/kg/day.^[8]

Notably absent from the protocol are calculations of receptor occupancy; predictions of in vivo ligand binding saturation levels; measures of target affinity; or assessment of the molecule’s activity in non-target tissues or non-target binding interactions as suggested by the European guidance for Phase I studies,^[16] assuming BIA 10-2474 could be considered ‘high risk’).^[8]

The trial protocol makes no reference to chimpanzee studies (only monkeys) which contradicts a previous statement to the media in which the French Health Minister stated that the drug had been tested on animals including chimpanzees.^[4]^[17] ^[18] Some experts had remarked that drug testing in chimpanzees was unlikely.^[19]

These findings provide no explanation for the type and severity of events observed in Rennes. In describing the rationale for the starting dose, the authors conclude that:

No target organ was identified during toxicology studies and few adverse clinical findings were observed at the highest dose tested. For the single ascending dose part [of the clinical trial], a starting dose of 0.25 mg was judged to be safe for a first-in-human administration. ^[8]

The protocol defines no starting dose for the multi-dose treatment groups, noting that this will be based on the outcome of the single dose portion of the trial (an approach known as adaptive trial design). The authors note that nonetheless, the starting dose will not exceed 33% of the maximum tolerated dose (MTD) identified in the single dose groups (or 33% of the maximum administered dose if the MTD is not reached).^[8]

Death and serious adverse events during phase I clinical trial

In July 2015 Biotrial, a contract research organization, began testing the drug in a human phase one clinical trial for the manufacturer. The study was approved by French regulatory authority, the Agence Nationale de Sécurité du Médicament (ANSM), on June 26, 2015, and by the Brest regional ethics committee on July 3, 2015.^[20] The trial commenced on July 9, 2015,^[21] in the city of Rennes, and recruited 128 healthy volunteers, both men and women aged 18 to 55. According to French authorities, the study employed a three-stage design with 90 of the volunteers having received the drug during the first two stages of the trial, with no serious adverse events being reported .^[17]^[20] Participants of the study were to receive €1,900 and, in turn, asked to stay at Biotrial’s facility for two weeks during which time they would take the drug for ten days and undergo tests.^[22]

In the third stage of the trial evaluating multiple doses, six male volunteers received doses by mouth, starting on 7 January 2016. The first volunteer was hospitalized at the Rennes University Hospital on January 10, became brain dead,^[17]^[23]^[24]^[25] and died on January 17.^[26] The other five men in the same dosage group were also hospitalized, in the period of January 10 through January 13^[27] four of them suffering injuries including deep hemorrhagic and necrotic lesions seen on brain MRI.^[7] The six men who were hospitalised were the group which received the highest dose.^[26] A neurologist at the University of Rennes Hospital Center, Professor Pierre-Gilles Edan, stated in a press conference with the French Minister for Health, that 3 of the 4 men who were displaying neurological symptoms “already have a severe enough clinical picture to fear that even in the best situation there will be an irreversible handicap” and were being given corticosteroids to control the inflammation.^[27] The sixth man from the group was not showing adverse effects but had been hospitalized for observation.^[25]^[28]^[29] Biotrial stopped the experiment on January 11, 2016.^[4]

No details of the trial have been made public by the manufacturer Bial. The study does not appear in searches of any of the key clinical trial registries, including EudraCT and ClinicalTrials.gov which would normally contain details of approved clinical studies.^[30]^[31]^[32]^[33] The trial protocol published by Le Figaro provides extensive detail on what was planned for the study, but many details of the key multi-dose part are not included and were to have been finalised at the conclusion of the single-dose part of the trial.^[8]

The French health minister Marisol Touraine called the event “an accident of exceptional gravity” and promised to investigate the matter.^[4] On January 18 it was reported authorities were investigating if a manufacturing or transport error might be involved.^[34]

Le Figaro posted a 96-page clinical study protocol for BIA 10-2474 that the French newspaper procured from an unnamed source.

According to the document, BIA 10-2474 is 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide.

BIA 10-2474 “is designed to act as a long-active and reversible inhibitor of brain and peripheral FAAH,” notes the protocol. The compound “increases anandamide levels in the central nervous system and in peripheral tissues.”

The clinical trial protocol also notes that the company tested BIA 10-2474 on mice, rats, dogs, and monkeys for effects on the heart, kidneys, and gastrointestinal tract, among other pharmacological and toxicological evaluations.

09404-notw1-cliniccxd

Six men in a Phase I clinical trial were admitted to the University Hospital Center of Rennes, France, (shown here) because of adverse reactions.Six men in a Phase I clinical trial were admitted to the University Hospital Center of Rennes, France, (shown here) because of adverse reactions.

Credit: Mathieu Pattier/SIPA/Newscom

One man is dead and five men were hospitalized after participating in a Phase I clinical trial in Rennes, France

The clinical trial, conducted by the company Biotrial on behalf of the Portuguese pharmaceutical firm Bial, was evaluating a pain relief drug candidate called BIA 10-2474 that inhibits fatty acid amide hydrolase (FAAH) enzymes. Blocking these enzymes prevents them from breaking down cannabinoids in the brain, a family of compounds that includes the euphoria-inducing neurotransmitter anandamide and Δ⁹-tetrahydrocannabinol, the major psychoactive component of marijuana.

Phase I clinical trials are conducted to check a drug candidate’s safety profile in healthy, paid volunteers. In this case, the drug caused hemorrhagic and necrotic brain lesions in five out of six men in a group who received the highest doses of the drug, said Gilles Edan, a neurologist at the University Hospital Center of Rennes.

The most severely affected man was pronounced brain-dead after hospitalization and then died on Jan. 17. Four men remain in the hospital in stable condition. The only man in the high-dose group who had no adverse symptoms has been released from the hospital.

Clinical trials are an essential part of the drug development process. In order to get life-improving and life-saving medicines to patients, they first have to go through an extensive series of tests. Even before a drug makes it to Phase 1 testing, where its safety, dosage amount, and side effects are tested in a small group of humans, it will undergo testing in animals. As a result, it is not common for a medicine undergoing clinical tests to have a very serious adverse effect on a human. This makes you wonder what happened to a group of patients involved in a clinical study in Rennes, France.

According to news reports, a drug undergoing testing in a French clinic has left one person dead, two others with what may be permanent brain damage, and and two others critically ill. The drug has thus far been unnamed, but it appears to have been produced by the Portuguese company Bial. The French health minister has stated the drug acted on natural receptors found in the body known as endocannibinoids, which regulate mood and appetite. It did not contain cannabis or anything derived from it, as was originally reported. All six trial participants were administered the doses simultaneously.

The trial was being performed at Biotrial, a French-based firm that was formed in 1989 and has conducted thousands of trials. A message on the company’s website stated that they are working with health authorities to understand the cause of the accident, while extending thoughts to the patients and their families. Bial has disclosed the drug was a FAAH (fatty acid amide hydrolase) inhibitor, which is an enzyme produced in the brain and elsewhere that breaks down neurotransmitters called endocannabinoids. Two scientists from the Nottingham Medical School who have worked with FAAH tried over the weekend to try and identify the drug by examining a list of drugs Bial currently has in its pipeline. They believe the culprit is one identified by the codename BIA 10-2474. That same codename appeared on a recruitment form that was given to a volunteer, which was published in a French newspaper. Little more is known about it, and there does not appear to be any entry for it in clinical trial registries.

The French health ministry is reporting the six patients were all in good health prior to taking the oral medicine, which was administered to 90 volunteers. The trial recruited 128 individuals, and the remaining participants received a placebo. Health minister Marisol Touraine, describing the situation as a very serious accident, noted the patients were taking part in a trial in Brittany, Rennes involving a medicine developed by a “European laboratory”, refusing to comment further until additional information became available. She has also asked the Inspector General of Social Affairs to lead an investigation into the circumstances around the trial, which has obviously been suspended. She notes the drug had been tested on animals, including chimpanzees. France’s National Agency for Medicine and Health Products Safety approved the trial on in June 2015.

One thing we do know is that the trial was a Phase 1 clinical study that included 90 healthy volunteers. Regulations that oversee all clinical trials in Europe do attempt to minimize the risk associated with trials, but there is always a risk involved with administering an unapproved medicine to humans. At this time the chief neuroscientist at the hospital where the patients are being treated has said there is no known antidote for the drug.

The drug, administered to men between the ages of 28 and 49, was intended to treat mood disorders such as anxiety. While the men were administered varying doses, the patients who are hospitalized were taking the drug “regularly”.

Old 2006 case

While safety issues like this are rare, they are not unheard of. In 2006, a clinical trial in London left six men ill. All were taking part in a study testing a drug designed to fight auto-immune disease and leukemia. Within hours of taking the drug TGN1412, all experienced a serious reaction, were admitted to intensive care, and had to be treated for organ failure. Two became critically ill, with one eventually losing all of his fingers and toes. All were told they would have a higher risk of developing cancers or auto-immune diseases.

This of course led many to wonder about the future of trials, and whether the situation could happen again. The Duff Report, written in response to the TGN1412 trial, noted the medicine should have been tested in one person at a time. It also helped to put additional safety measures in place. The Medicines and Health Products Regulatory Agency (MHRA) now requires committees to look at pre-clinical data to determine the proper initial dose, and rules are in place to stop the trial if unintended reactions occur.

However, since patients can fall ill immediately after being administered a medication, certain risks will still exist.

The company that manufactured TGN1412, TeGenero Immuno Therapeutics, later went bankrupt. However the drug was later purchased by a Russian investor and renamed TABO8. TheraMAB, a Russian biotech company, then conducted a new trial of the drug in a much lower dose. A later Phase 2 study was started in patients with Rheumatoid Arthritis.

Other pharmaceutical companies, including Merck, Pfizer, Johnson & Johnson, Sanofi and Vernalis, have previously taken other FAAH inhibitors into clinical trials without experiencing such adverse events (e.g. respectively, MK-4409,^[35]^[36] PF-04457845, JNJ-42165279,^[37] SSR411298 and V158866.^[38]^[39] Related enzyme inhibitor compounds such as URB-597 and LY-2183240 have been sold illicitly as designer drugs,^[40]^[41] all without reports of this type of toxicity emerging, so the mechanism of the toxicity observed with BIA 10-2474 remains poorly understood.

Following the events in Rennes, Janssen announced that it was temporarily suspending dosing in two Phase II clinical trials with its own FAAH inhibitor JNJ-42165279, headlining the decision as “precautionary measure follows safety issue with different drug in class”. Janssen was emphatic that no serious adverse events had been reported in any of the clinical trials with JNJ-42165279 to date. The suspension is to remain in effect until more information is available about the BIA 10-2474 study.^[42]

References

1 “BRIEF-Bial says firmly committed to ensure wellbeing of test participants”. Reuters. 15 January 2016.
2 “BIA 10-2474”. Biocentury.com. Retrieved 2016-01-17.
3 “BIA 102474”. Adisinsight/springer.com. Retrieved 2016-01-17.
4 Adamson B (January 15, 2016). “Botched Drug Trial Leaves 1 Brain Dead, 5 in Hospital”. ABC, AP. Retrieved January 16, 2016.
5 Angeline Benoit,Makiko Kitamura (15 January 2016). “France Ties Brain-Dead Person to Tests of Bial-Portela Drug”. Bloomberg.com.
6 “France/Monde – Essai thérapeutique : 90 personnes ont pris la molécule”. Ledauphine.com. Retrieved 2016-01-17.
7 Enserink, Martin (2016). “More Details Emerge on Fateful French Drug Trial” (online). Science (January 16). Retrieved 16 January 2016.
8 “Drame de Rennes : le protocole de l’essai clinique en accusation”. sante.lefigaro.fr. Retrieved 2016-01-21.
9 “News Release – Phase I Clinical Trial Rennes”. http://www.bial.com. Retrieved 2016-01-21.
10 Debora Mackenzie. “Six in hospital after French pain relief drug trial goes wrong”. New Scientist.
11 “The Discovery and Development of Inhibitors of Fatty Acid Amide Hydrolase (FAAH)”. PubMed Central.
12 “Patent WO2015012708A1 – Imidazolecarboxamides and their use as faah inhibitors – Google Patents”. Google.com. Retrieved 2016-01-17.
13 “Patent WO2015016729A1 – Urea compounds and their use as faah enzyme inhibitors”. Google.com. Retrieved 2016-01-17.
14 “PT2014000052 UREA COMPOUNDS AND THEIR USE AS FAAH ENZYME INHIBITORS”. Patentscope.wipo.int. Retrieved 2016-01-17.
15 WO application 2015016729, Laszlo Erno KISS, Rita GUSMÃO DE NORONHA, Carla Patrícia ROSA DA COSTA PEREIRA, Rui PINTO, “Urea compounds and their use as faah enzyme inhibitors”, published Feb 5, 2015, assigned to BIAL
16″Strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products (CHMP/SWP/28367/07)” (PDF). European Medicines Agency. 1 September 2007. Retrieved 22 January 2016.
17 “Accident grave dans le cadre d’un essai clinique – Intervention de Marisol Touraine à Rennes”. Ministère des Affaires Sociales, de la Santé et des Droits des Femmes, France. 15 January 2016.
18Barbara Casassus (23 January 2016). “France investigates drug trial disaster” (PDF). The Lancet 387 (10016): 326. doi:10.1016/S0140-6736(16)00154-9.
19
“Expert reaction to French drug trial – reports of one patient dying and five others in hospital and of the Paris prosecutor’s office having opened an investigation into what happened”. Science Media Centre, London. 16 January 2016. Retrieved 21 January 2016.
20
“La survenue d’effets graves ayant entraîné l’hospitalisation de 6 patients, dont un en état de mort cérébrale, a conduit à l’arrêt prématuré d’un essai clinique du laboratoire BIAL – Point d’information”. Agence Nationale de Sécurité du Médicament, France (ANSM). 15 January 2016.
21
“Six hospitalized in Bial clinical trial in France”. BioWorld.com. Retrieved 2016-01-17.
22
Enserink M (January 16, 2016). “More details emerge on fateful French drug trial”. Science Magazine. Retrieved January 18, 2016.
23
“France clinical trial: 90 given drug, one man brain-dead”. BBC. January 15, 2016. Retrieved January 16, 2016.
24
“Ce que l’on sait de l’accident survenu lors d’un essai clinique à Rennes”, Le Monde, 15 January 2016
25
Blamont M (January 15, 2016). “French drug trial disaster leaves one brain dead, five injured”. Reuters. Retrieved January 16, 2016.
26
Clarisse Lucas, AFP (January 17, 2016). “Man dies after being left brain-dead in French drug trial”. Yahoo. Retrieved January 17, 2016.
27
“Accident “inédit” lors d’un essai clinique: un homme en état de mort cérébrale, cinq hospitalisés”. La Depeche. January 15, 2016. Retrieved January 18, 2016.
28
“France clinical trial: ‘No known antidote’ to drug”. BBC News. 15 January 2016. Retrieved 18 January 2016.
29
Enserink, Martin (2016). “What We Know So Far About the Clinical Trial Disaster in France” (online). Science (January 15). Retrieved 16 January 2016.
30
“EU Clinical Trials Register”. European Medicines Agency. Retrieved 20 January 2016.
31
“WHO International Clinical Trials Registry Platform”. World Health Organization. Retrieved 20 January 2016.
32
“ANSM – Répertoire public des essais cliniques de médicaments”. Agence Nationale de Sécurité du Médicament et des Produits de Santé, France. Retrieved 20 January 2016.
33
“ClinicalTrials.gov Registry”. U.S. National Institutes of Health. Retrieved 20 January 2016.
34
“Drug trial volunteer dies as ‘manufacturing error’ theory investigated”. The Independent. January 18, 2016. Retrieved January 18, 2016.
35
Chobanian; et al. (10 April 2014). “Discovery of MK-4409, a Novel Oxazole FAAH Inhibitor for the Treatment of Inflammatory and Neuropathic Pain”. ACS Med. Chem. Lett.
36
Merck (15 October 2009). “Merck Pipeline, Oct 2009” (PDF). Merck.
37
“Seven studies found for: jnj-42165279”. Clinicaltrials.gov. Retrieved 2016-01-19.
38
“2 studies found for: V158866”. Clinicaltrials.gov. Retrieved 2016-01-19.
39
Bisogno T, Maccarrone M. Latest advances in the discovery of fatty acid amide hydrolase inhibitors. Expert Opin Drug Discov. 2013 May;8(5):509-22. PMID 23488865 doi: 10.1517/17460441.2013.780021.
40
Shanks KG, Behonick GS, Dahn T, Terrell A. Identification of novel third-generation synthetic cannabinoids in products by ultra-performance liquid chromatography and time-of-flight mass spectrometry. J Anal Toxicol. 2013 Oct;37(8):517-25. PMID 23946450 doi: 10.1093/jat/bkt062.
41
Uchiyama N, Matsuda S, Kawamura M, Shimokawa Y, Kikura-Hanajiri R, Aritake K, Urade Y, Goda Y. Characterization of four new designer drugs, 5-chloro-NNEI, NNEI indazole analog, α-PHPP and α-POP, with 11 newly distributed designer drugs in illegal products. Forensic Sci Int. 2014 Oct;243:1-13. PMID 24769262 doi: 10.1016/j.forsciint.2014.03.013.

“Janssen Research & Development, LLC Voluntarily Suspends Dosing in Phase 2 Clinical Trials of Experimental Treatment for Mood Disorders”. Janssen.com. 17 January 2016. Retrieved 21 January 2016.

External links

Bial pipeline
Urea compounds and their use as faah enzyme inhibitors – Patent
Selection patent

WO2005073199A1 *	Jan 15, 2005	Aug 11, 2005	Aventis Pharma Gmbh	Indazole derivatives as inhibitors of hormone-sensitive lipases
WO2010074588A2	Dec 23, 2009	Jul 1, 2010	BIAL – PORTELA & Cª, S.A.	Pharmaceutical compounds
WO2012015324A1	Jul 28, 2011	Feb 2, 2012	Bial – Portela & Ca, S.A.	Process for the synthesis of substituted urea compounds
US4051252 *	Nov 24, 1975	Sep 27, 1977	Bayer Aktiengesellschaft	3-aminoindazole-1 and 2-carboxylic acid derivatives
US4331678 *	Jan 14, 1980	May 25, 1982	Fbc Limited	Carbamoyl pyrazole compounds and their pesticidal application
US4973588 *	Feb 10, 1989	Nov 27, 1990	Mitsui Petrochemical Industries, Ltd.	Imidazole derivatives having anti-hypoxia properties
US5578627 *	Oct 27, 1993	Nov 26, 1996	Toyama Chemical Co., Ltd.	1,2-benzoisoxazole derivative or its salt and brain-protecting agent comprising the same

BIA 10-2474

Systematic (IUPAC) name

3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide

Clinical data

Legal status

Investigational New Medicine

Routes of
administration

Oral

Identifiers

PubChem

CID: 46831476

Chemical data

Formula

C₁₆H₂₀N₄O₂

Molecular mass	300.36 g·mol⁻¹
SMILES O=C(N1C=C(C2=C[N+]([O-])=CC=C2)N=C1)N(C3CCCCC3)C

/////////

C1C(CCCC1)N(C)C(=O)n2cc(nc2)c3ccc[n+](c3)O

FDA´s Emerging Technology Applications Program – Draft Guidance

January 21, 2016 4:35 am / Leave a comment

FDA´s Emerging Technology Applications Program – Draft Guidance

The FDA recently published a draft guidance for industry on the “Advancement of Emerging Technology Applications”. The draft guidance provides recommendations to pharmaceutical companies interested in participating in a program involving the submission of CMC information containing emerging manufacturing (including testing, packaging and labeling, and quality control) technology to FDA. Find out more about the draft guidance for industry “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“..

http://www.gmp-compliance.org/enews_05164_FDA%B4s-Emerging-Technology-Applications-Program—Draft-Guidance_15455,15149,15153,Z-PDM_n.html

On December 23, 2015, the FDA published a draft guidance for industry “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“. Comments and suggestions regarding this draft document should be submitted within 60 days of publication.

The draft guidance provides recommendations to pharmaceutical companies interested in participating in a program involving the submission of CMC (chemistry, manufacturing, and controls) information containing emerging manufacturing (including testing, packaging and labeling operations, and quality control) technology to FDA.

The program is open for new drug applications (INDs), original or supplemental new drug application (NDA), abbreviated new drug application (ANDA), or biologic license application (BLA). It only affects the quality section of a submission (CMC and facility-related information).

The development of emerging manufacturing technology, like, for example, aseptic manufacturing facilities with highly automated systems and isolators, may lead to improved manufacturing, and therefore improved product quality and availability throughout a product´s lifecycle.

Pharmaceutical companies can submit questions and proposals about the use of these technologies to a group within CDER (Emerging Technology Team – ETT).

The draft guidance is a follow-on to the FDA guidance for industry “PAT – A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance” which describes the concept that quality cannot be tested into products. It should be built-in or should be present by design. Through the ETT, FDA intends to encourage the adoption of innovative approaches by leveraging existing resources of FDA to facilitate regulatory reviews of submissions.

Examples of emerging technology elements include an innovative or novel:

Product manufacturing technology, such as the dosage form;
Manufacturing process (e.g., design, scale-up, and/or commercial scale);
Testing technology.

Interested parties should submit a written meeting request to participate in the ETT program at least three months prior to the planned application (IND, ANDA, BLA, NDA) submission date. In addition to the items outlined in the FDA guidance “Formal Meetings Between the FDA and Sponsors or Applicants” the request should also include the following items:

A brief description of the proposed testing, process, and/or proposed technology;
A brief explanation why the proposed testing, process, and/or technology are substantially novel and unique;
A description of how the proposed testing and/or technology could modernize pharmaceutical manufacturing and thus improve product safety, identity, strength, quality, or purity;
A summary of the development plan and any perceived roadblocks to technical or regulatory implementation;
A timeline for submission.

The request should generally not exceed five pages and FDA expects to notify companies of its decision regarding acceptance into the program within 60 days of receipt of the request. Once accepted into the program, the participant can engage with ETT and CMC in accordance with existing meeting procedures and guidances (e.g. above mentioned FDA guidance on Formal Meetings).

For further information, please find all the details in the draft guidance “Advancement of Emerging Technology Applications to Modernize the Pharmaceutical Manufacturing Base“.

Lupin Ltd, Patent, Pitavastatin, WO2014203045

January 20, 2016 4:59 am / Leave a comment

Lupin Ltd, Patent, Pitavastatin, WO2014203045

A NOVEL, GREEN AND COST EFFECTIVE PROCESS FOR SYNTHESIS OF TERT-BUTYL (3R,5S)-6-OXO-3,5-DIHYDROXY-3,5-O-ISOPROPYLIDENE-HEXANOATE

ROY, Bhairabnath; (IN).
SINGH, Girij, Pal; (IN).
LATHI, Piyush, Suresh; (IN).
AGRAWAL, Manoj, Kunjabihari; (IN).
MITRA, Rangan; (IN).
TRIVEDI, Anurag; (IN).
PISE, Vijay, Sadashiv; (IN).
RUPANWAR, Manoj; (IN)

The present invention describes an eco-friendly and cost effective process for the synthesis of teri-butyl (3R,5S)-6-oxo-3,5-dihydroxy-3,5-0-isopropylidene-hexanoate [I]

PITAVASTATIN

TEXT

tert-b tyl (3R,5S)-6-oxo-3,5-dihydroxy-3,5-0-isopropylidene-hexanoate [I] [CAS No. 124752-23-4] is key intermediate for the preparation of statins such as Atorvastatin (Tetrahedron 63, 2007, 8124 -8134), Cerivastatin (Journal of Labeled Compounds and Radiopharmaceuticals, 49, 2006 311-319), Fluvastatin [WO2007125547; US 4739073], Pitavastatin [WO2007/132482; US2012/22102 Al, WO2010/77062 A2; WO2012/63254 Al ; EP 304063; Tetrahedron Letters, 1993, 34, 513 – 516; Bulletin of the Chemical Society of Japan, 1995, 68, 364 – 372] and Rosuvastatin [WO2007/125547 A2; WO2011/132172 Al ; EP 521471]. Statins are used for treatment of hypercholesterolemia, which reduces the LDL cholesterol levels by inhibiting activity of HMG-CoA reductase enzyme, which is involved in the synthesis of cholesterol in liver.

[I]

Compound [I] is generally obtained by various methods of oxidation of teri-butyl 2- ((4R,65)-6-(hydroxymethyl)-2,2-dimethyl-l,3-dioxan-4-yl)acetate [compound II] and are discussed in details hereinafter. In addition, various methods for synthesis of compound [II] are also elaborated below.

[II]

A) tert-butyl2-((4«,6.S)-6-(hydroxymethyl)-2,2-dimethyl-l,3-dioxan-4-yl)acetate

[compound II]

US patent Number 5278313 describes a process for synthesis of compound [II]

(Schemel). In the said process, (5)-methyl 4-chloro-3-hydroxybutanoate has been obtained in only 70% yield through whole cell enzymatic reduction of methyl 4-chloro-3- oxobutanoate, which has a necessity of special equipment such as fermenters as well as other microbial facilities such as sterile area, autoclaves, incubator for growing seed culture, etc.

(S)-mefhyl 4-chloro-3-hydroxybutanoate upon reaction with teri-butyl acetate in presence of LiHMDS or LDA at -78°C, yielded (S)-ieri-butyl 6-chloro-5-hydroxy-3- oxohexanoate, which was further transformed to corresponding diol through syn selective reduction in presence of methoxydiethyl borane/sodium borohydride at -78°C. The diol thus obtained was converted to compound [II] .

The overall yield for this process is low and required special equipment such as fermenters, etc and in addition to that, this process is not cost effective due to use of costly reagent such as methoxydiethyl borane.

Moreover, methoxydiethylborane is highly pyrophoric (Encyclopedia for organic synthesis, editor in chief L. Paquette; 2, 5304; Published by John and Wiley Sons;

Organic Process Research & Development 2006, 10, 1292-1295) and hence safety is a major concern.

Scheme 1

EP 1282719 B l (PCT application WO 01/85975 Al ) discloses a process for synthesis of compound ( R, 5S)-tert-bv y\ 3,5,6-trihydroxyhexanoate from (S)-tert-b tyl-5,6-dihydroxy-3-oxohexanoate through a) asymmetric hydrogenation in presence of a chiral catalyst e.g. di-mu-chlorobis-[(p-cymene)chlororuthenium(II)] along with an auxiliary such as (IS, 2S)-(+)-N- (4-toluenesulfonyl)-l ,2-diphenylethylenediamine as ligand, which gave desired product only in 70% diastereomeric excess (de); b) Whole cell enzymatic reduction of (S)-tert- butyl 5,6-dihydroxy-3-oxohexanoate to obtain compound (3R, 5S)-tert-bv y\ 3,5,6-trihydroxyhexanoate in 99% de (80% yield).

It is needless to mention that it has necessity of fermenter and other microbiological equipment (Scheme 2).

Moreover, conversion of (2>R,5S)-tert-bv y\ 6-acetoxy-3,5-dihydroxyhexanoate to tert-bv yl 2-((4R,65)-6-(acetoxymethyl)-2,2-dimethyl-l ,3-dioxan-4-yl)acetate was accomplished in only 25% yield and also required the flash chromatography for isolation of desired product.

Thus, overall yield for this process is poor and process is not operation friendly especially at large scale hence cannot be considered feasible for commercial manufacturing.

Scheme 2

EP1317440 Bl (PCT Application WO 02/06266 Al) has disclosed the process for synthesis of compound [II] from 6-chloro-2,4,6-trideoxy-D-erythro-hexose (Scheme 3) .

In the said patent application 6-chloro-2,4,6-trideoxy-D-erythro-hexose was converted to (4R, 65)-4-hydroxy-6-chloromethyl-tetrahydropyran-2one with excess of bromine in presence of potassium bicarbonate, which liberates environmentally undesired gas i.e. carbon dioxide.

Moreover, starting material i.e. 6-chloro-2,4,6-trideoxy-D-erythro-hexose is not commercially available and conversion efficiency of starting material at large scale towards (4R, 65)-4-hydroxy-6-chloromethyl-tetrahydropyran-2-one is only 67%.

Scheme 3

US Patent No. 6689591 B2 has demonstrated the whole cell enzymatic reduction of teri-butyl 6-chloro-3,5-dioxohexanoate to compound [II] (Scheme 4).

In the said process, whole cell enzymatic reduction is not specific; yield for desired product is only 34% and other partially reduced products are also obtained.

Hence, further purification is required for obtaining the desired compound. Thus, this process is not suitable for commercial scale.

Scheme 4

Tatsuya et al (Tetrahedron Letters; 34, 1993,513 – 516) has reported synthesis of compound [I] from derivative of L-tartatric acid (Scheme 5).

In the said process, tartaric acid di-i^‘sopropyl ester is doubly protected by tert-butyldimethylsilyl group, which was reacted with dianion of teri-butyl acetoacetate to give β, δ-diketo ester compound.

β,δ-diketo ester was reacted with 2 equivalent of diisobutylaluminium hydride (which is a pyrophoric reagent) to afford -hydroxy,8-keto ester in only 60% yield.

This process is not industrially viable as overall yield is very low and also because of use of costly and pyrophoric reagents/chemicals.

Scheme 5

US7205418 (PCT application WO03/053950A1) has described the process for synthesis of compound [II] from (S)-ieri-butyl-3,4-epoxybutanoate (Scheme 6).

The overall yield for this process is very low and moreover, it required the diastereomeric separation of teri-butyl 2-(6-(iodomethyl)-2-oxo-l,3-dioxan-4-yl)acetate by flash chromatography.

Since overall requirement of title compound is very high, any operation involving flash chromatography will tend to render the process commercially unviable.

Scheme 6

Fengali et al (Tetrahedron: Asymmetry 17; 2006; 2907-2913) has reported the process for synthesis of compound [II] from racemic epichlorohydrin (Scheme 7).

In this process, racemic epichlorohydrin was converted to corresponding nitrile intermediate through reaction with sodium cyanide; nitrile intermediate thus obtained was further resolved through lipase catalyzed stereo-selective esterification to obtain (5)-4-(benzyloxy)-3-hydroxybutanenitrile and (R)-l-(benzyloxy)-3-cyanopropan-2-yl acetate;

separation of desired product i.e. (S)-4-(benzyloxy)-3-hydroxybutanenitrile having 98% de (40% yield) was done by column chromatography.

Needless to mention a commodity chemical like compound [I] cannot be manufactured by such a laboratory method, which involved number of steps.

Scheme 7

Bode et al (Organic letters, 2002, 4, 619-621) has reported diastereomer- specific hydrolysis of 1,3-diol-acetonides (Scheme 8).

In this publication, duration of the reaction for diastereomer- specific hydrolysis of 1,3, diol-acetonides is reported to be 4 h, however, in our hand it was observed that hardly any reaction took place in 4 h, which made it non-reproducible.

In addition to that, separation of desired product is achieved by flash chromatography and it is needless to mention that any process which involved flash chromatography would render the process to be commercially unviable.

Hence, additional innovation needs to be put in for making the process industrially viable.

Scheme 8

CN 101613341A has reported the process for synthesis of compound [II] (Scheme

9).

In the same patent application tert-b tyl (S)-6-chloro-5-hydroxy-3-oxohexanoate was synthesized through Blaise condensation of (5)-4-chloro-3-hydorxy-butanenitrile with zinc enolate of tert butyl bromo acetate.

In the literature, synthesis of tert-bv yl (S)-6-chloro-5-hydroxy-3-oxohexanoate was reported through Blaise condensation of silyl protected (5)-4-chloro-3-(trimethylsilyl)oxy-butanenitrile with zinc enolate of tert butyl bromo acetate, in good yield (Synthesis 2004, 16, 2629-2632). Thus, protection of hydroxy group in (5)-4-chloro-3-hydorxy-butanenitrile is imperative.

In the said Chinese patent application, in claim 7, it was mentioned that solvent used for conversion of tert-bv yl (5)-6-chloro-5-hydroxy-3-oxohexanoate to ( R,5S)-tert-butyl 6-chloro-3,5-dihydroxyhexanoate is anyone or mixture of more than one from tetrahydrofuran, ether, methanol, ethanol, n-propanol, /so-propanol and ethylene glycol.

However, in enablement the only example using mixture of solvent was that of THF-methanol (Experimental section, Example 4: The preparation of (R,5)-6-chloro-3,5- dihydroxyhexanoate) and same outcome was expected in other individual or mixture of solvents.

Claim 8 of CN 101613341A mentioned that reduction was carried out by any one or mixture of more than one reducing agents such as sodium borohydride, potassium borohydride, lithium aluminum hydride, diethylmethoxy borane, triethyl borane and tributyl borane.

It implies that either any one of the reducing agents or a mixture of the same can be employed. From reaction mechanism it is very much clear that diethylmethoxy borane, triethyl borane and tributyl borane form the six membered complex between optically active hydroxyl and carbonyl group, which gets reduced by sodium borohydride, signifying that individually diethylmethoxy borane, triethyl borane and tributyl borane are not reducing agents

Moreover, in claims 12 and 13 (Experimental section, Example 4: The preparation of (R,S)-6-chloro-3,5-dihydroxyhexanoate), it is mentioned that reduction should be carried out in temperature range -80 °C to -60 °C, implying that reaction would not work beyond this temperature range i.e. it would work in the temperature window of -80 °C to -60 °C only.

Summarizing, the teachings of the application are not workable.

Scheme 9

Wolberg et al (Angewandte Chemie International Edition, 2000, 4306) has reported that diastereomeric excess for syn selective reduction using mixture of diethyl methoxy borane/sodium borohydride of compound [VI] gave 93% de for compound [VIII], which required further re-crystallization to obtain compound [VIII] in 99% de and 70% yield.

Thus, all the reported methods for stereo-selective hydride reduction of compound [VI] were achieved through mixture of trialkyl borane or diethyl methoxy borane & sodium borohydride in THF, at -78°C. As mentioned earlier, trialkyl borane or diethyl methoxy borane are pyrophoric in nature; in addition to that anhydrous THF is costly and moreover, reaction required large dilution.

Hence, there is need for developing efficient, environment friendly, cost effective and green process for stereo-selective reduction compound [VI].

B) The process of Oxidation of compound [II] to compound [I] has been discussed in following literature processes.

1) Swern oxidation (US4970313; Tetrahedron Letters, 1990, 2545

Synthetic Communications, 2003, 2275 – 2284).

2) Parrkh-Doering oxidation (J. Am. Chem. Soc, 1967, 89, 5505-5507)

3) TEMPO/NaOCl oxidization (EP2351762)

4) Trichloroisocyanuric acid/ TEMPO (CN 101747313A)

5) Oxidation of compound [II] to compound [I] through IBX [CN101475558A].

It would be evident that most of the reported methods are not “green” and

environmentally benign; none of the reported methods use molecular oxygen as oxidizing agent in presence of metal catalyst/co-catalyst.

Example 18: Process for synthesis of tert-butyl 2-((4R,6S)-6-formyl-2,2-dimethyl-l,3-dioxan-4-yl)acetate [I]

A reactor was charged with 1.1 g of copper (I) chloride and 10 mL of acetonitrile. 2-2′ Bipyridyl (156 mg) and TEMPO (156 mg) were added to the reactor under oxygen environment at 25°C. A solution of (6-Hydroxymethyl-2,2-dimethyl-[l,3]dioxan-4-yl)-acetic acid tert-butyl ester 2.6 g in 26 mL DCM was added dropwise over a period of 10 min into it. The reaction mass was stirred at 40°C and progress of reaction was monitored on GLC, which shows that 90% conversion for desired product.

Example 19: Process for synthesis of tert-butyl 2-((4R,6S)-6-formyl-2,2-dimethyl-l,3-dioxan-4-yl)acetate [I]

A reactor was charged with 1.1 g of copper (I) chloride and 10 mL of dichlorome thane. 2-2′ Bipyridyl (156 mg) and TEMPO (156 mg) were added to the reactor under oxygen environment at 25°C. A solution of (6-Hydroxymethyl-2,2-dimethyl-[l,3]dioxan-4-yl)-acetic acid tert-butyl ester 2.6 g in 26 mL DCM was added dropwise over a period of 10 min into it. The reaction mass was stirred at 40°C and progress of reaction was monitored on GLC, which shows that 90% conversion for desired product.

AUTHORS

SEE………https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014203045&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCT+Biblio

///////

Breaking and Making of Olefins Simultaneously Using Ozonolysis: Application to the Synthesis of Useful Building Blocks and Macrocyclic Core of Solomonamides

January 19, 2016 6:43 am / Leave a comment

A simple and practical one-pot, two-directional approach to access olefinic esters through simultaneous breaking and making of olefins using ozonolysis of alkenyl aryl selenides is disclosed. The scope of the method with a variety of examples is demonstrated, and the end products obtained here are useful building blocks. As a direct application of the present method, the macrocyclic core of potent anti-inflammatory natural cyclic peptides, solomonamides, is synthesized.

Breaking and Making of Olefins Simultaneously Using Ozonolysis: Application to the Synthesis of Useful Building Blocks and Macrocyclic Core of Solomonamides

K. Kashinath, Santu Dhara, and D. Srinivasa Reddy^*

CSIR-National Chemical Laboratory, Division of Organic Chemistry, Dr. Homi Bhabha Road, Pune 411008, India

Org. Lett., 2015, 17 (9), pp 2090–2093

DOI: 10.1021/acs.orglett.5b00637

Publication Date (Web): April 14, 2015

*E-mail: ds.reddy@ncl.res.in.

http://pubs.acs.org/doi/abs/10.1021/acs.orglett.5b00637

http://pubs.acs.org/doi/suppl/10.1021/acs.orglett.5b00637/suppl_file/ol5b00637_si_001.pdf

GENERAL METHOD

Dr. D. Srinivasa Reddy

READ…….http://oneorganichemistoneday.blogspot.in/2015/02/dr-d-srinivasa-reddy.html

AT 9283

January 19, 2016 4:12 am / 1 Comment on AT 9283

AT9283, AT 9283

N-cyclopropyl-N’-[3-[6-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea

1-cyclopropyl-3-[(3Z)-3-[5-(morpholin-4-ylmethyl)benzimidazol-2-ylidene]-1,2-dihydropyrazol-4-yl]urea

896466-04-9
Molecular Weight	381.43
Molecular Formula	C19H23N7O2

CAS

896466-04-9, 896466-57-2 ((±)-Lactic acid), 896466-61-8 (HCl), 896466-55-0 (methanesulfonate) AT9283/AT-9283

MolFormulaC22H29N7O5

MolWeight471.5096

CAS 896466-76-5 L LACTATE

(2S)-2-Hydroxypropanoic acid compd. with N-cyclopropyl-N’-[3-[6-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]urea

Astex Therapeutics Ltd, INNOVATOR

AT-9283 is a potent AuroraA/AuroraB and multi-kinase inhibitor. AT-9283 has shown to inhibit growth and survival of multiple solid tumor cell lines and is efficacious in mouse xenograft models.

AT 9283 is a substance being studied in the treatment of some types of cancer. It is small molecule a multi-targeted c-ABL, JAK2, Aurora A and B inhibition with 4, 1.2, 1.1 ad approximate 3 nM for Bcr-Abl (T3151), Jak2 and Jak3 aurora A and B, respectively. It blocks enzymes (Aurora kinases) involved in cell division and may kill cancer cells

WO2006070195 to Astex Therapeuitcs discloses pyrazole compounds of the general structure shown below as kinase inhibitors.

The compound AT9283 is in phase II clinical trials for treating advanced or metastatic solid tumors or Non-Hodgkin’s Lymphoma. AT9283 is shown below.

a Reagents and conditions:

(a) SOCl2, THF, DMF; (b) morpholine, THF, Et3N; ………FORMATION OOF ACID CHLORIDE AND COUPLING WITH MORPHOLINE

(d) 10% Pd-C, H2, EtOH; TWO NITRO GPS TO TWO AMINO , REDN

(e) EDC, HOBt, DMF; (f) AcOH, reflux;COUPLING WITH 4-Nitro-lH-pyrazole-3-carboxylic acid

(g) 10%Pd-C, H2, DMF; NITRO GP TO AMINO

(h) standard amide and urea coupling methods

WO2006070195

https://www.google.co.in/patents/WO2006070195A1?cl=en

Stage 10: Synthesis of l-cvclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- beiizoimidazol-2-ylV 1 H-pyrazol-4-yli -urea.

To a mixture of 7-morpholin-4-ylmethyl-2,4-dihydro- 1 ,2,4,5a, 10- pentaaza- cyclopenta[a]fluoren-5-one (10.7 g, 32.9 mmol) in NMP (65 mL) was added cyclopropylamine (6.9 mL, 99 mmol). The mixture was heated at 100 ⁰C for 5 h. LC/MS analysis indicated -75% conversion to product, therefore a further portion of cyclopropylamine (2.3 mL, 33 mmol) was added, the mixture heated at 100 ⁰C for 4 h and then cooled to ambient. The mixture was diluted with water (100 mL) and extracted with EtOAc (100 niL). The organic portion was washed with sat. aq. NH₄Cl (2 x 50 mL) and brine (50 rnL) and then the aqueous portions re-extracted with EtOAc (3 x 100 mL). The combined organic portions were dried over MgSO₄ and reduced in vacuo to give l-cycloρropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea as an orange glassy solid (9.10 g).

Stage 11: Synthesis of l-cvclopropyl-S-P-fS-morpholin^-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yll-urea, L-lactate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea (9.10 g, 24 mmol) in EtOAc-iPrOH (1 :1, 90 mL) was added L-lactic acid (2.25 g, 25 mmol). The mixture was stirred at ambient temperature for 24 h then reduced in vacuo. The residue was given consecutive slurries using toluene (100 mL) and Et₂O (100 mL) and the resultant solid collected and dried (8.04 g).

This solid was purified by recrystallisation from boiling iPrOH (200 mL) to give after drying l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)- lH-pyrazol-4-yl]-urea, L-lactate salt (5.7 g) as a beige solid.

EXAMPLE 66

Stage 1: Preparation of (3,4-dinitrophenyl)-morpholin-4-yl-methanone

3,4-Dinitrobenzoic acid (1.000Kg, 4.71mol, l.Owt), tetiuhydrofuran (10.00L₅ lO.Ovol), and dimethylformamide (0.010L, O.Olvol) were charged to a flask under nitrogen. Thionyl chloride (0.450L, 6.16mol, 0.45vol) was added at 20 to 3O⁰C and the reaction mixture was heated to 65 to 7O⁰C. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO), typically in 3 hours. The reaction mixture was cooled to 0 to 5⁰C and triethylamine (1.25L, 8.97mol, 1.25vol) was added at 0 to 10⁰C. Morpholine (0.62L, 7.07mol, 0.62vol) was charged to the reaction mixture at 0 to 1O⁰C and the slurry was stirred for 30 minutes at 0 to 1O⁰C. Reaction completion was determined by H NMR analysis (d₆-DMSO). The reaction mixture was warmed to 15 to 2O⁰C and water (4.00L, 4.0vol) was added. This mixture was then charged to a 4OL flange flask containing water (21.0OL, 21.0vol) at 15 to 25⁰C to precipitate the product. The flask contents were cooled to and aged at 0 to 5⁰C for 1 hour and the solids were collected by filtration. The filter-cake was washed with water (4x 5.00L, 4x 5.0vol) and the pH of the final wash was found to be pH 7. The wet filter-cake was analysed by H NMR for the presence of triethylamine hydrochloride. The filter-cake was dried at 40 to 45⁰C under vacuum until the water content by KF <0.2%w/w, to yield (3,4-dinitrophenyl)-morpholin-4-yl-methanone (1.286Kg, 97.0%, KF 0.069%w/w) as a yellow solid.

Stage 2: Preparation of 4-(3,4-dinitro-benzyl)-morpholine

C₁₁H₁₁N₃O₆ C₁₁H₁₃N₃O₅

FW:281.22 FW:267.24

(3,4-DinitiOphenyl)-morpholin-4-yl-methanone (0.750Kg, 2.67mol, l.Owt) and tetrahydrofuran (7.50L, lO.Ovol) were charged to a flask under nitrogen and cooled to 0 to 5⁰C. Borontrifluoride etherate (0.713L, 5.63mol, 0.95vol) was added at 0 to 5⁰C and the suspension was stirred at this temperature for 15 to 30 minutes. Sodium borohydride (0.212Kg, 5.60mol, 0.282wt) was added in 6 equal portions over 90 to 120 minutes. (A delayed exotherm was noted 10 to 15 minutes after addition of the first portion. Once this had started and the reaction mixture had been re-cooled, further portions were added at 10 to 15 minute intervals, allowing the reaction to cool between additions). The reaction mixture was stirred at 0 to 5⁰C for 30 minutes. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). Methanol (6.30L, 8.4vol) was added drop wise at 0 to 1O⁰C to quench the reaction mixture (rapid gas evolution, some foaming). The quenched reaction mixture was stirred at 0 to 1O⁰C for 25 to 35 minutes then warmed to and stirred at 20 to 3O⁰C (exotherm, gas/ether evolution on dissolution of solid) until gas evolution had slowed. The mixture was heated to and stirred at 65 to 7O⁰C for 1 hour. The mixture was cooled to 30 to 4O⁰C and concentrated under vacuum at 40 to 45⁰C to give crude 4-(3,4-dinitro-benzyl)-morpholine (0.702Kg, 98.4%) as a yellow/orange solid.

4-(3,4-Dinitro-benzyl)-niorpholme (2.815kg, 10.53mol, l.Owt) and methanol (12.00L, 4.3vol) were charged to a flask under nitrogen and heated to 65 to 7O⁰C. The temperature was maintained until complete dissolution. The mixture was then cooled to and aged at 0 to 5⁰C for 1 hour. The solids were isolated by filtration. The filter-cake was washed with methanol (2x 1.50L, 2x 0.5vol) and dried under vacuum at 35 to 45°C to give 4-(3,4-dinitro-benzyl)-morpholine (2.353Kg, 83.5% based on input Stage 2, 82.5% overall yield based on total input Stage 1 material,) as a yellow solid.

Stage 3: Preparation of 4-morpholin-4-yl-methyl-benzene-L2-diamine

C₁₁H₁₃N₃O₅ C₁₁H₁₇N₃O

FW:267.24 FW:207.27

4-(3,4-Dinitro-benzyl)-morρholine (0.800Kg, 2.99mol, l.Owt), and ethanol (11.20L, 14.0vol) were charged to a suitable flask and stirred at 15 to 25⁰C and a vacuum / nitrogen purge cycle was performed three times. 10% Palladium on carbon (10%Pd/C, 50%wet paste, 0.040Kg, 0.05wt wet weight) was slurried in ethanol (0.80L, l.Ovol) and added to the reaction. The mixture was cooled to 10 to 2O⁰C and a vacuum / nitrogen purge cycle was performed three times. A vacuum / hydrogen purge cycle was performed three times and the reaction was stirred under a hydrogen atmosphere at 10 to 2O⁰C. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO), typically 14 to 20 hours. A vacuum / nitrogen purge cycle was performed three times and the reaction mixture was filtered through glass microfibre paper under nitrogen. The filter-cake was washed with ethanol (3x 0.80L, 3x l.Ovol) and the combined filtrate and washes were concentrated to dryness under vacuum at 35 to 45⁰C to give 4-morpholin-4-yl-methyl-benzene-l,2- diamine (0.61 IKg 98.6%) as a brown solid.

Stage 4: Preparation of 4-nitiO-lH-pyrazole-3-carboxγlic acid methyl ester

C₄H₃N₃O₄ C₅H₅N₃O₄

FW: 157.09 FW: 171.11

4-Nitro-lH-pyrazole-3-carboxylic acid (1.00kg, 6.37mol, l.Owt) and methanol (8.00L, 8.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. The suspension was cooled to 0 to 5°C under nitrogen and thionyl chloride (0.52L, 7.12mol, 0.52vol) was added at this temperature. The mixture was warmed to 15 to 25°C over 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). The mixture was concentrated under vacuum at 35 to 45°C. Toluene (2.00L, 2.0vol) was charged to the residue and removed under vacuum at 35 to 45⁰C. The azeotrope was repeated twice using toluene (2.00L, 2.0vol) to give 4-nitro-lH-pyrazole-3-carboxylic acid methyl ester (1.071Kg, 98.3%) as an off white solid.

Stage 5: Preparation of 4-amino-lH-pyrazole-3-carboxylic acid methyl ester. O₂Me

C₅H ₅N₃O₄ C₅H₇N₃O₂ FW: 171.11 FW: 141.13

A suspension of 4-nitro-lH-pyrazole-3-carboxylic acid methyl ester (1.084Kg, 6.33mol, l.Owt) and ethanol (10.84L, lO.Ovol) was heated to and maintained at 30 to 35°C until complete dissolution occurred. 10% Palladium on carbon (10% Pd/C wet paste, 0.152Kg, 0.14wt) was charged to a separate flask under nitrogen and a vacuum / nitrogen purge cycle was performed three times. The solution of 4-nitro- lH-pyrazole-3-carboxylic acid methyl ester in ethanol was charged to the catalyst and a vacuum / nitrogen purge cycle was performed three times. A vacuum / hydrogen purge cycle was performed three times and the reaction was placed under an atmosphere of hydrogen. The reaction mixture was stirred at 28 to 30°C until deemed complete by ¹H NMR analysis (d₆-DMSO). The mixture was filtered under nitrogen and concentrated under vacuum at 35 to 45⁰C to give 4-amino-lH- pyrazole-3-carboxylic acid methyl ester (0.883Kg, 98.9%) as a purple solid.

Stage 6: Preparation of 4-fert-butoxycarbonylamino-lH-pyrazole-3-carboxylic acid

C₅H₇N₃O₂ C₉H₁₃N₃O₄

FW: 141.13 FW:227.22

4-Amino-lH-pyrazole-3-carboxylic acid methyl ester (1.024Kg, 7.16mol, l.Owt) and dioxane (10.24L, lO.Ovol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. 2M aq. Sodium hydroxide solution (4.36L, 8.72mol, 4.26vol) was charged at 15 to 25⁰C and the mixture was heated to 45 to 55⁰C. The temperature was maintained at 45 to 55⁰C until reaction completion, as determined by ¹H NMR analysis (d₆-DMSO). Di-te/Y-butyl dicarbonate (Boc anhydride, 1.667Kg, 7.64mol, 1.628wt) was added at 45 to 55°C and the mixture was stirred for 55 to 65 minutes. ¹H NMR IPC analysis (d₆-DMSO) indicated the presence of 9% unreacted intermediate. Additional di-fert-butyl dicarbonate (Boc anhydride, 0.141Kg, 0.64mol, 0.14wt) was added at 55°C and the mixture was stirred for 55 to 65 minutes. Reaction completion was determined by ¹H NMR analysis (d₆-DMSO). The dioxane was removed under vacuum at 35 to 45⁰C and water (17.60L, 20.0vol) was added to the residue. The pH was adjusted to pH 2 with 2M aq. hydrochloric acid (4.30L, 4.20vol) and the mixture was filtered. The filter-cake was slurried with water (10.00L₃ 9.7vol) for 20 to 30 minutes and the mixture was filtered. The filter-cake was washed with heptanes (4.10L, 4.0vol) and pulled dry on the pad for 16 to 20 hours. The solid was azeodried with toluene (5x 4.00L, 5x 4.6vol) then dried under vacuum at 35 to 45°C to give 4-tert- butoxycarbonylamino-lH-pyrazole-3-carboxylic acid (1.389Kg, 85.4%) as a purple solid.

Stage 7: Preparation of [3-(2-amino-4-moipholin-4-ylmetliyl-phenylcarbamoviy lH-pyrazol-4-yl]-carbamic acid tert-butyl ester

C₉H₁₃N₃O₄ C₁₁H₁₇N₃O C₂₀H₂₈N₆O₄

FW: 227.22 FW: 207.27 FW: 416.48

+ regioisomer

4-førf-Butoxycarbonylamino-lH-pyrazole-3-carboxylic acid (0.750Kg, 3.30 mol, l.Owt), 4-morpholin-4yl-methyl-benzene-l,2-diamine (0.752Kg, 3.63mol, l.Owt) and N,N’-dimethylformamide (11.25L, 15.0vol) were charged under nitrogen to a flange flask equipped with a mechanical stirrer and thermometer. 1- Hydroxybenzotriazole (HOBT, 0.540Kg, 3.96mol, 0.72wt) was added at 15 to 25⁰C. N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, 0.759Kg, 3.96mol, 1.01 wt) was added at 15 to 25⁰C and the mixture was stirred at this temperature for 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis. The reaction mixture was concentrated under vacuum at 35 to 45°C. The residue was partitioned between ethyl acetate (7.50L, lO.Ovol) and sat. aq. sodium hydrogen carbonate solution (8.03L, 10.7vol) and the layers were separated. The organic phase was washed with brine (3.75L, 5.0vol), dried over magnesium sulfate (1.00Kg, 1.33wt) and filtered. The filter-cake was washed with ethyl acetate (1.50L, 2.0vol). The combined filtrate and wash were concentrated under vacuum at 35 to 45⁰C to give [3-(2-amino-4-morpholin-4-ylmethyl-phenylcarbamoyl)-lH-pyrazol- 4-yl]-carbamic acid tert-butyl ester (1.217Kg, 88.6%) as a dark brown solid.

Stage 8 : Preparation of 3 -f 5-morpholin-4-ylmethyl- 1 H-benzoimidazol-2-ylV 1 H- pyrazol-4-ylamme

C₁₅H₁₉N₆O

FW: 298.35

As a mixture of two regioisomers

[3-(2-Amino-4-morpholin-4-ylmethyl-phenylcarbamoyl)-lH-pyrazol-4-yl]- carbamic acid tert-butyl ester (1.350Kg, 3.24 mol, l.Owt) and ethanol (6.75L, 5.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. Cone. aq. hydrochloric acid (1.10L, 13.2 mol, 0.80vol) was added at 15 to 3O⁰C under nitrogen and the contents were then heated to 70 to 😯⁰C and maintained at this temperature for 16 to 24 hours. A second portion of hydrochloric acid (0.1 IL, 1.32 mol, O.OSOvol) was added at 70 to 😯⁰C and the reaction was heated for a further 4 hours. Reaction completion was determined by HPLC analysis. The reaction mixture was cooled to 10 to 20⁰C and potassium carbonate (1.355Kg, 9.08mol, l.Owt) was charged portionwise at this temperature. The suspension was stirred until gas evolution ceased and was then filtered. The filter-cake was washed with ethanol (1.35L, l.Ovol) and the filtrates retained. The filter-cake was slurried with ethanol (4.00L, 3.0vol) at 15 to 25⁰C for 20 to 40 minutes and the mixture was filtered. The filter-cake was washed with ethanol (1.35L₃ 1.Ovol) and the total combined filtrates were concentrated under vacuum at 35 to 45⁰C. Ethanol (4.00L, 3. Ovol) was charged to the residue and removed under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.90L, 4.4vol) was added to the residue and stirred for 10 to 20 minutes at 15 to 25°C. The resulting solution was filtered, the filter-cake was washed with tetrahydrofuran (1.35L, l.Ovol) and the combined filtrates were concentrated under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.40L, 4. Ovol) was charged to the concentrate and removed under vacuum at 35 to 45⁰C. Tetrahydrofuran (5.40L, 4. Ovol) was charged to the concentrate and removed under vacuum at 35 to 45°C to give the desired product, 3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-ylamine (0.924Kg, 95.5%, 82.84% by HPLC area) as a purple foam.

Stage 9: Preparation of 7-morpholin-4-ylmethyl-2,4-dihydro- 1,2,4,5a ,10-pentaaza- cyclopentaFal fluoren-5 -one

C₁₅H₁₈N₆O C₁₆H₁₆N₆O₂ FW: 298.35 FW: 324.34

As a mixture of two regioisomers

3-(5-Morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)-lH-pyrazol-4-ylamine (0.993Kg, 3.33 mol, l.Owt) and tetrahydrofuran (14.0L, 15.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. The contents were stirred under nitrogen at 15 to 25°C and l,l ‘-carbonyldiimidazole (0.596Kg, 3.67 mol, O.όOwt) was added. The contents were then heated to 60 to 70⁰C and stirred at this temperature for 16 to 24 hours. Reaction completion was determined by TLC analysis. The mixture was cooled to 15 to 20⁰C and filtered. The filter-cake was washed with tetrahydrofuran (4.00L, 4. Ovol) and pulled dry for 15 to 30 minutes. The solid was dried under vacuum at 35 to 45⁰C to yield 7- morpholin-4-ylmethyl-2,4-dihydro- 1 ,2,4,5a, 10-pentaaza-cyclopenta[a]fluoren-5- one (0.810Kg, 75.0%th, 92.19% by HPLC area) as a purple solid. Stage 10: Preparation of l-cvclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-vD- 1 H-pyrazol-4-yll -urea

C₁₆H₁₆N₆O₂ C₁₉H₂₃N₇O₂

FW: 324.34 FW: 381.44

As a mixture of two regioisomers

7-Morpholin-4-ylmethyl-2₅4-dihydro-l,2,4,5a,10-pentaaza-cyclopenta[a]fluoren-5- one (0.797Kg, 2.46mol, l.Owt) and l-methyl-2-pyrrolidinone (2.40L, 3.0vol) were charged to a flange flask equipped with a mechanical stirrer, condenser and thermometer. Cyclopropylamine (0.279Kg, 4.88mol, 0.35 lwt) was added at 15 to 30°C under nitrogen. The contents were heated to 95 to 105°C and stirred at this temperature for 16 to 24 hours. Reaction completion was determined by ¹H NMR analysis. The reaction mixture was cooled to 10 to 20⁰C and ethyl acetate (8.00L, lO.Ovol) and sat. aq. sodium chloride (2.50L, 3.0vol) were charged, the mixture was stirred for 2 to 5 minutes and the layers separated. The organic phase was stirred with sat. aq. sodium chloride (5.00L, ό.Ovol) for 25 to 35 minutes, the mixture filtered and the filter-cake washed with ethyl acetate (0.40L, 0.5vol). The filter-cake was retained and the filtrates were transferred to a separating funnel and the layers separated. The procedure was repeated a further 3 times and the retained solids were combined with the organic phase and the mixture concentrated to dryness under vacuum at 35 to 45⁰C. The concentrate was dissolved in propan-2-ol (8.00L, lO.Ovol) at 45 to 55°C and activated carbon (0.080Kg₅ O.lwt) was charged. The mixture was stirred at 45 to 55⁰C for 30 to 40 minutes and then hot filtered at 45 to 55°C. The filter-cake was washed with propan-2-ol (0.40L, 0.5vol). Activated carbon (0.080L, O.lwt) was charged to the combined filtrates and wash and the mixture stirred at 45 to 55⁰C for 30 to 40 minutes. The mixture was hot filtered at 45 to 55⁰C and the filter-cake washed with propan-2-ol (0.40L, 0.5vol). The filtrates and wash were concentrated under vacuum at 35 to 45⁰C. Ethyl acetate (8.00, lO.Ovol) and water (2.20L, 3.0vol) were charged to the concentrate at 25 to 35⁰C and the mixture stirred for 1 to 2 minutes. The layers were separated and the organic phase was concentrated under vacuum at 35 to 45°C. Ethyl acetate (4.00L, 5.0vol) was charged to the residue and concentrated under vacuum at 35 to 45⁰C. Ethyl acetate (4.00L, 5.0vol) was charged to the residue and the mixture was stirred for 2 to 20 hours at 15 to 25⁰C. The mixture was cooled to and aged at 0 to 5°C for 90 to 120 minutes and then filtered. The filter-cake was washed with ethyl acetate (0.80L, l.Ovol) and pulled dry for 15 to 30 minutes. The solid was dried under vacuum at 35 to 45⁰C to yield l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea (0.533Kg, 56.8%, 93.20% by HPLC area) as a brown solid.

Several batches of Stage 9 product were processed in this way and the details of the quantities of starting material and product for each batch are set out in Table IA.

Table IA – Yields from urea formation step – Stage 10

Stage 11 : Preparation of l-cyclopiOpyl-3-r3-(5-moipholin-4-ylmethyl-lH- benzoimidazol-2-yl^s)-lH-pyrazol-4-yll-urea £-lactic acid salt L-Lactic acid

acid

C₁₉H₂₃N₇O₂ C₂₂H₂₉N₇O₅

FW: 381.44 FW: 471.52 l-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2-yl)-lH-ρyrazol- 4-yl]-urea (1.859Kg, 4.872mol, l.Owt), propan-2-ol (9.00L₅ 5.0vol) and ethyl acetate (8.0OL, 4.5vol) were charged to a flange flask equipped with a mechanical stirrer and thermometer. The contents were stirred under nitrogen and L-lactic acid (0.504Kg, 5.59mol, 0.269wt) was added at 15 to 25°C followed by a line rinse of ethyl acetate (0.90L, 0.5vol). The mixture was stirred at 15 to 25°C for 120 to 140 minutes. The solid was isolated by filtration, the filter-cake washed with ethyl acetate (2x 2.00L, 2x l.Ovol) and pulled dry for 20 to 40 minutes. The filter-cake was dissolved in ethanol (33.00L, 17.7vol) at 75 to 85⁰C, cooled to 65 to 70⁰C and the solution clarified through glass microfibre paper. The filtrates were cooled to and aged at 15 to 25⁰C for 2 to 3 hours. The crystallised solid was isolated by filtration, the filter-cake washed with ethanol (2x 1.00L, 2x 0.5vol) and pulled dry for at least 30 minutes. The solid was dried under vacuum at 35 to 45°C to yield 1- cyclopropyl-3 – [3-(5 -morpholin-4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4- yl]-urea l-lactic acid salt (1.386Kg, 58.7%th, 99.47% by HPLC area,) as a dark pink uniform solid.

The infra-red spectrum of the lactate salt (KBr disc method) included characteristic peaks at 3229, 2972 and 1660 cm^“1.

Without wishing to be bound by any theory, it is believed that the infra red peaks can be assigned to structural components of the salt as follow:

Peak: Due to:

3229 cm^“1 N-H

2972 cm^“1 aliphatic C-H

1660 cm^“1 urea C=O EXAMPLE 67

Synthesis of Crystalline Free Base And Crystalline Salt Forms Of l-Cyclopropyl-3-

[3-(5-Morpholin-4-ylmethyl-lH-Benzoimidazol-2-vπ-lH-Pyrazol-4-yll-Urea

A. Preparation of l-Cvclopropyl-3-[3-f5-Moφholm-4-ylmethyl-lH- Benzoimidazol-2-yl)-lH-Pyrazol-4-yll-Urea free base

A sample of crude l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-lH-pyrazol-4-yl]-urea free base was prepared as outlined in Example 60 and initially purified by column chromatography on silica gel, eluting with EtOAc- MeOH (98:2 – 80:20). A sample of the free base obtained was then recrystallised from hot methanol to give crystalline material of l-cyclopropyl-3-[3-(5-morpholin- 4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4-yl] -urea free base.

B. Preparation of l-Cyclopropyl-S-rS-fS-Morpholin^-ylmethyl-lH-Benzoimidazol- 2-yl)-lH-Pyrazol-4-yl]-Urea free base dihydrate

A sample of crude l-cyclopropyl-3-[3-(5-moφholm-4-ylmethyl-lH-benzoimidazol- 2-yl)-l H-pyrazol-4-yl] -urea free base was dissolved in THF and then concentrated in vacuo to a minimum volume (~4 volumes). To the solution was added water dropwise (2 – 4 volumes) until the solution became turbid. A small amount of THF was added to re-establish solution clarity and the mixture left to stand overnight to give a crystalline material which was air-dried to give l-cyclopropyl-3-[3-(5- morpholin-4-ylmethyl- 1 H-benzoimidazol-2-yl)- 1 H-pyrazol-4-yl] -urea free base dihydrate.

C. Preparation of l-Cyclopl^pyl-3-[3-(5-Morpholm-4-ylmethyl-lH-Benzoimidazol- 2-ylVlH-Pyrazol-4-yl]-Urea hydrochloride salt

A sample of crude l-cyclopropyl-3-[3-(5-moφholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-l H-pyrazol-4-yl] -urea free base was dissolved in the minimum amount of MeOH and then diluted with EtOAc. To the solution at 0 °C was slowly added 1.1 equivalents of HCl (4M solution in dioxane). Following addition, solid precipitated from solution which was collected by filtration. To the solid was added MeOH and the mixture reduced in vacuo. To remove traces of residual MeOH the residue was evaporated from water and then dried at 60 ⁰C/ 0.1 mbar to give the hydrochloride salt.

D. Preparation of l-Cyclopropyl-3-[3-(^‘5-Morpholm-4-ylmethyl-lH- Benzoimidazol-2-yiyiH-Pyrazol-4-yl1-Urea ethanesulfonate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea free base in MeOH-EtOAc was added 1 equivalent of ethanesulfonic acid. The mixture was stirred at ambient temperature and then reduced in vacuo. The residue was taken up in MeOH and to the solution was added Et₂O. Mixture left to stand for 72 h and the solid formed collected by filtration and dried to give l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH- benzoimidazol-2-yl)-lH-pyrazol-4-yl]-urea ethanesulfonate salt.

E. Preparation of l-Cvclopropyl-3-[3-(^‘5-Morpholm-4-ylmethyl-lH-Benzoimidazol- 2-yl)-lH-Pyrazol-4-yl]-Urea methanesulfonate salt

To a solution of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea free base (394 mg) in MeOH-EtOAc was added 1 equivalent of methanesulfonic acid (67 μl). A solid was formed which was collected by filtration, washing with EtOAc. The solid was dissolved in the minimum amount of hot MeOH, allowed to cool and then triturated with Et₂O. The solid was left to stand for 72 h and then collected by filtration, washing with MeOH, to give l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol-2- yl)-lH-pyrazol-4-yl]-urea methanesulfonate salt.

EXAMPLE 68

Characterisation of l-Cvclopropyl-3-[3-(5-Morpholin-4-ylmethyl-lH-

Benzoimidazol-2-yl)-lH-Pyrazol-4-yll-Urea Free Base and Salts

Various forms of l-cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-lH-benzoimidazol- 2-yl)-lH-pyrazol-4-yl]-urea were characterised. The forms selected for characterisation were identified from studies which primarily investigated extent of polymorphism and salt stability. The salts selected for further characterisation were the L-lactate salt, Free base dihydrate, Esylate salt, Free base and Hydrochloride salt.

Paper

Fragment-Based Discovery of the Pyrazol-4-yl Urea (AT9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity^†

Astex Therapeutics Ltd., 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, U.K.

J. Med. Chem., 2009, 52 (2), pp 379–388

DOI: 10.1021/jm800984v

Publication Date (Web): December 30, 2008

†

Coordinates of the protein complexes with compounds 5, 7, 9, 10, and 16 have been deposited in the Protein Data Bank under accession codes 2w1d, 2w1f, 2w1c, 2w1e, 2w1g (Aurora A), 2w1h (CDK2), and 2w1i (JAK2).

, * To whom correspondence should be addressed. Phone: +44 (0)1223 226209. Fax: +44 (0)1223 226201. E-mail: s.howard@astex-therapeutics.com.

http://pubs.acs.org/doi/abs/10.1021/jm800984v

http://pubs.acs.org/doi/suppl/10.1021/jm800984v/suppl_file/jm800984v_si_001.pdf

Abstract

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC₅₀ ≈ 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

1-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-1H-benzoimidazol-2-yl)-1H-pyrazol-4-yl]urea (16)

16 as a pale-yellow solid (8.19 g, 87%). ¹H NMR (400 MHz, Me-d₃-OD): 8.07 (s, 1H), 7.58 (s, 2H), 7.26 (d, J = 8 Hz, 1H), 3.74−3.69 (m, 4H), 3.67 (s, 2H), 2.74−2.69 (m, 1H), 2.55−2.50 (m, 4H), 1.02−0.93 (m, 2H), 0.72−0.65 (m, 2H). LC/MS: t_R = 1.08 min, m/z = 382 [M + H]⁺.

1-Cyclopropyl-3-[3-(5-morpholin-4-ylmethyl-1H-benzoimidazol-2-yl)-1H-pyrazol-4-yl]urea (16), Hydrochloride Salt

¹H NMR (400 MHz, DMSO-d₆): 13.26−13.07 (m, 2H), 11.05−10.80 (m, 1H), 9.64 (s, 1H), 8.08 (s, 1H), 7.98−7.19 (4H, m), 4.44 (s, 2H), 3.94 (d, J = 12.4 Hz, 2H), 3.77 (t, J = 12.3 Hz, 2H), 3.28−3.20 (m, 2H), 3.17−3.05 (m, 2H), 2.65−2.57 (m, 1H), 0.96−0.79 (m, 2H), 0.63−0.51 (m, 2H).

Reference:

[1] J Med. Chem. 2009, 52, 379-388………http://pubs.acs.org/doi/pdf/10.1021/jm800984v

[2] Cell Cycle 2009, 8, 1921-1929.

[US2014336073]

///////////

C1CC1NC(=O)NC2=CNNC2=C3N=C4C=CC(=CC4=N3)CN5CCOCC5

	shivkr2 on SPSR Excellence Award 2025 – S…
	Bonkasaurus on Infigratinib phosphate
	PALOVAROTENE \| ORGAN… on Palovarotene
	Pfizer just purchase… on Temanogrel
	Pfizer To Cash In On… on Temanogrel

Category Archives: Uncategorized

SEARCH THIS BLOG

Blog Stats

Flag and hits

Follow Blog via Email

Pages

Archives

Categories

Recent Posts

ORGANIC SPECTROSCOPY

SUBSCRIBE

Follow Blog via Email

Verified Services

Pages

Archives

Categories

Recent Comments

SEARCH THIS BLOG

(R)-4-((4-(((4-(Tetrahydrofuran-3-yloxy)-1,2-benzisoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol

Two Routes to 4-Fluorobenzisoxazol-3-one in the Synthesis of a 5-HT4Partial Agonist

Development of a Multikilogram Scale Synthesis of a TRPV1 Antagonist

Abstract

Neurogen and Merck Agreement for Next-Generation Pain Drugs Consummated

History

References

Synthesis

PATENT

Structure and action

Death and serious adverse events during phase I clinical trial

Old 2006 case

References

External links

Dr. D. Srinivasa Reddy

Fragment-Based Discovery of the Pyrazol-4-yl Urea (AT9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity†

Abstract

Post navigation

SEARCH THIS BLOG

FLAGS AND HITS

Follow Blog via Email

Two Routes to 4-Fluorobenzisoxazol-3-one in the Synthesis of a 5-HT₄Partial Agonist

Fragment-Based Discovery of the Pyrazol-4-yl Urea (AT9283), a Multitargeted Kinase Inhibitor with Potent Aurora Kinase Activity^†