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Immunotherapy could help tackle tough liver cancer
Significant new data presented today at the International Liver Congress 2014 indicate that liver cancer (Hepatocellular Carcinoma (HCC)) may be treated by adoptive T-cell therapy.
This new therapeutic approach in the treatment of HCC could be very important as without treatment the 5 year survival rate is just 5%. Globally, HCC accounts for 746,000 deaths, and in the UK alone is responsible for over 4,000 deaths per year.
Glypican-3 (GPC3) is a tumour associated antigen expressed in up to 70% of HCC but not in healthy human tissue. Isolating GPC3-specific T-cell receptors and expressing them on patient’s T-cells can help treat HCC, as these T cells can recognise and eliminate GPC3-postive HCC.
The study detected and expanded MHC-multimer-positive CD8+ T-cells specific for targeted GPC3 epitopes and grew T-cell clones. From these clones, the most specific and active T-cell receptor was isolated. When this T-cell receptor was expressed on donor T…
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BI 224436 an investigational new drug under development for the treatment of HIV infection
(2S)-2-tert-butoxy-2-(4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-2- methylquinolin-3-yl)acetic acid
BI 224436
1155419-89-8 cas no
mw
| 442.51 |
3-Quinolineacetic acid, 4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-α-(1,1-dimethylethoxy)-2-methyl-, (αS,4R)-
hemi-succinate of (2S)-2-tert-butoxy-2-(4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-2-methylquinolin-3-yl)acetic acid)
BI 224436 is an investigational new drug under development for the treatment of HIV infection. BI 224436 is the first non-catalytic site integrase inhibitor (NCINI). It inhibits HIV replication via binding to a conserved allosteric pocket of the HIV integrase enzyme. This makes the drug distinct in mechanism of action compared to raltegravir and elvitegravir, which bind at the catalytic site.[2] In October 2011, Gilead Sciences purchased exclusive rights to develop BI 224436 and several related compounds under investigation in Boehringer Ingelheim’s noncatalytic site integrase inhibitor program.[3][4]
Novel hemi-succinate salt form of (2S)-2-tert-butoxy-2-(4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-2-methylquinolin-3-yl)acetic acid (presumed to be BI-224436) and its crystalline forms is desc in WO-2014055618.
Gilead, under license from BI, was developing BI-224436 for the oral treatment of HIV infection. In September 2011, this drug had entered phase 1 trials. Picks up from WO2012138670, claiming a process for the preparation of the same drug. Also see the concurrently published WO2014055603. This compound is claimed specifically in WO2009062285 and generically in WO2007131350.
BI 224436 has antiviral EC50 values ranging between 4 and 15 nM against different HIV-1 laboratory strains and CC50 values >90 μM in different cells, including peripheral blood mononuclear cells. BI 224436 also has a low, 2.2-fold shift in antiviral potency in the presence of 50% human serum and by virtue of a steep dose-response curve slope, BI 224436 exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Drug combination studies performed in cell-based antiviral assays have shown that BI 224436 displays, at the least, an additive effect in combination with any of the marketed antiviral classes including INSTIs. BI 224436 has drug-like ADME properties including a Caco-2 cell permeability of 14 .10-6 cm/sec, solubility > 24 mg/ml in the pH range 2.0-6.8 and low cytochrome P450 inhibition. Moreover BI 224436 shows excellent PK profiles in rat (CL=0.7% QH; F= 54%), monkey (CL= 23% QH; F= 82%) and dog (CL= 8%QH; F= 81%).
http://www.natap.org/2011/ICAAC/ICAAC_32.htm
……………………
Discovery of BI 224436, a Noncatalytic Site Integrase Inhibitor (NCINI) of HIV-1
ACS Med. Chem. Lett., 2014, 5 (4), pp 422–427
DOI: 10.1021/ml500002n
http://pubs.acs.org/doi/abs/10.1021/ml500002n
1H NMR: 12.4 (br, 1H), 8.52 (d, 1H, J = 4.4Hz), 7.94 (d, 1H, J = 7.9 Hz),7.65-7.61 (m, 1H), 7.45 (d,
1H, J = 8.2 Hz), 7.31-7.24 (m, 2H), 7.12 (d, 1H, J = 7.9 Hz), 6.94-6.92 (m, 1H), 4.99 (s, 1H), 4.57-4.47
(m, 2H), 3.37-3.30 (m, 2H), 2.86 (s, 3H), 0.82 (s, 9H).
13C NMR: 172.2, 158.4, 153.1, 150.1, 146.6,
146.1, 145.0, 141.0, 130.8 (br), 130.6 (br), 128.9, 128.0, 127.2, 127.1 (br) 126.4, 125.6, 118.0, 116.7,
109.1, 75.2, 70.8, 65.6, 27.7, 27.5, 24.9.
HRMS: m/z calc. for C27H26N2O4 + H+: 443.1965, m/z found:
443.1951 (-3.2 ppm).
UPLC-MS: rt = 0.68 min, m/z 443.3 [M + H]+, purity: >99.9% @ 254 nm.
http://pubs.acs.org/doi/suppl/10.1021/ml500002n/suppl_file/ml500002n_si_001.pdf
………………………….
http://www.google.com/patents/WO2012138670A1?cl=en
General Scheme IA:
G1 1001
wherein Y is I, Br or CI;
General Scheme 11 A:
wherein:
Example 1
1 a 1 b
1a (600 g, 4.1 mol) was charged into a dry reactor under nitrogen followed by addition of Ac20 (1257.5 g, 12.3 mol, 3 eq.). The resulting mixture was heated at 40 °C at least for 2 hours. The batch was then cooled to 30 °C over 30 minutes. A suspension of 1b in toluene was added to seed the batch if no solid was observed. After toluene (600 ml_) was added over 30 minutes, the batch was cooled to -5— 10 °C and was held at this temperature for at least 30 minutes. The solid was collected by filtration under nitrogen and rinsed with heptanes (1200 ml_). After being dried under vacuum at room temperature, the solid was stored under nitrogen at least below 20 °C. The product 1 b was obtained with 77% yield. 1H NMR (500 MHz, CDCI3): δ = 6.36 (s, 1 H), 3.68 (s, 2H), 2.30 (s, 3H). Example 2
2a 2b
2a (100g, 531 mmol) and 1b (95 g, 558 mmol) were charged into a clean and dry reactor under nitrogen followed by addition of fluorobenzene (1000 mL). After being heated at 35-37 °C for 4 hours, the batch was cooled to 23 °C. Concentrated H2S04 (260.82 g, 2659.3 mmol, 5 eq.) was added while maintaining the batch temperature below 35 °C. The batch was first heated at 30-35 °C for 30 minutes and then at 40- 45 °C for 2 hours. 4-Methyl morpholine (215.19 g, 2127 mmol, 4 eq.) was added to the batch while maintaining the temperature below 50 °C. Then the batch was agitated for 30 minutes at 40-50 °C. eOH (100 mL) was then added while maintaining the temperature below 55 °C. After the batch was held at 50-55 °C for 2 hours, another portion of MeOH (100 mL) was added. The batch was agitated for another 2 hours at 50-55 °C. After fluorobenzene was distilled to a minimum amount, water (1000 mL) was added. Further distillation was performed to remove any remaining fluorobenzene. After the batch was cooled to 30 °C, the solid was collected by filtration with cloth and rinsed with water (400 mL) and heptane (200 mL). The solid was dried under vacuum below 50 °C to reach KF < 0.1%. Typically, the product 2b was obtained in 90% yield with 98 wt%. 1H NMR (500 MHz, DMSO- d6): δ = 10.83 (s, 1 H), 9.85 (s, bs, 1 H), 7.6 (d, 1 H, J
Hz), 6.40 (s, 1 H), 4.00 (s, 2 H), 3.61 (s, 3 H). Example 3
2b 3a
2b (20 g, 64 mmol) was charged into a clean and dry reactor followed by addition of THF (140 mL). After the resulting mixture was cooled to 0 °C, Vitride® (Red-AI, 47.84 g, 65 wt%, 154 mmol) in toluene was added while maintaining an internal temperature at 0-5 °C. After the batch was agitated at 5-10 °C for 4 hours, IPA (9.24 g, 153.8 mmol) was added while maintaining the temperature below 10 °C. Then the batch was agitated at least for 30 minutes below 25 °C. A solution of HCI in IPA (84.73 g, 5.5 M, 512 mmol) was added into the reactor while maintaining the temperature below 40 °C. After about 160 mL of the solvent was distilled under vacuum below 40 °C, the batch was cooled to 20-25 °C and then aqueous 6M HCI (60 mL) was added while maintaining the temperature below 40 °C. The batch was cooled to 25 °C and agitated for at least 30 minutes. The solid was collected by filtration, washed with 40 mL of IPA and water (1V/1V), 40 mL of water and 40 mL of heptanes. The solid was dried below 60 °C in a vacuum oven to reach KF < 0.5%. Typically, the product 3a was obtained in 90-95% yield with 95 wt%. 1H NMR (400 MHz, DMSO-d6): δ = 10.7 (s, 1 H), 9.68 (s, 1 H), 7.59 (d, 1 H, J = 8.7 Hz), 6.64 (, 1 H, J = 8.7 Hz), 6.27 (s, 1 H), 4.62 (bs, 1 H), 3.69 (t, 2H, J = 6.3 Hz), 3.21 (t, 2H, J = 6.3 Hz).
Example 4
3a (50 g, 174.756 mmol) and acetonitrile (200 mL) were charged into a dry and clean reactor. After the resulting mixture was heated to 65 °C, POCI3 (107.18 g, 699 mmol, 4 eq.) was added while maintaining the internal temperature below 75 °C. The batch was then heated at 70-75 °C for 5-6 hours. The batch was cooled to 20 °C. Water (400 mL) was added at least over 30 minutes while maintaining the internal temperature below 50 °C. After the batch was cooled to 20-25 °C over 30 minutes, the solid was collected by filtration and washed with water (100 mL). The wet cake was charged back into the reactor followed by addition of 1 M NaOH (150 mL). After the batch was agitated at least for 30 minutes at 25-35 °C, it was verified that the pH was greater than 12. Otherwise, more 6M NaOH was needed to adjust the pH >12. After the batch was agitated for 30 minutes at 25-35 °C, the solid was collected by filtration, washed with water (200 mL) and heptanes (200 mL). The solid was dried in a vacuum oven below 50 °C to reach KF < 2%. Typically, the product 4a was obtained at about 75-80% yield. H NMR (400 MHz, CDCI3): δ = 7.90 (d, 1 H, J = 8.4 Hz), 7.16 (s, 1 H), 6.89 (d, 1 H, J = 8.4 Hz), 4.44 (t, 2 H, J = 5.9 Hz), 3.23 (t, 2 H, J = 5.9 Hz). 13C NMR (100 MHz, CDCI3): δ = 152.9, 151.9, 144.9, 144.1 , 134.6, 1 19.1 , 1 17.0, 1 13.3, 1 1 1.9, 65.6, 28.3.
Example 5
4a 5a
Zn powder (54 g, 825 mmol, 2.5 eq.) and TFA (100 mL) were charged into a dry and clean reactor. The resulting mixture was heated to 60-65 °C. A suspension of 4a (100 g, 330 mmol) in 150 mL of TFA was added to the reactor while maintaining the temperature below 70 °C. The charge line was rinsed with TFA (50 mL) into the reactor. After 1 hour at 65±5 °C, the batch was cooled to 25-30 °C. Zn powder was filtered off by passing the batch through a Celite pad and washing with methanol (200 mL). About 400 mL of solvent was distilled off under vacuum. After the batch was cooled to 20-25 °C, 20% NaOAc (ca. 300 mL) was added at least over 30 minutes to reach pH 5-6. The solid was collected by filtration, washed with water (200 mL) and heptane (200 mL), and dried under vacuum below 45 °C to reach KF ≤ 2%. The solid was charged into a dry reactor followed by addition of loose carbon (10 wt%) and toluene (1000 mL). The batch was heated at least for 30 minutes at 45-50 °C. The carbon was filtered off above 35 °C and rinsed with toluene (200 mL). The filtrate was charged into a clean and dry reactor. After about 1000 mL of toluene was distilled off under vacuum below 50 °C, 1000 mL of heptane was added over 30 minutes at 40-50 °C. Then the batch was cooled to 0±5 °C over 30 minutes. After 30 minutes, the solid was collected and rinsed with 200 mL of heptane. The solid was dried under vacuum below 45 °C to reach KF≤ 500 ppm. Typically, the product 5a was obtained in about 90-95 % yield. 1H NMR (400 MHz, CDCI3): δ = 8.93 (m, 1 H), 7.91 (dd, 1 H, J = 1.5, 8 Hz), 7.17 (m 1 H), 6.90 (dd, 1 H, J = 1 .6, 8.0 Hz), 4.46-4.43 (m, 2 H), 3.28-3.23 (m, 2 H). 13C NMR (100 MHz, CDCI3): δ = 152.8, 151 .2, 145.1 , 141.0, 133.3, 1 18.5, 1 18.2, 1 14.5, 1 1 1.1 , 65.8, 28.4.
Example 6
5a 6a
5a (1.04 kg, 4.16 mol) and toluene (8 L) were charged into the reactor. The batch was agitated and cooled to -50 to -55 °C. BuLi solution (2.5 M in hexanes, 1.69 L, 4.23 mol) was charged slowly while maintaining the internal temperature between – 45 to -50 °C. The batch was agitated at -45 °C for 1 hour after addition. A solution of triisopropyl borate (0.85 kg, 4.5 mol) in MTBE (1 .48 kg) was charged. The batch was warmed to 10 °C over 30 minutes. A solution of 5 N HCI in I PA (1 .54 L) was charged slowly at 10 °C, and the batch was warmed to 20 °C and stirred for 30 minutes. It was seeded with 6a crystal (10 g). A solution of aqueous concentrated HCI (0.16 L) in IPA (0.16 L) was charged slowly at 20 °C in three portions at 20 minute intervals, and the batch was agitated for 1 hour at 20 °C. The solid was collected by filtration, rinsed with MTBE (1 kg), and dried to provide 6a (943 g, 88.7 % purity, 80% yield). 1H NMR (400 MHz, D20): δ 8.84 (d, 1 H, J = 4 Hz)
1 H), 7.68 (d, 1 H, J = 6 Hz), 7.09 (m, 1 H), 4.52 (m, 2H), 3.47 (m, 2H).
Example 7
Iodine stock solution was prepared by mixing iodine (57.4 g, 0.23 mol) and sodium iodide (73.4 g, 0.49 mol) in water (270 mL). Sodium hydroxide (28.6 g, 0.715 mol) was charged into 220 mL of water. 4-Hydroxy-2 methylquinoline 7a (30 g, 0.19 mol) was charged, followed by acetonitrile (250 mL). The mixture was cooled to 10 °C with agitation. The above iodine stock solution was charged slowly over 30 minutes. The reaction was quenched by addition of sodium bisulfite (6.0 g) in water (60 mL). Acetic acid (23 mL) was charged over a period of 1 hour to adjust the pH of the reaction mixture between 6 and 7. The product was collected by filtration, washed with water and acetonitrile, and dried to give 7b (53 g, 98%). MS 286 [M + 1].
Example 8
7b 8a
4-Hydroxy-3-iodo-2-methylquinoline 7b (25 g, 0.09 mol) was charged to a 1-L reactor. Ethyl acetate (250 mL) was charged, followed by triethylamine (2.45 mL, 0.02 mol) and phosphorus oxychloride (12 mL, 0.13 mol). The reaction mixture was heated to reflux until complete conversion (~1 hour), then the mixture was cooled to 22 °C. A solution of sodium carbonate (3 .6 g, 0.3 mol) in water (500 mL) was charged. The mixture was stirred for 20 minutes. The aqueous layer was extracted with ethyl acetate (120 mL). The organic layers were combined and concentrated under vacuum to dryness. Acetone (50 mL) was charged. The solution was heated to 60 °C. Water (100 mL) was charged, and the mixture was cooled to 22 °C. The product was collected by filtration and dried to give 8a (25 g, 97.3 % pure, 91.4 % yield). MS 304 [M + 1].
(Note: 8a is a known compound with CAS # 1033931-93-9. See references: (a) J. Org Chem. 2008, 73, 4644-4649. (b) Molecules 2010, 15, 3171 -3178. (c) Indian J. Chem. Sec B: Org. Chem. Including Med Chem. 2009, 488(5), 692-696.)
Example 9
8a 9a
8a (100 g, 0.33 mol) was charged to the reactor, followed by copper (I) bromide dimethyl sulfide complex (3.4 g, 0.017 mol) and dry THF (450 mL). The batch was cooled to -15 to -12 °C. i-PrMgCI (2.0 M in THF, 173 mL, 0.346 mol) was charged into the reactor at the rate which maintained the batch temperature < -10 °C. In a 2nd reactor, methyl chlorooxoacetate (33 mL, 0.36 mol) and dry THF (150 mL) were charged. The solution was cooled to -15 to -10 °C. The content of the 1 st reactor (Grignard/cuprate) was charged into the 2nd reactor at the rate which maintained the batch temperature < -10 °C. The batch was agitated for 30 minutes at -10 °C. Aqueous ammonium chloride solution ( 0%, 300 mL) was charged. The batch was agitated at 20 – 25 °C for 20 minutes and allowed to settle for 20 minutes. The aqueous layer was separated. Aqueous ammonium chloride solution (10%, 90 mL) and sodium carbonate solution (10%, 135 mL) were charged to the reactor. The batch was agitated at 20 – 25 °C for 20 minutes and allowed to settle for 20 minutes. The aqueous layer was separated. Brine (10%, 240 mL) was charged to the reactor. The batch was agitated at 20 – 25 °C for 20 minutes. The aqueous layer was separated. The batch was concentrated under vacuum to -1/4 of the volume (about 80 mL left). 2-Propanol was charged (300 mL). The batch was concentrated under vacuum to -1/3 of the volume (about 140 mL left), and heated to 50 °C.
Water (70 mL) was charged. The batch was cooled to 20 – 25 °C, stirred for 2 hours, cooled to – 0 °C and stirred for another 2 hours. The solid was collected by filtration, washed with cold 2-propanol and water to provide 58.9 g of 9a obtained after drying (67.8 % yield). 1H NMR (400 MHz, CDCI3): δ 8.08 (d, 1 H, J = 12 Hz), 7.97 (d, 1 H, J = 12 Hz), 7.13 (t, 1 H, J = 8 Hz), 7.55 (t, 1 H, J= 8 Hz), 3.92 (s, 3H), 2.63 (s, 3H). 13C NMR (100 MHz, CDCI3): δ 186.6, 161.1 , 155.3, 148.2, 140.9, 132.0, 129.0, 128.8, 127.8, 123.8, 123.7, 53.7, 23.6.
Catalyst preparation: To a suitable sized, clean and dry reactor was charged dichloro(pentamethylcyclopentadienyl)rhodium (III) dimer (800 ppm relative to 9a, 188.5 mg) and the ligand (2000 ppm relative to 9a, 306.1 mg). The system was purged with nitrogen and then 3 ml. of acetonitrile and 0.3 ml_ of triethylamine was charged to the system. The resulting solution was agitated at room temperature for not less than 45 minutes and not more than 6 hours. Reaction: To a suitable sized, clean and dry reactor was charged 9a (1.00 equiv, 100.0 g (99.5 wt%), 377.4 mmol). The reaction was purged with nitrogen. To the reactor was charged acetonitrile (ACS grade, 4 L/Kg of 9a, 400 mL) and
triethylamine (2.50 equiv, 132.8 mL, 943 mmol). Agitation was initiated. The 9a solution was cooled to Tint= -5 to 0 °C and then formic acid (3.00 equiv, 45.2 mL, 1 132 mmol) was charged to the solution at a rate to maintain Tint not more than 20 °C. The batch temperature was then adjusted to Tint= -5 to -0 °C. Nitrogen was bubbled through the batch through a porous gas dispersion unit (Wiimad-LabGlass No. LG-8680-1 0, VWR catalog number 14202-962) until a fine stream of bubbles was obtained. To the stirring solution at Tint= -5 to 0 °C was charged the prepared catalyst solution from the catalyst preparation above. The solution was agitated at Tint= -5 to 0 °C with the bubbling of nitrogen through the batch until HPLC analysis of the batch indicated no less than 98 A% conversion (as recorded at 220 nm, 10-14 h). To the reactor was charged isopropylacetate (6.7 L/Kg of 9a, 670 ml_). The batch temperature was adjusted to Tint= 18 to 23 °C. To the solution was charged water (10 L/Kg of 9a, 1000 mL) and the batch was agitated at Tint= 18 to 23 °C for no less than 20 minutes. The agitation was decreased and or stopped and the layers were allowed to separate. The lighter colored aqueous layer was cut. To the solution was charged water (7.5 L/Kg of 9a, 750 mL) and the batch was agitated at Tint= 18 to 23 °C for no less than 20 minutes. The agitation was decreased and or stopped and the layers were allowed to separate. The lighter colored aqueous layer was cut. The batch was then reduced to 300 mL (3 L/Kg of 9a) via distillation while maintaining Text no more than 65 °C. The batch was cooled to Tint= 35 to 45 °C and the batch was seeded (10 mg). To the batch at Tint= 35 to 45 °C was charged heptane (16.7 L/Kg of 9a, 1670 mL) over no less than 1.5 hours. The batch temperature was adjusted to Tint= -2 to 3 °C over no less than 1 hour, and the batch was agitated at Tint= -2 to 3 °C for no less than 1 hour. The solids were collected by filtration. The filtrate was used to rinse the reactor (Filtrate is cooled to Tint= -2 to 3 °C before filtration) and the solids were suction dried for no less than 2 hours. The solids were dried until the LOD is no more than 4 % to obtain 82.7 g of 10a (99.6- 100 wt%, 98.5% ee, 82.5% yield). 1H-NMR (CDCI3, 400 MHz) δ: 8.20 (d, J= 8.4 Hz, 1 H), 8.01 (d, J= 8.4 Hz, 1 H), 7.73 (t, J= 7.4 Hz, 1 H), 7.59 (t, J= 7.7 Hz, H), 6.03 (s, 1 H), 3.93 (s, 1 H), 3.79 (s, 3H), 2.77 (s, 3H). 13C-NMR (CDCI3, 100 MHz) δ: 173.5, 158.3, 147.5, 142.9, 130.7, 128.8, 127.7, 127.1 , 125.1 , 124.6, 69.2, 53.4, 24.0.
Example 11
10a 6a
10a (2.45 kg, 96.8% purity, 8.9 mol), 6a (2.5 kg, 88.7% purity, 8.82 mol), tris(dibenzylideneacetone)dipalladium(0) (Pd2dba3, 40 g, 0.044 mol), (S)-3-ieri-butyl- 4-(2,6-dimethoxypheny1 )-2,3-dihydrobenzo[d][1 ,3]oxaphosphole (32 g, 0.01 1 mol), sodium carbonate (1.12 kg, 10.58 mol), 1-pentanol (16.69 L), and water (8.35 L) were charged to the reactor. The mixture was de-gassed by sparging with argon for 10-15 minutes, was heated to 60-63 °C, and was agitated until HPLC analysis of the reaction shows <1 A% (220 nm) of the 6a relative to the combined two atropisomer products (-15 hours). The batch was cooled to 18-23 °C. Water (5 L) and heptane (21 L) were charged. The slurry was agitated for 3 – 5 hours. The solids were collected by filtration, washed with water (4 L) and heptane/toluene mixed solvent (2.5 L toluene/5 L heptane), and dried. The solids were dissolved in methanol (25 L) and the resulting solution was heated to 50 °C and circulated through a CUNO carbon stack filter. The solution was distilled under vacuum to ~ 5 L. Toluene (12 L) was charged. The mixture was distilled under vacuum to ~ 5 L and cooled to 22 °C. Heptane (13 L) was charged to the contents over 1 hour and the resulting slurry was agitated at 20-25 °C for 3 – 4 hours. The solids were collected by filtration and washed with heptanes to provide 2.58 kg of 11a obtained after drying (73% yield). 1H NMR (400 MHz, CDCI3): δ 8.63 (d, 1 H, J = 8 Hz), 8.03 (d, 1 H, J = 12 Hz), 7.56 (t, 1 H, J = 8 Hz), 7.41 (d, 1 H, J = 8 Hz), 7.19 (t, 1 H, J = 8 Hz), 7.09 (m, 2H), 7.04 (d, 1 H, J = 8 Hz), 5.38 (d, 1 H, J = 8 Hz), 5.14 (d, 1 H, J = 8 Hz), 4.50 (t, 2H, J = 4 Hz), 3.40 (s, 3H), 3.25 (t, 2H, J = 4 Hz), 2.91 (s, 3H). 13C NMR (100 MHz, CDCI3): δ 173.6, 158.2, 154.0, 150.9, 147.3, 147.2, 145.7, 141.3, 132.9, 123.0, 129.4, 128.6, 127.8, 126.7, 126.4, 125.8, 1 18.1 , 1 17.3, 109.9, 70.3, 65.8, 52.3, 28.5, 24.0.
Example 12
11a 12a
To a suitable clean and dry reactor under a nitrogen atmosphere was charged 11a (5.47 Kg, 93.4 wt%, 1 .00 equiv, 12.8 mol) and fluorobenzene (10 vols, 51.1 kg) following by trifluoromethanesulfonimide (4 mol%, 143 g, 0.51 mol) as a 0.5 M solution in DCM (1.0 Kg). The batch temperature was adjusted to 35-41 °C and agitated to form a fine slurry. To the mixture was slowly charged i-butyl-2,2,2- trichloroacetimidate 12b as a 50 wt% solution (26.0 Kg of f-butyl-2,2,2- trichloroacetimidate (1 19.0 mol, 9.3 equiv), the reagent was -48-51 wt% with the remainder 52-49 wt% of the solution being – 1.8:1 wt:wt heptane: fluorobenzene) over no less than 4 hours at Tint= 35-41 °C. The batch was agitated at Tint= 35-41 °C until HPLC conversion (308 nm) was >96 A%, then cooled to Tint= 20-25 °C and then triethylamine (0.14 equiv, 181 g, 1 .79 mol) was charged followed by heptane (12.9 Kg) over no less than 30 minutes. The batch was agitated at Tint= 20-25 °C for no less than 1 hour. The solids were collected by filtration. The reactor was rinsed with the filtrate to collect all solids. The collected solids in the filter were rinsed with heptane (1 1 .7 Kg). The solids were charged into the reactor along with 54.1 Kg of DM Ac and the batch temperature adjusted to Tint= 70-75 °C. Water ( .2 Kg) was charged over no less than 30 minutes while the batch temperature was maintained at Tint= 65-75 °C. 12a seed crystals (34 g) in water (680 g) was charged to the batch at Tlnt= 65-75 °C. Additional water (46.0 Kg) was charged over no less than 2 hours while maintaining the batch temperature at Tint= 65-75 °C. The batch temperature was adjusted to Tint= 18-25 °C over no less than 2 hours and agitated for no less than 1 hour. The solids were collected by filtration and the filtrate used to rinse the reactor. The solids were washed with water (30 Kg) and dried under vacuum at no more than 45 °C until the LOD < 4% to obtain 12a (5.275 Kg, 99.9 A% at 220 nm, 99.9 wt% via HPLC wt% assay, 90.5% yield). 1H-NMR (CDCI3, 400
MHz) δ: 8.66-8.65 (m, 1 H), 8.05 (d, J= 8.3 Hz, 1 H), 7.59 (t, J= 7.3 Hz, 1 H), 7.45 (d, J= 7.8 Hz, 1 H), 7.21 (t, J= 7.6 Hz, 1 H), 7.13-7.08 (m, 3H), 5.05 (s, 1 H), 4.63-4.52 (m, 2H), 3.49 (s, 3H), 3.41 -3.27 (m, 2H), 3.00 (s, 3H), 0.97 (s, 9H). 13C-NMR (CDCI3, 100 MHz) δ: 172.1 , 159.5, 153.5, 150.2, 147.4, 146.9, 145.4, 140.2, 131.1 , 130.1 , 128.9, 128,6, 128.0, 127.3, 126.7, 125.4, 117.7, 117.2, 109.4, 76.1 , 71.6, 65.8, 51 .9, 28.6, 28.0, 25.4. Example 13
To a suitable clean and dry reactor under a nitrogen atmosphere was charged 12a (9.69 Kg, 21.2 mol) and ethanol (23.0 Kg). The mixture was agitated and the batch temperature was maintained at Τίηί= 20 to 25 °C. 2 M sodium hydroxide (17.2 Kg) was charged at Tint= 20 to 25 °C and the batch temperature was adjusted to Tint= 60- 65°C over no less than 30 minutes. The batch was agitated at Tint= 60-65°C for 2-3 hours until HPLC conversion was >99.5% area (12a is <0.5 area%). The batch temperature was adjuted to Tlnt= 50 to 55°C and 2M aqueous HCI (14.54 Kg) was charged. The pH of the batch was adjusted to pH 5.0 to 5.5 (target pH 5.2 to 5.3) via the slow charge of 2M aqueous HCI (0.46 Kg) at Tint= 50 to 55°C. Acetonitrile was charged to the batch (4.46 Kg) at Tint= 50 to 55°C. A slurry of seed crystals (1001 , 20 g in 155 g of acetonitrile) was charged to the batch at Tint= 50 to 55°C. The batch was agitated at Tint= 50 to 55°C for no less than 1 hour (1-2 hours). The contents were vacuum distilled to -3.4 vol (32 L) while maintaining the internal temperature at 45-55°C. A sample of the batch was removed and the ethanol content was determined by GC analysis; the criterion was no more than 10 wt% ethanol. If the ethanol wt% was over 10%, an additional 10% of the original volume was distilled and sampled for ethanol wt%. The batch temperature was adjusted to Tint= 18-22°C over no less than 1 hour. The pH of the batch was verified to be pH= 5 – 5.5 and the pH was adjusted, if necessary, with the slow addition of 2 M HCI or 2 M NaOH aqueous solutions. The batch was agitated at Tint= 18-22°C for no less than 6 hours and the solids were collected by filtration. The filtrate/mother liquid was used to remove all solids from reactor. The cake with was washed with water (19.4 Kg) (water temperature was no more than 20 °C). The cake was dried under vacuum at no more than 60 °C for 12 hours or until the LOD was no more than 4% to obtain 1001 (9.52 Kg, 99.6 A% 220 nm, 97.6 wt% as determined by HPLC wt% assay, 99.0% yield).
…………………
compd 1144
http://www.google.com/patents/WO2009062285A1?cl=en
……………………
http://www.google.com/patents/WO2012138669A1?cl=en
Compound (I), (2S)-2-tert-butoxy-2-(4-(2,3-dihydropyrano[4,3,2-de]quinolin-7-yl)-2- methylquinolin-3-yl)acetic acid, is an HIV non-catalytic site integrase inhibitor.
Compound (I) falls within the scope of the HIV inhibitors disclosed in WO
2007/131350. Compound (I) is disclosed specifically as compound no. 1144 in WO 2009/062285. Compound (I) can be prepared according to the general procedures found in WO 2007/13 350 and WO 2009/062285, which are hereby incorporated by reference.
Example 1
1 a 1b
1a (600 g, 4.1 mol) was charged into a dry reactor under nitrogen followed by addition of Ac20 (1257.5 g, 12.3 mol, 3 eq.). The resulting mixture was heated at 40 °C at least for 2 hours. The batch was then cooled to 30 °C over 30 minutes. A suspension of 1b in toluene was added to seed the batch if no solid was observed. After toluene (600 mL) was added over 30 minutes, the batch was cooled to -5 ~ -10 °C and was held at this temperature for at least 30 minutes. The solid was collected by filtration under nitrogen and rinsed with heptanes (1200 mL). After being dried under vacuum at room temperature, the solid was stored under nitrogen at least below 20 °C. The product 1b was obtained with 77% yield. 1H NMR (500 MHz, CDCI3): δ = 6.36 (s, 1 H), 3.68 (s, 2H), 2.30 (s, 3H).
Example 2
2a (100 g, 531 mmol) and 1 b (95 g, 558 mmol) were charged into a clean and dry reactor under nitrogen followed by addition of fluorobenzene ( 000 mL). After being heated at 35-37 °C for 4 hours, the batch was cooled to 23 °C. Concentrated H2S04 (260.82 g, 2659.3 mmol, 5 eq.) was added while maintaining the batch temperature below 35 °C. The batch was first heated at 30-35 °C for 30 minutes and then at 40- 45 °C for 2 hours. 4-Methyl morpholine (215.19 g, 2127 mmol, 4 eq.) was added to the batch while maintaining the temperature below 50 °C. Then the batch was agitated for 30 minutes at 40-50 °C. MeOH ( 00 mL) was then added while maintaining the temperature below 55 °C. After the batch was held at 50-55 °Cfor 2 hours, another portion of MeOH (100 mL) was added. The batch was agitated for another 2 hours at 50-55 °C. After fluorobenzene was distilled to a minimum amount, water (1000 mL) was added. Further distillation was performed to remove any remaining fluorobenzene. After the batch was cooled to 30 °C, the solid was collected by filtration with cloth and rinsed with water (400 mL) and heptane (200 mL). The solid was dried under vacuum below 50 °C to reach KF < 0.1 %. Typically, the product 2b was obtained in 90% yield with 98 wt%. 1H NMR (500 MHz, DMSO- cfe): δ = 10.83 (s, 1 H), 9.85 (s, bs, 1 H), 7.6 (d, 1 H, J = 8.7 Hz), 6.55 (d, 1 H, J = 8.7 Hz), 6.40 (s, 1 H), 4.00 (s, 2 H), 3.61 (s, 3 H).
Example 3
2b 3a
2b (20 g, 64 mmol) was charged into a clean and dry reactor followed by addition of THF (140 mL). After the resulting mixture was cooled to 0 °C, Vitride® (Red-AI, 47.84 g, 65 wt%, 154 mmol) in toluene was added while maintaining an internal temperature at 0-5 °C. After the batch was agitated at 5-10 °C for 4 hours, IPA (9.24 g, 153.8 mmol) was added while maintaining the temperature below 10 °C. Then the batch was agitated at least for 30 minutes below 25 °C. A solution of HCI in IPA (84.73 g, 5.5 M, 512 mmol) was added into the reactor while maintaining the temperature below 40 °C. After about 160 mL of the solvent was distilled under vacuum below 40 °C, the batch was cooled to 20-25 °C and then aqueous 6M HCI (60 mL) was added while maintaining the temperature below 40 °C. The batch was cooled to 25 °C and agitated for at least 30 minutes. The solid was collected by filtration, washed with 40 mL of IPA and water (1 V/1 V), 40 mL of water and 40 mL of heptanes. The solid was dried below 60 °C in a vacuum oven to reach KF < 0.5%. Typically, the product 3a was obtained in 90-95% yield with 95 wt%. 1H NMR (400 MHz, DMSO-c/e): 5 = 10.7 (s, 1 H), 9.68 (s, 1 H), 7.59 (d, 1 H, J = 8.7 Hz), 6.64 (, 1 H, J = 8.7 Hz), 6.27 (s, 1 H), 4.62 (bs, 1 H), 3.69 (t, 2H, J = 6.3 Hz), 3.21 (t, 2H, J = 6.3 Hz).
Example 4
3a 4a
3a (50 g, 174.756 mmol) and acetonitrile (200 mL) were charged into a dry and clean reactor. After the resulting mixture was heated to 65 °C, POC13 (107.18 g, 699 mmol, 4 eq.) was added while maintaining the internal temperature below 75 °C. The batch was then heated at 70-75 °C for 5-6 h. The batch was cooled to 20 °C. Water (400 mL) was added at least over 30 minutes while maintaining the internal temperature below 50 °C. After the batch was cooled to 20-25 °C over 30 minutes, the solid was collected by filtration and washed with water (100 mL). The wet cake was charged back into the reactor followed by addition of 1 M NaOH (150 mL). After the batch was agitated at least for 30 minutes at 25-35 °C, verify that the pH was greater than 12. Otherwise, more 6M NaOH was needed to adjust the pH >12. After the batch was agitated for 30 minutes at 25-35 °C, the solid was collected by filtration, washed with water (200 mL) and heptanes (200 mL). The solid was dried in a vacuum oven below 50 °C to reach KF < 2%. Typically, the product 4a was obtained at about 75-80% yield. 1H NMR (400 MHz, CDCI3): δ = 7.90 (d, 1 H, J = 8.4 Hz), 7.16 (s, 1 H), 6.89 (d, 1 H, J = 8.4 Hz), 4.44 (t, 2 H, J = 5.9 Hz), 3.23 (t, 2 H, J = 5.9 Hz). 13C NMR (100 MHz, CDCI3): δ = 152.9, 151.9, 144.9, 144.1 , 134.6, 119.1 , 1 17.0, 1 13.3, 1 1 1.9, 65.6, 28.3.
Example 5
4a 5a
Zn powder (54 g, 825 mmol, 2.5 eq.) and TFA (100 mL) were charged into a dry and clean reactor. The resulting mixture was heated to 60-65 °C. A suspension of 4a (100 g, 330 mmol) in 150 mL of TFA was added to the reactor while maintaining the temperature below 70 °C. The charge line was rinsed with TFA (50 mL) into the reactor. After 1 hour at 65±5 °C, the batch was cooled to 25-30 °C. Zn powder was filtered off by passing the batch through a Celite pad and washing with methanol (200 mL). About 400 mL of solvent was distilled off under vacuum. After the batch was cooled to 20-25 °C, 20% NaOAc (ca. 300 mL) was added at least over 30 minutes to reach pH 5-6. The solid was collected by filtration, washed with water (200 mL) and heptane (200 mL), and dried under vacuum below 45 °C to reach KF ≤ 2%. The solid was charged into a dry reactor followed by addition of loose carbon (10 wt%) and toluene (1000 mL). The batch was heated at least for 30 minutes at 45-50 °C. The carbon was filtered off above 35 °C and rinsed with toluene (200 mL). The filtrate was charged into a clean and dry reactor. After about 1000 mL of toluene was distilled off under vacuum below 50 °C, 1000 mL of heptane was added over 30 minutes at 40-50 °C. Then the batch was cooled to 0±5 °C over 30 minutes. After 30 minutes, the solid was collected and rinsed with 200 mL of heptane. The solid was dried under vacuum below 45 °C to reach KF≤ 500 ppm. Typically, the product 5a was obtained in about 90-95 % yield. 1H NMR (400 MHz, CDCI3): δ = 8.93 (m, 1 H), 7.91 (dd, 1 H, J = 1.5, 8 Hz), 7.17 (m 1 H), 6.90 (dd, 1 H, J = 1.6, 8.0 Hz), 4.46-4.43 (m, 2 H), 3.28-3.23 (m, 2 H). 13C NMR (100 MHz, CDCI3): δ = 152.8, 151 .2, 145.1 , 141.0, 133.3, 1 18.5, 1 18.2, 1 14.5, 1 1 1 .1 , 65.8, 28.4.
Example 6
5a (1.04 kg, 4.16 mol) and toluene (8 L) were charged into the reactor. The batch was agitated and cooled to -50 to -55 °C. BuLi solution (2.5 M in hexanes, 1.69 L, 4.23 mol) was charged slowly while maintaining the internal temperature between – 45 to -50 °C. The batch was agitated at -45 °C for 1 hour after addition. A solution of triisopropyl borate (0.85 kg, 4.5 mol) in MTBE (1.48 kg) was charged. The batch was warmed to 10 °C over 30 minutes. A solution of 5 N HCI in IPA (1.54 L) was charged slowly at 10 °C, and the batch was warmed to 20 °C and stirred for 30 minutes. It was seeded with 6a crystal (10 g). A solution of aqueous concentrated HCI (0.16 L) in IPA (0.16 L) was charged slowly at 20 °C in three portions at 20 minute intervals, and the batch was agitated for 1 hour at 20 °C. The solid was collected by filtration, rinsed with MTBE (1 kg), and dried to provide 6a (943 g, 88.7 % purity, 80% yield). 1H NMR (400 MHz, D20): δ 8.84 (d, 1 H, J = 4 Hz), 8.10 (m, 1 H), 7.68 (d, 1 H, J = 6 Hz), 7.09 (m, 1 H), 4.52 (m, 2H), 3.47 (m, 2H).
Example 7
7a 7b
Iodine stock solution was prepared by mixing iodine (57.4 g, 0.23 mol) and sodium iodide (73.4 g, 0.49 mol) in water (270 mL). Sodium hydroxide (28.6 g, 0.715 mol) was charged into 220 mL of water. 4-Hydroxy-2 methylquinoline 7a (30 g, 0.19 mol) was charged, followed by acetonitrile (250 mL). The mixture was cooled to 10 °C with agitation. The above iodine stock solution was charged slowly over 30 minutes. The reaction was quenched by addition of sodium bisulfite (6.0 g) in water (60 mL). Acetic acid (23 mL) was charged over a period of 1 hour to adjust the pH of the reaction mixture between 6 and 7. The product was collected by filtration, washed with water and acetonitrile, and dried to give 7b (53 g, 98%). MS 286 [M + 1].
7b 8a
4-Hydroxy-3-iodo-2-methylquinoline 7b (25 g, 0.09 mol) was charged to a 1 -L reactor. Ethyl acetate (250 mL) was charged, followed by triethylamine (2.45 mL, 0.02 mol) and phosphorus oxychloride (12 mL, 0.13 mol). The reaction mixture was heated to reflux until complete conversion (~1 hour), then the mixture was cooled to 22 °C. A solution of sodium carbonate (31.6 g, 0.3 mol) in water (500 mL) was charged. The mixture was stirred for 20 minutes. The aqueous layer was extracted with ethyl acetate (120 mL). The organic layers were combined and concentrated under vacuum to dryness. Acetone (50 mL) was charged. The solution was heated to 60 °C. Water (100 mL) was charged, and the mixture was cooled to 22 °C. The product was collected by filtration and dried to give 8a (25 g, 97.3 % pure, 91.4 % yield). MS 304 [M + 1].
(Note: 8a is a known compound with CAS # 1033931-93-9. See references: (a) J. Org Chem. 2008, 73, 4644-4649. (b) Molcules 2010, 15, 3171-3178. (c) Indian J. Chem. Sec B: Org. Chem. Including Med Chem. 2009, 48B(5), 692-696.)
8a (100 g, 0.33 mol) was charged to the reactor, followed by copper (I) bromide dimethyl sulfide complex (3.4 g, 0.017 mol) and dry THF (450 mL). The batch was cooled to – 5 to – 2 °C. i-PrMgCI (2.0 M in THF, 173 mL, 0.346 mol) was charged into the reactor at the rate which maintains the batch temperature < -10 °C.
In a 2nd reactor, methyl chlorooxoacetate (33 mL, 0.36 mol) and dry THF (150 mL) was charged. The solution was cooled to -15 to -10 °C. The content of the 1 st reactor (Grignard/cuprate) was charged into the 2nd reactor at the rate which maintained the batch temperature < -10 °C. The batch was agitated for 30 minutes at -10 °C. Aqueous ammonium chloride solution (10%, 300 mL) was charged. The batch was agitated at 20 – 25 °C for 20 minutes and allowed to settle for 20 minutes. The aqueous layer was separated. Aqueous ammonium chloride solution (10%, 90 mL) and sodium carbonate solution (10%, 135 mL) were charged to the reactor. The batch was agitated at 20 – 25 °C for 20 minutes and allowed to settle for 20 minutes. The aqueous layer was separated. Brine (10%, 240 mL) was charged to the reactor. The batch was agitated at 20 – 25 °C for 20 minutes. The aqueous layer was separated. The batch was concentrated under vacuum to -1/4 of the volume (about 80 mL left). 2-Propanol was charged (300 mL). The batch was concentrated under vacuum to -1/3 of the volume (about 140 mL left), and heated to 50 °C. Water (70 mL) was charged. The batch was cooled to 20 – 25 °C, stirred for 2 hours, cooled to -10 °C and stirred for another 2 hours. The solid was collected by filtration, washed with cold 2-propanol and water to provide 58.9 g of 9a obtained after drying (67.8 % yield). 1H NMR (400 MHz, CDCI3): δ 8.08 (d, 1 H, J = 12 Hz), 7.97 (d, 1 H, J = 12 Hz), 7.13 (t, 1 H, J = 8 Hz), 7.55 (t, 1 H, J = 8 Hz), 3.92 (s, 3H), 2.63 (s, 3H). 13C NMR (100 MHz, CDCI3): δ 186.6, 161.1 , 155.3, 148.2, 140.9, 132.0, 129.0, 128.8, 127.8, 123.8, 123.7, 53.7, 23.6.
Example 10
Catalyst preparation: To a suitable sized, clean and dry reactor was charged dichloro(pentamethylcyclopentadienyl)rhodium(lll) dimer (800 ppm relative to 9a, 188.5 mg) and the ligand (2000 ppm relative to 9a, 306.1 mg). The system was purged with nitrogen and then 3 ml_ of acetonitrile and 0.3 ml_ of triethylamine was charged to the system. The resulting solution was agitated at RT for not less than 45 minutes and not more than 6 hours.
Reaction: To a suitable sized, clean and dry reactor was charged 9a (1.00 equiv, 100.0 g (99.5 wt%), 377.4 mmol). The reaction was purged with nitrogen. To the reactor was charged acetonitrile (ACS grade, 4 L/Kg of 9a, 400 ml_) and
triethylamine (2.50 equiv, 132.8 ml_, 943 mmol). Agitation was initiated. The 9a solution was cooled to Tint= -5 to 0 °C and then formic acid (3.00 equiv, 45.2 ml_, 1 132 mmol) was charged to the solution at a rate to maintain Tint not more than 20 °C. The batch temperature was then adjusted to Tlnt= -5 to -0 °C. Nitrogen was bubbled through the batch through a porous gas dispersion unit (Wilmad-LabGlass No. LG-8680-1 10, VWR catalog number 14202-962) until a fine stream of bubbles was obtained. To the stirring solution at Jml= -5 to 0 °C was charged the prepared catalyst solution from the catalyst preparation above. The solution was agitated at Tint= -5 to 0 °C with the bubbling of nitrogen through the batch until HPLC analysis of the batch indicated no less than 98 A% conversion (as recorded at 220 nm, 10-14 h). To the reactor was charged isopropylacetate (6.7 L/Kg of 9a, 670 mL). The batch temperature was adjusted to Tint= 18 to 23 °C. To the solution was charged water (10 L/Kg of 9a, 1000 mL) and the batch was agitated at Tint= 18 to 23 °C for no less than 20 minutes. The agitation was decreased and or stopped and the layers were allowed to separate. The lighter colored aqueous layer was cut. To the solution was charged water (7.5 L/Kg of 9a, 750 mL) and the batch was agitated at Tint= 18 to 23 °C for no less than 20 minutes. The agitation was decreased and or stopped and the layers were allowed to separate. The lighter colored aqueous layer was cut. The batch was then reduced to 300 mL (3 L/Kg of 9a) via distillation while maintaining Text no more than 65 °C. The batch was cooled to Tint= 35 to 45 °C and the batch was seeded ( 0 mg). To the batch at Tint= 35 to 45 °C charged heptane (16.7 L/Kg of 9a, 1670 mL) over no less than 1.5 hours. Adjusted the batch temperature to Tint= -2 to 3 °C over no less than 1 hour, and agitated the batch at Tint= -2 to 3 °C for no less than 1 hour. Collected the solids by filtration. Used the filtrate to rinse the reactor (Filtrate is cooled to
-2 to 3 °C before filtration) and the solids were suction dried for no less than 2 hours. The solids were dried until the LOD was no more than 4 % to obtain 82.7 g of 10a (99.6-100 wt%, 98.5% ee, 82.5% yield). 1H- NMR (CDCI3, 400 MHz) δ: 8.20 (d, J= 8.4 Hz, 1 H), 8.01 (d, J= 8.4 Hz, 1 H), 7.73 (t, J= 7.4 Hz, 1 H), 7.59 (t, J= 7.7 Hz, 1 H), 6.03 (s, 1 H), 3.93 (s, 1 H), 3.79 (s, 3H), 2.77 (s, 3H). 13C-NMR (CDCI3, 100 MHz) δ: 173.5, 158.3, 147.5, 142.9, 130.7, 128.8, 127.7, 127.1 , 125.1 , 124.6, 69.2, 53.4, 24.0.
Example 11
10a 6a 11a
10a (2.45 kg, 96.8% purity, 8.9 mol), 6a (2.5 kg, 88.7% purity, 8.82 mol), tris(dibenzylideneacetone)dipalladium(0) (Pd2dba3, 40 g, 0.044 mol), (S)-3-iert-butyl-4-(2,6-dimethoxyphenyl)-2,3-dihydrobenzo[d][1 ,3]oxaphosphole (32 g, 0.01 1 mol), sodium carbonate (1.12 kg, 10.58 mol), 1 -pentanol (16.69 L), and water (8.35 L) were charged to the reactor. The mixture was de-gassed by sparging with argon for 10-15 minutes, was heated to 60-63 °C, and was agitated until HPLC analysis of the reaction shows <1 A% (220 nm) of the 6a relative to the combined two atropisomer products (-15 hours). The batch was cooled to 8-23 °C. Water (5 L) and heptane (21 L) were charged. The slurry was agitated for 3 – 5 hours. The solids were collected by filtration, washed with water (4 L) and heptane/toluene mixed solvent (2.5 L toluene/5 L heptane), and dried. The solids were dissolved in methanol (25 L) and the resulting solution was heated to 50 °C and circulated through a CUNO carbon stack filter. The solution was distilled under vacuum to ~ 5 L. Toluene (12 L) was charged. The mixture was distilled under vacuum to – 5 L and cooled to 22 °C. Heptane (13 L) was charged to the contents over 1 hour and the resulting slurry was agitated at 20-25 °C for 3 – 4 hours. The solids were collected by filtration and washed with heptanes to provide 2.58 kg of 11a obtained after drying (73% yield). 1H NMR (400 MHz, CDCI3): δ 8.63 (d, 1 H, J = 8 Hz), 8.03 (d, 1 H, J = 12 Hz), 7.56 (t, 1 H, J = 8 Hz), 7.41 (d, 1 H, J = 8 Hz), 7.19 (t, 1 H, J = 8 Hz), 7.09 (m, 2H), 7.04 (d, 1 H, J = 8 Hz), 5.38 (d, 1 H, J = 8 Hz), 5.14 (d, 1 H, J = 8 Hz), 4.50 (t, 2H, J = 4 Hz), 3.40 (s, 3H), 3.25 (t, 2H, J = 4 Hz), 2.91 (s, 3H). 13C NMR (100 MHz, CDCI3): δ 173.6, 158.2, 154.0, 150.9, 147.3, 147.2, 145.7, 141.3, 132.9, 123.0, 129.4, 128.6, 127.8, 126.7, 126.4, 125.8, 1 18.1 , 1 17.3, 109.9, 70.3, 65.8, 52.3, 28.5, 24.0.
To a suitable clean and dry reactor under a nitrogen atmosphere was charged 1a (5.47 Kg, 93.4 wt%, 1 .00 equiv, 12.8 mol) and fluorobenzene (10 vols, 51.1 kg) following by trifluoromethanesulfonimide (4 mol%, 143 g, 0.51 mol) as a 0.5 M solution in DCM (1.0 Kg). The batch temperature was adjusted to 35-41 °C and agitated to form a fine slurry. To the mixture was slowly charged i-butyt-2,2,2- trichloroacetimidate 12b as a 50 wt% solution (26.0 Kg of f-butyl-2,2,2- trichloroacetimidate (119.0 mol, 9.3 equiv), the reagent was -48-51 wt% with the remainder 52-49 wt% of the solution being ~ 1.8:1 wt:wt heptane: fluorobenzene) over no less than 4 hours at Tint= 35-41 °C. The batch was agitated at Tint= 35-41 °C until HPLC conversion (308 nm) was >96 A%, then cooled to Tlnt= 20-25 °C and then triethylamine (0.14 equiv, 181 g, 1.79 mol) was charged followed by heptane (12.9 Kg) over no less than 30 minutes. The batch was agitated at Tint= 20-25 °C for no less than 1 hour. The solids were collected by filtration. The reactor was rinsed with the filtrate to collect all solids. The collected solids in the filter were rinsed with heptane (1 1.7 Kg). The solids were charged into the reactor along with 54.1 Kg of DM Ac and the batch temperature adjusted to Tint= 70-75 °C. Water (1 1.2 Kg) was charged over no less than 30 minutes while the batch temperature was maintained at Tint= 65-75 °C. 12a seed crystals (34 g) in water (680 g) was charged to the batch at Tint= 65-75 °C. Additional water (46.0 Kg) was charged over no less than 2 hours while maintaining the batch temperature at Tint= 65-75 °C. The batch temperature was adjusted to Tint= 18-25 °C over no less than 2 hours and agitated for no less than 1 hour. The solids were collected by filtration and the filtrate used to rinse the reactor. The solids were washed with water (30 Kg) and dried under vacuum at no more than 45 °C until the LOD < 4% to obtain 12a (5.275 Kg, 99.9 A% at 220 nm, 99.9 wt% via HPLC wt% assay, 90.5% yield). H-NMR (CDCI3l 400 MHz) δ: 8.66-8.65 (m, 1 H), 8.05 (d, J= 8.3 Hz, 1 H), 7.59 (t, J= 7.3 Hz, 1 H), 7.45 (d, J= 7.8 Hz, 1 H), 7.21 (t, J= 7.6 Hz, 1 H), 7.13-7.08 (m, 3H), 5.05 (s, H), 4.63-4.52 (m, 2H), 3.49 (s, 3H), 3.41 -3.27 (m, 2H), 3.00 (s, 3H), 0.97 (s, 9H). 13C-NMR (CDCI3, 100 MHz) δ: 172.1 , 159.5, 153.5, 150.2, 147.4, 146.9, 145.4, 140.2, 131.1 , 130.1 , 128.9, 128.6, 128.0, 127.3, 126.7, 125.4, 1 17.7, 1 17.2, 109.4, 76.1 , 71.6, 65.8, 51.9, 28.6, 28.0, 25.4.
Example 13
To a suitable clean and dry reactor under a nitrogen atmosphere was charged 12a (9.69 Kg, 21.2 mol) and ethanol (23.0 Kg). The mixture was agitated and the batch temperature was maintained at Tjnt= 20 to 25 °C. 2 M sodium hydroxide (17.2 Kg) was charged at Tint= 20 to 25 °C and the batch temperature was adjusted to Tlnt= 60- 65°C over no less than 30 minutes. The batch was agitated at Tint= 60-65°C for 2-3 hours until HPLC conversion was >99.5% area (12a is <0.5 area%). The batch temperature was adjuted to Tint= 50 to 55°C and 2M aqueous HCI (14.54 Kg) was charged. The pH of the batch was adjusted to pH 5.0 to 5.5 (target pH 5.2 to 5.3) via the slow charge of 2M aqueous HCI (0.46 Kg) at Tint= 50 to 55°C. Acetonitrile was charged to the batch (4.46 Kg) at Τ,ηί= 50 to 55°C. A slurry of seed crystals (1001 , 20 g in 155 g of acetonitrile) was charged to the batch at Tint= 50 to 55°C. The batch was agitated at Tint= 50 to 55°C for no less than 1 hour (1-2 hours). The contents were vacuum distilled to -3.4 vol (32 L) while maintaining the internal temperature at 45-55°C. A sample of the batch was removed and the ethanol content was determined by GC analysis; the criterion was no more than 10 wt% ethanol. If the ethanol wt% was over 10%, an additional 10% of the original volume was distilled and sampled for ethanol wt%. The batch temperature was adjusted to Tint= 8-22°C over no less than 1 hour. The pH of the batch was verified to be pH= 5 – 5.5 and the pH was adjusted, if necessary, with the slow addition of 2 M HCI or 2 M NaOH aqueous solutions. The batch was agitated at Tint= 18-22°C for no less than 6 hours and the solids were collected by filtration. The filtrate/mother liquid was used to remove all solids from reactor. The cake with was washed with water (19.4 Kg) (water temperature was no more than 20 °C). The cake was dried under vacuum at no more than 60 °C for 12 hours or until the LOD was no more than 4% to obtain 1001 (9.52 Kg, 99.6 A% 220 nm, 97.6 wt% as determined by HPLC wt% assay, 99.0% yield). Example 14
Hydrochloride salt of Compound (I), Type A
Compound (I) (263 mg) was added to a vial of ethanol (1.5 ml_), and then 36.5% HCL aqueous solution (59 mg) was added. The mixture was heated to 70 °C; and stirred at this temperature until solid material was obtained. The mixture was cooled to 20 °C over a period of 10 hours. After cooling, isopropanol (400 μΙ_) was added over a period of 3 hours. The resulting solids were collected and characterized as the hydrochloride salt of Compound (I), Type A.
The hydrochloride salt of Compound (I), Type A was prepared analogously to the aforementioned procedure using methyl ethyl ketone, tetrahydrofuran, acetonitrile, ethyl acetate, dichloroethane and methyl-t-buyl ether instead of ethanol.
References
- Pharmacodynamics of BI 224436 for HIV-1 in an in vitro hollow fiber infection model system
- Levin, Jules. BI 224436, a Non-Catalytic Site Integrase Inhibitor, is a potent inhibitor of the replication of treatment-naïve and raltegravir-resistant clinical isolates of HIV-1. Conference Reports for NATAP. ICAAC Chicago Sept 17-20 2011.
- Gilead Negotiates Worldwide License to BI’s Early Clinical Stage HIV Program. Genetic Engineering and Biotechnology News. 6 Oct 2011.
- Highleyman, Liz. ICAAC: New Integrase Inhibitor BI 224436 Active against Raltegravir-Resistant HIV. HIVandHepatitis.com. 7 Oct 2011.
- WO-2014055618
Elbasvir, MK 8742 ……….Anti-Hepatitis C Virus Drug in phase 2
Elbasvir, MK 8742
1370468-36-2 cas
methyl N-[(2S)-1-[(2S)-2-[4-[(6S)-3-[2-[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-4H-imidazol-4-yl]-6-phenyl-6H-indolo[1,2-c][1,3]benzoxazin-10-yl]-2H-imidazol-2-yl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate
Methyl [(2S)-1-[(2S)-2-[4-[(6S)-3-[2-[(2S)-1-[(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-4-yl]-6-phenylindolo[1,2-c][1,3]benzoxazin-10-yl]-1H-imidazol-2-yl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate
Carbamic acid, N,N‘-[[(6S)-6-phenyl-6H-indolo[1,2-c][1,3]benzoxazine-3,10-diyl]bis[1H-imidazole-5,2-diyl-(2S)-2,1-pyrrolidinediyl[(1S)-1-(1-methylethyl)-2-oxo-2,1-ethanediyl]]]bis-, C,C‘-dimethyl ester
Carbamic acid, N,N’-(((6S)-6-phenyl-6H-indolo(1,2-c)(1,3)benzoxazine-3,10-diyl)bis(1H-imidazole-5,2-diyl-(2S)-2,1-pyrrolidinediyl((1S)-1-(1-methylethyl)-2-oxo-2,1-ethanediyl)))bis-, C,C’-dimethyl ester
Dimethyl N,N’-(((6S)-6-phenylindolo(1,2-c)(1,3)benzoxazine-3,10-diyl)bis(1H-imidazole-5,2-diyl-(2S)-pyrrolidine-2,1-diyl((2S)-3-methyl-1-oxobutane-1,2-diyl)))dicarbamate
Methyl ((1S)-1-(((2S)-2-(4-((6S)-10-(2-((2S)-1-((2S)-2-((methoxycarbonyl)amino)-3-methylbutanoyl)pyrrolidin-2-yl)-1H-imidazol-4-yl)-6-phenyl-6H-indolo(1,2-c)(1,3)benzoxazin-3-yl)-1H-imidazol-2-yl)pyrrolidin-1-yl)carbonyl)-2-methylpropyl)carbamate
MW 882.0171, C49 H55 N9 O7,
UNII-632L571YDK
MERCK-PHASE 2
HCV NS5A Inhibitors
patent….http://www.google.com/patents/WO2012040923A1?cl=en
MK-8742 is in phase II clinical development at Merck & Co. for the oral treatment of chronic hepatitis C infection in combination with MK-5172 and ribavirin. Phase I clinical trials are uongoing for the treatment of hepatitis C infected males. In 2013, breakthrough therapy designation was assigned to the compound.
MK-8742 is an inhibitor of Hepatitis C Virus (HCV) non-structural protein 5A (NS5A) that is being developed for the treatment of HCV infection. MK-8742 has broad, potent HCV genotypic activity in vitro against viral variants that are resistant to other NS5A inhibitors. MK-8742 exhibits potent antiviral activity during 5 days of monotherapy in patients with GT1 and GT3 chronic HCV infection. MK-8742 is currently in Phase IIB development.
MK-8742 is an inhibitor of Hepatitis C Virus (HCV) non-structural protein 5A (NS5A) that is being developed for the treatment of HCV infection. MK-8742 has broad, potent HCV genotypic activity in vitro against viral variants that are resistant to other NS5A inhibitors. MK-8742 exhibits potent antiviral activity during 5 days of monotherapy in patients with GT1 and GT3 chronic HCV infection. MK-8742 is currently in Phase IIB development.
http://www.natap.org/2012/EASL/EASL_46.htm
………………
http://www.google.com/patents/WO2012040923A1?cl=en
EXAMPLE 23
Preparation of Compound A
A mixture of Compound Int-19b (1.1 g, 3 mmol), (dibromomethyl)benzene (2.25 g, 9 mmol) and K2C03 (1.2 g, 9 mmol) in 15 mL of DMF was heated to 100 °C and allowed to stir at this temperature for 3 hours. The reaction mixture was cooled to room temperature, concentrated in vacuo and the residue obtained was dissolved with
dichloromethane and water. The aqueous phase was extracted with dichloromethane. The combined organic extracts were washed with brine, dried over Na2S04, filtered and concentrated in vacuo. The resulting residue was purified using flash column
chromatography on silica gel to provide Compound Int-23a (380 mg, 28 %) as a white solid. 1H MR (CDCI3): δ 7.72 (bs, 1 H), 7.44 – 7.46 (d, J= 8.4 Hz, 1 H), 7.21 – 7.28 (m, 3 H), 7.09 – 7.12 (m, 3 H), 7.04 (s, 1 H), 6.99 – 7.01 (bs, J= 6.8 Hz, 2 H), 6.78 (s, 1 H), 6.63 – 6.65 (d, J = 8.4 Hz, 1 H). MS (ESI)
m/e (M+H+): 456. Step B – Pre aration of Compound Int-23b
lnt-23a lnt-23b
To a solution of Int-23a (456 mg, 1.0 mmol) in 1,4-dioxane was added bis pinacol borate (2.2 mmol) , Pd(dppf)Cl2 (0.04 mmol) and KOAc (4 mmol). The reaction mixture was put under N¾ heated to 110°C and allowed to stir at this temperature for 3 hours. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the residue obtained was purified using column chromatography on silica gel to provide Compound Int- 23b (590 mg, 87 % yield). 1H MR (CDC13): δ 8.13 (s, 1 H), 7.60 (d, J= 7.6 Hz, 1 H), 7.52 (d, J= 8.0 Hz, 1H), 7.36 – 7.39 (m, 1 H), 7.14 -7.19 (m, 4 H), 6.93 – 6.95 (m, 3 H), 6.90 (s, 1 H), 1.26 – 1.29 (s, 24 H). MS (ESI) m / e (M+H+): 550.
– Pre aration of Compound Int-23c
lnt-23b lnt-23c
A suspension of Int-23b (550 mg, 1.0 mmol), tert-butyl 2-(2-bromo-lH- imidazol-5-yl) pyrrolidine- 1-carboxylate (2.4 mmol), Pd(dppf) Cl2 (200 mg), Na2C03 (3 mmol) and in THF/H20 (10: 1, 33 mL) was allowed to stir at reflux for about 15 hours under N2. The reaction mixture was cooled to room temperature and filtered, and the filtrate was washed with water (50 mL) and extracted with EtOAc (100 mL). The organic extract was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified using column chromatography on silica gel to provide Compound Int-23c (160 mg). MS (ESI) m / e (M+H+): 768.
Preparation of Compound Int-23d
Int-23c (0.10 g, 0.13 mmol) was added to HCl/CH3OH (5 mL, 3M) and the resulting reaction was allowed to stir at room temperature for about 3 hours. The reaction mixture was then concentrated in vacuo to provide Compound Int-23d, which was used without further purification. MS (ESI) m / e (M+H+): 568.
– Preparation of Compound A
To a solution of Int-23d (56.8 mg, 0.10 mmol), (S)-2- (methoxycarbonylamino)-3-methylbutanoic acid (35.0 mg, 0.20 mmol) and DIPEA (0.8 mmol) in CH3CN (1 mL) was added BOP (98 mg, 0.22 mmol). The resulting reaction was allowed to stir at room temperature and monitored using LCMS. After LCMS showed the starting material to be consumed, the reactionmixture was filtered, and the filtrate was purified using HPLC to provide Compound A as a white solid. 1H MR (MeOD): δ 7.94 (s,
1 H), 7.85 (d, J= 8.0 Hz, 1 H), 7.74 (s, 1 H), 7.63 (s, 1 H), 7.48 (s, 1 H), 7.35 – 7.37 (m, 2 H), 7.31 (s, 1 H), 7.17 – 7.18 (m, 4 H), 7.11 (s, 1 H), 6.96 – 6.98 (d, J = 7.6 Hz, 2 H), 5.09 – 5.17
(m, 2 H), 4.13 (t, J= 8.0 Hz, 2 H), 3.99 (bs, 2 H), 3.78 (bs, 2 H), 3.56 (s, 6 H), 2.44 – 2.47 (m,
2 H), 1.92 – 2.19 (m, 8 H), 0.77 – 0.85 (m, 12 H). MS (ESI) m / e (M+H+): 882.
The diastereomers were separated on a chiral SFC column: Isomer A: 1H NMR (MeOD): δ 8.08 (s, 1H), 7.91 – 7.93 (m, 1 H), 7.72 (s, 1 H), 7.56 (s, 1 H), 7.24 – 7.43 (m, 7 H), 7.19 (s, 1 H), 7.03 – 7.05 (m, 2 H), 5.16 – 5.24 (m, 2 H), 3.81 – 4.21 (m, 6 H), 3.62 (s, 6 H), 2.52 – 2.54 (m, 2 H), 2.00 – 2.25 (m, 8 H), 0.84 – 0.91 (m, 12 H). MS (ESI) m/z (M+H)+: 882.
Isomer B: 1H NMR (MeOD): δ 7.90 (s, 1 H), 7.81 – 7.83 (m, 1 H), 7.72 (s, 1 H), 7.62 (s, 1 H), 7.45 (s, 1 H), 7.14 – 7.33 (m, 6 H), 7.09 (s, 1 H), 6.93 – 6.95 (m, 2 H), 5.06 – 5.14 (m, 2 H), 3.71 – 4.11 (m, 6 H), 3.52 (s, 6 H), 2.41 – 2.44 (m, 2 H), 1.90 – 2.15 (m, 8 H), 0.74 – 0.86 (m, 12 H). MS (ESI) m/z (M+H)+: 882.
……………………..
Discovery of MK-8742: An HCV NS5A inhibitor with broad genotype activity
ChemMedChem 2013, 8(12): 1930
http://onlinelibrary.wiley.com/doi/10.1002/cmdc.201300343/abstract
The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.
see
Journal of Medicinal Chemistry (2014), 57(5), 1643-1672.
Want to know everything on vir series
click
http://drugsynthesisint.blogspot.in/p/vir-series-hep-c-virus-22.html
AND
http://medcheminternational.blogspot.in/p/vir-series-hep-c-virus.html
| WO2010111483A1 * | Mar 25, 2010 | Sep 30, 2010 | Merck Sharp & Dohme Corp. | Inhibitors of hepatitis c virus replication |
| US20070049593 * | Feb 23, 2005 | Mar 1, 2007 | Japan Tobacco Inc. | Tetracyclic fused heterocyclic compound and use thereof as HCV polymerase inhibitor |
CDRI planning to launch Phase-1 trials on 2 candidate drugs to fight malaria, diabetes
CDRI LUCKNOW INDIA
http://www.cdriindia.org/home.asp
CDRI planning to launch Phase-1 trials on 2 candidate drugs to fight malaria, diabetes
pharmabiz.com
The Central Drug Research Institute (CDRI), the public sector premier institution for drug discovery, will soon start Phase 1 clinical trials of a candidate …
Joseph Alexander, New Delhi
Monday, April 14, 2014, 08:00 Hrs [IST]
The Central Drug Research Institute (CDRI), the public sector premier institution for drug discovery, will soon start Phase 1 clinical trials of a candidate drug against malaria and another one to fight diabetes.
The institute has developed and licensed the anti-hyperglycemic candidate drug (CDR134F194) to TVC Sky Shop Ltd., Mumbai. The process of formulation of the drug in a GMP certified company is in progress. The single dose and multi-dose Phase- I clinical trial will be initiated soon at KEM Hospital & Seth GS Medical College in Mumbai. The permission for the trials was already given by the Drugs Controller General of India (DCGI), sources said.
Another candidate drug developed by the CDRI and waiting for the trials is in the therapeutic area of malaria. The single dose pharmacokinetic study in healthy volunteers as per revised protocol approved by DCGI was completed at PGIMER, Chandigarh for the CDRI compound 97/78 (Anti-malarial agent). A total of 16 volunteers completed the trial. The blood samples were analysed inthe Pharmacokinetics & Metabolism division and the final report on single dose pharmacokinetic study submitted to IPCA, Mumbai.
http://www.pharmabiz.com/NewsDetails.aspx?aid=81386&sid=1
DAREXABAN, TANEXABAN
DAREXABAN , TANEXABAN
N-(3-Hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}phenyl)-4-methoxybenzamide
365462-23-3 , 365462-24-4 (maleate)
Darexaban (YM150) is a direct inhibitor of factor Xa created by Astellas Pharma.[1] It is an experimental drug that acts as ananticoagulant and antithrombotic to prevent venous thromboembolism after a major orthopaedic surgery, stroke in patients with atrial fibrillation[2] and possibly ischemic events in acute coronary syndrome.[3] It is used in form of the maleate. The development of darexaban was discontinued in September 2011.
Factor Xa
Factor Xa (FXa) is an essential blood coagulation factor[2] that is responsible for the initiation of the coagulation cascade. FXa cleaves prothrombin to its active form thrombin, which then acts to convert soluble fibrinogen to insoluble fibrin and to activateplatelets. Stabilization of the platelet aggregation by fibrin mesh ultimately leads to clot formation.[4]
Metabolism
Darexaban is rapidly absorbed and extensively metabolized in the liver to its active metabolite, darexaban glucuronide (YM-222714) during first pass metabolism via glucuronidation.[5] The metabolism of darexaban also occurs in the small intestine but to a much lesser extent.[2] Glucuronidation of darexaban occurs quickly, thus the half life of darexaban itself is short. However, the resultant darexaban glucuronide metabolite has a long half life of approximately 14-18 hours, reaching its maximum levels in the blood 1-1.5 hour post dose.[2] As a result, darexaban glucuronide is the main determinant of the antithrombotic effects.[3] Darexaban shows minimal interaction with food and is excreted through the kidneys (urine) and feces.[6]
Mechanism of action
Darexaban and darexaban glucuronide selectively and competitively inhibit FXa, suppressing prothrombin activity at the sites of blood clot (thrombus) formation. This leads to a decrease in blood clot formation in a dose dependent manner.[2] Reducing blood clot formation will decrease blood flow blockages, thus possibly lowering the risk of myocardial infarction, unstable angina, venous thrombosis, and ischemic stroke.[7]
Clinical uses
Atrial fibrillation
Atrial fibrillation is an abnormal heart rhythm that causes a reduction in the cardiac output and blood flow to the brain. It also promotes the formation of blood clots in the atria.[4]Atrial fibrillation is associated with an increased risk of embolic stroke due to the increased risk of blood clot development.[8] Oral anticoagulant drugs such as Darexaban decrease the incidence and severity of stroke in patients with atrial fibrillation by preventing the formation of blood clots.[9]
Contraindictions
The RUBY-1 phase II trial results show that oral administration of darexaban in combination with the standard dual antiplatelet therapy used for ACS patients caused a two- to four-fold increase in bleeding rates and no effect on ACS.[6] Though there were no cases of fatal bleeding or intracranial haemorrhage, the results of this study questions the concept of adding an oral anticoagulant to standard of care dual antiplatelet therapy in order to prevent recurrent ischemic events after ACS. The developpement of darexaban was discontinued in september 2011.
References
- Eriksson, B., et al. “A dose escalation study of YM150, an oral direct factor Xa inhibitor, in the prevention of venous thromboembolism in elective primary hip replacement surgery.” Journal of Thrombosis and Haemostasis (2007): 1660-1665
- Yoshiyuki, I., et al. “Biochemical and pharmalogical profile of darexaban, an oral direct Xa inhibitor.” European Journal of Pharmacology (2011): 49-55
- Toshifumi, S., et al. “Identification of UDP-Glucuronosyltransferases Responsible for the Glucuronidation of Darexaban, an Oral Factor Xa Inhibitor, in Human Liver anD Intestine.” The American Society for Pharmacology and Experimental Therapeutics (2011): 278-282
- Katsung, B., S. Masters and A. Trevor. Basic and Clinical Pharmacology 11th Edition. United States of America: McGraw-Hill, 2009
- Turpie, A., et al. “Prevention of venous thromboembolism with an oral factor Xa inhibitor, YM150, after total hip arthoplasty. A dose finding study (ONYX-2).” Journal of Thrombosis and Haemostasis (2010): 714-721
- Steg, PG; Mehta, SR; Jukema, JW; Lip, GY; Gibson, CM; Kovar, F; Kala, P; Garcia-Hernandez, A; Renfurm, RW; Granger, CB; Ruby-1, Investigators (2011). “RUBY-1: A randomized, double-blind, placebo-controlled trial of the safety and tolerability of the novel oral factor Xa inhibitor darexaban (YM150) following acute coronary syndrome”.European heart journal 32 (20): 2541–54. doi:10.1093/eurheartj/ehr334.PMC 3295208. PMID 21878434.
- Hirayama, F., et al. “Discovery of N-[2-Hydroxy-6-(4-methoxybenamido)phenyl]-4-(4-methyl-1,4-diazepan-1-yl)benzamide (Darexaban, YM150) as a Potent and Orally Available Factor Xa Inhibitor.” Journal of Medicinal Chemistry (2011): 8051-8065
- Zhong, Y., et al. “Atrial Fibrillation as a Risk Factor for Stroke: A Retrospective Cohort Study of Hospitalized Medicare Beneficiaries.” American Journal of Public Health (1998): 395-400
- Hylek, E., et al. “Effect of intesity of oral anticoagulation on stroke severity and mortality in atrial fibrillation.” The New England Journal of Medicine (2003): 1019-26
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PHARMACEUTICAL COMPOSITION FOR ORAL ADMINISTRATION
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Diazepan derivatives or salts thereof
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Barley Grass Inhibits 73% of Leukemia Cells in Vitro
Barley Grass Inhibits 73% of Leukemia Cells in Vitro: An extract of green barley grass (Hordeum vlgare L.) powder was shown to inhibit the proliferation of human leukemia cells (Nalm-6) by up to 73% in vitro, and killed 62% of the cancer cells outright via apoptosis and necrosis.
The barley grass extract also potently inhibited three other types of leukemia cells, while leaving healthy non-cancerous cells alone. What’s really interesting with this study is that the extract was prepared from a common green barley powder supplement which was purchased at an online supplement retailer in the USA. This was the first-ever study to show the anti-leukemia activity of green barley.
Barley contains several unique compounds with potent anti-cancer effects such as the peptide lunacin, immune-stimulating glucans, and ribosome inactivating protein conjugates. Past studies have shown mature barley to be active against melanoma and cancers of the breast, skin, colon, liver, and lung. While mature barley contains gluten (albeit less than wheat), barley grass should be gluten-free if harvested before any seeds are produced.
And since younger plants often contain much higher concentrations of healthy phytochemicals and enzymes than the mature versions, barley grass might be a highly beneficial superfood for reducing cancer risk and supporting overall health.
http://www.ncbi.nlm.nih.gov/pubmed/24039967
Abstract 10693: Identification of Lunasin as the Active Component in Soy Protein Responsible for Reducing LDL Cholesterol and Risk of Cardiovascular Disease by Alfredo F Galvez, Missouri Plant Science Center, Mexico, MO
In the above referenced report, published by the American Heart Association, Dr Galvez notes that the FDA had originally approved the health claim that soy reduced LDL cholesterol and CVD risk, and then recinded part of that claim. The confusion, he notes, came from the lack of understanding at the time about what in the soy created the benefits the original research had shown. In the intervening years, he and his team had tested the hypothesis that the lunasin peptide was the active component in soy protein responsible for lowering LDL cholesterol.
What he found was that lunasin lowers LDL cholesterol levels by stopping the gene responsible from being active – it covers it over, and opens the genes that cover cholesterol management in the liver that had gotten covered by environmental and lifestyle-induced damage.
The lunasin soy peptide binds specifically to histone H3 and inhibits H3-Lysine 14 acetylation by PCAF histone acetylase enzyme. Transcriptional activation of HMG Co-A reductase, the rate-limiting enzyme for cholesterol biosynthesis requires the specific acetylation of histone H3 by PCAF. By inhibiting PCAF acetylation of H3-Lysine 14, lunasin was show to significantly reduce HMG Co-A reductase expression in HepG2 liver cells grown in cholesterol-free media. Westerns and RT-PCR experiments also revealed that the presence of lunasin increases LDL receptor expression, which can be attributed to the coordinate increase in expression of SP1 co-transcriptional activator.
Based on these results, his team found a way to extract the active lunasin from the rest of the soy. This lunasin-enriched soy extract (LSE) contained 100-200 fold more bioactive lunasin than soy protein isolates. Then they tested the LSE pigs bred to have high LDL cholesterol due to mutations in their LDL receptor genes. The pigs were fed casein-based diets and after two weeks their casein diet was supplemented with 250 mg LSE everyday for eight weeks. Results showed that casein diet increased LDL cholesterol levels in the LDL-R mutant pigs by an average of 6.7%. The addition of 250 mg of LES in casein diet reduced LDL cholesterol by 8.6% and and 6.4% after 4 and 8 weeks of treatment, respectively.
These results prove that lunasin is the active nutrient in soy protein responsible for LDL cholesterol lowering and its mechanism of action is by reducing cholesterol biosynthesis in the liver.
4 Surprising Foods Packed With Estrogen — The Chemical Linked to Obesity and Sexual Dysfunction
Estrogen is blamed for everything from breast and prostate cancer and other hormone-linked conditions to obesity, sexual dysfunction, dropping sperm counts and depression and mood disorders. In studies of women given prescribed hormone drugs, estrogen was linked to lung cancer, ovarian cancer, skin cancer, gall bladder cancer, cataracts urinary incontinence and joint degeneration.
Chinese Dodder Seeds, Tu Si Zi, Semen Cuscutae, 菟絲子
Chinese Dodder Seeds ( Tu Si Zi ) 菟絲子 , also known as Beggarweed, Cuscutae, Devil’s Guts, Dodder Of Thyme, Hellweed, Lesser Dodder, Scaldweed, Strangle Tare, Tu Si Zi, Tu Sizi. Cuscuta epithymum; Cuscuta chinensis. It belong to the “Convolvulaceae” family.
Chinese Dodder Seeds ( Tu Si Zi ) 菟絲子 has a sweet, pungent and neurtal properties. It is use for treating the kidney and liver.
In the term of traditional Chinese medicine (TCM),
Tu Si Zi is acrid, sweet, neutral.
The channels Tu Si Zi influences are Kidney, Liver.Action:
In the term of TCM, Tu Si Zi:
1: Tonifies Kidneys, Augments Yin, Secures Jing and Reserves Urine.
2: Tonifies Kidneys and Liver, Improves Vision.
3: Benefits Spleen and Kidneys, Stops Diarrhea.
4: Calms the Fetus.
Usage:
• tonify kidneys, strengthen yin, secures essence, reserves urine.
• tonify liver, improves vision.
• strengthen spleen, stops diarrhea.
• calms fetus, habitual/threatened miscarriage.
Other Use:
Orally, dodder is used for urinary tract, spleen, and hepatic disorders.
Cuscuta chinensis.Dodder.Dodder seed extract Pharmacological Actions.
-
- Botanical Basic Data of Cuscuta chinensis(Dodder).
- Archeology of Dodder.
- Description of Cuscuta chinensis(Dodder).
- Constituents and Phytochemicals of Dodder Seed.
- Historical Use of Dodder Seed.
- Pharmacological Actions of Dodder Seed.
- Dodder Seed extracts as male sexual enhancement material.
- Dodder plant extracts as good cancer inhibitor.
- Tinnitus Relief Formula Material.
- Polysaccharide of Cuscuta Chinesis and its Immunological Adjuvant Effect.
- Vision Formular for Cataract and Glaucoma and Dodder Seed Combination.
- Administration and Dosage.
- Adverse Effect, Side Effects and Cautions,Toxity.
- Research Update:Dodder Seed.
- Photo Gallery of Dodder.
Botanical Basic Data of Cuscuta chinensis(Dodder).:
Botanical Source:Cuscuta chinensis(The ripe seed of Cuscuta chinensis Lam.,an annual voluble parasitic herb of the family Convolvulaceae).
Latin Name: Semen Cuscutae
Family: Convolvulaceae.
Common Name: Cuscuta seed, Chinese Dodder seed,Huang Si,Huang Teng Zi,Dou Ji Sheng.Huang Shan Teng.Wu Gen Cao,Wu Niang Teng,Huang Shan Si,Lao Ya Si,Huang Si Teng.
Scientific Name: Cuscuta chinensis Lam
Pin Yin Name: Tu Si Zi
Cuscuta Classification in China:(1).Cuscuta chinensis Lam.;(2).Cuscuta australis R.Br.;(3).Cuscuta campestris Yunker;
Pinyin Name: Tu si zi,Also called Chinese Dodder Seed.
Pin yin description:tu is a character for this herb derived from the character meaning rabbit; si means silk, and zi means seeds, the part used; this plant is a parasitic weed that sets up a mat of hair-like fibers at its base and then rapidly sends fibrous stems upward; thus Tu Si refers to the quality of these fibers like silky rabbit hair; a common name for dodders in the West, based on the undesirable weed-like nature of these plants, is Devil’s Hair.
Part use:Dodder seed,Aerial parts.(whole plants are harvested in autumn when the seeds are ripe, and then threshed after dried to get the seeds)
Synoms:Dodder,love vine,strangleweed,devil’s-guts,goldthread,pull-down,devil’s-ringlet,hellbine,hairweed,devil’s-hair,Beggarweed, Cuscutae, Devil’s Guts, Dodder Of Thyme, Hellweed,Lesser Dodder,Scaldweed,Strangle Tare,Tu Si Zi,Tu Sizi,Cuscuta epithymum,Cuscuta chinensis and hailweed.
Habitat:Dodder grows throughout Europe, Asia, and southern Africa. Dodder prefers coastal and mountainous regions, and is gathered in summer.In China,mainly distributed in Jiangsu,Liaoning, Jilin, Hebei, Shandong and Henan provinces of China.
Taste:Pungent, Sweet,It is sweet in taste, warm in nature and manifests its therapeutic actions in the liver, kidney and spleen meridians.
Constitutents:Dodder contains flavonoids (including kaempferol and quercitin) and hydroxycinnamic acid.
Cuscuta , or Dodder plant, is a parasitic vine that wraps around other plants for nourishment.The ripe seed of Cuscuta chinensis Lam.; an annual voluble parasitic herb of the family Convolvulaceae.Cuscuta seed is used in China for kidney deficiency. Cuscuta has a high content of flavonoids and has strong antioxidant properties. Cuscuta seed has been found in studies to have positive effects on sperm health and motility, and invigorates the reproductive system.
The plant growns near seashores.Slim stems spread out,twist and yellow color,no leaf.flower blossom fascination on axil.flower bud and small bud squama shape,caylx shape cup,5 divide,white crown,bell shape,double length of calyx.The flowers are hermaphrodite (have both male and female organs).Stamen flower flat short,squama grow on base,shape square roundness,2 room germen. Capsule shape flat ball.Seed 2~4,florescence July to September,fruit august to october. It can grow in semi-shade (light woodland) or no shade and requires moist soil.
Dodder is distributed in most parts of China. It is collected in autumn when the seed is ripe, dried in the sun and used unprepared or boiled after removal of impurities.
Ambit Biosciences announces Phase 3 trial comparing quizartinib as monotherapy to chemotherapy regimens in relapsed/refractory acute myeloid leukemia (AML) patients with the FMS-like tyrosine kinase-3 (FLT3)-ITD mutation.
N-(5-tert-butyl-isoxazol-3-yl)-N’-{ 4- [7-(2-morpholin-4-yl-ethoxy)imidazo [2, 1 -b] [ 1 ,3 ]benzothiazol-2-yl]phenyl } urea
| CAS | 950769-58-1 (free base) 1132827-21-4 (2HCl) |
| Formula | C29H32N6O4S |
| MW | 560.7 |
| Synonim | AC220, AC-010220 ASP-2689 |
Quizartinib
Ambit Biosciences
Ambit Biosciences (NASDAQ:AMBI) is a biotech company that focuses on treatments that inhibit kinases, which are drivers for diseases such as cancer. Three drugs are in development, with the lead one being quizartinib — a Phase 2B trial treatment for acute myeloid leukemia. However, AMBI’s collaboration agreement with Astellas Pharma is set to expire in September, and if it is not replaced, it could mean a delay in Phase 3 trials for quizartinib. Keep in mind that AMBI generated $23.8 million in collaboration revenues last year.
Quizartinib (AC220) is a small molecule receptor tyrosine kinase inhibitor that is currently under development by Ambit Biosciencesfor the treatment of acute myeloid leukaemia. Its molecular target is FLT3, also known as CD135 which is a proto-oncogene.[1]
AC-220 is an angiogenesis inhibitor that antagonizes several proteins involved in vascularization. It was engineered by Ambit Biosciences using KinomeScan technology to potently target FLT3, KIT, CSF1R/FMS, RET and PDGFR kinases. Ambit is developing oral AC-220 in phase III clinical studies for the treatment of relapsed/refractory acute myeloid leukemia (AML) patients with the FMS-like tyrosine kinase-3 (FLT3)-ITD mutation. Early clinical trials are also ongoing for the treatment of advanced solid tumors, for the treatment of refractory or relapsed myelodysplasia, in combination with induction and consolidation chemotherapy for previously-untreated de novo acute myeloid leukemia, and as a maintenance therapy of AML following hematopoietic stem cell transplantation (HSCT). In 2009, orphan drug designation was received both in the U.S. and in the EU for the treatment of AML. In 2009, Ambit Biosciences and Astellas Pharma have entered into a worldwide agreement to jointly develop and commercialize the drug candidate for the treatment of cancer and non-oncology indications. This agreement was terminated in 2013.
Flt3 mutations are among the most common mutations in acute myeloid leukaemia due to internal tandem duplication of Flt3. The presence of this mutation is a marker of adverse outcome.
| Quizartinib is a small molecule with potential anticancer activity. Quizartinib is a selective inhibitor of class III receptor tyrosine kinases, including FMS-related tyrosine kinase 3 (FLT3/STK1), stem cell factor receptor (SCFR / KIT), colony-stimulating factor 1 receptor (CSF1R/FMS) and platelet-derived growth factor receptors (PDGFRs .) Able to inhibition of ligand-independent cell proliferation and apoptosis. Mutations in FLT3 are the most frequent genetic alterations in acute myeloid leukemia (AML) and occur in approximately 30% of cases of AML. | |
| Quizartinib представляет собой малую молекулу с потенциальной противораковой активностью. Quizartinib является селективным ингибитором класса III рецепторов тирозин киназ, в том числе FMS-связанных тирозинкиназы 3 (FLT3/STK1), фактор стволовых клеток рецепторов (SCFR / KIT), колониестимулирующий фактор 1 рецепторов (CSF1R/FMS) и тромбоцитарный рецепторов фактора роста (PDGFRs). Способен к торможению лиганд-независимой клеточной пролиферации и апоптоза. Мутации в FLT3 являются наиболее частыми генетическими изменениями в остром миелобластном лейкозе (ОМЛ) и встречаются примерно в 30% случаев ОМЛ. |
Mechanism
Specifically, Quizartinib selectively inhibits class III receptor tyrosine kinases, including FMS-related tyrosine kinase 3 (FLT3/STK1), colony-stimulating factor 1 receptor (CSF1R/FMS), stem cell factor receptor (SCFR/KIT), and platelet derived growth factor receptors (PDGFRs).
Mutations cause constitutive action of Flt3 leading to resulting in inhibition of ligand-independent leukemic cell proliferation and apoptosis.
Clinical trials
It had good results in a phase II clinical trial for refractory AML – particularly in patients who went on to have a stem cell transplant.[2]
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WO 2007109120 COMPD B1
EXAMPLE 3: PREPARATION OF N-(5-TERT-BUTYL-ISOXAZOL-3-YL)-N’-{4-[7-(2- MORPHOLIN-4-YL-ETHOXY)IMIDAZO[2,1 -B3[1 ,3]BENZOTHIAZOL-2-YL]PHENYL}UREA [Compound B1]
[00426] A. The intermediate 2-amino-1,3-benzothiazol-6-ol was prepared according to a slightly modified literature procedure by Lau and Gompf. J. Org. Chem. 1970, 35, 4103-4108. To a stirred solution of thiourea (7.6 g, 0.10 mol) in a mixture of 200 ml_ ethanol and 9 ml_ concentrated hydrochloric acid was added a solution of 1 ,4-benzoquinone (21.6 g, 0.20 mol) in 400 mL of hot ethanol. The reaction was stirred for 24 hours at room temperature and then concentrated to dryness. The residue was triturated with hot acetonitrile and the resulting solid was filtered and dried.
[00427] The free base was obtained by dissolving the hydrochloride salt in water, neutralizing with sodium acetate, and collecting the solid by filtration. The product (2-amino-1 ,3-benzothiazol-6-ol) was obtained as a dark solid that was pure by LCMS (M+H = 167) and NMR. Yield: 13.0 g (78 %). NMR (DMSOd6) £7.6 (m, 2H ), 6.6 (d, 1H).
[00428] B. To prepare the intermediate 2-(4-nitrophenyl)imidazo[2,1- b][1 ,3]benzothiazoI-7-ol, 2-amino-1 ,3-benzothiazol-6-ol, (20.0 g, 0.12 mol) and 2-bromo-4′-nitroacetophenone (29.3 g, 0.12 mol) were dissolved in 600 mL ethanol and heated to reflux overnight. The solution was then cooled to 00C in an ice-water bath and the product was collected by vacuum filtration. After drying under vacuum with P2O5 , the intermediate (2-(4- nitrophenyl)imidazo[2,1-_D][1,3]benzothiazol-7-ol) was isolated as a yellow solid. Yield: 17.0 g (46 %) NMR (DMSO-CT6) δ 10 (s, 1 H), 8.9 (s, 1H), 8.3 (d, 2H), 8.1 (d, 2H), 7.8 (d, 1 H), 7.4 (s, 1 H), 6.9 (d, 1 H). [00429] C. To make the 7-(2-morpholin-4-yl-ethoxy)-2-(4-nttro- phenyl)imidazo[2,1-£>][1 ,3]benzothiazo!e intermediate: 2-(4- nitrophenyl)imidazo[2,1-jb][1 ,3]benzothiazol-7-ol, (3.00 g, 9.6 mmol) was suspended in 100 mL dry DMF. To this mixture was added potassium carbonate (4.15 g, 30 mmol, 3 eq), chloroethyl morpholine hydrochloride (4.65 g, 25 mmol, 2.5 eq) and optionally tetrabutyl ammonium iodide (7.39 g, 2 mmol). The suspension was then heated to 900C for 5 hours or until complete by LCMS. The mixture was cooled to room temperature, poured into 800 mL water, and allowed to stand for 1 hour. The resulting precipitate was collected by vacuum filtration and dried under vacuum. The intermediate, (7-(2- morpholin-4-yl-ethoxy)-2-(4-nitro-phenyl)imidazo[2,1-jb][1 ,3]benzothiazole) was carried on without further purification. Yield: 3.87 g (95 %) NMR (DMSO-Cf6) δ 8.97 (s, 1 H), 8.30 (d, 2H), 8.0 (d, 2H), 7.9 (d, 1 H), 7.7 (s, 1 H), 7.2 (d, 1 H), 4.1 (t, 2H), 5.6 (m, 4H), 2.7 (t, 2H).
[00430] D. To make the intermediate 7-(2-morpholin-4-yl-ethoxy)-2-(4- amino-phenyl)!midazo[2, 1 -b][1 ,3]benzothiazole: To a suspension of 7-(2- morpholin-4-yl-ethoxy)-2-(4-nitro-phenyl)imidazo[2,1-ib][1 ,3]benzothiazole (3.87g, 9.1 mmol) in 100 ml_ isopropyl alcohol/water (3:1 ) was added ammonium chloride (2.00 g, 36.4 mmol) and iron powder (5.04 g, 90.1 mmol). The suspension was heated to reflux overnight with vigorous stirring, completion of the reaction was confirmed by LCMS. The mixture was filtered through Celite, and the filtercake was washed with hot isopropyl alcohol (150 ml_). The filtrate was concentrated to approximately 1/3 of the original , volume, poured into saturated sodium bicarbonate, and extracted 3 times with dichloromethane. The combined organic phases were dried over MgSO4 and concentrated to give the product as an orange solid containing a small amount (4-6 %) of starting material. (Yield: 2.75 g 54 %). 80% ethanol/water may be used in the place of isopropyl alcohol /water — in which case the reaction is virtually complete after 3.5 hours and oniy traces of starting material are observed in the product obtained. NMR (DMSO-d6) δ 8.4 (s, 1 H), 7.8 (d, 1 H), 7.65 (d, 1 H), 7.5 (d, 2H), 7.1 (d, 1 H), 6.6 (d, 2H), 4.1 (t, 2H)1.3.6 (m, 4H), 2.7 (t, 2H).
[00431] E. A suspension of 7-(2-morpholin-4-yl-ethoxy)-2-(4-amino- phenyl)imidazo[2,1-b][1 ,3]benzothiazole (4.06 g, 10.3 mmol) and 5-tert- butylisoxazole-3-isocyanate (1.994 g, 12 mmol) in toluene was heated at 120 0C overnight. The reaction was quenched by pouring into a mixture of methylene chloride and water containing a little methanol and neutralized with saturated aqueous NaHCO3 solution. The aqueous phase was extracted twice with methylene chloride, the combined organic extracts were dried over MgSO4 and filtered. The filtrate was concentrated to about 20 ml volume and ethyl ether was added resulting in the formation of a solid. The precipitate was collected by filtration, washed with ethyl ether, and dried under vacuum to give the free base. Yield: 2.342 g (41 %) NMR (DMSO-Cf6) £9.6 (br, 1H), 8.9 (br, 1H), 8.61 (s, 1H), 7.86 (d, 1 H), 7.76 (d, 2H), 7.69 (d, 1 H), 7.51 (d, 2H), 7.18 (dd, 1H), 6.52 (s, 1H), 4.16 (t, 2H), 3.59 (t, 4H), 3.36 (overlapping, 4H), 2.72 (t, 2H), 1.30 (s, 9H). NMR (CDCI3) £9.3 (br, 1H), 7.84 (m, 4H), 7.59 (d, 2H), 7.49 (d, 1 H), 7.22 (d, 1 H), 7.03 (dd, 1 H)1 5.88 (s, 1 H), 4.16 (t, 2H), 3.76 (t, 4H), 2.84 (t, 2H), 2.61 (t, 4H), 1.37 (s, 9H).
[00432] F. For the preparation of the hydrochloride salt, N-(5-tert-butyl- isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2, 1 – b][1 ,3]benzothiazol-2-yl]phenyI}urea hydrochloride, the free base was dissolved in a mixture of 20 ml methylene chloride and 1 ml methanol. A solution of 1.0 M HCI in ethyl ether (1.1 eq.) was added dropwise, followed by addition of ethyl ether. The precipitate was collected by filtration or centrifugation and washed with ethyl ether to give the hydrochloride salt. Yield: 2.44 g (98 %) NMR (DMSO-d6) £11-0 (br, 1 H), 9.68 (s, 1H), 9.26 (s, 1H), 8.66 (s, 1 H), 7.93 (d, 1H), 7.78 (m, 3H), 7.53 (d, 2H), 7.26 (dd, 1H), 6.53 (S, 1 H), 4.50 (t, 2H), 3.97 (m, 2H), 3.81 (t, 2H), 3.6 (overlapping, 4H)13.23 (m, 2H)1 1.30 (s, 9H).
[00433] G. Alternatively, Compound B1 may be made by taking the intermediate from Example 4B and reacting it with chloroethyl morpholine hydrochloride under conditions described in Step C. [00434] H . Λ/-(5-tert-butyl-isoxazol-3-yl)-Λ/’-{4-[5-(2-morpholin-4-yl- ethoxy)imidazo[2,1-6][1 ,3]benzothiazol-2-yl]phenyl}urea hydrochloride, a compound having the general formula (I) where R1 is substituted on the 5 position of the tricyclic ring, was prepared in the manner described in Steps A- F but using the cyciization product 2-amino-benzothiazol-4-ol with 2-bromo-4′- nitroacetophenone in Step A. 1H NMR (DMSO-d6) δ 11.6 (br, 1 H)1 9.78 (br, 1H), 9.56 (br, 1 H), 8.64 (s, 1H)1 7.94 (d, 2H), 7.70 (s, 1H)1 7.56 (d, 2H), 7.45 (t, 1 H), 7.33 (d, 1H), 6.54 (s, 1 H), 4.79 (t, 2H), 3.87 (m, 6H), 3.60 (m, 2H), 3.34 (m, 2H)1 1.30 (s, 9H); LC-MS: ESI 561 (M+H)+. [Compound B11] [00435] I. N-(5-tert-butyl-isoxazol-3-yl)-N’-{4-[6-(2-morpholin-4-yl- ethoxy)imidazo[2,1-b][1 ,3]benzothiazol-2-yl]phenyl}urea hydrochloride [Compound B12] was also prepared by first preparing the benzothiazole starting material, 5 methoxy-benzothiazol-2yl~amine: [00436] To prepare the 5-methoxy-benzothiazol-2-ylamine starting material: To a suspension of (3-methoxy-phenyl)-thiourea (1.822g, 10 mmol) in CH2CI2 (20 ml_) at 0 0C was added dropwise a solution of bromine (1.76 g, 11 mmol) in 10 ml of trichloromethane over a period of thirty minutes. The reaction was stirred for 3 hours at room temperature then heated to 3 hours to reflux for one hour. The precipitate was filtered and washed with dichloromethane. The solid was suspended in saturated NaHCOsand extracted with CH2CI2. The extract was dried over MgSO4 and concentrated to give a white solid (1.716 g, 95%).
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WO 2011056939
N-(5-ieri-butyl- isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l-&][l,3]benzothiazol-2- yl]phenyl}urea (I), or a pharmaceutically acceptable salt, solvate, hydrate, or polymorph thereof. N-(5-ieri-Butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- / ][!, 3]benzo
N- (5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- &][l,3]benzo-thiazol-2-yl]phenyl}urea (I), or a pharmaceutically acceptable salt, solvate, hydrate, or polymorph thereof, comprising any one, two, three, four, five, six, seven of the steps of:
(A) converting 2-amino-6-alkoxybenzothiazole (II), wherein R1 is a suitable phenolic hydroxyl protecting ;
(II) (III)
(B) reacting 2-amino-6-hydroxybenzothiazole (III) with compound (IV), wherein X is a leaving group, to yield 2-(4-nitrophenyl)imidazo[2,l-b]benzothiazol-7-ol (V);
(C) reacting 2-(4-nitrophenyl)imidazo[2,l-b]benzothiazol-7-ol (V) with compound (VI), wherein X2 is a leaving group, to yield 7-(2-morpholin-4-yl-ethoxy)-2-(4- nitrophenyl)imidazo[ -b]benzothiazole (VII);
(D) reducing 7-(2-morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2, 1- bjbenzothiazole (VII) to yield 7-(2-morpholin-4-yl-ethoxy)-2-(4- am
(E) converting 3-amino-5-£er£-butyl isoxazole (IX) to a 5-?er?-butylisoxazol-3- ylcarbamate derivative (X), wherein R2 is optionally substituted aryl, heteroaryl, alkyl, or cycloalkyl;
(IX) (X)
(F) reacting 7-(2-morpholin-4-yl-ethoxy)-2-(4-aminophenyl)imidazo[2,l- bjbenzothiazole (VIII) with a 5-£er£-butylisoxazol-3-ylcarbamate derivative (X) to yield N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- &][l,3]benzo-
(G) converting N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2,l-&][l,3]benzo-thiazol-2-yl]phenyl}urea to an acid addition salt of N- (5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- b] [ 1 ,3]benzo-thiazol-2-yl]phenyl } urea.
[00128] In certain embodiments, provided herein are processes for the preparation of N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- &][l,3]benzo-thiazol-2-yl]phenyl}urea, or a pharmaceutically acceptable salt, solvate, hydrate, or polymorph thereof, as depicted in Scheme 1, wherein R1, R2, X1, and X2 are defined herein elsewhere. In specific embodiments, provided herein are processes for the preparation of N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2,l-&][l,3]benzo-thiazol-2-yl]phenyl}urea (I), or a pharmaceutically acceptable salt, solvate, hydrate, or polymorph thereof, comprising any one, two, three, four, five, six, seven, of the Steps A, B, C, D, E, F, and G, as depicted in Scheme 1.
Scheme 1 :
A. Preparation of 2-amino-6-hydroxybenzothiazole
1. Example A-l[00252] To a 1-L 3-necked round bottom flask fitted with a condenser, heating mantle, and mechanical stirrer was charged aqueous hydrobromic acid (48%, 632 mL, 5.6 mol, 10 equiv). 2-Amino-6-methoxybenzothiazole (100 g, 0.55 mol, 1 equiv) was added to the above flask over 15 minutes. The reaction temperature was raised slowly to reflux (105-110 °C). A clear dark brown colored solution was observed at about 80 °C. The reflux was continued at 105-110 °C for about 4 hr. The progress of the reaction was monitored by HPLC. When 2-amino-6-methoxybenzothiazole was less than 2%, the reaction was substantially complete.
[00253] The reaction mass was cooled to 0-5 °C and at this point precipitation of a solid was observed. The mixture was maintained at 0-5 °C for 0.5 hr and filtered, and the cake was pressed to remove HBr. The wet cake was transferred to a 2-L round bottom flask fitted with a mechanical stirrer. Saturated aqueous sodium bicarbonate solution (-1500 mL) was added slowly at ambient temperature, whereupon considerable frothing was observed. The pH of the solution was found to be about 6.5 to 7. The mixture was stirred for 0.5 hr at ambient temperature and filtered. The filter cake was washed with water (500 mL), dried on the filter and then under vacuum at 30-35 °C for 10-12 hr to give the product 2-amino-6-hydroxybenzothiazole (82 g, 89% yield, HPLC purity = 99%). JH NMR (DMSO-if6, 500 MHz): δ 7.12 (d, 1H), 7.06 (S, 2H, NH2), 7.01 (d, 1H), 6.64 (dd, 1H); MS (m/z) = 167.1 [M+ + 1].
[00254] Table: Summary of Plant Batches
[00255] HPLC chromatographic conditions were as follows: The column used was XTerra RP8, 250 X 4.6 mm, 5μ or equivalent. Mobile Phase A was buffer, prepared by mixing 3.48 g of dipotassium hydrogen phosphate in 1.0 L of water, and adjusting the pH to 9.0 with phosphoric acid. Mobile Phase B was methanol. The flow rate was 1.0 mL/minute. Detection was set at UV 270 nm. The injection volume was 20 μΐ^, and the sample was diluted with a diluent (Mobile Phase A : Mobile Phase B = 70:30). Test solution was prepared by weighing accurately about 25 mg of sample and transferring it into a 100 mL volumetric flask, dissolving with 20-30 mL of diluent, making up the volume to the mark with diluent, and mixing. The HPLC was performed by separately injecting equal volumes of blank and test solution, and recording the chromatogram for all injections. The purity was calculated by area normalization method.
[00256] Table: HPLC Method
2. Example A-2
[00257] 2-Amino-6-methoxybenzothiazole was reacted with hot aqueous HBr at a temperature of >70 °C for about 3 hours and then the clear solution was cooled to ambient temperature overnight. The precipitated solids were collected, dissolved in hot water and the pH was adjusted to between 4.5-5.5. The resultant solids were collected, dried and re-crystallized from isopropanol. Second crop material was collected. The solids were vacuum dried to give 2-amino-6-hydroxybenzothiazole.
[00258] The reaction progress was monitored by thin layer chromatography (TLC). The product was isolated as a white solid, with 99.4% purity (HPLC area %). JH NMR (300 MHz, DMSO-if6) was collected, which conformed to structure.
3. Example A-3
[00259] A 22-L 3-neck round bottom flask was equipped with a mechanical agitator, thermocouple probe, a reflux condenser, and a heating mantle. The flask was charged with hydrobromic acid (14 L, 123.16 mol, 13.10 equiv). Heating was initiated and 2- amino-6-methoxybenzothiazole was added (1.7 kg, 9.4 mol, 1.00 equiv) over 10 minutes with stirring. The heating of the reaction mixture was continued to reflux, and maintained (>107 °C) for approximately 5 hours. The reaction mixture turned into a clear solution between 75 °C and 85 °C. The reaction progress was monitored by TLC until no starting material was observed (A -0.5 mL reaction mixture aliquot was diluted with -0.5 mL water as a clear solution, neutralized with sodium acetate to pH -5 and extracted with 1 mL dichloromethane. The organic layer was spotted: 5%
methanol/dichloromethane; Rf (product) = 0.35; Rf (starting material) = 0.40).
[00260] The reaction mixture was cooled to – 20 °C (overnight). White solids precipitated. The solids were filtered on a polypropylene filter and pressed to remove as much hydrobromic acid from the solids as possible to facilitate the subsequent pH adjustment step. The slightly wet crude product was dissolved in hot (50 °C) water (5 L). The clear solution was filtered to remove any insoluble material present, and the solids were washed with 50 °C water. The filtrate was cooled to 10 °C. The cooled filtrate was neutralized with sodium acetate (- 1.0 kg) to pH 4.5 to 5.5 with vigorous stirring. A thick white solid precipitated. The solids were collected by filtration, and washed with cool (-10 °C) water (2 x 1.0 L) and air dried.
[00261] The wet crude product was slurried in hot (50 °C) isopropanol (3 L) briefly and allowed to stand in a cool room (-5 °C) overnight. The solids were collected by filtration and washed with methyl ferf-butylether (2 x 500 mL). The solids were dried in a vacuum oven overnight (<30 mm Hg) at 30 °C (first crop). Yield: 1068 g (68%), white solid. HPLC: 99.4% (area). JH NMR (300 MHz, DMSO- ) conformed to structure.
[00262] The organic filtrate was collected in a total volume of 1.0 L, cooled to 10 °C. The off-white solids were precipitated and collected by filtration. The solids were dried in a vacuum oven overnight (<30 mm Hg) at 30 °C (second crop). Yield: 497 g (32%), off-white solid. HPLC: 99.8% (area).
[00263] The overall yield combining the first crop and the second crop was 1565 g, (99%).
B. Preparation of 2-(4-nitrophenyl)imidazo[2,l-b]benzothiazol-7-ol
1. Example B-l[00264] A 3-L 3-neck round bottom flask fitted with a condenser, a heating mantle, and a mechanical stirrer was charged with H-butanol (1.5 L), followed by 2-amino-6- hydroxybenzothiazole (75 g, 0.45 mol, 1.0 equiv), 2-bromo-4′-nitroacetophenone (121 g, 0.50 mol, 1.1 equiv), and sodium bicarbonate (41.6 g, 0.50 mol, 1.0 equiv). The reaction temperature was gradually raised to reflux and maintained at reflux (110-115 °C) for 2-3 hr. During the temperature increase, the reaction mass turned into a clear solution and then immediately changed into an orange colored suspension at 65-75 °C. The progress of the reaction was monitored by HPLC analysis every 1 hr (reaction mass sample was submitted to QC). When the level of 2-amino-6-hydroxybenzothiazole was less than 2%, the reaction was substantially complete.
[00265] The reaction mass was slowly cooled to 50-60 °C and then further cooled to 0-5 °C and stirred for 15 min. The precipitated solids were collected by filtration, and dried on the filter. The wet cake was transferred in to a 1-L round bottom flask, and water (600 mL) was added. The suspension was stirred for 0.5 hr and filtered, and the solid was dried on the filter. The wet cake was again taken in to a 1-L round bottom flask and stirred with acetone (200 mL). The slurry was filtered and washed with acetone (2 X 100 mL), and the solid was dried on the filter, unloaded and further dried in a vacuum oven at ambient temperature to give the product 2-(4-nitrophenyl)imidazo[2,l- b]benzothiazol-7-ol (V) (120 g, 85.7% yield, HPLC purity = 98.7%). JH NMR (DMSO- d6, 500 MHz): δ 9.96 (s, 1H, OH), 8.93 (s, 1H), 8.27 (d, 2H), 8.06 (d, 2H), 7.78 (d, 1H), 7.38 (d, 1H), 6.97 (dd, 1H); MS (m/z) = 312 [M+ + 1].
[00266] Table: Summary of Plant Batches
* Input of 2-amino-6-hydroxybenzothiazole (III)
[00267] HPLC chromatographic conditions were as follows: The column used was XTerra RP8, 250 X 4.6 mm, 5μ or equivalent. Mobile Phase A was buffer, prepared by mixing 3.48 g of dipotassium hydrogen phosphate in 1.0 L of water, and adjusting the H to 9.0 with phosphoric acid. Mobile Phase B was methanol. The flow rate was 1.0 mL/minute. Detection was set at UV 235 nm. The injection volume was 10 μΐ^. The blank was prepared by transferring 200 μΐ. of DMSO and 200 μΐ. of 2M NaOH into a 10 mL volumetric flask, making up the volume to the mark with methanol, and mixing. The test solution was prepared by weighing accurately about 10 mg of sample and transferring it into a 50 mL volumetric flask, dissolving with 1 mL of DMSO and 1 mL of 2M NaOH, sonicating to dissolve, making up the volume to the mark with methanol, and mixing. The HPLC was performed by separately injecting equal volumes of blank and test solution, and recording the chromatogram for all injections. The purity was calculated by area normalization method.
[00268] Table: HPLC Method
2. Example B-2
[00269] A 50-L 3-neck round bottom flask was equipped with a mechanical agitator, a thermocouple probe, a reflux condenser, and a heating mantle. The flask was charged with 2-amino-6-hydroxybenzothiazole (1068 g, 6.43 mol, 1.0 equiv) and ethanol (200 proof, 32.0 L), and the suspension was stirred for 10 minutes. 2-Bromo-4- nitroacetophenone (1667 g, 6.49 mol, 1.01 equiv) was added in one portion. The reaction mixture was heated to reflux (78 °C). The reflux was maintained for approximately 25 hours, resulting in a yellow suspension. The reaction progress was monitored by TLC (20% methanol/ethyl acetate; Rf (product) = 0.85; Rf (starting material) = 0.30). TLC indicated -50% 2-amino-6-hydroxybenzothiazole after 24 hours of reflux. Tetrabutylammonium iodide (10 g) was added and reflux was maintained for an additional 12 hours. TLC indicated -50% starting material still present. Coupling was seen to occur at both the thiazole and the amine.
[00270] The reaction mixture was cooled to 0-5 °C. The solids were collected by filtration, and the yellow solid was washed with ethanol (200 proof, 2 X 1.0 L) and diethyl ether (2 X 1.5 L). The solids were dried in a vacuum oven (<10 mm Hg) at 40 °C. Yield: 930 g (46%), yellow solid. HPLC: 99.5% (area). JH NMR (300 MHz, DMSO-i¾) conformed to structure.
3. Example B-3
[00271] The reaction of Step B was carried out on multiple runs, varying solvents, temperature, and base. The results were summarized in the table below. The product (V) was isolated as yellow or green solids, with 1H NMR consistent with the structure and a purity of greater than about 98% by HPLC analysis.
[00272] Table: Reaction Condition Screening
TBAI = Tetrabutylammonium Iodide
C. Preparation of 7-(2-morpholin-4-yl-ethoxy)-2-(4- nitrophenyl)imidazo[2, 1 -bjbenzothiazole
1. Example C-l
[00273] To a 2000-L glass-lined (GL) reactor was charged DMF (298 kg), and the agitator was started. Under a nitrogen blanket, the reactor was charged with 2-(4- nitrophenyl)imidazo[2,l-&]benzothiazol-7-ol (36.8 kg, 118.2 mol, 1.0 equiv), 4-(2- chloroethyl)morpholine hydrochloride (57.2-66.0 kg, 307.3-354.6 mol, 2.6-3.0 equiv), tetrabutylammonium iodide (8.7 kg, 23.6 mol, 0.2 equiv) and potassium carbonate (49.0 kg, 354.6 mol, 3.0 equiv). The resulting yellow suspension was heated and stirred at 90 + 5 °C for at least 15 minutes, then the temperature was increased to 110 + 5 °C. The mixture was stirred for at least 1 hour and then sampled. The reaction was deemed complete if 2-(4-nitrophenyl) imidazo[2,l-&]benzothiazol-7-ol was <1% by HPLC. If the reaction was not complete, the heating was continued and the reaction sampled. If the reaction was incomplete after 6 hours, additional 4-(2-chloroethyl)morpholine hydrochloride may be charged. In general, additional charges of 4-(2- chloroethyl)morpholine hydrochloride had not been necessary at the given scale.
[00274] The reactor was cooled to 20 + 5 °C and charged with water (247 kg) and acetone (492 kg). The mixture was agitated at 20 + 5 °C for at least 1 hour. The product (VII) was isolated by filtration or centrifuge, and washed with water and acetone, and then dried in a vacuum oven at 45 °C to constant weight to give a yellow solid (46.2 kg, 92% yield, HPLC purity = 97.4% by area). JH NMR (300 MHz, DMSO- ) conformed to structure.
2. Example C-2
[00275] 2-(4-Nitrophenyl)imidazo[2, l-b]benzothiazol-7-ol, 4-(2-chloroethyl)- morpholine hydrochloride, potassium carbonate, and tetrabutylammonium iodide were added to N,N-dimethylformamide forming a yellow suspension that was heated at a temperature of >50 °C for over 3 hours. The reaction was cooled and the solids were collected, slurried into water, filtered, slurried into acetone, filtered and washed with acetone to give yellow solids that were dried under vacuum to give 7-(2-morpholin-4-yl- ethoxy)-2-(4-nitrophenyl)imidazo[2,l-b]benzothiazole.
[00276] The reaction progress was monitored by thin layer chromatography (TLC). The product was isolated as a yellow solid, with 99% purity (HPLC area %), and a water content of 0.20%. Infrared (IR) spectrum was collected, which conformed to structure.
3. Example C-3
[00277] A 50-L 3-neck round bottom flask was equipped with a mechanical agitator, a thermocouple probe, a drying tube, a reflux condenser, and a heating mantle. The flask was charged with 2-(4-nitrophenyl)imidazo [2,l-&]benzothiazol-7-ol (1.770 kg, 5.69 mol, 1.0 equiv), N,N-dimethylformamide (18.0 L), 4-(2-chloroethyl)morpholine hydrochloride (2.751 kg, 14.78 mol, 2.6 equiv), potassium carbonate (2.360 kg, 17.10 mol, 3.0 equiv), and tetrabutylammonium iodide (0.130 kg, 0.36 mol, 0.06 equiv) with stirring. The resulting yellow suspension was heated to about 90 °C to 95 °C, maintaining the temperature for approximately 5 hours. The reaction was monitored by TLC until no starting material was observed (20% methanol / ethyl acetate; Rf (product) = 0.15; Rf (starting material) = 0.85).
[00278] The reaction mixture was cooled to -10 °C, and the yellow solids were collected by filtration on a polypropylene filter pad. The solids were slurried in water (2 X 5 L) and filtered. The crude wet product was slurried in acetone (5 L), filtered, and the solids were rinsed with acetone (2 X 1.5 L). The solids were dried in a vacuum oven (<10 mm Hg) at 45 °C. Yield: 2.300 kg (95%), yellow solid. TLC: R/ = 0.15 (20% methanol / EtOAc). HPLC: 95.7% (area). JH NMR (300 MHz, DMSO-i¾) conformed to the structure.
[00279] Table: Yields from multiple batch runs
4. Example C-4
[00280] To a reactor were added 2-(4-nitrophenyl)imidazo [2,l-&]benzothiazol-7-ol (1.0 kg), 4-(2-chloroethyl)morpholine hydrochloride (1.6 kg), tetrabutylammonium iodide (0.24 kg), and potassium carbonate (1.3 kg, anhydrous, extra fine, hydroscopic). N,N-Dimethylformamide (DMF) (8.6 L) was added into the reactor. The DMF used had water content of no more than 0.05% w/w. The mixture was stirred for between 15 and 30 minutes to render a homogeneous suspension, which was heated to between 85 °C and 95 °C and stirred at between 85 °C and 95 °C for 15 to 30 minutes. The mixture was then heated to between 105 °C and 120 °C and stirred at between 105 °C and 120 °C (e.g. , 115 °C) until the reaction was complete (as determined by HPLC to monitor the consumption of starting material). In some embodiments, if necessary (e.g. , if after 6 hours the reaction was not complete as indicated by HPLC analysis), additional 4-(2- chloroethyl)morpholine hydrochloride (0.03 kg) may be added and the reaction mixture stirred at between 105 °C and 120 °C (e.g. , 115 °C) until reaction completion.
[00281] The reaction mixture was cooled to between 20 °C and 30 °C (e.g. , over a period of 3 hours). To another reactor was added deionized water (7.6 L) and acetone (15 L). The mixture of water and acetone was then added into the reaction mixture while maintaining the temperature at between 20 °C and 30 °C. The mixture was then stirred for 1 to 2 hours at a temperature of between 20 °C and 30 °C. The mixture was filtered, and the solid was washed with deionized water (e.g. , about 45x deionized water) until pH of washes was below 8. The solid was then washed with acetone (9.7 L). The solid was dried under vacuum at a temperature of less than 50 °C until the water content by Karl-Fischer was less than 0.30% w/w and TGA curve showed a mass loss of less than 0.30% w/w at about 229 °C (sampling approximately every 6 hours). The desired product was obtained in about 89% yield having about 99% purity by HPLC.
5. Example C-5
[00282] To a reactor were added 2-(4-nitrophenyl)imidazo [2, l-&]benzothiazol-7-ol (1.0 kg), 4-(2-chloroethyl)morpholine hydrochloride (1.6 kg), and potassium carbonate (1.3 kg, anhydrous, extra fine, hydroscopic). N,N-Dimethylformamide (DMF) (8.6 L) was added into the reactor. The DMF used had water content of no more than 0.05% w/w. The mixture was stirred for between 15 and 30 minutes to render a homogeneous suspension, which was heated to between 95 °C and 120 °C (e.g. , between 100 °C and 105 °C) and stirred at between 95 °C and 120 °C (e.g. , 105 °C) until the reaction was complete (as determined by HPLC to monitor the consumption of starting material). In some embodiments, if necessary (e.g. , if after 6 hours the reaction was not complete as indicated by HPLC analysis), additional 4-(2-chloroethyl)morpholine hydrochloride (0.03 kg) and potassium carbonate (0.024 kg) may be added and the reaction mixture stirred at between 100 °C and 120 °C (e.g. , 105 °C) until reaction completion.
[00283] The reaction mixture was cooled to between 60 °C and 70 °C over a period of at least 60 minutes. Industrial water (6 L) was added to the reactor. The reaction mixture was cooled to between 20 °C and 30 °C. Acetone (6 L) was added to the reactor. The mixture was stirred for 1 to 2 hours at a temperature of between 20 °C and 30 °C. The mixture was filtered, and the solid was washed with industrial water (e.g. , about 45 x industrial water) until pH of washes was below 8. The solid was then washed with acetone (9.7 L). The solid was dried under vacuum at a temperature of less than 50 °C, until the water content by Karl-Fischer was less than 0.30% w/w and TGA curve showed a mass loss of less than 0.30% w/w at about 229 °C (sampling approximately every 6 hours).
6. Example C-6
[00284] To a reactor is added 2-(4-nitrophenyl)imidazo [2, l-&]benzothiazol-7-ol (1.0 kg), 4-(2-chloroethyl)morpholine hydrochloride (1.6 kg), and potassium carbonate (1.3 kg, anhydrous, extra fine, hydroscopic). N,N-Dimethylformamide (DMF) (8.6 L) is added into the reactor. The DMF has a water content of no more than 0.05% w/w. The mixture is stirred for between 15 and 30 minutes to render a homogeneous suspension, which is heated to between 95 °C and 120 °C (e.g. , between 100 °C and 105 °C) and stirred at between 95 °C and 120 °C (e.g. , 105 °C) until the reaction is complete (as determined by HPLC to monitor the consumption of starting material). In some embodiments, if necessary (e.g. , if after 6 hours the reaction is not complete as indicated by HPLC analysis), additional 4-(2-chloroethyl)morpholine hydrochloride (0.03 kg) and potassium carbonate (0.024 kg) may be added and the reaction mixture stirred at between 100 °C and 120 °C (e.g. , 105 °C) until reaction completion.
[00285] The reaction mixture is cooled to between 20 °C and 30 °C (e.g. , over a period of 3 hours). To another reactor is added deionized water (7.6 L) and acetone (15 L). The mixture of water and acetone is then added into the reaction mixture while maintaining the temperature at between 20 °C and 30 °C. The mixture is then stirred for 1 to 2 hours at a temperature of between 20 °C and 30 °C. The mixture is filtered, and the solid is washed with deionized water (e.g. , about 45x deionized water) until pH of washes is below 8. The solid is then washed with acetone (9.7 L). The solid is dried under vacuum at a temperature of less than 50 °C until the water content by Karl-Fischer is less than 0.30% w/w and TGA curve shows a mass loss of less than 0.30% w/w at about 229 °C (sampling approximately every 6 hours). D. Preparation of 7-(2-morpholin-4-yl-ethoxy)-2-(4- aminophenyl)imidazo [2, 1 -bjbenzothiazole
[00286] To a 200-L high pressure (HP) reactor was charged a slurry of 7-(2- morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo [2,l-&]benzothiazole (VII) (7.50 kg, 17.7 mol, 1.0 equiv) in methanol (30 kg). The container was rinsed with additional methanol (10 kg) and the rinse was charged to the reactor. The reactor was then charged with THF (67 kg) and methanol (19 kg). The contents were agitated and the reactor was flushed with nitrogen by alternating nitrogen and vacuum. Vacuum was applied to the reactor and Raney Ni catalyst (1.65 kg, 0.18 wt. equiv) was charged through a sample line. Water (1 kg) was charged through the sample line to rinse the line. The reactor was flushed with nitrogen by alternating nitrogen and vacuum. The reactor was then vented and heated to 50 °C. The reactor was closed and pressurized with hydrogen gas to 15 psi keeping the internal temperature below 55 °C. The reactor was vented and re- pressurized a total of 5 times, then pressurized to 150 psi with hydrogen gas. The contents were agitated at 50 °C for at least 4 hours. At this point a hydrogen uptake test was applied: The reactor was re-pressurized to 150 psi and checked after 1 hour. If a pressure drop of more than 5 psi was observed, the process was repeated. Once the pressure drop remained < 5 psi, the reactor was vented and sampled. The reaction was deemed complete when 7-(2-morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo [2,1- 6]benzothiazole (VII) was < 0.5% by HPLC.
[00287] The reactor was flushed with nitrogen as shown above. The 200-L HP reactor was connected to the 2000-L GL reactor passing through a bag filter and polish filter. The bag filter and polish filter were heated with steam. The 200-L HP reactor was pressurized (3 psi nitrogen) and its contents were filtered into the 2000-L reactor. The filtrates were hot. The 200-L reactor was vented and charged with THF (67 kg) and methanol (59 kg), the reactor agitated, and filtered into the 2000-L GL reactor.
[00288] A total of 6 reductions (46.2 kg processed) were carried out and the combined batches were concentrated by vacuum distillation (without exceeding an internal temperature of 40 °C) to a volume of -180 L. The reactor was cooled to 20 °C and charged with heptane (250 kg) and again vacuum distilled to a volume of -180 L. The reactor was charged with heptane (314 kg) and agitated at 20 °C for at least 1 hour, and then the product was isolated by centrifugation or collection on a Nutsche filter, washing with heptanes (2-5 kg per portion for centrifugation, followed by a 10-20 kg heptanes rinse of the reactor; or 94 kg for Nutsche filtration, rinsing the reactor first). The cake was blown dry, transferred to a vacuum oven and dried to constant weight maintaining a temperature < 50 °C to give the desired product (VIII) (34.45 kg, 80% yield, HPLC purity = 97.9%).
2. Example D-2
[00289] 7-(2-Morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,l-b]benzothiazole was dissolved into methanol and THF and placed in a hydrogenator. Raney nickel was added and the vessel was pressurized with hydrogen and stirred for >24 hours. The reaction mixture was concentrated to a thick paste and diluted with methyl ferf-butyl ether. The resulting solids were filtered and washed with methyl ferf-butyl ether and dried under vacuum to give 7-(2-morpholin-4-yl-ethoxy)-2-(4-aminophenyl) imidazo [2, 1 -bjbenzothiazole.
[00290] The reaction progress was monitored by thin layer chromatography (TLC). The product was isolated as a yellow solid, with 99% purity (HPLC area %). IR was collected, which conformed to structure.
3. Example D-3
[00291] Into a 5-gallon autoclave, 7-(2-morpholin-4-yl-ethoxy)-2-(4-nitrophenyl) imidazo[2,l-&]benzothiazole (580 g, 1.37 mol, 1.0 equiv), THF (7.5 L), methanol (7.5 L, AR) and -55 g of decanted Raney nickel catalyst were added. The reaction vessel was purged with nitrogen (3 X 50 psi) and hydrogen (2 X 50 psi), with stirring briefly under pressure and then venting. The autoclave was pressurized with hydrogen (150 psi). The mixture was stirred and the hydrogen pressure was maintained at 150 psi for over 24 hours with repressurization as necessary. The reaction progress was monitored by TLC (10% methanol / chloroform with 1 drop ammonium hydroxide; Rf (product) 0.20; Rf (SM) 0.80). The reaction was substantially complete when the TLC indicated no starting material present, typically after 24 hours of stirring at 150 psi. The hydrogenation was continued at 150 psi for a minimum of 4 hours or until completion if starting material was still present after the initial 4 hours.
[00292] The reaction mixture was filtered through a Buchner funnel equipped with a glass fiber filter topped with a paper filter. Unreacted starting material was removed together with the catalyst. The filtrate was concentrated to a total volume of 0.5 L, and the residue was triturated with methyl ferf-butyl ether (0.5 L). The resultant solids were collected by filtration, and washed with methyl ferf-butyl ether (0.3 L) (first crop).
[00293] The filtrate was concentrated to dryness and the residue was diluted with methyl ferf-butyl ether (2 L). The resultant solids were collected by filtration, washing with methyl ferf-butyl ether (0.5 L) (second crop).
[00294] The solids were dried in a vacuum oven (<10 mm Hg) at 25 °C. Yield: 510 g (95%), beige solid. TLC: R/ 0.2 (10% methanol / chloroform with one drop of ammonium hydroxide). HPLC: 99.0% (area). JH NMR (300 MHz, DMSO-i¾) conformed to the structure.
[00295] Table: Yields from multiple batch runs
4. Example D-4
[00296] The reaction of Step D was carried out in multiple runs under various conditions, such as, e.g. , varying catalyst loading, concentration of reactant, reaction temperature, and/or workup procedures. The results are summarized in the table below.
Description Run # l Run # 2 Run # 3 Run # 4 Run # 5Rxn Temp (°C) RT RT RT RT RT
Rxn Time (Hr) 24 hr 24 hr 24 hr 24 hr 24 hr
Filtered the Filtered the solution
Filtered the Filtered the Filtered the
solution through through celite. The solution through solution through solution through
celite, washed celite filter cake celite, celite, celite,
with THF, refluxed in THF concentrated, concentrated, concentrated,
concentrated, washed with hot solvent exchanged solvent exchanged solvent exchanged
Work Up solvent exchanged THF, concentrated, with heptane, with heptane, with heptane,
with heptane, solvent exchanged stirred the solids stirred the solids stirred the solids
stirred the solids with heptane, stirred and filtered and filtered and filtered
and filtered the solids and washed with washed with washed with
washed with filtered washed with heptane heptane heptane
heptane heptane
Produce (VIII) 1.9 g 3.88 g 1.11 g 2.6 g 4.4 g
Yield 88% 83.4% 56 94.6%
HPLC purity 95.6% 77.5% 91% 93.8%
5. Example D-5
[00297] To a pressure reactor under nitrogen atmosphere was added a slurry of Raney Nickel in water (0.22 kg) (e.g. about 0.14 kg dry catalyst in water) and the charging line was rinsed with deionized water (0.13 L). To another reactor (Reactor B) were added methanol (5.05 L) and 7-(2-morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo [2, 1- &]benzothiazole (1.0 kg), and the mixture was stirred for between 15 and 30 minutes to render a homogenous suspension. The suspension was transferred to the pressure reactor. Reactor B was washed with methanol (4.88 L) and the wash was transferred to the pressure reactor. Tetrahydrofuran (10.1 L) was added to the pressure reactor.
Hydrogen was charged to the pressure reactor to a pressure of between 2.0 bar and 3.0 bar. The reactor was heated to a temperature of between 45 °C and 55 °C. Hydrogen was then charged to the pressure reactor to a pressure of between 6.0 bar and 7.0 bar. The mixture was stirred at a temperature of between 45 °C and 55 °C (e.g. , 50 °C), while maintaining the hydrogen pressure between 6.0 bar and 7.0 bar until reaction completion (as determined by HPLC to monitor the consumption of starting material).
[00298] The mixture was filtered while maintaining the temperature at between 35 °C and 50 °C. The pressure reactor and the filter were washed with a mixture of THF (10.1 L) and methanol (9.93 L) preheated to a temperature of between 45 °C and 55 °C (e.g. , 50 °C). The combined filtrate was concentrated to a volume of between 2.4 L and 2.8 L under vacuum at a temperature of no more than 40 °C, and a precipitate was formed. Methanol (7.5 L) was added. The slurry was cooled to a temperature of between 5 °C and -5 °C (e.g. , over 3 hours) and stirred for at least 1 hour (e.g. , for 3 hours) while maintaining the temperature at between 5 °C and -5 °C. The suspension was filtered. The solid was washed with methanol (2 X 1.2 L). The solid was then dried under vacuum at a temperature of less than 50 °C until the methanol and THF contents were each less than 3000 ppm as analyzed by GC (e.g. , less than 1500 ppm). The desired product was obtained in about 88.5% yield having about 99% purity by HPLC.
E. Preparation of phenyl 5-£er£-butylisoxazol-3-ylcarbamate
[00299] The jacket temperature of a 200-L glass-lined (GL) reactor was set to 20 °C. To the reactor was charged 5-ieri-butylisoxazole-3-amine (15.0 kg, 107.0 mol, 1.0 equiv), then K2C03 (19.5 kg, 141.2 mol, 1.3 equiv) and anhydrous THF (62 kg).
Agitation was started and then phenyl chloroformate (17.6 kg, 112.4 mol, 1.05 equiv) was charged. The charging line was rinsed with additional anhydrous THF (5 kg). The reaction was agitated at 20 + 5 °C for at least 3 hours then sampled. The reaction was deemed complete if 5-£er£-butylisoxazole-3-amine was < 2% by HPLC. If the reaction was not complete after 6 hours, additional K2CO3 and phenyl chloroformate may be added to drive the reaction to completion.
[00300] Once complete, the reaction was filtered (Nutsche). The filter was rinsed with THF (80 kg). The filtrate was vacuum distilled without exceeding an internal temperature of 40 °C until -50 L remained. Water (188 kg) and ethanol (45 L) were charged, and the mixture was agitated for at least 3 hours with a jacket temperature of 20 °C. The resulting solid was isolated by centrifugation or collection on a Nutsche filter, rinsed with water (2-5 kg for each centrifugation portion; 30 kg for Nutsche filtration) and blow-dried. The solid was then dried to constant weight in a vacuum oven (45 °C) to give the desired product (19.4 kg, 92% yield, HPLC purity = 97.4%). On an 800 g scale, 1559 g of the desired product (98% yield) was obtained with a 99.9% HPLC purity. JH NMR (DMSO-i¾) δ 11.17 (s, 1H); 7.4 (m, 2H); 7.2 (m, 3H); 1.2 (s, 9H). LCMS (M+H)+ 261.
F. Preparation of N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2, 1 -b] [ 1 ,3 ]benzothiazol-2-yl]phenyl } urea
1. Example F-l
[00301] The jacket of a 2000-L GL reactor was set to 20 °C and the reactor was charged with 7-(2-morpholin-4-yl-ethoxy)-2-(4-aminophenyl)imidazo[2,l- &]benzothiazole (26.7 kg, 67.8 mol, 1.0 equiv), 3-amino-5-?-butylisoxazole phenyl carbamate (19.4 kg, 74.5 mol, 1.1 equiv), DMAP (0.5 kg, 4.4 mol, 0.06 equiv), and DCM (anhydrous, 260 kg). Agitation was started, triethylamine (1.0 kg, 10.2 mol, 0.15 equiv) was charged followed by additional DCM (5 kg) through the charging line. The reaction was heated to reflux (-40 °C) and agitated for at least 20 hours with complete dissolution observed followed by product crystallizing from solution after -30 minutes. The reaction was sampled and deemed complete when HPLC analysis showed a ratio of compound (VIII) to compound (I) < 1%.
[00302] The reactor was cooled to 0 °C and stirred for at least 2 hours. The content of the reactor were isolated by centrifuge. Each portion was rinsed with 2-3 kg of cold (0 °C) DCM and spun dry for at least 5 minutes with a 10 psi nitrogen purge. For the final portion, the reactor was rinsed with 10 kg of cold (0 °C) DCM and transferred to the centrifuge where it was spun dry for at least 5 minutes with a 10 psi nitrogen purge. The combined filter cakes were transferred to a vacuum tray dryer and dried to constant weight at 50 °C and at least >20 inches of Hg to give the desired product (I) (35.05 kg, 92% yield, HPLC purity = 98.8%). Phenol was the major impurity detected (0.99%); and three other impurities (<0.10%) were detected. JH NMR (300 MHz, DMSO- ) conformed to structure.
2. Example F-2
[00303] A variety of solvents were used in the reaction of Step F to optimize for better yields and purity profiles. The contents of the symmetrical urea impurity (XI) were compared and summarized in the table below:
http://www.google.com/patents/WO2011056939A1?cl=en SE THIS FOR DELETED CLIPS
4. Example F-4
[00305] To Reactor A under a nitrogen atmosphere was added 7-(2-morpholin-4-yl- ethoxy)-2-(4-aminophenyl)imidazo[2,l-&]benzothiazole (1 kg) and DMAP (0.02 kg). To Reactor B under a nitrogen atmosphere was added 3-amino-5-?-butylisoxazole phenyl carbamate (0.73 kg) and DCM (5.6 L). The DCM used had a water content of less than 0.05 % w/w. The mixture in Reactor B was stirred until dissolution. The solution was transferred into Reactor A (the solution can be filtered into Reactor A to remove any insoluble impurities in the carbamate starting material), and the mixture was stirred in Reactor A. Reactor B was washed with DCM (0.8 L) and the wash was transferred into Reactor A. Reactor A was washed with DCM (0.9 L). To Reactor A was added triethylamine (0.1 L) and the charging vessel and lines were rinsed with DCM (0.1 L) into Reactor A. The mixture was then heated to reflux and stirred at reflux until reaction completion (as determined by HPLC to monitor the consumption of starting material).
[00306] The reaction mixture was cooled (e.g. , over 2 to 3 hours) to a temperature of between -5 °C and 5 °C (e.g. , 0 °C). The mixture was then stirred for 2 to 3 hours at a temperature of between -5 °C and 5 °C (e.g. , 0 °C). The suspension was filtered. The solid was washed with cool DCM (2 X 1.5 L) (pre-cooled to a temperature of between -5 °C and 5 °C). The solid was dried under vacuum at a temperature of less than 45 °C until the DCM content was less than 1000 ppm (e.g., below 600 ppm) as analyzed by GC. The desired product was obtained having about 99% purity by HPLC.
G. Preparation of N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2, l-b] [1 ,3]benzothiazol-2-yl]phenyl }urea dihydrochloride
1. Example G-l
[00307] The jacket of a 2000-L GL reactor was set to 20 °C and the reactor was charged with N-(5-ieri-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo [2, 1-&][1, 3]benzothiazol-2-yl]phenyl}urea (35.0 kg, 62.4 mol, 1.0 equiv) followed by methanol (553 kg). Agitation was started and the reaction mixture was heated to reflux (-65 °C). Concentrated aqueous HC1 (15.4 kg, 156.0 mol, 2.5 equiv) was charged rapidly (<5 minutes) and the charge line was rinsed into the reactor with methanol (12 kg). Addition of less than 2.0 equivalents of HC1 normally resulted in the formation of an insoluble solid. The reaction mixture was heated at reflux for at least 1 hour. Upon HC1 addition, the slurry dissolved and almost immediately the salt started to crystallize, leaving insufficient time for a polish filtration.
[00308] The reactor was cooled to 20 °C and the product was isolated by filtration (Nutsche) rinsing the reactor and then the cake with methanol (58 kg). The solid was then dried in a vacuum oven (50 °C) to constant weight to give the desired
dihydrochloride salt (35 kg, 89% yield, HPLC purity = 99.94%). JH NMR (300 MHz, DMSO-i¾) conformed to structure.
2. Example G-2
[00309] Concentrated HC1 was added to a suspension of N-(5-ieri-butyl-isoxazol-3- yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l-&][l,3]benzothiazol-2- yl]phenyl}urea in warm methanol forming a solution that slowly began to precipitate. The reaction mixture was refluxed for over 2 hours and then stirred overnight at ambient temperature. The dihydrochloride salt was collected and dried under vacuum.
3. Example G-3
[00310] A 50-L 3-neck round bottom flask was equipped with a mechanical agitator, a thermocouple probe, a nitrogen inlet, a drying tube, a reflux condenser, an addition funnel, and a heating mantle. The flask was charged with N-(5-ieri-butyl-isoxazol-3-yl)- N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l-&][l,3]benzothiazol-2-yl]phenyl}urea (775 g, 1.38 mol, 1.0 equiv) and MeOH (40 L, AR). The resulting off-white suspension was heated to reflux (68 °C). A clear solution did not form. HC1 (37% aqueous) (228 mL, 3.46 mol, 2.5 equiv) was added over 5 minutes at 68 °C. The reaction mixture turned into a clear solution and then a new precipitate formed within approximately 3 minutes. Heating was continued at reflux for approximately 5 hours. The reaction mixture was allowed to cool to ambient temperature overnight. The off-white solids were collected by filtration on a polypropylene filter, washing with MeOH (2 X 1 L, AR). [00311] Two lots of material prepared in this manner were combined (740 g and 820 g). The combined solids were slurried in methanol (30 L) over 30 minutes at reflux and allowed to cool to the room temperature. The solids were collected by filtration on a polypropylene filter, rinsing with methanol (2 X 1.5 L). The solids were dried in a vacuum oven (<10 mm Hg) at 40 °C. Yield: 1598 g (84%), off-white solid. HPLC: 98.2% (area). MS: 561.2 (M+l)+. JH NMR (300 MHz, DMSO-i¾) conformed to the structure. Elemental Analysis (EA): Theory, 54.97 %C; 5.41 %H; 13.26 %N; 5.06 %S; 11.19 %C1; Actual, 54.45 %C; 5.46 %H; 13.09 %N; 4.99 %S; 10.91 %C1.
4. Example G-4
[00312] Into a 50-L 3-neck round bottom flask equipped with a mechanical stirrer, a heating mantle, a condenser and a nitrogen inlet, were charged N-(5-ieri-butyl-isoxazol- 3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l-&][l,3]benzothiazol-2- yl]phenyl}urea (1052.4 g, 1.877 mol, 1.0 equiv) and methanol (21 L). The reactor was heated and stirred. At an internal temperature of > 50 °C, cone. HC1 (398.63 mL, 4.693 mol, 2.5 equiv) was charged over 5 minutes through an addition funnel. During the addition, the mixture changed from a pale yellow suspension to a white suspension. The internal temperature was 55 °C at the conclusion of the addition. The mixture was heated to reflux for 1 hour, then heating was discontinued and the mixture was allowed to cool to room temperature. The mixture was filtered in two portions, and each filter cake was washed with methanol (2 X 1 L), transferred to trays and dried in a vacuum oven (45 °C) to constant weight. The dried trays were combined to produce 1141.9 g of the salt (96% yield, 99.1 % HPLC purity, 10.9% chloride by titration).
H. Analytical Data
1. N-(5-ieri-butyl-isoxazol-3-yl)-N’-{ 4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2, l-&] [l ,3]benzothiazol-2-yl]phenyl}urea
dihydrochloride
[00314] A batch of about 30 grams of N-(5-ieri-butyl-isoxazol-3-yl)-N’- {4-[7-(2- morpholin-4-yl-ethoxy)imidazo[2, l-&] [l ,3]benzothiazol-2-yl]phenyl}urea
dihydrochloride was prepared using the methods described herein. This lot was
prepared in accordance with the requirements for production of clinical Active
Pharmaceutical Ingredients (APIs) under GMP conditions. The analytical data for this batch was obtained, and representative data were provided herein. [00315] Summary of analytical data for the dihydrochloride salt.
………………………
EXAMPLE 1. SYNTHESIS OF N-(5-TERT-BUTYL-ISOXAZOL-3-YU- N>-{4-f7-(2-MORPHOLIN-4- YL-ETHOXY)IMID AZO[2,1- BlH,31BENZOTHIAZOL-2-YL|PHENYLiUREA (“COMPOUND Bl”)
[00357] A. The intermediate 2-amino-l,3-benzothiazol-6-ol was prepared according to a slightly modified literature procedure by Lau and Gompf: J. Org. Chem. 1970, 35, 4103- 4108. To a stirred solution of thiourea (7.6 g, 0.10 mol) in a mixture of 200 mL ethanol and 9 mL concentrated hydrochloric acid was added a solution of 1 ,4-benzoquinone (21.6 g, 0.20 mol) in 400 mL of hot ethanol. The reaction was stirred for 24 hours at room temperature and then concentrated to dryness. The residue was triturated with hot acetonitrile and the resulting solid was filtered and dried.
[00358] The free base was obtained by dissolving the hydrochloride salt in water, neutralizing with sodium acetate, and collecting the solid by filtration. The product (2- amino-l,3-benzothiazol-6-ol) was obtained as a dark solid that was pure by LCMS (M+H = 167) and NMR. Yield: 13.0 g (78 %). NMR (DMSO-^) Sl.6 (m, 2H), 6.6 (d, IH). [00359] B. To prepare the 2-(4-nitrophenyl)imidazo[2,l-b][l,3]benzothiazol-7-ol intermediate, 2-amino-l,3-benzothiazol-6-ol (20.0 g, 0.12 mol) and 2-bromo-4′- nitroacetophenone (29.3 g, 0.12 mol) were dissolved in 600 mL ethanol and heated to reflux overnight. The solution was then cooled to O0C in an ice-water bath and the product was collected by vacuum filtration. After drying under vacuum with P2O5 , the intermediate (2- (4-nitrophenyl)imidazo[2,l-£][l,3]benzothiazol-7-ol) was isolated as a yellow solid. Yield: 17.0 g (46 %) NMR (DMSO-(I6) δ 10 (s, IH), 8.9 (s, IH), 8.3 (d, 2H), 8.1 (d, 2H), 7.8 (d, IH), 7.4 (s, IH), 6.9 (d, IH).
[00360] C. To make the 7-(2-morpholin-4-yl-ethoxy)-2-(4-nitro-phenyl)imidazo[2,l-
6][l,3]benzothiazole intermediate: 2-(4-nitrophenyl)imidazo[2,l-6][l,3]benzothiazol-7-ol,
NYI-4144519vl 84 (3.00 g, 9.6 mmol) was suspended in 100 mL dry DMF. To this mixture was added potassium carbonate (4.15 g, 30 mmol, 3 eq), chloroethyl morpholine hydrochloride (4.65 g, 25 mmol, 2.5 eq) and optionally tetrabutyl ammonium iodide (7.39 g, 2 mmol). The suspension was then heated to 900C for 5 hours or until complete by LCMS. The mixture was cooled to room temperature, poured into 800 mL water, and allowed to stand for 1 hour. The resulting precipitate was collected by vacuum filtration and dried under vacuum. The intermediate, (7-(2-morpholin-4-yl-ethoxy)-2-(4-nitro-phenyl)imidazo[2, 1 – b][\, 3]benzothiazole) was carried on without further purification. Yield: 3.87 g (95 %) NMR (DMSO-d6) δ 8.97 (s, IH), 8.30 (d, 2H), 8.0 (d, 2H), 7.9 (d, IH), 7.7 (s, IH), 7.2 (d, IH), 4.1 (t, 2H), 5.6 (m, 4H), 2.7 (t, 2H).
[00361] D. To make the intermediate 7-(2-morpholin-4-yl-ethoxy)-2-(4-amino- phenyl)imidazo[2,l-b][l,3]benzothiazole: To a suspension of 7-(2-morpholin-4-yl-ethoxy)- 2-(4-nitro-phenyl)imidazo[2,l -b][\ , 3]benzothiazole (3.87g, 9.1 mmol) in 100 mL isopropyl alcohol/water (3:1) was added ammonium chloride (2.00 g, 36.4 mmol) and iron powder (5.04 g, 90.1 mmol). The suspension was heated to reflux overnight with vigorous stirring, completion of the reaction was confirmed by LCMS. The mixture was filtered through Celite, and the filtercake was washed with hot isopropyl alcohol (150 mL). The filtrate was concentrated to approximately 1/3 of the original volume, poured into saturated sodium bicarbonate, and extracted 3 times with dichloromethane. The combined organic phases were dried over MgSO4 and concentrated to give the product as an orange solid containing a small amount (4-6 %) of starting material. (Yield: 2.75 g 54 %). 80% ethanol/water may be used in the place of isopropyl alcohol /water – in which case the reaction is virtually complete after 3.5 hours and only traces of starting material are observed in the product obtained. NMR (DMSO-Λfc) δ 8.4 (s, IH), 7.8 (d, IH), 7.65 (d, IH), 7.5 (d, 2H), 7.1 (d, IH), 6.6 (d, 2H), 4.1 (t, 2H), 3.6 (m, 4H), 2.7 (t, 2H).
[00362] E. A suspension of 7-(2-morpholin-4-yl-ethoxy)-2-(4-amino- phenyl)imidazo[2,l-b][l,3]benzothiazole (4.06 g, 10.3 mmol) and 5-tert-butylisoxazole-3- isocyanate (1.994 g, 12 mmol) in toluene was heated at 120 0C overnight. The reaction was quenched by pouring into a mixture of methylene chloride and water containing a little methanol and neutralized with saturated aqueous NaHCO3 solution. The aqueous phase was extracted twice with methylene chloride, the combined organic extracts were dried over
NYI-4144519vl 85 MgSO4 and filtered. The filtrate was concentrated to about 20 ml volume and ethyl ether was added resulting in the formation of a solid. The precipitate was collected by filtration, washed with ethyl ether, and dried under vacuum to give the free base of Compound B 1. Yield: 2.342 g (41 %) NMR (DMSO-J6) £9.6 (br, IH), 8.9 (br, IH), 8.61 (s, IH), 7.86 (d, IH), 7.76 (d, 2H), 7.69 (d, IH), 7.51 (d, 2H), 7.18 (dd, IH), 6.52 (s, IH), 4.16 (t, 2H), 3.59 (t, 4H), 3.36 (overlapping, 4H), 2.72 (t, 2H), 1.30 (s, 9H). NMR (CDCl3) £9.3 (br, IH), 7.84 (m, 4H), 7.59 (d, 2H), 7.49 (d, IH), 7.22 (d, IH), 7.03 (dd, IH), 5.88 (s, IH), 4.16 (t, 2H), 3.76 (t, 4H), 2.84 (t, 2H), 2.61 (t, 4H), 1.37 (s, 9H).
6.2 EXAMPLE 2. ALTERNATIVE SYNTHESIS QF N-(5-TERT-BUTYL- ISOXAZQL-3- YL)-N -{4-[7-q-MORPHOLIN-4- YL- ETHOXYUMID AZOf2,l-BUl,31BENZOTHIAZOL-2- YLIPHENYLIUREA (“COMPOUND Bl”)
[00363] A. To a suspension of the intermediate 2-(4-Nitrophenyl)imidazo[2,l- b][l,3]benzothiazol-7-ol from Example IB (2.24 g, 7.2 mmol) in ethanol (40 mL) was added SnCl2 1H2O (7.9Og, 35 mmol) and heated to reflux. Concentrated HCl was added to the reaction mixture and the precipitate formed gradually. The reaction mixture was heated to reflux for 20 hours and then allowed to cool to room temperature. The solution was poured into ice and neutralized with 10% NaOH and adjusted to approximately pH 6. The organic phase was extracted three times with ethyl acetate (80 mL x 3). Extracts were dried over MgSθ4 and concentrated to give a yellow solid. (1.621 g, 80%). The solid was recrystallized from methanol to give a pure product (1.355 g, 67%).
[00364] B. To a suspension of the intermediate from Step 2A (0.563 g, 2 mmol) in toluene (30 mL) was added 5-tert-butylisoxazole-3-isocyanate (0.332g, 2 mmol) and heated to reflux overnight. LC-MS analysis showed presence of the intermediate but no trace of 5- tert-butylisoxazole-3-isocyanate and an additional 0.166 g of the isocyanate was added. The reaction was again heated to reflux overnight. Completion of reaction was verified by LC- MS. The solvent was removed and the resulting mixture was dissolved in methanol which was removed to give the second intermediate as a solid.
[00365] The mixture was dissolved in CH2Cl2 (150 mL) and washed with saturated
NaHCO3. The organic layer was dried over MgSO4, concentrated, and purified by silica gel chromatography three times, first using a methanol/CH2Cl2 gradient, the second time using a
NYI-4144519vl 86 hexane/ethyl acetate gradient followed by a methanol/ethyl acetate gradient, and a third time using a methanol/CH2Cl2 gradient.
[00366] C. To a suspension of the intermediate from Step 2B (0.1 10 g, 0.25 mmol) in
THF (5mL) was added Ph3P (0.079g, 0.3 mmol), diisopropylazodicarboxylate (0.06 Ig, 0.3 mmol) and 4-morpholinoethanol (0.039 g, 0.3 mmol). The reaction mixture was stirred at room temperature overnight. Completion of the reaction was verified by LC-MS. The solvent was removed and the final product was purified using silica gel chromatography, with methanol in CH2Cl2 (0.030g, 21%).
6.3 EXAMPLE 3. BULK SYNTHESIS OF N-(5-TERT-BUTYL- ISOXAZOL-3-YL)-N’-f4-[7-(2-MORPHOLIN-4-YL- ETHOXY^IMID AZO[2α-BUlJlBENZOTHIAZOL-2- YLlPHENYLiUREA (“COMPOUND Bl”)
[00367] A multi-step reaction scheme that was used to prepare bulk quantities of
Compound Bl is depicted in FIG. 66a and FIG. 66b, and is described further below. [00368] Step 1 : Preparation of 2- Amino-6-hydroxybenzothiazole (Intermediate 1). 2-
Amino-6-methoxybenzothiazole is reacted with hot aqueous HBr for about 3 hrs and then the clear solution is cooled to ambient temperature overnight. The precipitated solids are collected, dissolved in hot water and the pH is adjusted to between 4.5-5.5. The resultant solids are collected, dried and recrystallized from Isopropanol. Second crop material is collected. The solids are vacuum dried to give Intermediate 1.
[00369] Step 2: Preparation of 2-(4-Nitrophenyl) imidazo [2J-b]benzothiazol-7-ol
(Intermediate 2). 2-Amino-6-hydroxybenzothiazole, 2-Bromo-4-nitroacetophenone and absolute Ethanol are added together and heated to reflux for approximately 24 hours. Tetrabutylammonium iodide is added and the reaction is refluxed an additional 12 hours. The resulting yellow suspension is cooled and the solids collected and washed with Ethanol and Diethyl ether. The solids are dried under vacuum to give Intermediate 2. [00370] Step 3: Preparation of 7-(2-Morpholin-4-yl-ethoxy)-2-(4-nitrophenyl) imidazo
[2,1-b] benzothiazole (Intermediate 3). Intermediate 2, 4-(2-Chloroethyl)morpholine hydrochloride, Potassium carbonate and Tetrabutylammonium iodide are added to N,N- Dimethylformamide forming a yellow suspension that is heated for over 3 hours. The reaction is cooled and the solids are collected, slurried into water, filtered, slurried into
NYl-4 l4451′)v l 87 acetone, filtered and washed with Acetone to give yellow solids that are dried under vacuum to give Intermediate 3.
[O0371] Step 4: Preparation of 7-(2-Moφholin-4-yl-ethoxy)-2-(4-aminophenyl) imidazo [2,1 -b] benzothiazole (Intermediate 4). Intermediate 3 is dissolved into Methanol and THF and placed in a Hydrogenator. Raney Nickel is added and the vessel is pressurized with Hydrogen and stirred for >24 hrs. The reaction mixture is concentrated to a thick paste and diluted with Methyl tert-butyl ether. The resulting solids are filtered and washed with Methyl tert-butyl ether and dried under vacuum to give Intermediate 4. [O0372] Step 5: Preparation of {[5-(tert-Butyl) isoxazol-3-vnatnino}-N-{4-r7-(2- morpholin-4-yl-ethoxy)(4-hvdroimidazolo[2J-blbenzothiazol-2-yl)]phenyl|carboxamide (Compound Bl). 3 -Amino- 5 -tert-butyl isoxazole in Methylene chloride is added to a vessel containing toluene which is cooled to approx 0 0C. Triphosgene is then added and the reaction mixture is cooled to below -15 0C. Triethylamine is added, followed by Intermediate 4. The mixture is heated to distill off the Methylene chloride and then heated to over 60 0C for over 12 hours and cooled to 50-60 °C. The resulting solids are filtered, washed with Heptane, slurried with 4% sodium hydroxide solution, and filtered. The solids are then washed with Methyl tert-butyl ether followed by Acetone and dried under vacuum to give Compound Bl.
6.4 EXAMPLE 4. EXAMPLES OF PREPARATION OF COMPOUND Bl HCL SALT
[00373] Example A: For the preparation of a hydrochloride salt of Compound Bl5 N-
(5-tert-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,l- b][l,3]benzothiazol-2-yl]phenyl}urea hydrochloride, the free base was dissolved in a mixture of 20 ml methylene chloride and 1 ml methanol. A solution of 1.0 M HCl in ethyl ether (1.1 eq.) was added dropwise, followed by addition of ethyl ether. The precipitate was collected by filtration or centrirugation and washed with ethyl ether to give a hydrochloride salt of Compound Bl. Yield: 2.44 g (98 %) NMR (DMSO-^) S X 1.0 (br, IH), 9.68 (s, IH), 9.26 (s, IH), 8.66 (s, IH), 7.93 (d, IH), 7.78 (m, 3H), 7.53 (d, 2H), 7.26 (dd, IH), 6.53 (s, IH), 4.50 (t, 2H), 3.97 (m, 2H), 3.81 (t, 2H), 3.6 (overlapping, 4H), 3.23 (m, 2H), 1.30 (s, 9H). [00374] Example B: Concentrated HCl is added to a suspension of Compound Bl in warm methanol forming a solution that slowly begins to precipitate. The reaction mixture is
NYI-4144519vl 88 refluxed for over 2 hrs and then stirred overnight at ambient temperature. The HCl salt is collected and dried under vacuum.
[00375] Example C: Materials: {[5-(tert-Butyl) isoxazol-3-yl]amino}-N-{4-[7-(2- morpholin-4-yl-ethoxy)(4-hydroimidazolo[2,l-6]benzothiazol-2-yl)] phenyl }carboxamide (775 g, 1.38 mol, 1.0 eq); HCl 37% aqueous (288 mL, 3.46 mol, 2.5 eq); Methanol (MeOH, AR) (40L). Procedure: (Step 1) Equipped a 5OL 3-neck round bottom flask with a mechanical agitator, thermocouple probe, Nitrogen inlet, drying tube, reflux condenser, addition funnel and in a heating mantle. (Step 2) Charged the flask with {[5-(tert-Butyl) isoxazol-3-yl] amino}-N-{4-[7-(2-morpholin-4-yl-ethoxy)(4-hydroimidazolo[2,l- b]benzothiazol-2-yl)] phenyl jcarboxamide (775g) and MeOH, AR (40L). Heat the resulting off-white suspension to reflux (680C). A clear solution did not form. (Step 3) Added HCl (37% aqueous) (228 mL) over 5 minutes at 68°C. The reaction mixture turned into a clear solution and then a new precipitate formed within approximately 3 minutes. Continued heating at reflux for approximately 5 hours. Allowed the reaction mixture to cool to ambient temperature overnight. (Step 4) Collected the off-white solids by filtration onto a polypropylene filter, washing the solids with MeOH, AR (2 x 1 L). (Step 5) Combined two lots of material prepared in this manner (74Og and 82Og). Slurried the combined solids in Methanol (30 L) over 30 minutes at reflux and cool to the room temperature. (Step 6) Collected the solids by filtration onto a polypropylene filter, rinsing with Methanol (2 x 1.5L). (Step 7) Dried the solids in a vacuum oven (<10mniHg) at 400C. Yield: 1598 g (84%), off-white solid; HPLC: 98.2% (area); MS: 561.2 (M+l); IH NMR: conforms (300 MHz, DMSO-d6); Elemental Analysis (EA): Theory = 54.97 %C; 5.41 %H; 13.26 %N; 5.06 %S; 11.19 %C1; Actual = 54.45 %C; 5.46 %H; 13.09 %N; 4.99 %S; 10.91 %C1.
NYl-4I44519v! 89 [00376] Examples of Compound Bl HCl salt synthesis
[00377] Example D: In a 50-L 3-neck round bottom flask equipped with a mechanical stirrer, heating mantle, condenser and nitrogen inlet was charged Compound Bl (1052.4 g, 1.877 mol, 1.00 equiv.) and methanol (21 L). The reactor was heated and stirred. At an internal temperature > 50 0C, cone. HCl (398.63 mL, 4.693 mol, 2.5 equiv.) was charged over 5 minutes through an addition funnel. With the addition, the reaction changed from a pale yellow suspension to a white suspension. The internal temperature was 55 0C at the conclusion of the addition. The reaction was heated to reflux for 1 hour, then heating discontinued and the reaction allowed to cool to room temperature. The reaction was filtered in two portions, each filter cake washed with methanol (2 x 1 L), transferred to trays and dried in a vacuum oven (45 0C) to constant weight. The dried trays were combined to produce 1141.9 g, 96% yield, 99.1 % HPLC purity, 10.9% chloride by titration.
Solid Forms Comprising the HCl Salt of Compound Bl 6.6.2.1 Preparation of Solid Forms
6.6.2.2 Cold Precipitation Experiments
NYl-4144519vl 102 6.6.2.3 Slurry Experiments
NYI-41445 l9vl 103 6.6.2.4 Additional Preparation of Solid Forms Comprising the HCI Salt of Compound Bl
NYl-4144519v l 104
NYM 144519vl 105
N Y l -4 1 4 4 5 1 9 v l 1 0 6
NYI-4I44519vi 107
N V I 4 1 4 4 5 1 9 1 0 8
“Abbreviations in Table: CC = crash cool, CP = crash precipitation, EtOAc = ethyl acetate, FE = fast evaporation, VD = vapor diffusion, IPA = isopropanol, MEK = methyl ethyl ketone (2-butanone), RE = rotary evaporation, RT = room (ambient) temperature, SC = slow cool, SE = slow evaporation, THF = tetrahydrofuran, TFE = 2,2,2=trifluoroethanol.
6.6.2.5 Scale-up Experiments of Involving Crystal Forms Comprising the HCl Salt of Compound Bl
NYI-4144519v l 109
Abbreviations in Table: CC = crash cool, CP = crash precipitation, EtOAc = ethyl acetate, FE = fast evaporation, IPA = isopropanol, MEK = methyl ethyl ketone (2-butanone), RE = rotary evaporation, RT = room (ambient) temperature, SC = slow cool, SE = slow evaporation, THF = tetrahydrofuran, TFE = 2,2,2=trifluoroethanol.
……………………
Identification of N-(5-tert-butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (AC220), a uniquely potent, selective, and efficacious FMS-like tyrosine kinase-3 (FLT3) inhibitor
J Med Chem 2009, 52(23): 7808
http://pubs.acs.org/doi/full/10.1021/jm9007533
References
- Chao, Qi; Sprankle, Kelly G.; Grotzfeld, Robert M.; Lai, Andiliy G.; Carter, Todd A.; Velasco, Anne Marie; Gunawardane, Ruwanthi N.; Cramer, Merryl D.; Gardner, Michael F.; James, Joyce; Zarrinkar, Patrick P.; Patel, Hitesh K.; Bhagwat, Shripad S. (2009). “Identification of N-(5-tert-Butyl-isoxazol-3-yl)-N’-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihydrochloride (AC220), a Uniquely Potent, Selective, and Efficacious FMS-Like Tyrosine Kinase-3 (FLT3) Inhibitor”. Journal of Medicinal Chemistry 52 (23): 7808–7816.
- Drug Tames Refractory AML. ASH Dec 2012
- NMR……….http://file.selleckchem.com/downloads/nmr/S152601-AC-220-HNMR-Selleck.pdf
- HPLC………http://file.selleckchem.com/downloads/hplc/S152601-AC-220-HPLC-Selleck.pdf
DR ANTHONY MELVIN CRASTO
ORGANIC SPECTROSCOPY
New Drug Approvals 