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Pevonedistat

August 26, 2015 8:05 am / Leave a comment

Millennium Pharmaceuticals, Inc. INNOVATOR

Millennium Pharmaceuticals, Inc., a subsidiary of Takeda Pharmaceutical Company Limited,

MLN4924, MLN 4924-003, TAK-924

905579-51-3 BASE

1160295-21-5 HcL

A potent and selective inhibitor of NAE. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. The ubiquitin-proteasome pathway mediates the destruction of unwanted proteins.

(((1S,2S,4R)-4-{4-[(S)-2,3-Dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate hydrochloride) (pevonedistat), a novel NEDD8-activating enzyme (NAE) inhibitor, has demonstrated in vitro cytotoxic activity against a variety of human malignancies and is currently being developed by Takeda Pharmaceuticals Company Limited as a clinical candidate for the treatment of cancer

In 2011, orphan drug designation was assigned to MLN-4924 for the treatment of MDS and for the treatment of acute myelogenous leukemia.

PHASE 1…….CANCER SOLID TUMOR

………………….

PATENT

http://www.google.com/patents/US20120330013

preparing a compound represented by the following formula 1 by reacting the compound of formula 11 with TFA (step 9):

The retrosynthetic analysis of MLN4924 (1), as the final desired nucleoside, is shown in the following.

MLN 4924 (1) can be synthesized by condensing cyclic sulfate 3 as the glycosyl donor with a purine base. The glycosyl donor 3 can be produced from diol 4, which in turn can be obtained from cyclopentanone 5 via a stereoselective reduction and a regioselective cleavage of the isopropylidene moiety. The cyclopentanone 5 can be synthesized from cyclopentenone 6 by stereoselective reduction. The intermediate cyclopentenone 6 can be easily derived from D-ribose according to our previously published procedure (Jeong, L. S. et al., J. Org. Chem. 2004, 69, 2634-2636).

The synthetic route for the glycosyl donor 3 is shown in the following scheme 1.

Example 1 Preparation of MLN4924 Step 1: Preparation of 6-(tert-butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-cyclopenta[1,3]dioxol-4-one (Compound 5)

To a suspension of the compound 6 (20.0 g, 47.1 mmol) in methanol (400 ml) was added 10% palladium on activated carbon (1.0 g), and the mixture was stirred at room temperature overnight under H₂atmosphere. After filtration of the reaction mixture, the solvent was removed and the residue was dissolved in methylene chloride and then filtered through short pad silica gel. Then, the solvent was evaporated to give the compound 5 (20.1 g, 100%) as a colorless syrup.

[α]²⁰ _D−28.32 (c 1.49, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₂NaO₄Si [M+Na]⁺447.1968, Found 447.1956; ¹H NMR (400 MHz, CDCl₃) δ 7.69 (m, 4H), 7.40 (m, 6H), 4.84 (t, J=4.4 Hz, 1H), 4.22 (dd, J=1.2, 4.8 Hz, 1H), 3.96 (dd, J=8.0, 10.0 Hz, 1H), 3.82 (dd, J=6.8, 10.0 Hz, 1H), 2.37 (m, 1H), 2.30 (ddd, J=1.2, 8.4, and 18.4 Hz, 1H), 2.20 (ddd, J=1.2, 12.0, and 18.4 Hz, 1H), 1.37 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 112.6, 80.5, 77.6, 77.2, 76.9, 63.6, 38.1, 36.9, 27.1, 27.02, 27.01, 25.3, 19.5; Anal. Calcd for C₂₅H₃₂O₄Si: C, 70.72; H, 7.60. Found: C, 70.79; H, 7.75.

Step 2: Preparation of 6-(tert-butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-cyclopenta[1,3]dioxol-4-ol (Compound 7)

To a suspension of the compound 5 (20.1 g, 47.1 mmol) in methanol (500 ml) were added sodium borohydride (2.17 g, 57.4 mmol) and cerium (III) chloride heptahydrate (21.3 g, 57.2 mmol) at 0° C., and the mixture was stirred at room temperature for 30 min. After the solvent was removed, the residue was partitioned between ethyl acetate and water. The organic layer was then washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=5/1) to give the compound 7 (20.86 g, 98%) as a colorless syrup.

[α]²⁰ _D+34.55 (c 0.55, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₄NaO₄Si [M+Na]⁺: 449.2124; Found: 449.2110; ¹H NMR (400 MHz, CDCl₃) δ 7.69 (m, 4H), 7.39 (m, 6H), 4.62 (t, J=5.6 Hz, 1H), 4.44 (t, J=5.6 Hz, 1H), 3.89 (dd, J=6.0, 7.6 Hz, 1H), 3.84 (m, 1H), 3.68 (dd, J=6.4, 10.0 Hz, 1H), 1.91 (m, 2H), 1.26 (m, 1H), 1.42 (s, 3H), 1.33 (s, 3H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.9, 135.8, 134.2, 134.1, 129.8, 129.7, 127.8, 127.7, 110.6, 79.4, 78.9, 77.6, 77.2, 76.9, 72.5, 62.9, 41.6, 33.4, 27.0, 25.9, 27.0, 25.9, 24.4, 19.5; Anal. Calcd for C₂₅H₃₄O₄Si: C, 70.38; H, 8.03. Found: C, 70.41; H, 8.08.

Step 3: Preparation of 3-tert-butoxy-4-(tert-butyl-diphenyl-silanyloxymethyl)-cyclopentane-1,2-diol (Compound 4)

To a solution of the compound 7 (20.86 g, 47.12 mmol) in methylene chloride was added trimethylaluminum (2.0 M in toluene, 132.1 ml) at 0° C., and the mixture was stirred at room temperature for 2 days. The mixture was cooled to 0° C., slowly quenched with an aqueous saturated ammonium chloride solution, filtered, and evaporated. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=2/1) to give the compound 4 (13.42 g, 62%) as a colorless syrup.

[α]²⁰ _D+3.30 (c 0.55, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₈NaO₄Si [M+Na]⁺: 465.2437; Found: 465.2423; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (m, 4H), 7.41 (m, 6H), 4.05 (dd, J=4.4, 7.2 Hz, 1H), 3.93 (m, 1H), 3.72 (m, 2H), 3.59 (dd, J=3.6, 12.0 Hz, 2H), 2.70 (d, J=20.8 Hz, 1H), 2.10 (m, 2H), 1.60 (m, 1H), 1.20 (s, 9H), 1.06 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.9, 133.5, 130.0, 129.9, 127.9, 127.9, 77.6, 77.2, 76.9, 74.9, 73.8, 72.7, 72.1, 63.3, 42.1, 34.0, 28.5, 27.0, 19.4; Anal. Calcd for C₂₆H₃₈O₄Si: C, 70.55; H, 8.65. Found: C, 70.61; H, 8.70.

Step 4: Preparation of (4-tert-butoxy-2,2-dioxo-tetrahydro-2-yl-6-cyclopenta[1,3,2]-dioxathiol-5-ylmethoxy)-tert-butyl-diphenyl-silane (Compound 3)

To a solution of the compound 4 (13.42 g, 30.3 mmol) in methylene chloride were added triethyl amine (14.5 ml, 101.0 mmol) and thionyl chloride (3.7 ml, 47.4 mmol) at 0° C., and the reaction mixture was stirred at 0° C. for 10 minutes. The reaction mixture was partitioned between methylene chloride and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=6/1) to give the cyclic sulfite (14.37 g, 97%) as a white foam.

[α]²⁰ _D+20.00 (c 0.05, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₆NaO₅SSi [M+Na]⁺: 511.1950; Found: 511.1929; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (m, 4H), 7.40 (m, 6H), 5.23 (m, 1H), 5.04 (dd, J=4.4, 6.0 Hz, 1H), 4.01 (t, J=4.8 Hz, 1H), 3.68 (dd, J=3.6, 10.4 Hz, 1H), 3.56 (dd, J=8.0, 10.4 Hz, 1H), 2.07 (m, 2H), 1.96 (m, 1H), 1.14 (s, 9H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.8, 135.7, 133.9, 133.8, 129.9, 129.9, 127.9, 127.8, 85.7, 83.2, 77.6, 77.2, 76.9, 75.0, 71.1, 62.7, 44.7, 31.4, 28.5, 27.1, 19.4; Anal. Calcd for C₂₆H₃₆O₅SSi: C, 63.90; H, 7.42; S, 6.56. Found: C, 63.94; H, 7.45; S, 6.61.

To a solution of the cyclic sulfite obtained above (14.37 g, 29.4 mmol) in the mixture of carbon tetrachloride, acetonitrile and water (1:1:1.5, 210 ml) were added sodium metaperiodate (18.56 g, 56.4 mmol) and ruthenium chloride (1.72 g, 8.25 mmol), and the reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was partitioned between methylene chloride and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=4/1) to give the compound 3 (13.36 g, 90%) as a white solid.

mp 101-104° C.; [α]²⁰ _D−80.00 (c 0.05, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₆NaO₆SSi [M+Na]⁺: 527.1900; Found: 527.1881; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (m, 4H), 7.41 (m, 6H), 5.13 (m, 1H), 4.83 (dd, J=4.4, 6.8 Hz, 1H), 4.13 (t, J=4.0 Hz, 1H), 3.92 (dd, J=6.4, 10.4 Hz, 1H), 3.69 (dd, J=5.2, 10.4 Hz, 1H), 2.11 (m, 2H), 2.02 (m, 1H), 1.15 (s, 9H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.7, 135.0, 133.8, 133.7, 130.0, 128.0, 127.9, 83.5, 82.2, 77.6, 77.2, 76.9, 75.4, 70.4, 70.4, 62.2, 43.9, 31.3, 28.2, 27.1, 26.8, 19.4; Anal. Calcd for C₂₆H₃₆O₆SSi: C, 61.87; H, 7.19; S, 6.35. Found: C, 61.91; H, 7.14; S, 6.30.

Step 5: Preparation of 2-tert-butoxy-3-(tert-butyl-diphenyl-silanyloxymethyl)-5-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentanol (Compound 8)

A suspension of N⁶-indanyl-7-deazaadenine (8.80 g, 35.2 mmol), sodium hydride (1.38 g, 45.7 mmol) and 18-crown-6 (9.11 g, 45.7 mmol) in THF (200 ml) was stirred at 80° C. To the reaction mixture was added a solution for the compound 3 (13.36 g, 26.5 mmol) in THF (150 ml), and the stirring was continued at 80° C. overnight. The reaction mixture was cooled down to 0° C., and conc. HCl was added slowly until pH reaches 1-2. Then the reaction mixture was further stirred at 80° C. for 2 hours. After neutralized with saturated aqueous NaHCO₃solution, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=2/1) to give the compound 8 (11.62 g, 65%) as a white foam.

UV (CH₂Cl₂) λ_max272.5 nm; [α]²⁰ _D−8.89 (c 0.45, MeOH); HR-MS (ESI): m/z calcd for C₄₁H₅₁N₄O₃Si [M+H]⁺: 675.3730; Found: 675.3717; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.70 (m, 4H), 7.41 (m, 6H), 6.92 (d, J=3.6 Hz, 1H), 6.29 (d, J=3.2 Hz, 1H), 5.91 (dd, J=7.6, 14.8 Hz, 1H), 5.14 (br d, J=6.8 Hz, 1H), 4.77 (m, 1H), 4.36 (t, J=6.0 Hz, 1H), 4.22 (dd, J=5.2, 10.8 Hz, 1H), 3.84 (dd, J=5.6, 10.4 Hz, 1H), 3.73 (dd, J=8.4, 10.4 Hz, 1H), 3.37 (d, J=5.6 Hz, 1H), 3.06 (m, 1H), 2.95 (m, 1H), 2.75 (m, 1H), 2.75 (m, 1H), 2.58 (m, 1H), 2.38 (m, 1H), 2.15 (m, 1H), 1.98 (m, 1H), 1.65 (s, 1H), 1.55 (s, 1H), 1.16 (s, 9H), 1.07 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 156.4, 151.8, 150.3, 144.1, 143.8, 135.9, 134.0, 129.9, 128.2, 127.9, 127.9, 127.0, 125.1, 124.4, 123.3, 103.8, 97.4, 77.8, 77.6, 77.2, 76.9, 74.9, 72.4, 63.5, 62.1, 56.3, 43.9, 34.9, 30.5, 30.5, 28.5, 27.2, 19.5; Anal. Calcd for C₄₁H₅₀N₄O₃Si: C, 72.96; H, 7.47; N, 8.30. Found: C, 73.01; H, 7.45; N, 8.36.

Step 6: Preparation of {7-[3-tert-butoxy-4-(tert-butyl-diphenyl-silanyloxymethyl)-cyclopentyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-indan-1-yl-amine (Compound 9)

To a solution of the compound 8 (11.62 g, 17.2 mmol) in methylene chloride (300 ml) were added N,N-dimethylaminopyridine (5.64 g, 51.6 mmol) and phenyl chlorothionocarbonate (4.3 ml, 34.4 mmol), and the reaction mixture was stirred at room temperature overnight. After the solvent was removed, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=6/1) to give the thiocarbonate (13.82 g, 99%) as a white foam.

UV (MeOH) λ_max271.50 nm; [α]²⁰ _D+10.00 (c 0.15, MeOH); HR-MS (ESI): m/z calcd for C₄₈H₅₅N₄O₄SSi [M+H]⁺: 811.3713; Found: 811.3687; ¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.61 (dd, J=1.6, 7.6 Hz, 4H), 7.34 (m, 5H), 7.26 (m, 4H), 7.18 (m, 6H), 6.86 (s, 1H), 6.25 (d, J=3.2 Hz, 1H), 6.00 (dd, J=3.2, 8.4 Hz, 1H), 5.83 (d, J=6.8 Hz, 1H), 5.19 (m, 1H), 5.07 (br s, 1H), 4.48 (t, J=3.6 Hz, 1H), 3.82 (dd, J=7.2, 10.4 Hz, 1H), 3.52 (dd, J=7.2, 10.0 Hz, 1H), 2.99 (m, 1H), 2.88 (m, 2H), 2.69 (m, 2H), 2.18 (dd, J=11.2, 13.6 Hz, 1H), 1.94 (m, 2H), 1.12 (s, 9H), 0.98 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 194.9, 153.5, 152.1, 143.9, 135.9, 135.8, 134.1, 129.9, 129.6, 128.3, 127.9, 127.0, 126.7, 125.1, 124.6, 123.2, 122.0, 87.9, 77.6, 77.2, 76.9, 74.6, 70.4, 63.5, 57.3, 42.8, 35.0, 30.7, 30.5, 29.9, 28.7, 27.1, 19.4; Anal. Calcd for C₄₈H₅₄N₄O₄SSi: C, 71.08; H, 6.71; N, 6.91; S, 3.95. Found: C, 71.14; H, 6.75; N, 6.95; S, 4.01.

To a solution of the thiocarbonate obtained above (13.82 g, 17.0 mmol) in toluene (200 ml) were added tri-n-butyltinhydride (9.4 ml, 34.1 mmol) and 2,2′-azo-bis-isobutyronitrile (4.32 g, 26.3 mmol), and the reaction mixture was stirred at 110° C. for 1 hour. After the mixture was cooled down, the solvent was removed. The resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate=3/1) to give the compound 9 (9.21 g, 82%) as a white foam.

UV (MeOH) λ_max272.50 nm; [α]²⁰ _D−10.00 (c 0.20, MeOH); HR-MS (ESI): m/z calcd for C₄₁H₅₁N₄O₂Si [M+H]⁺: 659.3781; Found: 659.3757; ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.69 (m, 4H), 7.41 (m, 6H), 7.29 (m, 2H), 7.23 (m, 2H), 6.92 (d, J=3.6 Hz, 1H), 6.31 (d, J=3.6 Hz, 1H), 5.90 (dd, J=7.2, 14.8 Hz, 1H), 5.38 (m, 1H), 5.15 (br s, 1H), 4.33 (dd, J=5.2, 8.4 Hz, 1H), 3.88 (dd, J=6.4, 10.0 Hz, 1H), 3.68 (dd, J=7.2, 10.4 Hz, 1H), 3.05 (m, 1H), 2.96 (dd, J=7.6, 15.6 Hz, 1H), 2.76 (m, 1H), 2.45 (d, J=5.2 Hz, 1H), 2.29 (m, 2H), 2.06 (m, 1H), 1.95 (m, 2H), 1.55 (s, 1H), 1.13 (s, 9H), 1.06 (s, 9H);¹³C NMR (100 MHz, CDCl₃) δ 156.3, 151.9, 144.1, 143.9, 135.9, 135.8, 134.3, 129.8, 128.2, 127.8, 127.0, 125.1, 124.6, 121.8, 77.6, 77.2, 76.7, 73.5, 72.2, 63.6, 56.4, 52.8, 46.8, 42.8, 34.9, 34.5, 30.5, 28.6, 27.2, 28.7, 19.4; Anal. Calcd for C₄₁H₅₀N₄O₂Si: C, 74.73; H, 7.65; N, 8.30. Found: C, 74.79; H, 7.61; N, 8.25.

Step 7: Preparation of 2-tert-butoxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentanol (Compound 10)

To a solution of the compound 9 (9.21 g, 13.97 mmol) in the mixture of THF and pyridine (1:1, 160 ml) was added dropwise pyridine hydrofluoride (18.42 ml, 190.0 mmol) at 0° C., and the reaction mixture was stirred at room temperature for 1 hour. The mixture was neutralized with saturated aqueous NaHCO₃solution and partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. Then, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=1/3) to give the compound 10 (5.63 g, 99%) as a white foam.

UV (MeOH) λ_max273.00 nm; [α]²⁰ _D−6.36 (c 1.10, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₃N₄O₂[M+H]⁺: 421.2604; Found: 421.2599; ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.30 (d, J=7.6 Hz, 1H), 7.22 (d, J=7.2 Hz, 2H), 7.15 (t, J=6.8 Hz, 1H), 6.88 (d, J=3.2 Hz, 1H), 6.23 (d, J=3.6 Hz, 1H), 5.83 (dd, J=7.2, 15.2 Hz, 1H), 5.28 (m, 1H), 5.06 (m, 1H), 4.47 (dd, J=5.6, 10.4 Hz, 1H), 3.78 (m, 1H), 3.70 (m, 1H), 3.24 (t, J=5.2 Hz, 1H), 2.98 (m, 1H), 2.87 (m, 1H), 2.68 (m, 1H), 2.46 (m, 1H), 2.37 (m, 2H), 1.93 (m, 2H), 1.18 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 156.2, 151.8, 147.9, 143.9, 143.9, 128.3, 126.9, 125.1, 124.5, 121.9, 97.7, 77.6, 77.2, 76.9, 75.5, 74.9, 63.4, 56.4, 53.8, 44.2, 42.2, 34.9, 33.2, 30.5, 28.6; Anal. Calcd for C₂₅H₃₂N₄O₂: C, 71.40; H, 7.67; N, 13.32. Found: C, 71.46; H, 7.60; N, 13.35.

Step 8: Preparation of sulfamic acid 2-tert-butoxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentylmethyl ester (Compound 11)

Preparation of 2.0 M solution of chlorosulfonamide in acetonitrile: Formic acid (14.15 ml, 166.0 mmol) was added dropwise to chlorosulfonyl isocyanate (32.0 ml, 162.5 mmol) under nitrogen atmosphere at 0° C. When the addition was completed, the mixture was solidified. To the mixture was added acetonitrile (61.3 ml), and the resulting solution was left to stand under nitrogen source at room temperature overnight.

To a solution of the compound 10 (5.63 g, 13.83 mmol) and triethyl amine (9.7 ml, 0.74 mmol) in acetonitrile (278 ml) was added 2.0 M solution of chlorosulfonamide in acetonitrile (13.83 ml, 27.76 mmol) at 0° C., and the reaction mixture was stirred at room temperature for 45 minutes. Additional 2.0 M chlorosulfonamide solution in acetonitrile (13.83 ml, 27.76 mmol) was added and the mixture was stirred at room temperature for 15 minutes. The reaction was quenched with methanol, and the solvent was removed. The residue was purified by silica gel column chromatography (methylene chloride/methanol=20/1) to give the compound 11 (6.37 g, 92%) as a white foam.

UV (MeOH) λ_max273.00 nm; [α]²⁰ _D−18.00 (c 0.50, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₄N₅O₄S [M+H]⁺: 500.2332; Found: 500.2331; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.36 (d, J=7.2 Hz, 1H), 7.29 (d, J=7.2 Hz, 1H), 7.22 (m, 2H), 6.95 (d, J=3.6 Hz, 1H), 6.31 (d, J=3.2 Hz, 1H), 5.89 (d, J=6.4 Hz, 1H), 5.10 (s, 2H), 4.41 (m, 2H), 4.26 (m, 1H), 3.05 (m, 1H), 2.94 (m, 1H), 2.76 (m, 2H), 2.27 (m, 3H), 2.06 (m, 1H), 1.97 (m, 1H), 1.76 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 156.4, 151.9, 149.9, 143.9, 143.8, 128.3, 126.9, 125.1, 124.5, 121.9, 121.9, 103.5, 97.9, 77.4, 77.2, 76.9, 74.3, 71.9, 71.3, 56.4, 53.1, 49.0, 42.3, 34.9, 34.3, 30.5, 28.6; Anal. Calcd for C₂₅H₃₃N₅O₄S: C, 60.10; H, 6.66; N, 14.02; S, 6.42. Found: C, 60.15; H, 6.71; N, 13.98; S, 6.39.

Step 9: Preparation of sulfamic acid 2-hydroxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentylmethyl ester (Compound 1)

A solution of the compound 11 (6.37 g, 12.72 mmol) in 70% trifluoroacetic acid (149.24 ml) was stirred at room temperature for 2 hours. The solvent was removed and the residue was purified by silica gel column chromatography (hexane/ethylene acetate=1/2) to give the compound 1 (5.08 g, 90%) as a white foam. BASE

UV (MeOH) λ_max279.50 nm; [α]²⁰ _D−6.41 (c 2.34, MeOH);

HR-MS (ESI): m/z calcd for C₂₁H₂₆N₅O₄S [M+H]⁺: 444.1705; Found: 444.1706;

¹H NMR (400 MHz, CD₃OD) δ 8.17 (d, J=1.6 Hz, 1H), 7.25 (m, 2H), 7.18 (m, 2H), 6.64 (d, J=3.6 Hz, 1H), 5.86 (t, J=7.6 Hz, 1H), 5.46 (m, 1H), 4.49 (d, J=2.8 Hz, 1H), 3.07 (m, 1H), 2.92 (m, 1H), 2.80 (m, 1H), 2.64 (m, 1H), 2.35 (m, 1H), 2.25 (m, 2H), 2.03 (m, 2H);

¹³C NMR (100 MHz, CD₃OD) δ 152.1, 145.3, 144.6, 128.8, 127.6, 125.7, 125.2, 122.6, 100.5, 73.1, 70.9, 56.9, 54.0, 44.8, 43.6, 34.9, 34.6, 31.1;

Anal. Calcd for C₂₁H₂₅N₅O₄S: C, 56.87; H, 5.68; N, 15.79; S, 7.23. Found: C, 56.91; H, 5.73; N, 15.82; S, 7.26.

…………………….

http://www.google.com/patents/WO2010132110A1?cl=en

((lS,2S,4R)-4-{4-[(lS)-2,3-dihydro-lH-inden-l-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl }-2-hydroxycyclopentyl)methyl sulfamate (//) is described in Intl. App. Pub. No. WO 07/092213, U.S. App. Pub. No. 2007/0191293, and U.S. App. Pub. No. 2009/0036678. The potassium salt of ((lS,2S,4R)-4-{4-[( 1 S)-2,3-dihydro- 1 H-inden- 1 -ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl } -2-hydroxycyclopentyl)methyl sulfamate is disclosed in Intl. App. Pub. No. WO 07/092213 and U.S. App. Pub. No. 2007/0191293.

(H)

((lS,2S,4R)-4-{4-[(lS)-2,3-dihydro-lH- inden-l-ylamino]-7H-pyπOlo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate (/):

Step 3: Synthesis of ((lS,2S.4R)-4-(4-r(lS)-2,3-dihydro-lH-inden-l-ylaminol-7H-pyrrolor2.3-dlpyrimidin-7-yl}-2-hvdroxycvclopentyl)methyl sulfamate hydrochloride Form 1

[0158] A reactor was charged with ((lS,2S,4R)-4-{4-[(lS)-2,3-dihydro-lH-inden-l-ylarnino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl }-2-hydroxycyclopentyl)methyl sulfamate (13.4 Kg, 30.2 mol) and 200-proof ethanol (106.2 Kg). The mixture was heated to reflux to afford a clear solution. The mixture was cooled to 50 ± 5 ⁰C and passed through a cartridge filter. 200 proof ethanol (8.9 Kg) was used to rinse the filter. 1.27M hydrogen chloride in ethanol (10.2 Kg) was added via a cartridge filter at a rate to maintain a temperature of 50 ± 5 ⁰C. The mixture was then seeded with Form 1 (67 g). Further 1.27M HCl (10.2 Kg) was added via a cartridge filter at a rate to maintain a temperature of 50 ± 5 ⁰C. The mixture was then stirred at 50 ± 5 ⁰C for about 3 hours. The mixture was then cooled to 20 ± 5 ⁰C over about 3 hours and then stirred for about 2.5 hours. The solid product was then isolated by filtration and washed with 200-proof ethanol (I x 20.4 Kg and 1 x 21.2 Kg). The solids were dried by aspiration on the filter until no supernatant was seen to be collected, and then further dried under reduced pressure at <30 ⁰C to afford the title compound (12.2 Kg) as a white solid determined to be Form 1 by XRPD. IH NMR (300MHz, DMSO, δ): 9.83 (s, IH), 8.34 (s, IH), 7.62 (s, IH), 7.44 (s, 2H), 7.30 (m, 3H), 7.22 (t, IH), 7.07 (s, IH), 5.86 (dd, IH), 5.42 (m, IH), 4.32 (m, IH), 4.21 (dd, IH), 4.02 (dd, IH), 3.04 (m, IH), 2.88 (m, IH), 2.67 (m, 2H), 2.15 (m, 2H), 2.08 (m, 2H), 1.94 (m, IH). XRPD data for Form 1 is shown in FIGURE 1 and Table 1; DSC data is shown in FIGURE 2, and TGA data for Form 1 is shown in FIGURE 3.

…………..

http://www.google.com/patents/WO2007092213A2?cl=en

Example 70: Diastereoisomeric mixture of (lS_/2R_/4R)-4-{4-[(lS)-2_/3-dihydro-lH-inden-l- ylaimnol-ZH-pyrrolop^-dlpyxirnidin-Z-ylJ^-hydroxycyclopentyl s ulf amate and (lR_f2S_/4S)-4-{4-[(lS)-2,3-dihydro-lH-inden-l-ylaminol-7H-pyrrolo[2,3d]- pyrimidin-7-yl}-2-hydroxycyclopentyl sulfamate (Compounds 1-77 and 1-78)

Step a: Cyclopent-3-en-l-yl methanesulfonate

[0335] 3-Cydopentene-l-ol (0.500 g, 5.94 mmol) was stirred in DCM (95 mL).

Pyridine (2.40 mL), N,N-dimethylaminopyridine (0.10 g, 1.00 mmol) and methanesulfonyl chloride (0.690 mL, 8.92 mmol) were added, and the reaction mixture was stirred at 35⁰C for 4 h. N,N-Dimethylarrιinopyridirιe (0.14 g, 1.2 mmol) and methanesulfonyl chloride (0.69 mL, 8.92 mmol) were added, and the reaction was stirred overnight. TLC indicated complete conversion. The reaction mixture was cooled and concentrated. The residue was purified by silica gel chromatography, eluting with DCM, to afford the title compound as a clear oil (0.660 g, 68%).

Step b: 7-Cyclopent-3-en-l-yl-N-r(lSV2,3-dihydro-lH-inden-l-yn-7H-pyrrolor2,3-rfl- pyrmτidin-4-arnine

[0336] N-[(lS)-2,3-DihydrcHlH-mden-l-yl]-7H-pyrrolo[2_/3-d]p3αimidin-4-amine (1.32 g, 5.29 mmol) was azeotroped with toluene and placed under high vacuum for 30 min. N,N-Dimethylformamide (17.7 mL) was added, followed by cesium carbonate (1.99 g, 6.10 mmol). The mixture was stirred at 70⁰C for 10 min. Cyclopent-3-en-l-yl methanesulfonate (0.660 g, 4.07 mmol) in N,N-dimethylformarnide (12.6 mL) was added dropwise. The reaction mixture was heated to 110⁰C for 1 h. The reaction mixture was cooled, quenched with brine and diluted with H₂O. The aqueous layer was extracted with EtOAc (3x), washed with H₂O and brine, dried (Na₂SO₄), filtered, and concentrated. The residue -was purified by via silica gel chromatography, eluting with a gradient of 0 to 5% MeOH in DCM followed by 25 to 50% EtOAc in hexanes, to afford the title compound as a pale brown solid (0.684 g, 53%). LC/MS: R₁ = 1.38 min, ES⁺ 317 (FA standard). Step c: (lR,2S,45)-4-{4-r(lS)-2,3-dihydro-lH-inden-l-ylaininol-7H-pyrrolof2.3- rf1pyrimidin-7-yl}cyclopentane-l,2-diol

[0337] 7-Cyclopent-3-en-l-yl-N-[(lS)-2^-dihyrdo-lH-inden-l-yl]-7H-pyrrolo[2,3- d]pyτimidin-4-amine (0.312 g, 0.986 mmol) was stirred in tert-butyl alcohol (4.9 mL) and H₂O (4.9 mL). AD-mix-α (Sigma- Aldrich, 1.4 g) was added, and the suspension was stirred at rt overnight. TLC indicated complete conversion. The reaction was quenched with sodium sulfite (1.48 g, 11.7 mmol), and the mixture was stirred for 5 h. The reaction mixture was diluted with EtOAc and H₂O, and the aqueous layer was extracted with EtOAc (2x). The organic layer was dried (Na₂SO₄), filtered, and concentrated. The residue was purified via silica gel chromatography, eluting with EtOAc, to afford the title compound as a white solid (0.190 g, 55%).

Step d: Diastereoisomeric mixture of (lS,2R,4R)-4-{4-r(15)-23-dihydro-lH-inden-l- ylarninoi^jH-pyrrolofΣ^dlpyrirnidin-y-yll-l-hydroxycyclopentyl sulfamate and (lR,2S,4S)-4-{4-iαSV2,3-dihydro-lH-inden-l-ylarninol-7H-pyrrolor2,3- rf1pyrimidm-7-yl)-2-hydroxycyclopenryl sulfamate (Compounds 1-77 and 1-78)

[0338] (lR,2S,4S)-4-{4-[(lS)-2,3-Dihydro-lH-inden-l-ylarnino]-7H-pyrrolo[2_/3- d]pyrimidin-7-ylJcyclopentane-l,2-diol (0.080 g, 0.23 mmol) was azeotroped with toluene and then was dissolved in anhydrous acetonitrile (2.3 mL). Pyridine (0.0369 mL, 0.458 mmol) was added. The reaction mixture was cooled to 0⁰C, and a 2N solution of chlorosulfonamide in acetonitrile (0.144 mL) was added dropwise. The reaction was stirred for 1 h, and then additional 2N chlorosulfonamide in acetonitrile (0.028 mL) was added. After 30 min, additional 2N chlorosulfonamide in acetonitrile (0.0342 mL) was added, and the reaction mixture was stirred for 2 h. The reaction was quenched with methanol, and the mixture was concentrated in vacuo. The residue was purified by preparative thin layer chromatography using DCM:AcCN:MeOH (50:45:5). The relevant band was cut, washed with acetone, filtered, and concentrated to give a mixture of diastereomers as a white solid. (11 mg, 11%). ¹H NMR (CDCl₃, 400 NMR, δ): 8.36-8.27 (m, IH); 7.38-7.09 (m, 5H); 6.90-6.80 (m, IH); 6.36- 6.20 (m, IH); 5.95-5.76 (m, IH); 5.51-5.22 (m, 2H); 4.83-4.68 (m, IH); 3.87-3.72 (m, IH); 3.12- 2.83 (m, 2H); 2.75-2.53 (m, IH); 2.50-2.14 (m, 2H); 2.08-1.79 (m, 2H) ppm. LC/MS: R, = 1.16 min, ES⁺ 430 (FA standard).

…………

WO 2012061551

http://www.google.im/patents/WO2012061551A1?cl=en

The compound ((lS,2S,4R)-4-(4-((lS)-2,3-dihydro-lH-inden-l-ylamino)-7H-pyrrolo[2,3-d]- pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate:

also known as MLN4924, is an inhibitor of NEDD8-activating enzyme (NAE). Inhibition of NAE has been shown to induce cancer cell death and inhibit the growth of tumors in xenograft models. See, e.g., T.A. Soucy et al., Nature, 2009, 458, 732-737; T.A. Soucy ei al., Clin. Cancer Res., 2009, 15 (12), 3912-3916; and J.E. Brownell et al., Mol. Cell., 2010, 37 (1), 102-111, each of which is hereby incorporated by reference herein in its entirety. MLN4924, pharmaceutical compositions of MLN4924, processes for its synthesis, and polymorphic forms have been described previously. See, e.g., US Patent Appl. Nos. 11/700,614 (Publ. No. 2007/0191293), 12/221,399 (Publ. No. 2009/0036678) and 12/779,331 (Publ. No. 2011/0021544),

……………

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00209, http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00209

A practical synthesis of a novel NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924) is described. Key steps include an enantioselective synthesis of an amino-diol cyclopentane intermediate containing three chiral centers and a novel, regioselective sulfamoylation using N-(tert-butoxycarbonyl)-N-[(triethylenediammonium)sulfonyl]azanide. The linear process, involving six solid isolations, has been carried out in multiple cGMP productions on 15–30 kg scale to produce pevonedistat in 98% (a/a) chemical purity and 25% overall yield.

((1S,2S,4R)-4-(4-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl Sulfamate (1)

The reaction yielded 1 (0.285 kg, 58.5%, 93.0% a/a) as an off-white solid.

HPLC retention time of 1 BASE(Method C): 22.6 min;

¹H NMR (400 MHz, DMSO) δ 8.19 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.45 (s, 2H), 7.31–7.26 (m, 2H), 7.22 (t, J = 6.6 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 6.66 (d, J = 3.5 Hz, 1H), 5.92 (q, J = 8.0 Hz, 1H), 5.39 (qd, J = 8.8, 5.7 Hz, 1H), 4.95 (d, J = 3.9 Hz, 1H), 4.42–4.31 (m, 1H), 4.25 (dd, J = 9.7, 7.0 Hz, 1H), 4.07 (dd, J = 9.6, 8.0 Hz, 1H), 3.01 (ddd, J = 15.7, 8.7, 3.0 Hz, 1H), 2.95–2.81 (m, 1H), 2.81–2.65 (m, 1H), 2.58–2.49 (m, 1H), 2.31–1.86 (m, 5H);

¹³C NMR (100 MHz, DMSO) δ 155.91, 151.18, 149.02, 144.66, 142.98, 127.30, 126.28, 124.49, 124.11, 121.68, 102.83, 98.86, 70.82, 69.37, 54.48, 52.15, 42.58, 42.25, 33.50, 33.26, 29.72;

m/z: 444.4 (M + H)⁺;

mp: 164–166 °C.

((1S,2S,4R)-4-(4-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl Sulfamate·Hydrochloride (Pevonedistat)

Pevonedistat (14.0 g, 92.5%, 99.0% a/a) as a white solid.

HPLC retention time of pevonedistat (Method C): 22.6 min;

¹H NMR (400 MHz, DMSO) δ 9.70 (s, 1H), 8.39 (s, 1H), 7.63 (s, 1H), 7.45 (s, 2H), 7.41–7.20 (m, 4H), 7.04 (s, 1H), 5.78 (s, 1H), 5.44 (s, 1H), 4.42–4.28 (m, 1H), 4.24 (dd, J = 9.7, 6.9 Hz, 1H), 4.05 (dd, J = 9.6, 8.0 Hz, 1H), 3.18–2.99 (m, 1H), 2.91 (dt, J = 15.6, 7.7 Hz, 1H), 2.81–2.57 (m, 2H), 2.24–1.86 (m, 6H).

¹³C NMR (100 MHz, DMSO) δ 149.12, 145.71, 143.23, 142.11, 141.30, 128.28, 126.64, 124.97, 124.82, 124.49, 102.57, 101.74, 70.67, 69.22, 57.38, 53.14, 42.52, 42.40, 33.57, 32.56, 29.80;

m/z: 444.4 (M + H)⁺;

mp: 155–157 °C.

……………..

http://pubs.acs.org/doi/abs/10.1021/jo2001897

http://pubs.acs.org/doi/suppl/10.1021/jo2001897/suppl_file/jo2001897_si_001.pdf

J. Org. Chem., 2011, 76 (9), pp 3557–3561

DOI: 10.1021/jo2001897

MLN4924 (1), which is in clinical trials as an anticancer agent, was stereoselectively synthesized from d-ribose via a route involving stereoselective reduction, regioselective cleavage of an isopropylidene moiety, and selective displacement of a cyclic sulfate moiety as key steps.

Sulfamic Acid 2-Hydroxy-4-[4-(indan-1-ylamino)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentylmethyl Ester (1) BASE

purified by silica gel column chromatography (hexane/ethyl acetate = 1/2) to give 1 (5.08 g, 90%) as a white foam:

UV (MeOH) λ_max 279.50 nm;

[α]²⁰_D −6.41 (c 2.34, MeOH);

HR-MS (ESI) m/z calcd for C₂₁H₂₆N₅O₄S [M + H]⁺ 444.1705, found 444.1706;

¹H NMR (400 MHz, CD₃OD) δ 8.17 (d, J = 1.6 Hz, 1H), 7.25 (m, 2H), 7.18 (m, 2H), 6.64 (d, J = 3.6 Hz, 1H), 5.86 (t, J = 7.6 Hz, 1H), 5.46 (m, 1H), 4.49 (d, J = 2.8 Hz, 1H), 3.07 (m, 1H), 2.92 (m, 1H), 2.80 (m, 1H), 2.64 (m, 1H), 2.35 (m, 1H), 2.25 (m, 2H), 2.03 (m, 2H);

¹³C NMR (100 MHz, CD₃OD) δ 152.1, 145.3, 144.6, 128.8, 127.6, 125.7, 125.2, 122.6, 100.5, 73.1, 70.9, 56.9, 54.0, 44.8, 43.6, 34.9, 34.6, 31.1. Anal. Calcd for C₂₁H₂₅N₅O₄S: C, 56.87; H, 5.68; N, 15.79; S, 7.23. Found: C, 56.91; H, 5.73; N, 15.82; S, 7.26.

NMR FROM CHEMIETEK

MLN4924, Current Lot, Certificate of Analysis

MLN4924, Current Lot, NMR

MLN4924, Current Lot, HPLC-MS, Chiral HPLC

WO2012061551A1 *	Nov 3, 2011	May 10, 2012	Millennium Pharmaceuticals, Inc.	Administration of nedd8-activating enzyme inhibitor
WO2013028832A2 *	Aug 23, 2012	Feb 28, 2013	Millennium Pharmaceuticals, Inc.	Inhibitors of nedd8-activating enzyme
WO2013028832A3 *	Aug 23, 2012	May 2, 2013	Millennium Pharmaceuticals, Inc.	Inhibitors of nedd8-activating enzyme
US8809356	Aug 23, 2012	Aug 19, 2014	Millennium Pharmaceuticals, Inc.	Inhibitors of NEDD8-activating enzyme

Reference
1	*	Lee, et. al., Journal of Organic Chemistry (2011), 76(9), 3557-3561.

(1) Lee, Hyuk Woo; Journal of Organic Chemistry 2011, 76(9), P3557-3561
(2) Soucy, Teresa A.; Nature (London, United Kingdom) 2009, 458(7239), P732-736

1H NMR PREDICT

13 C NMR

//////////Pevonedistat, MLN4924, Millennium Pharmaceuticals, TAKEDA, TAK-924 , PHASE 1, orphan drug designation

ASLAN Pharmaceuticals Gains Orphan Designation for Rare Cancer Drug ASLAN001 (varlitinib)

August 24, 2015 10:35 am / Leave a comment

(R)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

ASLAN001 , Varlitinib

C22H19ClN6O2S

Molecular Weight: 466.94

Elemental Analysis: C, 56.59; H, 4.10; Cl, 7.59; N, 18.00; O, 6.85; S, 6.87

CAS: 845272-21-1 (Varlitinib); 1146629-86-8 (Varlitinib tosylate).

ASLAN001; ASLAN-001; ASLAN 001; AR 00334543; ARRY-334543; ARRY334543; ARRY-543; ARRY543; ARRY 543.

(R)-N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)-N6-(4-methyl-4,5-dihydrooxazol-2-yl)quinazoline-4,6-diamine.

(R)-4-[[3-Chloro-4-[(thiazol-2-yl)methoxy]phenyl]amino]-6-[(4-methyl-4,5-dihydrooxazol-2-yl)amino]quinazoline

4,6-Quinazolinediamine, N4-[3-chloro-4-(2-thiazolylmethoxy)phenyl]-N6-[(4R)-4,5-dihydro-4-methyl-2-oxazolyl]-

ASLAN Pharmaceuticals, a Singapore-based drugmaker, announced The Food and Drug Administration (FDA) gave an orphan drug designation on August 13 to its pan-HER inhibitor ASLAN001 (varlitinib), a drug candidate created to treat a destructive form of bile duct cancer called cholangiocarcinoma that has no known cure. ………http://www.dddmag.com/news/2015/08/aslan-pharmaceuticals-gains-orphan-designation-rare-cancer-drug

Current developer: Array Biopharma Inc,

Varlitinib, also known as ARRY-543 and ASLAN001, is an orally bioavailable inhibitor of the epidermal growth factor receptor family with potential antineoplastic activity.

Varlitinib (ASLAN-001) is an oncolytic drug in phase II clinical trials at ASLAN Pharmaceuticals for the treatment of gastric cancer and for the treatment of metastatic breast cancer in combination with capecitabine. Clinical development is also ongoing for the treatment of solid tumors in combination with cisplatin/FU and cisplatin/capecitabine. The product had been in phase I/II clinical trials at Array BioPharma for the treatment of patients with advanced pancreatic cancer. Phase II clinical trials had also been ongoing for the treatment of solid tumors. No recent development has been reported for this research

Varlitinib selectively and reversibly binds to both EGFR (ErbB-1) and Her-2/neu (ErbB-2) and prevents their phosphorylation and activation, which may result in inhibition of the associated signal transduction pathways, inhibition of cellular proliferation and cell death. EGFR and Her-2 play important roles in cell proliferation and differentiation and are upregulated in various human tumor cell types. Due to the dual inhibition of both EGFR and Her-2, this agent may be therapeutically more effective than agents that inhibit EGFR or Her-2 alone.

The drug is a dual inhibitor of the ErB-2 and EGFR receptor kinases, both of which have been shown to stimulate aberrant growth, prolong survival and promote differentiation of many tumor types. The compound behaves as a reversible ATP-competitive inhibitor with nanomolar potency both in vitro and in cell-based proliferation assays.

In 2011, the compound was licensed to Aslan Pharmaceuticals by Array BioPharma worldwide for the treatment of solid tumors, initially targeting patients with gastric cancer through a development program conducted in Asia.

In 2015, orphan drug designation was assigned to the compound in the U.S. for the treatment of cholangiocarcinoma.

SEE NMR ………….http://www.medkoo.com/Product-Data/Varlitinib/Varlitinib-QC-KB20121128web.pdf

……………..

https://www.google.co.in/patents/US20050043334

Example 52

(R)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

Prepared using (R)-2-aminopropan-1-o1. MS APCI (+) m/z 467, 469 (M+1, Cl pattern) detected; ¹H NMR (400 mHz, DMSO-D6) δ 9.53 (s, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.86 (d, 1H), 7.81 (d, 1H), 7.77 (d, 1H), 7.69 (m, 3H), 7.32 (d, 1H), 7.02 (s, 1H), 5.54 (s, 2H), 4.47 (m, 1H), 3.99 (m, 1H), 3.90 (m, 1H), 1.18 (d, 3H).

Example 53

(S)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

Prepared using (S)-2-amino-propan-1-o1. MS APCI (+) m/z 467, 469 (M+1, Cl pattern) detected; ¹H NMR (400 mHz, DMSO-D6) δ 9.53 (s, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.86 (d, 1H), 7.81 (d, 1H), 7.77 (d, 1H), 7.69 (m, 3H), 7.32 (d, 1H), 7.02 (s, 1H), 5.54 (s, 2H), 4.47 (m, 1H), 3.99 (m, 1H), 3.90 (m, 1H), 1.18 (d, 3H).

………………

PATENT

http://www.google.co.in/patents/WO2005016346A1?cl=en

Example 52

R VN4-r3-Chloro-4-(^‘thiazol-2-v-metho-xy)-phenyll-N6-(4-methyl-4,5-dihvdro-oxazol- 2-yl)-quinazoUne-4,6-diamine

[00194] Prepared using (R)-2-aminopropan- 1 -ol. MS APCI (+) m/z 467, 469

(M+l, CI pattern) detected; 1H NMR (400 mHz, DMSO-D6) δ 9.53 (s, IH), 8.47 (s, IH), 8.09 (s, IH), 7.86 (d, IH), 7.81 (d, IH), 7.77 (d, IH), 7.69 (m, 3H), 7.32 (d, IH), 7.02 (s, IH), 5.54 (s, 2H), 4.47 (m, IH), 3.99 (m, IH), 3.90 (m, IH), 1.18 (d, 3H). Example 53

(S)-N4-|^“3-Chloro-4- thiazol-2-ylmethoxy)-phenyll-N6-(^‘4-methyl-4,5-dihvdro-oxazol- 2-yl)-quinazoline-4,6-diamine [00195] Prepared using (S)-2-amino-propan- 1 -ol. MS APCI (+) m z 467, 469

………

CAUTION a very similar molecule but not same

NOTE……..METHYL NEXT TO OXYGEN ATOM

Design, Synthesis and Bioactivities Evaluation of Novel Quinazoline Analogs Containing Oxazole Units

Xuehui Hou¹,
Jingyu Zhang²,
Xuan Zhao²,
Liming Chang²,
Ping Hu^1,* and
Hongmin Liu^3,*

DOI: 10.1002/cjoc.201400271,…………http://onlinelibrary.wiley.com/doi/10.1002/cjoc.201400271/abstract;jsessionid=04211D7526D97240F44E4355C6C57F50.f01t01

http://onlinelibrary.wiley.com/store/10.1002/cjoc.201400271/asset/supinfo/cjoc_201400271_sm_suppl.pdf?v=1&s=8dd0bf6d7ee4d3d6bf8503fef49bafc69220c931

CAUTION …THIS IS NOT SAME

A novel type of quinazoline derivatives, which were designed by the combination of quinazoline as the backbone and oxazole scaffold as the substituent, have been synthesized and their biological activities were evaluated for anti-proliferative activities and EGFR inhibitory potency. Compound 12b demonstrated the most potent inhibitory activity (IC₅₀=0.95 µmol/L for EGFR), which could be optimized as a potential EGFR inhibitor in the further study. The structures of the synthesized quinazoline analogs and all intermediates were comfirmed by ¹H and ¹³C NMR, 2D NMR spectra, IR spectra and MS spectra.

12c: Employing the same method as above, compound 12c was prepared and the amino alcohol was (S)-2-amino-propan-1-ol. Yellow solid, yield 52 %. m.p. 243-244 °C; [α] 20D =﹢22.5 ° (c 1.0, CH3CN); 1 H NMR (DMSO-D6): δ 9.54 (s, 1 H), 8.46 (s, 1 H), 8.06 (s, 2 H), 7.85 (d, 2 H, J=3.3 Hz), 7.79 (d, 2 H, J=3.3 Hz), 7.75 (d, 1 H, J=8.9 Hz), 7.64 (d, 1 H, J=8.3 Hz), 7.30 (d, 1 H, J=9.0 Hz), 5.54 (s, 2 H), 4.76 (m, 1 H), 3.72 (s, 1 H), 3.19 (s, 1 H), 1.34 (d, 3 H, J=6.15 Hz). 13C NMR (DMSO-D6) δ: 165.8, 156.9, 152.0, 148.8, 145.3, 142.6, 134.3, 128.7, 128.0, 123.5, 121.7, 121.3, 121.0, 115.6, 114.6, 72.5, 67.7, 63.0, 29.8, 29.0, 20.0, 13.9. IR (KBr) ν: 3439, 3278, 3101, 2925, 1660, 1631, 1601, 1557, 1500, 1428, 1404, 1384, 1329, 1291, 1257, 1225, 1052 cm-1. Anal. calcd for C22H19N6O2SCl: C 55.59, H 4.10, N 18.00, O 6.85; found C 55.55, H 4.13, N 18.02, O 6.78; MS (ESI) m/z: 467.2 (M+H).

12d: Employing the same method as above, compound 12d was prepared and the amino alcohol was (R)-2-amino-propan-1-ol. Yellow solid, yield 60%. m.p. 242-243 °C; [α] 20D = ﹣22.3 ° (c 1.0, CH3CN); 1 H NMR (DMSO-D6): δ 9.52 (s, 1 H), 8.80 (s, 1 H), 8.52 (dd, 1 H, J=2.7 Hz, J=8.9 Hz), 8.45 (s, 1 H), 8.30 (s, 1 H), 8.07 (s, 1 H), 7.85 (d, 1 H, J=3.2 Hz), 7.79 (d, 1 H, J=3.2 Hz), 7.75 (s, 1 H), 7.63 (d, 1 H, J=8.2 Hz), 7.31 (d, 1 H, J=9.0 Hz), 5.53 (s, 2 H), 4.76 (m, 1 H), 3.81 (s, 1 H), 3.19 (s, 1 H), 1.34 (d, 3 H, J=6.2 Hz). 13C NMR (DMSO-D6) δ: 165.8, 156.9, 152.0, 148.8, 145.3, 142.6, 134.3, 128.7, 128.0, 123.5, 121.7, 121.3, 121.0, 115.6, 114.6, 72.5, 67.7, 63.0, 29.8, 29.0, 20.0, 13.9. IR (KBr) ν: 3439, 3278, 3101, 2925, 1660, 1631, 1601, 1557, 1500, 1428, 1404, 1384, 1329, 1291, 1257, 1225, 1052 cm-1. Anal. calcd for C22H19N6O2SCl: C 55.59, H 4.10, N 18.00, O 6.85; found C 55.55, H 4.13, N 18.02, O 6.78; MS (ESI) m/z: 467.20 (M+H).

The above paper allows you to synthesize the key amino int 11 ………N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)quinazoline-4,6-diamine (11)

this can be applied to varlitinib till int 11

6-Nitro-4-hydroxyquinazoline (3)

2-amino-5-nitrobenzoic acid (5.46 g, 30 mmol) was added to a 250 mL flask equipped with a reflux condenser. Then 50 mL formamide was added. The mixture was heated with vigorous stirring at 160 °C for 3 h. After cooling the solution was poured in ice-water to give 3 in almost pure form (Yellow solid 4.70 g, yield 82.0%). m.p. 317-318 °C; 1 H NMR (DMSO-d6): δ 12.74 (1 H, s, OH, exchangeable), 8.78 (1 H, d, J=2.4 Hz), 8.53 (1 H, dd, J=2.6 Hz, 9.0 Hz), 8.30 (s, 1 H), 7.84 (1 H, d, J=9.0 Hz); 13C NMR (DMSO-d6) δ: 160.1, 152.9, 148.9, 145.0, 129.1, 128.3, 122.7, 121.9. IR (KBr) ν: 3172, 3046, 2879, 1674, 1615, 1577, 1514, 1491, 1469, 1343, 1289, 1242, 1167, 1112, 928, 920, 901, 803, 753, 630, 574, 531 cm-1. Anal. calcd for C8H5N3O3: C 50.27, H 2.64, N 21.98; found C 50.30, H 2.65, N 21.96; MS (ESI) m/z: 189.97 (M-H).

13C NMR OF 3 IN DMSOD6

4-chloro-6-Nitroquinazoline (4)

In a 100 mL flask equipped with a reflux condenser, 6-nitroquinazolin-4-one (2.86 g, 15 mmol) and thionyl chloride (SOCl2) 25 mL were added. The mixture was heated under reflux with vigorous stirring for 2 h. After the solution was clear, the reaction mixture was heated for another 2 h. Then, 150 mL of ice MeOH was dropped into it carefully, the mixture was extracted with CH2Cl2. The organic layer was S3 dried under MgSO4, filtered and the solvent removed to give 4-chloro-6-nitroquinazoline (4). Yellow solid 2.45 g, yield 78%. m.p. 134-135 °C; 1 H NMR (DMSO-d6): δ 8.80 (1 H, d, J=3.0 Hz), 8.54(1 H, dd, J=2.7 Hz, 9.0 Hz), 8.35(s, 1 H), 7.87 (1 H, d, J= 9.0 Hz); 13C NMR (DMSO-d6) δ: 160.0, 152.5, 149.1, 145.1, 128.7, 128.4, 122.7, 122.0. IR (KBr) ν: 3431, 3082, 3038, 2664, 2613, 2567, 1724, 1685, 1676, 1646, 1617, 1578, 1526, 1468, 1359, 1346, 1269 cm-1. Anal. calcd for C8H4N3O2Cl: C 45.84, H 1.92, N 20.05, O 15.27; found C 45.81, H 1.97, N 20.02, O 15.21; MS (ESI) m/z: 207.96 (M-H).

nmr1 4 nmr dmsod6

13C NMR OF4 IN DMSOD6

Thiazol-2-yl-methano1 (6)

Sodium borohydride (16.0 g, 140 mmol) was added to a stirred solution of thiazole-2-carbaldehyde (24.2 g, 214 mmol) in MeOH (400 mL) at 0 °C . The reaction mixture was warmed to room temperature. After 1 hour, the reaction mixture was quenched by the addition of water and the organics were removed by concentration. The resulting aqueous mixture was extracted with EtOAc. The combined organic extracts were dried under Na2SO4 and concentrated to give thiazol-2-yl-methano1 (23.39 g, 95%). bp:75-76 °C (0.2 mmHg) [lit.[19] bp:70-80 °C (0.2 mmHg)]; m. p. 63-64 °C. 1 H NMR (CDCl3) δ 4.91 (s, 2 H), 5.1(br, l H), 7.28(d, 1 H, J=3.2 Hz), 7.68 (d, 1 H, J=2.9 Hz). IR (KBr) ν: 3135, 3099, 3082, 2814, 1509, 1446, 1351, 1189, 1149, 1073, 1050, 977, 775, 745, 613, 603 cm-1. Anal. calcd for C4H5NOS: C 41.72, H 4.38, N 12.16; found C 41.74, H 4.33, N 12.18; MS (ESI) m/z: 116.11 (M+H).

nmr1 6 in dmsod6 1H NMR

2-((2-Chloro-4-nitrophenoxy)methyl)thiazole (8)

2-(2-chloro-4-nitro-phenoxymethy1)-thiazole was prepared by adding thiazol-2-yl-methanol (5.48 g, 47.65 mmol) to a slurry of sodium hydride (2.42 g of a 60% dispersion in oil, 60.5 mmol) in THF (50 ml) at 0 °C After several minutes, 2-chloro-1-fluoro- 4-nitro-benzene (7.58 g, 43.60 mmol) was added and the reaction mixture warmed to room temperature. The reaction mixture was stirred at room temperature for 3 h, and 60 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into 300 mL water. The resulting precipitate was collected by filtration, washed with water, and dried in vacuo to give 2-(2- chloro-4-nitrophenoxymethy1)-thiazole (11.06 g, 86%) which was used in next step without further purification. m.p. 170-171 °C; 1 H NMR (DMSO-d6): δ 8.35 (1 H, d, J=2.8 Hz), 8.25 (1 H, dd, J=2.8 Hz, 9.15 Hz), 7.87 (1 H, d, J=3.3 Hz), 7.83(1 H, d, J=3.3 Hz), 7.54 (1 H, d, J=9.2 Hz), 5.73(s, 1 H); 13C NMR (DMSO-d6) δ: 164.2, 158.5, 143.2, 141.7, 125.9, 124.9, 122.4, 122.2, 114.6, 68.4; IR (KBr) ν: 3112, 3009, 1587, 1509, 1500, 1354, 1319, 1284, 1255, 1154, 1125, 1054, 1006, 894, 780, 746, 728 cm-1. Anal. calcd for C10H7N2O3SCl: C 44.37, H 2.61, N 10.35, O 17.73; found C 44.31, H 2.67, N 10.29; MS (ESI) m/z: 268.89 (M-H).

1H NMR 8 DMSOD6

13C NMR OF 8 IN DMSOD6

3-Chloro-4-(thiazol-2-ylmethoxy)aniline (9)

In a flask equipped with a reflux condenser, the compound 8 15.00 g (55.6 mmol), reduced zinc powder 14.44 g (222.0 mmo1, 4 eq), saturated ammonia chloride (5 mL) and methanol (100 mL) were mixed. The mixture was stirred at a temperature of 40 °C for 1.5 h. Then the zinc powder was filtered off, the filtrate was concentrated to obtain yellow solid 13.21 g, yield 99%. m.p. 60-61 °C; 1 H NMR (DMSO-d6): δ 7.80 (1 H, d, J=3.3 Hz), 7.75 (1 H, d, J=3.3 Hz), 6.96 (1 H, d, J=8.8 Hz), 6.64(1 H, d, J=2.7 Hz), 6.46 (1 H, dd, J=2.7 Hz, J=8.7 Hz), 5.30 (s, 2 H), 5.04 (s, 2 H, NH2, exchangeable); 13C NMR (DMSO-d6) δ: 166.8, 145.1, 144.1, 142.80, 123.1, 121.5, 117.7, 115.2, 113.6, 69.1. IR (KBr) ν: 3322, 3192, 3112, 1607, 1499, 1457, 1436, 1291, 1274, 1221, 1191, 1144, 1057, 1027, 857, 797, 767, 733, 584 cm-1. Anal. calcd for C10H9N2OSCl: C 49.90, H 3.77, N 11.64, O 6.65; found C 49.95, H 3.76, N 11.66, O 6.60; MS (ESI) m/z: 239.01 (M-H).

1H NMR DMSOD6 OF 9

13C NMR OF 9 IN DMSOD6

N-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)-6-nitro- quinazolin-4-amine（10）

In a flask equipped with a reflux condenser, 6-nitro-4-chloro- quinazoline 8.0 g (38.3 mmol) and 3-Chloro-4-(thiazol-2-ylmethoxy)aniline 8.9 g (37.0 mmol) were dissolved into 150 mL of THF, and the solution was refluxed for 3 h.Then a lot of yellow solid was deposited. Then it was filtered affording to yellow solid 12.8 g, yield 81%. m.p. 183-184 °C (decompose); 1 H NMR (DMSO-d6): δ 11.97(s, 1 H, exchangeable), 9.84 (s, 1 H), 9.00 (s, 1 H), 8.76 (1 H, d, J=9.1 Hz), 8.12-8.14 (m, 1 H), 7.94 (1 H, d, J=2.3 Hz), 7.87 (1 H, d, J=3.2 Hz), 7.81 (1 H, d, J=3.2 Hz), 7.44 (1 H, d, J=9.0 Hz), 7.69 (1 H, dd, J=2.5 Hz, J=8.9 Hz), 5.61 (s, 2 H); 13C NMR (DMSO-d6) δ: 166.8, 145.1, 144.1, 142.8, 123.1, 121.5, 117.7, 115.2, 113.7, 69.1. IR (KBr) ν: 3442, 3100, 1636, 1618, 1570, 1552, 1523, 1492, 1442, 1400, 1377, 1344, 1301, 1267, 1069, 805 cm-1. Anal. calcd for C18H12N5O3SCl: C 52.24, H 2.92, N 16.92, O 11.60; found C 52.26, H 2.93, N 16.96, O 11.58; MS (ESI) m/z: 412.84 (M-H).

1H NMR DMSOD6 OF 10

13C NMR OF 10 IN DMSOD6

N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)quinazoline-4,6-diamine (11)

In a flask equipped with a reflux condenser, the compound 10 5.00 g (12.1 mmol), reduced zinc powder 3.2 g (48.5 mmo1, 4 eq), saturated ammonia chloride (3 mL) and methanol (60 mL) were mixed. The mixture was stirred at room temperature for 30 min. Then the zinc powder was filtered off, the filtrate was concentrated to obtain yellow solid 4.58 g, yield 98%. m.p. 197-198 °C (decompose); 1 H S4 NMR (DMSO-d6): δ 9.33(s, 1 H, exchangeable), 8.31 (s, 1 H), 8.05 (d, 1 H, J=2.6 Hz), 7.85 (d, 1 H, J=3.3 Hz), 7.79 (1 H, d, J=3.3 Hz), 7.73 (1 H, dd, J=2.5 Hz, J=9.0 Hz), 7.51 (1 H, d, J=8.9 Hz), 7.30 (1 H, d, J=2.4 Hz), 7.29 (1 H, d, J=4.7 Hz), 7.23 (1 H, dd, J=2.3 Hz, J=8.9 Hz), 5.57 (s, 2 H, exchangeable), 5.52 (s, 2 H); 13C NMR (DMSO-d6) δ: 165.9, 155.8, 149.7, 148.5, 147.3, 142.6, 142.5, 134.6, 128.7, 123.6, 123.2, 121.4, 121.3, 121.1, 116.5, 114. 7, 100.9, 67.8. IR (KBr) ν: 3443, 3358, 3211, 3100, 1631, 1596, 1577, 1560, 1530, 1494, 1431, 1383, 1217, 910 cm-1. Anal. calcd for C18H14N5OSCl: C 56.32, H 3.68, N 18.24, O 4.17; found C 56.34, H 3.70, N 18.22, O 4.14; MS (ESI) m/z: 382.66 (M-H).

11 1HNMR DMSOD6

13C NMR OF 11 IN DMSOD6

Construction finally as per patent ……….US20050043334

Treatment of N4-[3-chloro-4-(thiazol-2-ylmethoxy)phenyl]quinazoline-4,6-diamine (11) with 1,1′-thiocarbonyldiimidazole , followed by condensation with 2(R)-amino-1-propanol in THF/CH2Cl2 affords thiourea derivative , which finally undergoes cyclization in the presence of TsCl and NaOH in THF/H2O to furnish varlitinib .

ASLAN Pharmaceuticals
Address: 10 Bukit Pasoh Rd, Singapore 089824

Phone:+65 6222 4235

Map of ASLAN Pharmaceuticals

carl fith

Mr Carl Firth, CEO, Aslan Pharmaceuticals, Singapore (left) and Mr Dan Devine, CEO, Patrys, Australia (right)

///////ASLAN001, varlitinib, ASLAN Pharmaceuticals, Orphan Designation, ARRY-534, ARRY-334543 , PHASE 2, ORPHAN DRUG DESIGNATION, array

AXITINIB

July 9, 2015 12:08 pm / Leave a comment

Axitinib (AG013736; trade name Inlyta) is a small molecule tyrosine kinase inhibitor developed by Pfizer. It has been shown to significantly inhibit growth of breast cancer in animal (xenograft) models^[2] and has shown partial responses in clinical trials with renal cell carcinoma (RCC)^[3] and several other tumour types.^[4] It was approved by the U.S. Food and Drug Administration after showing a modest increase in progression-free survival,^[5] though there have been reports of fatal adverse effects.^[6]

Axitinib, a small-molecule indazole derivative chemically known as (E)-N-methyl-2-(3-(2-(pyridin-2-yl)-vinyl)-1H-indazol-6-ylthio)benzamide developed by Pfizer, was approved in January 2012 by the U.S. FDA with the trade name Inlyta. It selectively inhibits vascular endothelial growth factor receptors for the treatment of renal cell carcinoma

On January 27, 2012, axitinib was approved with the trade name INLYTA for treatment of patients in the United States with advanced renal cell carcinoma after failure of one prior systemic therapy.

It has received FDA (27 January 2012), EMA (13 September 2012), MHRA (3 September 2012) and TGA (26 July 2012) approval for use as a treatment for renal cell carcinoma.^[11]^[12]^[13]^[14]

A study published in 2015^[15] showed that axitinib effectively inhibits a mutated gene (BCR-ABL1[T315I]) that is common in chronic myeloid leukemias and adult acute lymphoblastic leukemias which have become resistant to other tyrosine kinase inhibitors likeimatinib. This is one of the first examples of a new indication for an existing drug being discovered by screening known drugs using a patient’s own cells.

Abstract Image

The discovery and development of an efficient synthesis route to axinitib is reported. The first-generation route researched by Pfizer implemented two Pd-catalyzed coupling reactions as key steps. In this work, the development of Heck-type and C–S coupling reactions catalyzed by CuI is briefly described, using an economial and practical protocol. Aspects of this route, such as selecting optimal ligands, solvent, and other conditions, are discussed in detail. The scale-up experiment was carried out to provide more than 300 g of active pharmaceutical ingredients of axitinib in Form XLI with 99.9% purity in 39% yield. In short, we provide a new choice of synthesis route to axitinib, through two copper-catalyzed coupling reactions with good yield.

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00123

http://pubs.acs.org/doi/full/10.1021/acs.oprd.5b00123

(E)-N-Methyl-2-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-ylthiol)benzamide (Axitinib) Form XLI (326.4 g in 96% yield with purity 99.91%). Residual Cu content was determined to be 2.2 ppm by atomic absorption spectroscopy: mp 227.7 °C;

1H NMR (300 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 16.4 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.63–7.44 (m, 3H), 7.29 (p, J = 7.4, 6.6 Hz, 3H), 7.19 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 7.4 Hz, 1H), 2.78 (d, J = 4.6 Hz, 3H);

13C NMR (75 MHz, DMSO-d6) δ 167.89, 154.86, 149.54, 142.01, 141.86, 136.92, 136.88, 135.67, 132.52, 130.32, 129.99, 129.25, 127.80, 126.15, 125.59, 123.66, 122.68, 122.50, 121.79, 120.29, 114.76, 26.13.

………………………..

Axitinib (Axitinib, AG-013736, CAS: 319460-85-0) is a Pfizer research and development by the United States of new, mainly targeting VEGFR kinase GABA, inhibiting angiogenesis anticancer small molecule drug, trade name Inlyta, for other systems therapy for advanced renal cell carcinoma (Renal Cell Carcinoma, RCC), 2008 has been approved in the domestic clinical, and Pfizer’s cancer drug Sutent another similar imatinib (Sunitinib) , Axitinib also potent and selective multi-targeted tyrosine kinase inhibitor, can inhibit the vascular endothelial growth factor receptor (Vascular EndothelialGrowth Factor Rec India tor, VEGFR), including VEGFl receptor, VECF2 receptors and VECF3 receptor, can inhibit platelet-derived growth factor receptor (Platelet-derived growth factor receptor, PDGFR) and c_KIT. Axitinib is called sunitinib second generation, better than sunitinib adverse reactions.

Axitinib (II) chemical name 6- [2_ (methylcarbamoyl) phenylsulfanyl] -3-E- [2_ (Batch-2-yl) ethenyl] indazole structural formula as follows:

Axitinib (II)

Assi synthesis method for Nepal mainly in the following three ways:

(I) Patent US20060094881 (Agouron Pharmaceuticals), EP2163544 (Pfizer) reported the first synthesis method Axitinib to 3,6-diiodo-indazole as a starting material, first-iodo-6-position is substituted mercapto group, protection of the NH group, then the Heck reaction occurs (pyridine-2-yl) vinyl 3-position, after deprotection Axitinib whole synthesis route is as follows:

Axitinib Scheme I

This method although the synthesis route is shorter, but the catalyst and reagents used relatively expensive and require purified through the column, the total yield is low, is not conducive to industrial production.

[0004] (2) The second method of synthesis Axitinib e.g. W00102369 (Agouron Pharmaceuticals), US6531491 (Agouron Pharmaceuticals) reported in 6-nitro-indazole as a starting material, the 3-position first iodo, followed by the protecting group NH, Suzuki coupling reaction with boronic acid to give 3- styryl styryl-position, a nitro group reduced to an amino group, an amino diazotization reaction was iodo, the 3-position of the styrene-based ozone of the obtained aldehyde, followed by Wittig reaction to give the 3-position (pyridin-2-yl) ethenyl, 6-position is substituted mercapto iodine, alkaline hydrolysis then amidated, and finally deprotection Axitinib, the entire reaction formula as follows:

Axitinib Scheme 2

The method of synthesis route is long, harsh reaction conditions, complex process, the total yield is low, does not apply to industrial production.

[0005] (3) The third method is W02006048745 (Pfizer) discloses to 6-nitro-indazole as a starting material, the 3-position iodo first, followed by the protecting group NH, 3- bits Heck coupling reaction, a nitro group reduced to an amino group, an amino diazotization reaction was iodo, iodo-6-position is substituted mercapto group, and finally deprotected to give Axitinib, the entire reaction is as follows:

This method has an advantage over the first two methods, it is possible to enlarge the production, but the reaction was not complete in the reaction step, will generate new impurities through the column needs to be purified.

SYNTHESIS

aReagents and conditions: (a) I2, K2CO3, DMF; (b) CH2Cl2, CH3SO3H, dihydrofuran; (c) compound B, i-Pr2EtN, Pd(OAc)2, (o-Tol)3P, DMF; (d) iron, EtOH, NH4Cl; (e) AcOH, NaNO2, CH2Cl2, I2/KI; (f) compound C, Pd(dppf)Cl2, Cs2CO3, DMF; (h) 1, p-TsOH, MeOH; 2, NaHCO3; (i) AcOH, MeOH, Pd removal, recrystallization.

http://www.google.com/patents/WO2006048745A1?cl=en

Example 15: Final deprotectioπ step to produce 6-r2-(methylcarbamoyl)phenylsulfanyll-3-E-f2- (pyridine-2-yl)ethenyllindazole

N-1 THP 6-[2-(methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole (355 g) was suspended in 2,485 ml_ of methanol, after which p-toluenesulfonic acid monohydrate (718 g) was added. The mixture was then heated to 65 ⁰C (hard reflux) for 4 hours under argon while the reaction was monitored by HPLC (gluco method). Heating continued until less than 1% of the N-1 THP protected starting material persisted. The heating was then removed and the reaction was cooled to room temperature. The solid was filtered and the wet cake was washed with methanol (2 volumes, 710 mL) then the solids were rinsed with ethyl acetate (2 volumes, 710 mL). The wet cake was transferred to a reactor containing sodium bicarbonate (126.84 g), deionized water (1800 mL), and ethyl acetate (975 mL), which was then stirred for 2 hours at 2O⁰C. The solids were filtered and washed with 5 volumes of deionized water (1800 mL), then with 2 volumes of ethyl acetate (760 mL), and then dried in a vacuum oven at 40⁰C for 16 hours. The isolated yield for the reaction was 92.5% (274 g). The isolated material was identified as crystalline Form III free base (0.5 ethyl acetate solvate). ¹H NMR, 300 MHz, (DMSO-D6), ppm; 13.35 (1 H, s), 8.60 (1 H, d, J=3.8 Hz), 8.39 (1 H, m), 8.23 (1 H, d, J=8.5 Hz), 7.95 (1 H, d, J=16.4 Hz), 7.82 (1 H, ddd, J=7.7, 7.6, 1.8 Hz), 7.67 (1 H, d, J=7.8 Hz), 7.60 (a H, s), 7.57 (1 H, d, J=16.4 Hz), 7.49 (1 H, dd, J=7.1 , 1.6 Hz), 7.35-7.26 (3 H, m), 7.19 (1 H, d, J=8.4 Hz), 7.04 (1 H, d, J=7.8 Hz), 2.77 (3 H, d, J=4.6 Hz). ¹³C NMR, 75 MHz, (DMSO-D6) ppm: 168.23, 155.18, 149.81 , 142.35, 142.22, 137.31 , 136.00, 132.89, 130.64, 130.36, 129.51 , 128.14, 126.50, 125.93, 124.08, 123.01 , 122.85, 122.12, 120.642, 115.08, 26.45.

Example 21 : Preparation of 6-F2-(methylcarbamovDphenylsulfanyll-3-Z-r2-(pyridine-2- vDethenyllindazole

To a 100 ml_ 3-neck flask containing a solution of 0.95 g of 6-[2- (methylcarbamoyl)phenylsulfanyl]-3-[2-(pyridine-2-yl)ethynyl]indazole was added 2.5 g of phenyliodide diacetate followed by 1.0 mL of H₂NNH₂ H₂O. After the bubbling had settled, more phenyliodide diacetate and H₂NNH₂ H₂O were added in small portions, until LC/MS indicated the disappearance of 6-[2-(methylcarbamoyl)phenylsulfanyl]-3-[2-(pyridine-2-yl)ethynyl]indazole and the formation of 6-[2-(methylcarbamoyl)phenylsuIfanyl]-3-Z-[2-(pyridine-2-yl)ethenyl]indazole. Example 22: Palladium removal and polymorph control of 6-[2-(methylcarbamoyl)phenylsulfanvn- 3-E-r2-(pyridine-2-vDethenyllindazole

4) MeOH, reflux

Polymorph Form IV

5) HOAc/Xylenes

To a 12 L 3-neck flask, equipped with a mechanical stirrer, was added 160.20 g of 6-[2- (methylc’arbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole and 1.6 L of DMA and 1.6 L of THF. After stirring for 20 minutes, the mixture became homogeneous. To the clear solution was added 800.99 g of 10% cysteine-silica and the resulting mixture was allowed to stir at room temperature overnight.

The mixture was filtered through a medium sintered glass fritted funnel, and the cake was washed with a solution of 500 mL of DMA and 500 mL of THF. The cake was further washed with 2.0 L of THF and the filtrate was collected into a separate flask. The volatile parts in the latter filtrate were removed in vacuo and the residue was combined with the main filtrate. The combined filtrate was recharged back into the 12 L flask, followed by 800 g of 10% cysteine-silica. The flask was equipped with a mechanical stirrer and stirred over the weekend at room temperature. The mixture was then filtered through a medium sintered glass fritted funnel and the silica was washed with a mixture of solvents of 500 ml. of DMA and 500 ml_ of THF, followed by 3.0 L of THF. The volatile parts in the filtrate were removed in vacuo and the remaining solution was transferred to a 22 L 3-neck flask and treated with 12 L of water (added over a 20 minute period of time), a thick precipitate formed at this stage. After stirring overnight, the mixture was filtered and the cake was washed with 2.0 L of water and sucked dry.

The cake was charged to a 5 L 3-neck flask, followed by 1.6 L of THF and 160 mL of DMF. The flask was equipped with a mechanical stirrer, a reflux condenser and the mixture was heated at reflux for 8 hours. After cooling overnight, the mixture was filtered through sharkskin filter paper and sucked dry. The cake was charged to a 5 L 3-neck flask and 1.6 L of MeOH was added. The flask was equipped with a mechanical stirrer, a water condenser and the contents were heated at reflux for 6 hours. After cooling overnight, the mixture was filtered through sharkskin filter paper and sucked dry.

The cake was dissolved into 1.6 L of HOAc with the assistance of gentle heating in the water bath of a rotary evaporator. The solution was filtered through #3 filter paper and the total volume of the filtrate was reduced to ~500 mL in volume on the rotary evaporator at 60 °C/60 mmHg. At this stage, the bulk of the mixture remained a yellow solution and a small amount of precipitate formed. To the flask was charged 500 mL of xylenes (precipitate formed) and the total volume was reduced to -500 mL in volume on the rotary evaporator at 60°C/60 mmHg. The process was repeated two more times. After cooling, the mixture was filtered, the cake was washed with 500 mL of xylenes and sucked dry. The cake was transferred to a glass dish and further dried at 80°C/27 inch vacuum overnight.

The cake was off-white in color and weighed 108.38g. X-ray powder diffraction analysis indicated that a crystalline form was present, which was characterized as Form IV by a powder X- ray diffraction pattern comprising peaks at the following approximate diffraction angles (20): 8.9, 12.0, 14.6, 15.2, 15.7, 17.8, 19.2, 20.5, 21.6, 23.2, 24.2, 24.8, 26.2, and 27.5.

While the invention has been illustrated by reference to specific and preferred embodiments, those skilled in the art will recognize that variations and modifications may be made through routine experimentation and practice of the invention. Thus, the invention is intended not to be limited by the foregoing description, but to be defined by the appended claims and their equivalents.

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Chekal, B. P.; Guinness, S. M.; Lillie, B. M.; McLaughlin, R. W.; Palmer, C. W.; Post, R. J.; Sieser, J. E.; Singer, R. A.; Sluggett, G. W.; Vaidyanathan, R.; Withbroe, G. Org. Process Res. Dev. 2014, 18, 266 http://pubs.acs.org/doi/abs/10.1021/op400088k

The manufacturing process of axitinib (1) involves two Pd-catalyzed coupling reactions, a Migita coupling and a Heck reaction. Optimization of both of these pivotal bond-formation steps is discussed as well as the approach to control impurities in axitinib. Essential to the control strategy was the optimization of the Heck reaction to minimize formation of impurities, in addition to the development of an efficient isolation of crude axitinib to purge impurities.

Babu, S.; Dagnino, R., Jr.; Ouellette, M. A.; Shi, B.; Tian, Q.; Zook, S. E. PCT Int. Appl. WO/2006/048745, 2006.

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http://www.google.com/patents/CN103570696A?cl=en

formula:

A Axitinib intermediate (1) production method, based on 6-nitro-indazole as a starting material, in the first catalyst is reacted with 3,4-dihydro -2H- pyran, bits of NH the protecting group tetrahydro -2H- pyran-2-yl, then the three iodide, to give the key intermediate in high yield 3-iodo-6-nitro-1- (tetrahydro -2H- pyrazol pyran-2-yl) -1H- indazole (I), comprising the following synthetic steps:

(1) 6-nitro-indazole dissolved in an aprotic solvent, and 3,4-dihydro -2H- pyran catalyst, 6-nitro-indazole in the catalyst and the 3,4-dihydro -2H – pyran reaction, the protecting group NH-position, was prepared to give 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, the reaction equation is:

Wherein the 3,4-dihydro -2H- pyran an amount of 3 equivalents wide;

Aprotic solvent is acetonitrile, ethyl acetate, toluene or xylene;

The catalyst is 2,3-dichloro-5,6-dicyano-p-benzoquinone, p-toluenesulfonic acid or methanesulfonic acid;

The reaction temperature is 7 (T90 ° C, the reaction time is 1 to 4 hours;

(2) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole dissolved in a polar aprotic solvent, iodine was added and the acid-binding agent, an inorganic base, to afford 3- iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I), the reaction equation is:

Wherein the polar aprotic solvent is N, N- dimethylformamide (DMF), N, N- dimethylacetamide, N, N- diethylformamide, N, N- diethyl-acetamide ;

Inorganic base acid binding agent is potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, potassium bicarbonate, sodium bicarbonate, cesium carbonate, lithium hydroxide;

The reaction temperature is 2 (T40 ° C, the reaction time is 8 to 20 hours.

[0009] A Axitinib intermediate (1) in preparation for the Nepalese Asif application, based on intermediate (1) and 2-vinyl pyridine Heck coupling reaction, followed sequentially nitro reduction and the diazotization reaction of iodine, and finally with a 2-mercapto–N- methylbenzamide was prepared by deprotection docking axitinib, including the following synthetic steps:

(I) Intermediate (1) and be given 2_ vinylpyridine Jie Heck coupling reaction to give (E) _6_ nitro _3- [2_ (P than-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, the reaction equation is:

(2) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- nitro indazole group reduction reaction, to give (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, The reaction equation is:

(3) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole diazo of the iodide to give (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole The reaction equation is:

(4) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole with 2- mercapto-methylbenzamide reaction -N-, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyrazol pyran-2-yl) -1H- indazol-6-yl] thio} benzamide, the reaction equation is:

(5) (E) -N- methyl-2- {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio} benzamide deprotected Axitinib (II), the reaction equation is:

Example 1

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (I) 6- nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added acetonitrile (2L), followed by addition of 6-nitro-indazole (163.1g, 1.0mol), 3, 4- dihydro -2H- pyran (168.2g, 2.0mol), 2,3- dichloro-5,6-dicyano-p-benzoquinone (22.7g, 0.1mol), was heated to 820C under reflux for 2 hours to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirring I hour, delamination, the organic phase washed with brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile, recrystallized from ethanol, 60 ° C and dried in vacuo 12 hours to give an off-white solid, 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 236.3 g, yield 95.6%, m.p. 110 ~ 120 ° C, 1Η NMR (CDCl3): δ 1.30-1.83 (m, 6Η, Η3, _Η5,), 3.82-3.93 (m, 2Η, Η6 ‘), 5.86 (m , 1Η, Η2 ‘), 8.10-8.12 (m, 2Η, Η3, Η5), 8.31 (m, 1Η; Η4), 8.55 (s, 1Η, Η7);

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (225.0g, 0.91mol, l.0eq) and potassium carbonate ( 251.6g, 1.82mol, 2.0eq), ice-cooled (10 ° C or less), followed by stirring, iodine (415.8g, 1.64mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system, addition time 2 hours , the reaction system was stirred at 25 ° C for 16 hours to complete the reaction, sodium thiosulfate was added (223.0g, 1.41mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the internal temperature 30 ° C Hereinafter, stirred for 30 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 30 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid (Ι), 326.5g, yield 96.2%, m.p. 135 ~ 137 ° C / H NMR (DMS0_d6): δ 1.60-1.61 (m, 2H, H4,, H5 ‘), 1.73-1.76 (m, 1H, H5’), 2.01-2.04 (m, 2H, H3 ‘, H4’), 2.35-2.38 (m, 1H, H3 ‘), 3.81-3.87 (m, 2H, H6’), 6.11-6.14 (dd, 1H, H2 ‘), 7.70-7.72 (d , 1H, H4),

8.05-8.07 (dd, 1H, H5), 8.79 (s, 1H, H7).

The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis of (I) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin-2 – yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2Η-) -1H- indazole

5L reaction flask in ethanol HOOmLdjC 1000mL and ammonium chloride (300.0g, 5.61mol), was dissolved with stirring, followed by addition of (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (255.0g, 0.73mol), was added iron powder (162.6g, 2.91mol), heated to 50 ° C the reaction was stirred for 2 hours to completion of the reaction, was cooled to 22 ° C, tetrahydrofuran 2L, stirred for I hour at room temperature, filtered through Celite, the filter cake washed with tetrahydrofuran and the filtrate was rotary evaporated to dryness, cooled to room temperature, water was added 2L, stirred for I hour at room temperature, pumping filtered, the filter cake washed with petroleum ether, 50 ° C and dried under vacuum for 12 hours to give a pale yellow solid 206.5g, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole, yield 88.6%, m.p. 162 ~ 164 ° C / H NMR (CDCl3): δ 1.63-1.77 (m, 2H, H4 ‘, H5 ‘), 2.02-2.06 (m, 1H, H5’), 2.17-2.18 (m, 1H, H4 ‘), 2.55-2.60 (m, 1H, H3’) 3.70-3.72 (m, 2H, H3 ‘, H6 ‘), 3.91 (s, 2H, NH2), 4.04-4.07 (m, 1H, H6’), 5.57-5.60 (dd, 1H, H2 ‘), 6.64-6.66 (dd, 1H, H5), 6.74-6.75 (d, 1H, H7), 7.13-7.16 (dd, 1H, pyridine H5), 7.48-7.50 (d, 1H, pyridine H4), 7.49-7.53 (d, 1H, J = 16Hz, vinyl H2), 7.64 -7.68 (m, 1H, pyridine H3), 7.78-7.82 (d, 1H, J = 16Hz, vinyl Hl), 7.82-7.83 (d, 1H, H4), 8.60-8.61 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

A 5L reaction flask was added 600mL of water and sodium nitrite (70.2g, 1.02mol), stirred and dissolved, and cooled to (TC, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl ] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g, 0.62mol) was dissolved in glacial acetic acid 1.3L, dropwise added to the system dropwise over I h, a solution process maintain an internal temperature of 0 ° C, the same temperature for I hour, dropping HCl solution (concentrated hydrochloric acid 112mL, water 200mL) at O ° C, the dropping time of 10 minutes, with the temperature for I h, TLC plate tracking point diazonium salt formation reaction (PE: EA = 1: 1). dropwise 800mL dichloromethane between 0 ° C, the dropping time of 5 minutes, potassium iodide (207.3g, l.25mol) and iodine (79.2g, 0.31mol) was dissolved water 600mL, in (TC dropwise added to the system at the same temperature for 2 hours to complete the reaction. The reaction mixture was poured into the system to 20% sodium thiosulfate solution (2L) and dichloromethane SOOmL and stirred, layered , the aqueous phase was extracted with dichloromethane frozen (2x800mL), dichloromethane phases were combined burning, 3M sodium hydroxide solution was added dropwise 3.5L, adjust the aqueous phase pH = 9 ~ 12, and water was added ammonia 200mL 400mL, stirred for 30 minutes , separated and the aqueous phase was extracted with dichloromethane (2×1.2L), the organic phases were combined, rotary evaporated to dryness, and purified through silica gel to give (E) -6- iodo-3- [2- (pyridin-2-yl ) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 176.0g, 65.4% yield, m.p. 142 ~ 143 ° C, 1H NMR (DMS0_d6): δ 1.58- 1.61 (m, 2H, H4 ‘, H5,) 1.72-1.78 (m, 1H, H5,), 1.97-2.04 (m, 2H, H3,, H4,), 2.38-2.44 (m, 1H, H3,) , 3.79-3.81 (m, 1H, H6,), 3.88-3.90 (m, 1H, H6,), 5.91-5.94 (dd, 1H, H2,), 7.29-7.31 (m, 1H, pyridine H5), 7.56 -7.60 (d, 1H ,, J = 16Hz, vinyl H2), 7.57-7.59 (m, 1H, pyridine H4), 7.69-7.71 (d, 1H, pyridine H3), 7.80-7.84 (m, 1H, H4 ), 7.89-7.93 (d, 1H, J = 16Hz, vinyl HI), 8.01-8.03 (d, 1H, H5), 8.25 (s, 1H, H7), 8.61-8.62 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

A 5L reaction flask was added DMF (1750mL) and (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1H- indazole (175.0g, 0.41mol), nitrogen, was added [1, I, – bis (diphenylphosphino) ferrocene] dichloropalladium dichloromethane complex (14.9g, 0.018mmol ), cesium carbonate (198.3g, 0.61mol) and dichloromethane 20mL, was added 2-mercapto -N- methylbenzamide (84.9g, 0.5Imol), heated to 80 ° C for 16 hours to complete the reaction, spin distilled was removed DMF, cooled to room temperature, ethyl acetate was added 3L, water 4L, stirred for 40 minutes, the organic phase was separated, washed with brine, layered, dried over sodium sulfate, filtered, and rotary evaporated to dryness, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio } benzamide, 165.6g, a yield of 86.7%, the melting point of 142 ~ 143 ° C;

The reaction equation is as follows:

(5) Synthesis of axitinib

In a 2L reaction flask was added (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H – indazol-6-yl] thio} benzamide (150.0g, 0.32mol), p-toluenesulfonic acid monohydrate (303.2g, 1.59mol), methanol (800mL) and water (150mL), nitrogen, heated to 65 ° C for 4 hours, spin evaporated to dryness and ethanol (800mL), 65 ° C was stirred for I hour, the ethanol was removed by rotary evaporation, then repeated three times, TLC spot plate tracking reaction (petroleum ether: ethyl acetate = 1: 1). Completion of the reaction, cooled to room temperature, rotary evaporated to dryness, water was added 500mL, stirred for I h, filtered, and the filter cake was washed with methanol and ice, and then added to the reaction vessel, ethyl acetate was added 450mL, stirred at 65 ° C 30 minutes. cooled to room temperature, suction filtered, the filter cake washed with ethyl acetate and freeze paint, water paint, 50 ° C and dried under vacuum for 12 hours to give a white solid 117.5g, Axitinib (II), yield 95.4%, HPLC purity 98.8 % / H NMR (DMS0_d6): δ 2.78 (d, 3H, CH3), 7.05 (dd, 1H), 7.19 (dd, 1H), 7.36-7.23 (m, 3H), 7.50 (dd, 1H), 7.58 ( d, 1H), 7.61 (s, 1H), 7.66 (d, 1H), 7.85-7.76 (m, 1H), 7.96 (d, 1H, J = 16Hz), 8.21 (d, 1H), 8.39 (q, 1H), 8.61 (d, 1H), 13.35 (s, 1H).

The reaction equation is as follows:

Example 2

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (1) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added ethyl acetate (2L), followed by addition of 6-nitro-indazole (163.14g, 1.0mol), 3, 4- dihydro -2H- pyran (210.3g, 2.5mol), toluene acid (20.7g, 0.12mol), heated to 78 ° C under reflux for 3 hours to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirred for I hour, stratification, the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile, recrystallized from ethanol , 60 ° C and dried in vacuo 12 hours to give an off-white solid 223.3g, 6- nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, yield 90.3%, m.p. 110 ^ 11 TC;

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g, 0.81mol, l.0eq) and sodium hydroxide (64.7g, 1.62mol, 2.0eq), ice-cooled (10 ° C or less), followed by stirring, iodine (369.6g, 1.46mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system, addition time 2 hours, the reaction system was stirred at 25 ° C for 12 hours to complete the reaction, sodium thiosulfate was added (198.2g, 1.25mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the temperature of 30 ° C or less, and stirred for 30 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 30 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid

(1), 294.3g, 97.5% yield, m.p. 136 ~ 137. . .

[0014] The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis (1) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2Η-) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin _2 _-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

The reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

The reaction equation is as follows:

(5) Synthesis of axitinib

The reaction equation is as follows:

Example 3

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (1) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

5L reaction flask in toluene (2L), followed by addition of 6-nitro-indazole (163.lg, 1.0mol), 3,4- dihydro -2H- pyran (193.5g, 2.3mol), methanesulfonic acid (14.4g, 0.15mol), heated to 85 ° C under reflux for 3.5 hours, to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirred for I hour, stratification, the organic phase was washed with brine wash, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile and paint, and recrystallized from ethanol , 60 ° C and dried in vacuo 12 hours to give an off-white solid, 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 234.4g, 94.8% yield, m.p. 111 ~ 112.. ;

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (225.0g, 0.91mol, 1.0eq) and potassium hydroxide ( 102.lg, 1.82mol, 2.0eq), ice-cooled below 10 ° C, with stirring, iodine (415.8g, 1.64mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system dropwise over 2 hours, The reaction system was stirred at 30 ° C for 10 hours to complete the reaction, sodium thiosulfate was added (223.0g, 1.41mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the internal temperature below 30 ° C , stirred for 45 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 45 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid

(1), 317.2g, 93.4% yield, m.p. 135 ~ 136 ° C.

The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis (1) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin _2 _-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

0.62mol) was dissolved in glacial acetic acid 1.3L, dropwise added to the system dropwise over I hour, added dropwise to maintain the internal temperature process 0 ° C, the same temperature for I h, HCl solution was added dropwise at O ° C (112mL of concentrated hydrochloric acid , water 200mL), was added dropwise for 10 minutes, with the temperature for I h, TLC plate tracking point diazonium salt formation reaction (PE: EA = 1: 1). Solution of methylene chloride at 0 ° C and 800mL, dropping time of 5 minutes, potassium iodide (207.3g, l.25mol) and iodine (79.2g, 0.31mol) dissolved in water 600mL, at (TC dropwise added to the system, same temperature for 2 hours to complete the reaction. The reaction system was poured into a mixture of 20% sodium thiosulfate solution (2L) and dichloromethane SOOmL and stirred, layers were separated, the aqueous phase was extracted with dichloromethane frozen (2x800mL ), methylene chloride phases were combined burning, 3M sodium hydroxide solution was added dropwise 3.5L, adjust the aqueous phase pH = 9 ~ 12, and water was added ammonia 200mL 400mL, stirred for 30 minutes, separated and the aqueous phase extracted with dichloromethane ( 2×1.2L), the organic phases were combined, rotary evaporated to dryness, and purified through silica gel to give (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H – pyran-2-yl) -1H- indazole, 176.0g, 65.4% yield, m.p. 142 ~ 143 ° C, 1H NMR (DMS0_d6): δ 1.58-1.61 (m, 2H, H4 ‘, H5,) 1.72-1.78 (m, 1H, H5,), 1.97-2.04 (m, 2H, H3,, H4,), 2.38-2.44 (m, 1H, H3,), 3.79-3.81 (m, 1H, H6,) , 3.88-3.90 (m, 1H, H6,), 5.91-5.94 (dd, 1H, H2,),

7.29-7.31 (m, 1H, pyridine H5), 7.56-7.60 (d, 1H ,, J = 16Hz, vinyl H2), 7.57-7.59 (m, 1H, pyridine H4), 7.69-7.71 (d, 1H, pyridine H3), 7.80-7.84 (m, 1H, H4), 7.89-7.93 (d, 1H, J = 16Hz, vinyl HI), 8.01-8.03 (d, 1H, H5), 8.25 (s, 1H, H7 ), 8.61-8.62 (d, 1H, pyridine H6); reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

The reaction equation is as follows:

(5) Synthesis of axitinib

yield 95.4%, HPLC purity 98.8 % / H NMR (DMS0_d6): δ 2.78 (d, 3H, CH3), 7.05 (dd, 1H), 7.19 (dd, 1H), 7.36-7.23 (m, 3H), 7.50 (dd, 1H), 7.58 ( d, 1H), 7.61 (s, 1H), 7.66 (d, 1H), 7.85-7.76 (m, 1H), 7.96 (d, 1H, J = 16Hz), 8.21 (d, 1H), 8.39 (q, 1H), 8.61 (d, 1H), 13.35 (s, 1H).

The reaction equation is as follows:

…………………….

http://www.scielo.br/scielo.php?pid=S0103-50532014001202186&script=sci_arttext&tlng=en

………………………

http://emuch.net/html/201202/4061970.html

SEE NMR……….

http://orgspectroscopyint.blogspot.in/2015/07/axitinib.html

………..

NMR source apexbt

http://dmd.aspetjournals.org/content/suppl/2014/03/07/dmd.113.056531.DC1/Supplemental__Data_Figures_56531.pdf

NMR..SEE……..http://www.abmole.com/download/axitinib-hnmr.pdf

MASS

References

“Inlyta (axitinib) dosing, indications, interactions, adverse effects, and more”. Medscape Reference. WebMD. Retrieved 25 January 2014.
Wilmes, LJ; Pallavicini, MG; Fleming, LM; Gibbs, J; Wang, D; Li, KL; Partridge, SC; Henry, RG; Shalinsky, DR; Hu-Lowe, D; Park, JW; McShane, TM; Lu, Y; Brasch, RC; Hylton, NM (April 2007). “AG-013736, a novel inhibitor of VEGF receptor tyrosine kinases, inhibits breast cancer growth and decreases vascular permeability as detected by dynamic contrast-enhanced magnetic resonance imaging”. Magnetic Resonance Imaging 25 (3): 319–27. doi:10.1016/j.mri.2006.09.041. PMID 17371720.
Rini, B; Rixe, O; Bukowski, R; Michaelson, MD; Wilding, G; Hudes, G; Bolte, O; Steinfeldt, H; Reich, SD; Motzer, R (June 2005). “AG-013736, a multi-target tyrosine kinase receptor inhibitor, demonstrates anti-tumor activity in a Phase 2 study of cytokine-refractory, metastatic renal cell cancer (RCC)”. Journal of Clinical Oncology ASCO Annual Meeting Proceedings 23 (16S): 4509.
Rugo, HS; Herbst, RS; Liu, G; Park, JW; Kies, MS; Steinfeldt, HM; Pithavala, YK; Reich, SD; Freddo, JL; Wilding, G (August 2005). “Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results”(PDF). Journal of Clinical Oncology 23 (24): 5474–83. doi:10.1200/JCO.2005.04.192.PMID 16027439.
“FDA Approves Inlyta for Advanced Renal Cell Carcinoma”. Drugs.com. January 27, 2012.
John Fauber, Elbert Chu (Oct 27, 2014). “The Slippery Slope: Is a Surrogate Endpoint Evidence of Efficacy?”. Milwaukee Journal Sentinel/MedPage Today.
Spano, JP; Chodkiewicz, C; Maurel, J; Wong, R; Wasan, H; Barone, C; Létourneau, R; Bajetta, E; Pithavala, Y; Bycott, P; Trask, P; Liau, K; Ricart, AD; Kim, S; Rixe, O (June 2008). “Efficacy of gemcitabine plus axitinib compared with gemcitabine alone in patients with advanced pancreatic cancer: an open-label randomised phase II study”. Lancet 371(9630): 2101–2108. doi:10.1016/S0140-6736(08)60661-3. PMID 18514303.
“Pfizer pancreatic cancer drug fails, trial halted”. Reuters. January 30, 2009.
“Pfizer’s Phase III Trial in mRCC Turns Up Positive Results”. 19 Nov 2010.
“ODAC Unanimously Supports Axitinib for Renal Cell Carcinoma”. 7 Dec 2011.
“INLYTA (axitinib) tablet, film coated [Pfizer Laboratories Div Pfizer Inc]”. DailyMed. Pfizer Laboratories Div Pfizer Inc. September 2013. Retrieved 25 January 2014.
“Inlyta : EPAR – Product Information” (PDF). European Medicines Agency. Pfizer Ltd. 17 December 2013. Retrieved 25 January 2014.
“Inlyta 1 mg 3mg, 5 mg & 7mg film-coated tablets – Summary of Product Characteristics (SPC)”. electronic Medicines Compendium. Pfizer Limited. 5 December 2013. Retrieved25 January 2014.
“PRODUCT INFORMATION INLYTA (axitinib)” (PDF). TGA eBusiness Services. Pfizer Australia Pty Ltd. 5 July 2013. Retrieved 25 January 2014.
Tea Pemovska,Eric Johnson,Mika Kontro,Gretchen A. Repasky,Jeffrey Chen,Peter Wells,Ciarán N. Cronin,Michele McTigue,Olli Kallioniemi,Kimmo Porkka,Brion W. Murray & Krister Wennerberg. “Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation”. Nature. doi:10.1038/nature14119.
“FDA Prescribing Information” (PDF). 30 Jan 2012.
Escudier, B; Gore, M. “Axitinib for the Management of Metastatic Renal Cell Carcinoma” (PDF). Drugs in R&d 11 (2): 113–126. doi:10.2165/11591240-000000000-00000. PMC 3585900. PMID 21679004.
Zhang Y (Jan 2014). “Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways.”. J Mol Med Rep 9 (1): 83–90.doi:10.3892/mmr.2013.1781. PMID 24213221.
[1]  http://www.cancer.gov/cancertopics/druginfo/axitinib[2]  http://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm289439.htm[3] Kosugi M, Shimizu T, T. Migita, Chemistry Letters , 1978 , pp 13-14.[4] Organic Process Research & Development 2008 , 12, 869? 876.[5] Furstner A.  Chem. Commun ., 2008 , 2873? 2875.[6] Thorarensen A. ,  Synlett ,    2010  , 2 pp 219 – 222.
[7]  http://en.wikipedia.org/wiki/Heck_reaction  – where you can find the reaction mechanism and many other useful information.
[8] Aoyama, T.,  Synthesis ,    2004  , 8 pp 1183-1186.

http://www.cosmoscience.org/blog/wp-content/uploads/2014/08/GSluggett-Application-of-Topochemical-Principles-and-Solid-State-Photoreactivity.pdf

Honokiol, from magnolia bark, shuts down cancer cells in lab

June 29, 2015 6:57 am / Leave a comment


Magnolia virginiana

Honokiol, from magnolia bark, shuts down cancer cells in lab

Compound in magnolia may combat head and neck cancers

Honokiol, from magnolia bark, shuts down cancer cells in lab

Magnolias are prized for their large, colorful, fragrant flowers. Does the attractive, showy tree also harbor a potent cancer fighter?

Yes, according to a growing number of studies, including one from VA and the University of Alabama at Birmingham that is now online in the journal Oncotarget.

The study focused on squamous cell head and neck cancers, a scourge among those who use tobacco and alcohol. According to the National Cancer Institute, at least 3 in 4 head and neck cancers are caused by the use of tobacco and alcohol. The cancers have only a 50 percent survival rate, killing some 20,000 Americans each year.

Enter honokiol–chemical formula C18H18O2. As one of the major active compounds in magnolia extract, the phytochemical has been used for centuries in traditional Chinese and Japanese medicine to treat anxiety and other conditions. More recently, scientists have been discovering that the compound, found in magnolia bark, is a wily and versatile adversary of cancer. It seems to exploit many biochemical pathways to shrink tumors of various types, or to keep them from growing in the first place.

The Alabama scientists have now shown how it works against head and neck cancers: It blocks a protein called epidermal growth factor receptor, or EGFR. Prior research has found that almost all head and neck cancer cells display an over-abundance of the protein, and it had been suggested in the literature as a potential target.

The VA-UAB team says, based on its lab studies, that honokiol binds more strongly with EGFR than does the drug gefitinib (sold as Iressa), which is commonly used to treat head and neck cancers.

The researchers tested honokiol on cell lines derived from human cancers of the oral cavity, larynx, tongue, and pharynx. In all cases, the botanical shut down the aberrant cells. The team also tested it against tumors implanted into mice, with similar results.

Senior author Dr. Santosh K. Katiyar and his colleagues wrote, “Conclusively, honokiol appears to be an attractive bioactive small molecule phytochemical for the management of head and neck cancer which can be used either alone or in combination with other available therapeutic drugs.”

Katiyar has published extensively in the past on other natural substances that work against tumors, especially skin cancer. Some of his recent work has focused on compounds in green tea, for example, and grape seed proanthocyanidins.

Purification

There are several methods for purifying and isolating honokiol. In nature, honokiol exists with its structural isomer magnolol, which differs from honokiol only by the position of onehydroxyl group. Because of the very similar properties of magnolol and honokiol, purification has often been limited to a HPLC or electromigration. However, methods developed in 2006 by workers in the lab of Jack L. Arbiser, took advantage of the proximity of the phenolic hydroxyl groups in magnolol, which form a protectable diol, to generate amagnolol acetonide (Figure 1), with a subsequent simple purification via flash chromatography over silica.^[4]

Figure 1

Magnolol and Honokiol are normally inseparable. Honokiol is easily separable from the protected magnolol acetonide

Additionally a rapid separation approach was published in the Journal of Chromatography A in 2007. The process uses high-capacity high-speed countercurrent chromatography(high-capacity HSCCC).^[5] Through this method honokiol can be separated and purified to above 98% purity with a high yield in under an hour.

Honokiol is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus Magnolia. It has been identified as one the chemical compounds in some traditional eastern herbal medicines along with magnolol, 4-O-methylhonokiol, andobovatol.

Traditional medicine

Seed Cone

Extracts from the bark or seed cones of the Magnolia tree have been widely used in traditional medicine in China, Korea, and Japan.^[2]

Houpu has traditionally been used in Eastern medicine as analgesic and to treat anxiety and mood disorders.^[2]^[6] However, it has been shown to treat a number of other conditions. In China, magnolia bark is called Houpu and is most commonly taken from the Magnolia obovata and the Magnolia officinalis species.^[7] Some Chinese traditional formulas containing Houpu include Banxia Houpu Tang (半夏厚朴丸), Xiao Zhengai Tang, Ping Wei San(平胃散) and Shenmi Tang.^[2] Japanese Kampo formulas include, Hange-koboku-to (半夏厚朴湯) and Sai-boku-to (柴朴湯).^[2]^[6]

Seeds

Modern medicine

In the late 1990s, honokiol saw a revival in interest as a potent and highly tolerable antitumorigenic and neurotrophiccompound.

Alternative medicine

Currently there are a large number of supplements containing honokiol on the market, and its use has been widely well received among practitioners of new age, homeopathic, and holistic medicine

Stereo image

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Mature Magnolia fruit just starting to open, with a few seeds visible

Honokiol
Names

IUPAC name 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol
Other names houpa, hnk
Identifiers
CAS Registry Number	35354-74-6
ChEMBL	ChEMBL16901
ChemSpider	65254
InChI [show]
Jmol-3D images	Image
KEGG	C10630
PubChem	72303
SMILES [show]
Properties
Chemical formula	C₁₈H₁₈O₂
Molar mass	266.334 g/mol
Appearance	White solid
Solubility in water	sparingly (25 °C)
Related compounds
Related biphenols	diethylstilbestrol, dihydroxyeugenol
Related compounds	magnolol. 4-O-Methylhonokiol
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

Magnolia seeds and fruit on a tree in northern Argentina

Magnolia grandiflora

http://www.google.com/patents/WO2006107451A2?cl=en

The root and stem bark of Magnolia has been used as a traditional Chinese medicine for the treatment of thrombotic stroke, gastrointestinal complaints, and anxiety. Honokiol (HNK), a substituted biphenyl and an active component isolated and purified from Magnolia, has anti-oxidant, antithrombosis, antibacterial, neurotrophic, xanthine oxidase inhibitory, and anxiolytic effects (Taira et al., Free Radic Res Commun. 1993;19 Suppl l:S71-77; Teng et al. Thromb Res. 1988;50:757-765; Clark et al., J. Pharm. Sci. 1981;70:951-952; Chang et al., Anticancer Res. 1994;14:501-506; Kuribara et al., J. Pharm Pharmacol. 1998;50:819-826; Esumi et al., Bioorg & Medicinal Chem Let 2004, 14: 2621-25).

In the early 1990s, reports of HNK’s anticancer effects were published. In 1994, Hirano et al (Life Sci. 1994;55(13): 1061-9) examined the anti leukemic-cell efficacy of 28 naturally occurring and synthetic flavonoids and 11 naturally occurring ligands on human promyelocytic leukemic cell line HL-60, and cytotoxicity of these compounds was compared with four clinical anti-cancer agents. HNK was identified as one of the most potent compounds in this screen, with an IC₅₀ value less than 100 ng/ml. In 1998, Hibasami et al. demonstrated that HNK induced apoptosis in human lymphoid leukemia Molt 4B cells (Hibasami et al., Int. J. MoI. Med. 1998).

HNK has also been found to induce apoptosis in human squamous cell lung cancer CH27 cells (Yang SE, et al Biochem Pharmacol. 2002;63:1641-1651) and in human colorectal RKO cells (Wang et al World J Gastroenterol. 2004; 10:2205-2208). In 2004, Chen et al. (World J Gastroenterol. 2004; 10: 3459-3463) reported that HNK was effective in an in vivo animal model of human colon cancer by inhibiting tumor growth and prolonging the lifespan of tumor bearing mice.

Honokiol is an inhibitor of angiogenesis and antitumor activity in vivo. HNK can cause apoptosis in tumor cells and inhibit angiogenesis through blocking phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), the major mitogenic and chemoattractant endothelial growth factor (Bai et al. (2003) J. Biol. Chem. 278, 35501- 35507). Honokiol also exhibits direct antitumor activity through induction of apoptosis through tumor necrosis factor apoptosis-inducing ligand (TRAIL/ Apo2L) signaling and has been found to be highly effective against angiosarcoma in nude mice in vivo (Bai et al. (2003) J. Biol. Chem. 278, 35501-35507).

Esumi et al. (Biorganic & Medicinal Chemistry Letters (2004) 14: 2621-2625) describe a synthesis method to produce HNK. This report also evaluates the structure activity relationship of O-methylated and/or its hydrogenated analogs of HNK in an in vitro neurotrophic assay. Esumi et al. conclude that the 5-allyl and 4′-hydroxyl groups are essential for the neurotrophic activity of HNK.

PCT Publication No. WO 02/076393 and U.S. Publication No. 2004/0105906 to Emory University describe pharmaceutical compositions and methods of treating conditions such angiogenic-, neoplastic-, and cancer-related conditions and skin conditions by administration of honokiol-type and/or magnolol-type compounds, as shown in Figures 1-4. For example, such compositions comprise at least one compound of formula Al :

AI wherein R₁, R₂, R₃, R₄, R₅, R₁, R₂, R₃, R₄, and R’₅ can be independently selected from groups that include, but are not limited to, hydrogen, hydroxyl groups, amides, amines, hydrocarbons, halogenated hydrocarbons, cyclic hydrocarbons, cyclic heterocarbons, halogenated cyclic heterocarbons, benzyl, halogenated benzyl, organo selenium compounds, sulfides, carbonyl, thiol, ether, dinitrogen ring compounds, thiophenes, pyridines, pyrroles, imidazoles, and pyrimidines. Honokiol-type and magnolol-type compounds are shown to inhibit SVR cell proliferation.

In November of 2004, Arbiser et al. reported that honokiol inhibited the growth of multuple myeloma cell lines via induction of Gl growth arrest, followed by apoptosis with IC50 values at 48h of 5 to 10 μg/mL. It was also reported that honokiol inhibited growth of doxorubin (Dox)-resistant (RPMI-Dox40), mephalan resistant (RPMI-LR5) and dexamethasone (Dex)-resistant (MM. IR) cell lines. It was suggested that the mechanism of honokiol triggered cytotoxicity is the honokiol induced increased expressin of Bax and Bad, down-regulated Mc-I protein expression, followed by caspase-8/9/3 cleavage, (Arbiser, J. et al. Poster at the American Society of Hematology Annual Meeting, 2004. Abstract published online November 4, 2004).

In July of 2005, Battle et al. reported that honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells (Blood. July 2005; 106:690- 697). Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined, which were primary tumor cells derived from B-CLL patients, and was more toxic toward B- CLL cells than to normal mononuclear cells. The honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). It was also reported that honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil.

In September 2005, Ishitsuka et al reported that honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and – independent apoptosis (Blood, 1 September 2005, Vol. 106, No. 5, pp.1794-1800). HNK induced cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM through induction of apoptosis via both caspase- dependent and -independent pathways. HNK also enhanced MM cell cytotoxicity and apoptosis induced by bortezomib.

It is an object of the present invention to provide new compounds, compositions, methods and uses for the treatment of disorders associated with angiogenesis, cell proliferation, tumor growth, tumorogenesis, and myeloma.

the intermediates for the synthesis of honokiol are 3-allyl-4- hydroxybenzeneboronate 5 and 4-allyl-2-bromophenol 9. The boronate 5 can be prepared from 2-iodophenol 1 by bromination, followed by Suzuki coupling to introduce the allyl group, and boronation under Suzuki conditions. Compound 9 can be prepared from 4- iodophenol 6 by bromination and allylation (Suzuki coupling). The coupling of 5 and 9 under Suzuki conditions can yield honokiol from Suzuki coupling, not other allyl-oriented products from the Heck reaction, as it was shown that Suzuki coupling can succeed in the presence of C=C double bond (see Miyaura, N.; Suzuki, A. (1995), Chem. Rev. 95, 2457- 2483; and Suzuki, A. (1999), J. Organometal. Chem. 576, 147-168, and the references cited therein). Thus, honokiol and derivatives can be synthesized from commercially available starting materials in 6 steps (Scheme 1). Scheme 1

Treatment of honokiol with TMS-diazomethane in methanol results in mono- and di- methylated compounds I-III, and hydrogenation of honokiol with Wilkinson’s catalyst yields di- and tetrahydrohonokiols VI-VIII, as reported by Esumi, T. et al. (2004), Bioorg. Med. Chern. Lett. 14, 2621-2625. The amino and fluoro analogues (IV and V) can be constructed from iodoacetanilide under Suzuki coupling conditions. From 2-iodoacetanilide 10, after bromination, allylation, and boronation, the boronated intermediate 13 can be prepared. The other bromo intermediate 16 can be prepared from 4-iodoacetanilide 14 via bromination and allylation. The coupling of boronate 13 and bromide 16 under Suzuki conditions can afford, after deprotection, the compound IV. Diazotization followed by Schiemann reaction can convert the amino analogue TV to fluoro analogue V (Scheme 2).

The dimethoxy honokiol derivative, III, can also be prepared, for example, by the treatment of honokiol with potassium carbonate, iodomethane. (Scheme 2a). The hydrogenated honokiol analog can alternatively be prepared by the hydrogenation of honokiol with sodium borohydride and nickel(II) chloride to yields tetrahydrohonokiols VI- Vπi. (Scheme 2a). Scheme 2a

The preparation of the vinyl analogue IX is based on combining the Wittig reaction with Suzuki coupling. The intermediate aldehyde 18 can be prepared from 4-iodophenol 17 via the Reimer-Tiemann reaction, while 3-bromo-4-hydroxybenzenealdehyde 23 can be prepared from para-hydroxybenzoic ester 21 via bromination and reduction. The Wittig reaction of these two aldehydes can yield the corresponding vinyl substituted benzenes 19 and 24. Compound 19 can afford the boronate 20, which can be coupled with 24, to yield the compound IX (Scheme 3).

Reagents and conditions: (a) CHCl₃, aq. NaOH, 70 °C; (b) Ph₃PCH₃Br₁ n-BuLi, THF; (c) PdC!₂(dppf), dppf, KOAc, dioxane, bis(pinacoato)diboron, 80 °C; (d) DIBALH, -70 °C; (e) PdCI₂(dppf), dppf, K₃PO₄, dioxane, reflux.

For the synthesis of honokiol analogues with changed positions of the allyl or hydroxyl groups, the boronate 5, and the bromophenols 4 and 9 can be used as intermediates. Suzuki coupling of one of these intermediates with an appropriate halide or boronate can provide the compounds X-XVII. Compounds X-XII and XTV-XV can be prepared by Suzuki coupling of boronate 5 with an appropriate halide. Halide 25, needed for compound X, can be prepared from 2-bromo-6-iodophenol 2 via allylation, while the intermediate, 5-allyl-2- bromophenol 29 for compound XI, can be furnished from 3-iodophenol 26 via bromination and allylation. The preparation of halide 5-allyl-3-bromophenol 33, an intermediate for the synthesis of compound XIV, requires an organothallium reagent. The thallation of 3- bromophenol 30 followed by treatment with iodide can yield 3-bromo-5-iodophenol 32. After allylation, the allyl-substituted intermediate 33 can be prepared. The synthesis of compound XII can begin with 2-iodoacetanilide 10, via sulfonation, nitration, and reduction to obtain the intermediate 36. Aniline 36, after diazotization, followed by acid and base treatments, will afford 2-amino-3-iodophenol 37. Diazotization, Sandmeyer reaction, and allylation of compound 37 will yield halide 39. By a coupling reaction of these halides (25, 29, 33, and 39) with boronate 5, these compounds (X-XII, and XTV) can be prepared. Compound XV can be synthesized by Suzuki coupling of halide 4 with boronate 5 (Scheme 4).

Scheme 4

Alternatively, compounds X, XV, and XVII can be synthesized by an allylation- Claisen pathway. Biphenol compounds can be reacted first with potassium carbonate and allyl bromide, followed by reaction with BCl₃ to yield honokiol-like compounds, for example, X, XV, and XVII. (Scheme 4a). To a cooled solution (O⁰C with an ice bath) of diallyl starting material (1 eq.) in dry diehloromethane (Concentration of the solution : 0.1 mol.L^“1) was added dropwise a solution Of BCl₃ (IM in diehloromethane; 1.5 eq. = 0.75 eq for each allyl group). The reaction is then stirred at O⁰C until disappearance of the starting material on TLC (If after 15 minutes, the reaction is not complete, 1 more equivalent of BCl₃ can be added). After hydrolysis with water (about same volume than diehloromethane), the two layers are separated. The organic layer is washed again with water, dried under MgSO₄ then evaporated under vacuum. The residue is finally purified by column chromatography to give the di- hydroxy derivative . Scheme 4a

Bromide 9 is also a useful intermediate for coupling with some boronates. For example, Suzuki coupling of bromide 9 with boronate 42, which is prepared from 4-bromo-3- iodophenol 40 via allylation and boronation, can yield the compound XIII. Similarly, the coupling between bromide 9 and boronate 43 can afford the compound XVT. The compound XVII can be prepared from 4-allyl-2-bromophenol 9 via boronation followed by Suzuki coupling with 2-allyl-6-bromophenol 25 (Scheme 5).

The compounds XVIII and XIX can be synthesized from commercially available bisphenol 45 and the dihydroxynaphthalene-disulfuric acid salt 47. Thus, the bisphenol 45, through the Williamson reaction and Claisen rearrangement, can be converted to compound XVπi. Similarly, desulfonation of dihydroxynaphthalene-disulfuric acid salt .47, followed by the Williamson reaction and Claisen rearrangement, can produce the compound XIX (Scheme 6).Scheme 6

Dioxolane compounds can be prepared from magnoliol by reaction of magnoliol with 2,2′-dimethoxypropane and p-toluenesulfonic acid. (Scheme 7). This synthesis also provides a method of separating mixtures of honokiol and magnoliol. Scheme 7

The following examples are offered by way of illustration and not by way of limitation.

……..

http://www.google.com/patents/US20080300298

BEZ 235 (NVP-BEZ235), Dactolisib

June 12, 2015 3:57 am / Leave a comment

BEZ235 (NVP-BEZ235)Dactolisib

4-[2,3-dihydro-3-methyl-2-oxo-8-(3-quinolinyl)-1H-imidazo[4, 5-c]quinolin-1-yl]-α,α-dimethyl-benzeneacetonitrile

2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-1H,2H,3H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]-propionitrile

Chemical Formula: C₃₀H₂₃N₅O

CAS Number: 915019-65-7

Molecular Weight: 469.54

PHASE 2, NOVARTIS

CANCER, BLADDER

NVP-BEZ235 is a dual inhibitor of phosphatidylinositol 3-kinase (P13K)and the downstream mammalian target of rapamycin (mTOR) by binding to the ATP-binding cleft of these enzymes. It specifically blocks the dysfunctional activation of the P13K pathway and induce G(1) arrest. NPV-BEZ235 has been shown to inhibit VEGF induced cell proliferation and survival in vitro and VEGF induced angiogenesis in vivo. It has also been shown to inhibit the growth of human cancer in animal models.

BEZ-235 is an orally active phosphatidylinositol 3-kinase (PI3K) inhibitor in early clinical trials at Novartis for the treatment of advanced breast cancer, renal cell carcinoma, solid tumors and castration-resistant prostate cancer. Phase I clinical trials were also under way at the company for the treatment of glioma, however, no developments in this indication has been reported. Phase II clinical trials are ongoing at Johann Wolfgang Goethe Universität for the treatment of relapsed or refractory acute leukemia.
PI3Ks perform various functions, promoting cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Mutations leading to increased activity of PI3Ks, including faulty production or action of PI3K antagonists, have been found in many cancers.

……………………………..

WO 2006122806

http://www.google.com/patents/WO2006122806A2?cl=en

…………………………..

WO 2008064093

2-methyl-2-[4-(3-methyl- 2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile of formula I (compound I),

Example 1

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]-propionitrile

In a suitable lab glass reactor are placed 45.0 g of starting 2[4-(8-bromo-3-methyl-2-oxo-2,3- dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]2-methyl-propionitrile together with 2.25 g of bistriphenylphosphine’palladium dichloride in 445 ml N,N-dimethylformamide. This mixture is heated to 95 ⁰C and then a solution of 22.2 g of 3-quinoline boronic acid in a mixture of 225 ml DMF, 300 ml H₂O and 60 g of KHCO₃ is added. This mixture is heated for 2 h at 95 ⁰C. Then 1080 ml H₂O are added. The product 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl- 2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile precipitates. The mixture is cooled within 1.5 h to 0 – 5 °C. After stirring at that temperature for 2 h the crude product is filtered and washed with 300 ml H₂O. This product is dried in vacuo at 60 ⁰C for 18 h, to yield crude product.

40 g of this crude product is dissolved in 200 ml formic acid at 60 ⁰C. 8 g of active charcoal and Smopex 234 are added. The mixture is stirred at 60 ⁰C for 1 h, the charcoal is filtered, the residue washed with 80 ml formic acid and then 175 ml formic acid are distilled off in vacuo. Then 320 ml methanol are added and the mixture is heated at reflux for 3 h. The purified product precipitates from the reaction mixture. The mixture is cooled to 0 – 5 ⁰C within 1 h, then stirred 2 h at that temperature is finally filtered and washed with 80 ml cold methanol. This recrystallisation procedure is repeated again. Finally the twice recrystallised material is dried in vacuo at 60 ⁰C to yield purified 2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin- 3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile.

Example 1a 5-Bromo-2-(2-nitro-vinylamino)-benzoic acid

A suspension of 25 g (16 mmol) of 2-amino-5-bromo-benzoic acid (Fluka, Buchs, Switzerland) in H₂O-HCI (37%) (10:1) is stirred for 8 h and then filtered (solution A). 8.17 g (255 mmol) of nitromethane (Fluka, Buchs, Switzerland) are added over 10 min to an ice- bath cooled mixture of 35 g of ice and 15.3 g (382 mmol) of NaOH. After stirring for 1 h at 0 ⁰C and 1 h at rt, the solution is added at 0 ⁰C to 28 g of ice and 42 ml of HCI (37%) (solution B). Solutions A and B are combined and the reaction mixture is stirred for 18 h at rt. The yellow precipitate is filtered off, washed with H₂O and dried in vacuo at 40⁰C to give the title compound. ES-MS: 287, 289 (M + H)⁺, Br pattern; ¹H NMR (DMSO-d₆): δ 13.7-14.6/br s (1 H), 12.94/d (1 H), 8.07/d (1 H), 8.03/dd (1 H), 7.83/dd (1 H), 7.71/d (1 H), 6.76/d (1 H).

Example 1b 6-Bromo-3-nitro-quinolin-4-ol

29 g (101 mmol) of 5-bromo-2-(2-nitro-vinylamino)-benzoic acid (Example 1a) and 11.9 g (121 mmol) of potassium acetate in 129 ml (152 mmol) of acetic anhydride are stirred for 1.5 h at 120 ⁰C. The precipitate is filtered off and washed with acetic acid until the filtrate is colorless, then is washed with H₂O and dried in vacuo to give the title compound. ES-MS: 269, 271 (M + H)⁺, Br pattern; analytical HPLC: W= 2.70 min (Grad 1).

Example 1c 6-Bromo-4-chloro-3-nitro-quinoline

20 g (74.3 mmol) of 6-bromo-3-nitro-quinolin-4-ol (Example 1b) in 150 ml (1.63 mol) of POCI₃ are stirred for 45 min at 120 °C. The mixture is cooled to rt and poured slowly into ice- water. The precipitate is filtered off, washed with ice-cold water, and dissolved in CH₂CI₂. The organic phase is washed with cold brine, and the aqueous phase is discarded. After drying over MgSO₄, the organic solvent is evaporated to dryness to provide the title compound. ¹H NMR (CDCI₃): J9.20/S (1H), 8.54/d (1H), 8.04/d (1H), 7.96/dd (1H); analytical HPLC: W= 4.32 min (Grad 1).

Example 1d 2-Methyl-2-(4-nitro-phenyl)-propionitrile

O .

To 15 g (92.5 mmol) of (4-nitro-phenyl)-acetonitrile (Fluka, Buchs, Switzerland), 1.64 mg (5.09 mmol) of tetrabutylammonium bromide (Fluka, Buchs, Switzerland) and 43.3 g (305 mmol) of iodomethane in 125 mL of CH₂CI₂ are added 1O g (250 mmol) of NaOH in 125 ml of water. The reaction mixture is stirred for 20 h at RT. After this time, the organic layer is separated, dried over MgSO₄, and evaporated to dryness. The residue is dissolved in diethylether and treated with black charcoal for 30 min, filtered over Celite and evaporated in vacuo to give the title compound as a pale yellow solid. Analytical HPLC: t_ret= 3.60 minutes (Grad 1).Example 1e (2-(4-Amino-phenyl)-2-methyl-propionitrile

16 g (84.1 mmol) of 2-methyl-2-(4-nitro-phenyl)-propionitrile (Example 1d) and 4.16 g of Raney-Ni are shacked in 160 ml of THF-MeOH (1:1) under 1.1 bar of H₂ for 12 h at rt. After completion of the reaction, the catalyst is filtered-off and the filtrate is evaporated to dryness. The residue is purified by flash chromatography on silica gel (hexane-EtOAc 3:1 to 1:2) to provide the title compound as an oil. ES-MS: 161 (M + H)⁺; analytical HPLC: t_ret= 2.13 minutes (Grad 1).

Example 1f 2-[4-(6-Bromo-3-nitro-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile

18 g (62.6 mmol) of 6-bromo-4-chloro-3-nitro-quinoline (Example 1c) and 11 g (68.9 mmol) of (2-(4-amino-phenyl)-2-methyl-propionitrile (Example 1e) are dissolved in 350 ml of acetic acid and stirred for 2 h. After this time, water is added and the yellow precipitate is filtered off and washed with H₂O. The solid is dissolved in EtOAc-THF (1 :1), washed with sat. aqueous NaHCO₃ and dried over MgSO₄. The organic phase is evaporated to dryness to give the title compound as a yellow solid. ES-MS: 411 , 413 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.69 min (Grad 1).

Example 1q 2-[4-(3-Amino-6-bromo-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile

24 g (58.4 mmol) of 2-[4-(6-bromo-3-nitro-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile (Example 1e) is shacked in 300 ml of MeOH-THF (1:1) under 1.1 bar of H₂ in the presence of 8.35 g of Raney-Ni for 1 h. After completion of the reaction, the catalyst is filtered off and the filtrate is evaporated to dryness to give the title compound as a yellow foam. ES-MS: 381 , 383 (M + H)⁺, Br pattern; analytical HPLC: W= 3.21 min (Grad 1).

Example 1h

2-[4-(8-Bromo-2-oxo-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-2-methyl- propionitrile

A solution of 5 g (13.1 mmol) of 2-[4-(3-amino-6-bromo-quinolin-4-ylamino)-phenyl]-2- methyl-propionitrile (Example 1g) and 1.59 g (15.7 mmol) of triethylamine in 120 ml CH₂CI₂ is added over 40 min to a solution of 2.85 g (14.4 mmol) of trichloromethyl chloroformate (Fluka, Buchs, Switzerland) in 80 ml of CH₂CI₂ at 0⁰C with an ice-bath. The reaction mixture is stirred for 20 min at this temperature then is quenched with sat. aqueous NaHCO₃, stirred for 5 min and extracted with CH₂CI₂. The organic layer is dried over Na₂SO₄, filtered and evaporated in vacuo to give crude title compound as a brownish solid. ES-MS: 407, 409 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.05 min (Grad 1). Example 1i

2-[4-(8-Bromo-3-methyl-2-oxo-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-2- methyl-propionitrile

To a solution of 3.45 g (8.47 mmol) of 2-[4-(8-bromo-2-oxo-2,3-dihydro-imidazo[4,5- c]quinolin-1-yl)-phenyl]-2-methyl-propionitrile (Example 1h), 1.8 g (12.7 mmol) of iodomethane (Fluka, Buchs, Switzerland) and 273 mg (0.847 mmol) of tetrabutylammonium bromide (Fluka, Buchs, Switzerland) in 170 ml of CH₂CI₂ is added a solution of 508 mg (12.7 mmol) of NaOH (Fluka, Buchs, Switzerland) in 85 ml of H₂O. The reaction mixture is stirred for 2 days and 900 mg (6.35 mmol) of iodomethane and 254 mg (6.35 mmol) of NaOH in 5 ml of H₂O are added. The reaction mixture is stirred for 1 day at rt . After this time, the reaction is quenched with H₂O and extracted with CH₂CI₂ (2*). The organic layer is washed with brine, dried over Na₂SO₄, filtered and evaporated in vacuo to give the title compound as a beige solid. ES-MS: 421 , 423 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.15 min (Grad 1).

Example 2

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]propionitrile p-toluenesulfonate salt

26.5 g of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1- yl)-phenyl]propionitrile are placed together with 55 ml formic acid into a glass reactor. This mixture is heated to 60 ⁰C to get a clear solution. This solution is clearfiltered and washed with 36 ml formic acid. Then formic acid is distilled off until the volume of the residual solution is 55 ml. Then a solution of 11.3 g of p-toluenesulfonic acid in 228 ml acetone is added at 50 ⁰C, followed by further addition of 822 ml acetone within 30 minutes. The salt precipitates from the reaction mixture. The mixture is cooled to 0 ⁰C within 2 h, stirred at that temperature for 3 h, is then filtered and washed with 84 ml acetone. The product^‘ is dried at 60 ⁰C in vacuo for 18 h to yield 29.8 g (82.4 %) of the 2-Methyl-2-[4-(3-methyl-2-oxo-8- quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile p-toluenesulfonate salt (crystalline form A). The crystalline forms of the present invention are synthesized in accordance with the following examples which are illustrative without limiting the scope of the present invention.

Example 3:

Preparation of form A of 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro- imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile

Form A of compound I can be manufactured in the following way: 241 g of free base are dissolved 2.4 I acetic acid at 50 ⁰C. The solution is clearfiltered, washed with 250 ml acetic acid and then at 50 ⁰C 7.2 I of water are added. The free base starts precipitating. The mixture is cooled within 1 h to 25 ⁰C, is then filtered and washed with 10 I H₂O. The free base is then dried in vacuo at 50 ⁰C over night to yield 204 g of free base.

References

Maira et al. (2008) Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther. 7(7):1851-63.

Schnell et al. (2008) Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical imaging. Cancer Res. 68(16):6598-607.

Cho et al. (2010) The efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 compared with rapamycin in renal cell carcinoma. Clin Cancer Res. 16(14):3628-38.

WO2005054237A1	19 Nov 2004	16 Jun 2005	Hans-Georg Capraro	1h-imidazoquinoline derivatives as protein kinase inhibitors
WO2006122806A2	18 May 2006	23 Nov 2006	Novartis Ag	1,3-dihydro-imidazo [4,5-c] quinolin-2-ones as lipid kinase inhibitors
CL11872006A				Title not available

Citing Patent	Filing date	Publication date	Applicant	Title
WO2009118324A1 *	24 Mar 2009	1 Oct 2009	Novartis Ag	5imidazoquinolines and pyrimidine derivatives as potent modulators of vegf-driven angiogenic processes
WO2013049300A1 *	27 Sep 2012	4 Apr 2013	Dana-Farber Cancer Institute, Inc.	Method of treating mucoepidermoid carcinoma
WO2013152717A1	9 Apr 2013	17 Oct 2013	Shanghai Yunyi Healthcare Management Co., Ltd.	Fused pyrimidine compound, and preparation method, intermediate, composition， and uses thereof
EP2474323A2 *	24 Mar 2009	11 Jul 2012	Novartis AG	Imidazoquinolines and pyrimidine derivatives as potent modulators of vegf-driven angiogenic processes
US8476294	2 Jun 2010	2 Jul 2013	Novartis Ag	1H-imidazo[4,5-c]quinolinone derivatives

ZSTK 474

May 20, 2015 7:32 am / 1 Comment on ZSTK 474

ZSTK474

4-[4-[2-(difluoromethyl)benzimidazol-1-yl]-6-morpholin-4-yl-1,3,5-triazin-2-yl]morpholine

ZSTK474; 475110-96-4; 4,4′-(6-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine; ZSTK-474; ZSTK 474; TCMDC-137004;

2-(2-Difluoromethylbenzimidazol-1-yl)-4,6-bis(morpholino)-1,3,5-triazine

2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine

Zenyaku Kogyo (Innovator)

phase2………Treatment of Solid Tumors Therapy

ZSTK474 is a cell permeable and reversible P13K inhibitor with an IC₅₀ at 6nm. It was identified as part of a screening library, selected for its ability to block tumor cell growth. ZSTK474 has shown strong antitumor activities against human cancer xenographs when administered orally to mice without a significant toxic effect.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases.

However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002.

Other Names: ZSTK-474

Chemical Formula: C₁₉H₂₁F₂N₇O₂

CAS Number: 475110-96-4

Molecular Weight: 417.41

WO 2002088112

http://www.google.co.in/patents/EP1389617A1?cl=en

The condensation of 2,4-dichloro-6-(4-morpholinyl)-1,3,5-triazine

with 2-(difluoromethyl)-1H-benzimidazole by means of K2CO3 in DMF gives

2-chloro-4-[2-(difluoromethyl)-1H-benzimidazol-1-yl]-6-(4-morpholinyl)-1,3,5-triazine ,

which is then condensed with morpholine by means of K2CO3 in DMF to afford the target trisubstituted triazine.

ZSTK474

^aReagents and conditions: (i) K₂CO₃, DMF, room temp; (ii) morpholine, DMF or THF, room temp; (iii) NaH or K₂CO₃, DMF or DMSO, 120 °C.

2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine(compound 19)
Melting point: 211-214°C
NMR(CDCl₃) δ : 3.79(8H, t, J=4Hz), 3.88(8H, t, J=4Hz), 7.3-7.4(2H, m), 7.56(1H, t, J=53Hz), 7.88(1H, d, J=7Hz), 8.32(1H, d, J=7Hz)
MS m/z: 417(M⁺

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J. Med. Chem., 2011, 54 (20), pp 7105–7126

DOI: 10.1021/jm200688y

http://pubs.acs.org/doi/abs/10.1021/jm200688y

1 (0.35 g, 84% yield): mp (EtOH) 217–219 °C (lit. 211–214 °C);

¹H NMR (CDCl₃) δ 8.33 (dd, J = 7.3, 1.4 Hz, 1H), 7.89 (dd, J = 7.2, 1.5 Hz, 1H), 7.56 (t, J_HF= 53.6 Hz, 1H), 7.46–7.37 (m, 2H), 3.91–3.86 (m, 8H), 3.81–3.76 (m, 8H).

Kawashima, S.; Matsuno, T.; Yaguchi, S.; Sasahara, H.; Watanabe, T.Preparation of Heterocyclic Compounds as Antitumor Agents. PCT Int. Appl. WO 02088112, 2002;

Chem. Abstr. 2002, 137, 370113.

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2-(difluoromethyl)-1H-benzimidazole

A mixture of o-phenylenediamine (5.41 g, 50 mmol) and difluoroacetic acid (9.6 g, 100 mmol) in 4 M HCl (20 mL) was heated under reflux for 1 h and diluted with hot water (50 mL). The solution was treated with charcoal and filtered through Celite before being neutralized with aqueous NH₃. The resulting white precipitate was collected, washed with water, and dried to give 2-(difluoromethyl)-1H-benzimidazole (6.07 g, 72% yield): mp 156–158 °C; ¹H NMR (DMSO-d₆) δ 13.28 (br, 1H), 7.76–7.68 (m, 1H), 7.61–7.54 (m, 1H), 7.36–7.26 (m, 2H), 7.26 (t,J_HF= 53.3 Hz, 1H).

Ge, F.; Wang, Z.; Wan, W.; Lu, W.; Hao, J.One-pot synthesis of 2-trifluoromethyl and 2-difluoromethyl substituted benzo-1,3-diazoles Tetrahedron Lett. 2007, 48, 3251–3254

TRIAZINE, PYRIMIDINE AND PYRIDINE ANALOGS AND THEIR USE AS THERAPEUTIC AGENTS AND DIAGNOSTIC PROBES [US2011275762]2011-11-10

Patent	Submitted	Granted
Heterocyclic compound and antitumor agent containing the same as active ingredient [US7071189]	2004-06-17	2006-07-04
Treatment of prostate cancer, melanoma or hepatic cancer [US2007244110]	2007-10-18
Heterocyclic compound and antitumor agent containing the same as effective ingredient [US7307077]	2006-11-02	2007-12-11
IMMUNOSUPPRESSIVE AGENT AND ANTI-TUMOR AGENT COMPRISING HETEROCYCLIC COMPOUND AS ACTIVE INGREDIENT [US7750001]	2008-05-15	2010-07-06
PYRIMIDINYL AND 1,3,5-TRIAZINYL BENZIMIDAZOLES AND THEIR USE IN CANCER THERAPY [US2011009405]	2011-01-13
SUBSTITUTED PYRIMIDINES AND TRIAZINES AND THEIR USE IN CANCER THERAPY [US2011053907]	2011-03-03
IMMUNOSUPPRESSIVE AGENT AND ANTI-TUMOR AGENT COMPRISING HETEROCYCLIC COMPOUND AS ACTIVE INGREDIENT [US2010267700]	2010-10-21
AMORPHOUS BODY COMPOSED OF HETEROCYCLIC COMPOUND, SOLID DISPERSION AND PHARMACEUTICAL PREPARATION EACH COMPRISING THE SAME, AND PROCESS FOR PRODUCTION OF THE SAME [US8227463]	2010-09-30	2012-07-24
PYRAZOLO[1,5-a]PYRIDINES AND THEIR USE IN CANCER THERAPY [US2010226881]	2010-09-09
PYRIMIDINYL AND 1,3,5-TRIAZINYL BENZIMIDAZOLE SULFONAMIDES AND THEIR USE IN CANCER THERAPY [US2010249099]	2010-09-30

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Zenyaku Kogyo

Sector: Health Care

Industry: Biotech & Pharma

Sub-Industry: Specialty Pharma

Zenyaku Kogyo Co. Ltd. produces pharmaceuticals. The Company manufactures and sells over-the-counter drugs, health foods, and prescription medicines, as well as skin care products.

Address:

5-6-15 Otsuka

Bunkyo, 112-8650

Japan

Phone: 81-3-3946-1111

Fax: 81-3-3946-1130

Web url: www.zenyaku.co.jp

Otsuka

Bunkyo

……

Evofosfamide

April 28, 2015 4:38 am / 1 Comment on Evofosfamide

Evofosfamide, HAP-302 , TH-302

Evofosfamide
Names

IUPAC name (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate
Other names TH-302; HAP-302
Identifiers
CAS Registry Number	918633-87-1
ChemSpider	10157061
InChI [show]
Jmol-3D images	Image
PubChem	11984561
SMILES [show]
Properties
Molecular formula	C₉H₁₆Br₂N₅O₄P
Molar mass	449.04 g·mol⁻¹
Solubility in water	6 to 7 g/l

TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs

evofosfamide (first disclosed in WO2007002931), useful for treating cancer.

Threshold Pharmaceuticals and licensee Merck Serono are codeveloping evofosfamide, the lead in a series of topoisomerase II-inhibiting hypoxia-activated prodrugs and a 2-nitroimidazole-triggered bromo analog of ifosfamide, for treating cancer, primarily soft tissue sarcoma and pancreatic cancer (phase 3 clinical, as of April 2015).

In November 2014, the FDA granted Fast Track designation to the drug for the treatment of previously untreated patients with metastatic or locally advanced unresectable soft tissue sarcoma.

Evofosfamide (INN,^[1] USAN;^[2] formerly known as TH-302) is an investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. The prodrug is activated only at very low levels of oxygen (hypoxia). Such levels are common in human solid tumors, a phenomenon known as tumor hypoxia.^[3]

Evofosfamide is being evaluated in clinical trials for the treatment of multiple tumor types as a monotherapy and in combination with chemotherapeutic agents and other targeted cancer drugs
Discovered at Threshold, TH-302 is a hypoxia-activated prodrug (HAP) designed to exploit low oxygen levels in hypoxic tumor regions. Therapeutics that specifically target resistant hypoxic zones could provide significant additional antitumor activity and clinical benefit over current chemotherapeutic and radiation therapies.

Evofosfamide (TH-302) was developed by Threshold Pharmaceuticals Inc. (Threshold).^[4] The company is located in South San Francisco, CA, USA.

In 2012, Threshold signed a global license and co-development agreement for evofosfamide with Merck KGaA, Darmstadt, Germany, which includes an option for Threshold to co-commercialize eofosfamide in the United States. Threshold is responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA are developing evofosfamide together.^[5] From 2012 to 2013, Merck KGaA paid 110 million US$ for upfront payment and milestone payments to Threshold. Additionally, Merck KGaA covers 70% of all evofosfamide development expenses.^[6]
Discovered at Threshold, TH-302 is a hypoxia-activated prodrug (HAP) designed to exploit low oxygen levels in hypoxic tumor regions. Therapeutics that specifically target resistant hypoxic zones could provide significant additional antitumor activity and clinical benefit over current chemotherapeutic and radiation therapies.

History

Date	Event
Jun 2005	Threshold files evofosfamide (TH-302) patent applications in the U.S.^[49]
Jun 2006	Threshold files a evofosfamide (TH-302) patent application in the EU and in Japan^[50]
Sep 2011	Threshold starts a Phase 3 trial (TH-CR-406) of evofosfamide in combination with doxorubicin in patients with soft tissue sarcoma
Feb 2012	Threshold signs an agreement with Merck KGaA to co-develop evofosfamide
Apr 2012	A Phase 2b trial (TH-CR-404) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer meets primary endpoint

SEE

WO2007002931

http://www.google.com/patents/WO2007002931A2?cl=en

Example 8

Synthesis of Compounds 25, 26 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at – 1O⁰C was added a solution OfPOCl₃ (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3×25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

Isophosphoramide mustard

can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).

The phosphoramidate alkylator toxin:

was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.

Example 25

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

A suspension of the nitro ester (39.2 g, 196.9 rnmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HCl and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS O4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%). Example 26

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 niL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetonitrile bath (temperature < -20 ⁰C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than O⁰C. The reaction mixture was warmed up to O⁰C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

……………………………………….

WO-2015051921

EXAMPLE 1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (R_t = 7,7 min): 97,9% (a/a).

REFERENCES

WHO Drug Information; Recommended INN: List 73
2
Adopted Names of the United States Adopted Names Council
3
Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q. PMID 18257544.
4
Website of Threshold Pharmaceuticals Inc.
5
Threshold Pharmaceuticals and Merck KGaA Announce Global Agreement to Co-Develop and Commercialize Phase 3 Hypoxia-Targeted Drug TH-302 – Press release from 3 February 2012
6

Threshold Pharmaceuticals Form 8-K from 3 Nov 2014

DHAKA BANGLADESH

Steamers and ferries in Sadarghat Port

Kawran Bazar

Dry fish sellers at the Karwan Dry Fish Market (Bazar), Dhaka, Bangladesh.

Tazemetostat

April 27, 2015 3:59 am / Leave a comment

Tazemetostat

Current developer: Epizyme, Inc., Cambridge, MA 02139.

EPZ-6438 (Tazemetostat)
CAS: 1403254-99-8

HBR 1467052-75-0

タゼメトスタット臭化水素酸塩

Current developer: Epizyme, Inc., Cambridge, MA 02139.

EPZ-6438 (Tazemetostat)
CAS: 1403254-99-8

HBR

Chemical Formula: C34H44N4O4
Exact Mass: 572.33626

USFDA APPROVED 23/1/2020 AS HBR SALT, TAZVERIK, EPIZYME

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide
SIMLES: O=C(C1=CC(C2=CC=C(CN3CCOCC3)C=C2)=CC(N(CC)C4CCOCC4)=C1C)NCC5=C(C)C=C(C)NC5=O

(1,1′-Biphenyl)-3-carboxamide, N-((1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(4-morpholinylmethyl)-

N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(oxan-4-yl)amino)-4-methyl-4′-((morpholin-4-yl)methyl)(1,1′-biphenyl)-3-carboxamide

UNII-Q40W93WPE1

Tazemetostat, sold under the brand name Tazverik, is a medication used for the treatment of adults and adolescents aged 16 years and older with metastatic (when cancer cells spread to other parts of the body) or locally advanced (when cancer has grown outside the organ it started in, but has not yet spread to distant parts of the body) epithelioid sarcoma not eligible for complete resection (surgically removing all of a tissue, structure, or organ).^[1]

Tazemetostat is a cancer drug that acts as a potent selective EZH2 inhibitor.^[2]

Tazemetostat blocks activity of the EZH2 methyltransferase, which may help keep the cancer cells from growing.^[1] Most cases of epithelioid sarcoma begin in the soft tissue under the skin of an extremity, though it can start in other areas of the body.^[1] Surgical removal is considered the main treatment when the cancer is localized to one area of the body.^[1] Chemotherapy or radiation may also be given.^[1] However, there is a high likelihood for local and regional spread of the disease even with treatment and approximately 50% of patients have metastatic disease at the time of diagnosis.^[1] Metastatic disease is considered life-threatening to the patient.^[1]

The most common side effects are pain, fatigue, nausea, decreased appetite, vomiting and constipation.^[1] People taking tazemetostat are at increased risk of developing secondary malignancies including: T-cell lymphoblastic lymphoma (a type of blood cancer that affects the lymphatic system usually found in the lymph nodes), myelodysplastic syndrome (a disorder resulting from poorly formed or dysfunctional blood cells) and acute myeloid leukemia (a cancer of the blood and bone marrow).^[1]

According to the NCI Drug Dictionary, “tazemetostat is an orally available, small molecule selective and S-adenosyl methionine (SAM) competitive inhibitor of histone methyl transferase EZH2, with potential antineoplastic activity. Upon oral administration, tazemetostat selectively inhibits the activity of both wild-type and mutated forms of EZH2. Inhibition of EZH2 specifically prevents the methylation of histone H3 lysine 27 (H3K27). This decrease in histone methylation alters gene expression patterns associated with cancer pathways and results in decreased tumor cell proliferation in EZH2 mutated cancer cells. EZH2, which belongs to the class of histone methyltransferases (HMTs), is overexpressed or mutated in a variety of cancer cells and plays a key role in tumor cell proliferation.”^[3]

History

The U.S. Food and Drug Administration (FDA) approved tazemetostat in January 2020,^[1] based on the results of a clinical trial (NCT02601950) enrolling 62 subjects with metastatic or locally advanced epithelioid sarcoma.^[1]^[4] During the clinical trial, subjects received 800 milligrams (mg) of tazemetostat twice a day until the disease progressed or the subject reached an unacceptable level of toxicity.^[1]^[4] Tumor response assessments were performed every eight weeks during the clinical trial.^[1] The trial measured how many subjects experienced complete or partial shrinkage (by a certain amount) of their tumors during treatment (overall response rate).^[1] The overall response rate was 15%, with 1.6% of subjects having a complete response and 13% having a partial response.^[1] Of the nine subjects that had a response, six (67%) subjects had a response lasting six months or longer.^[1]

The trial was conducted at 22 sites in France, United Kingdom, Taiwan, Italy, Canada, Belgium, and the United States.^[4]

The FDA granted the application for tazemetostat accelerated approval and orphan drug designation.^[1] The FDA granted the approval of Tazverik to Epizyme Inc.^[1]

PATENT

PRODUCT PAT

US 8410088 EXP 21/1/2034

WO 2012142504

US 9090562 EXP 13/4/32

SEE Proceedings of the National Academy of Sciences of the United States of America (2013), 110(19), 7922-7927, S7922/1-S7922/5….http://www.pnas.org/content/110/19/7922.abstract

http://www.epizyme.com/wp-content/uploads/2014/11/Ribrag-ENA-FINAL.pdf

2D chemical structure of 1403254-99-8

Tazemetostat, also known as EPZ-6438, is a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity. EPZ-6438 induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells.

Treatment of xenograft-bearing mice with EPZ-6438 leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation.

These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers. EPZ-6438 is currently in clinical trials.

Epizyme, Inc., Eisai R&D Management Co.Ltd.

Epizyme is developing tazemetostat, a lead from several small molecule EZH2 inhibitors, for treating cancer (phase 1 clinical, as of April 2015). Japanese licensee Eisai was developing the program for the potential oral treatment of cancers, including non-Hodgkin’s lymphoma; however, in March 2015, Epizyme regained worldwide, ex-Japan, rights to the program.

It appeared that Eisai was planning to investigate the program in Japan .

WO-2015057859 From, Eisai Research Institute; Epizyme Inc, indicates Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

see WO2013155317, claiming novel hydrobromide salt of tazemetostat.

PREDICT

………………………………….

PATENT

WO 2012142504

http://www.google.com/patents/WO2012142504A1?cl=en

Example 44: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(moφholinomethyl)-[l , – biphenyl]-3-carboxamide

Compound 44

[Step 1 : Synthesis of 5-brom -2-methyl-3-nitrobenzoic acid

To stirred solution of 2-methyl-3-nitrobenzoic acid ( 100 g, 552 mmol) in cone. H₂S0₄ (400 mL), 1 ,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid ( 140 g, 98%). The isolated compound was taken directly into the next step. Ή NMR (DMSO-4$, 400 MHz) δ 8.31 (s, 1 H), 8.17 (s, 1 H), 2.43 (s, 3H).

Step 2: Synthesis of methyl -bromo-2-methyl-3-nitrobenzoate

To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1 105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate ( 1 L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g,

1058 mmol) in ethanol (1 .5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in-vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 7.37 (s, 1 H), 6.92 (s, 1 H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Step 4: Synthesis of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate

To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (15 g, 61 .5 mmol) and dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300 mL) was added acetic acid (22 g, 369 mmol) and the reaction mixture stirred at room temperature for 15 minutes, then the reaction mixture was cooled to 0°C and sodium triacetoxyborohydnde (39 g, 184 mmol) was added. The reaction mixture was stirred overnight at room temperature. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH of 7-8 was obtained. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a solid ( 14 g, 69%). ‘H NMR (DMSO-<fc, 400 MHz) δ 7.01 (s, 1 H), 6.98 (s, 1 H), 5.00 (d, 1 H, J=7.6 Hz), 3.84-3.87 (m, 2H), 3.79 (s, 31 1), 3.54-3.56 (m_f 1 H), 3.43 (L 21 1, J 12 Hz), 2.14 (s. 31 1). 1 . 1 – 1 .84 (m_: 211). 1 .47- 1 .55 (m, 2H).

Step 5: Synthesis of methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate

triacetoxyborohydnde (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). Ή NMR (DMSO-cfo 400 MHz) δ 7.62 (s, 1 H), 7.52 (s, 1 H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1 H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1 .37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3 -(ethyl (tetrahydro-2H-pyra -4-yl) amino)-2-methylbenzamide

To a stirred solution of 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate (14 g, 39.4 mmol) in ethanol ( 100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol in 25mL water) and the resulting mixture was stirred at 60 °C for 1 h. Upon completion of the reaction as determined by TLC, the solvent was removed under reduced pressure and the residue obtained was acidified with IN HC1 until a pH 7 was obtained and then aqueous citric acid solution was added until a pH 5-6 was obtained. The aqueous layer was extracted with 10% MeOH in DCM (200mL X 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the respective acid (14 g, 100%).

The above acid (14 g, 40.9 mmol) was then dissolved in DMSO (70 mL) and 3- (amino methyl)-4, 6-dimethylpyridin-2( l H)-one ( 12.4 g, 81 .9 mmol) was added to it. The reaction mixture was stirred at room temperature for 15 minutes, then PYBOP (31.9 g, 61.4 mmol) was added and stirring was continued for overnight at room temperature. Upon completion of the reaction as determined by TLC, the reaction mixture was poured onto ice- cold water (700 mL), stirred for 30 minutes and the precipitated solid was collected by filtration, washed with water (500 mL) and air dried. The solid obtained was stirred with acetonitrile (75mL X 2), filtered and air dried. The solid obtained was again stirred with 5% MeOH in DCM ( l OOmL), filtered and dried completely under vacuum to afford the title compound as a solid ( 14 g, 74 %). Ή NMR (DMSO- ₆, 400 MHz) δ 1 1.47 (s, 1 H), 8.23 (t, 1 H), 7.30 (s, 1 H), 7.08 (s, 1 H), 5.85 (s, 1 H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=l 0.4 Hz), 3.20-3.26 (m, 2H), 3.00-3.07 (m, I H), 2.91 -2.96 (m, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m, 2H), 0.78 (t, 3H, J=6.8 Hz).

Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl) amino)-4-methyl-4′-(morpholinomethyl)-[l , l ‘-biphenyl]-3- carboxamide

TITLE COMPD

To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/ water mixture (70 mL/ 14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl- l , 3, 2- dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol) followed by addition of Na₂C0₃ (1 1 .2 g, 106.1 mmol). The solution was purged with argon for 15 minutes and then Pd (PPh₃)₄ (3.40 g, 2.94 mmol) was added and the solution was again purged with argon for a further 10 min. The reaction mixture was heated at 100°C for 4 h. After completion (monitored by TLC), the reaction mixture was diluted with water and extracted with 10% MeOH/DCM.

The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with methanol: DCM to the title compound as a solid (12 g, 71 %).

Analytical Data: LCMS: 573.35 (M + 1 )⁺; HPLC: 99.5% (@ 254 nm) (R,;3.999; Method: Column: YMC ODS-A 1 50 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol : 10 μΐ, Col. Temp.: 30 °C; Flow rate: 1 .4 mL/min.;

Gradient: 5% B to 95% B in 8 min, Hold for 1 .5 min, 9.51 -12 min 5% B);

Ή NMR (DMSO-i ₆, 400 MHz) 5 1 1 .46 (s, I H), 8. 19 (t, 1 H), 7.57 (d, 2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, I H), 5.85 (s, I H), 4.28 (d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57 (bs, 4H), 3.48 (s, 2H), 3.24 (t, 2H, J=10.8Hz), 3.07-3.09 (m, 2H), 3.01 (m, I H), 2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1 .64-1 .67 (m, 2H), 1 .51 – 1 .53 (m, 2H), 0.83 (t, 3H, J=6.4 Hz).

TRIHYDROCHLORIDE

Step 8: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3-yl)methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[ 1 , 1 ‘-biphenyl]-3- carboxamide trihydrochloride

N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5-(ethyl (tetrahydro- 21 l-pyran-4-yl) amino)-4-methyI-4′-(niorpholinornethyl)-[ 1 , 1 ‘-biphenyl]-3-carboxamide ( 12 g, 21.0 mmol) was dissolved in methanolic HC1 (200 mL) and stirred at room temperature for 3 h. After three hours of stirring, the reaction mixture was concentrated under reduced pressure. The solid obtained was stirred with ether ( l OOmL X 2) to afford the desired salt as a solid ( 1 1 g, 77 %).

Analytical Data of the tri-HCl salt: LCMS: 573.40 (M + 1 )⁺; HPLC: 99.1 % (@ 254 nm) (R,;3.961 ; Method: Column: YMC ODS-A 150 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol: 10 pL, Col. Temp.: 30 °C; Flow rate: 1.4 mL/min.; Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51 -12 min 5% B);

Ή NMR (D₂0 400 MHz) δ 7.92 (bs, I H,) 7.80 (s, I H), 7.77 (d, 2H, J=8 Hz), 7.63 (s, I H), 7.61 (s, I H), 6.30 (s, I H), 4.48 (s, 2H), 4.42 (s, 2H), 4.09-4.1 1 (m, 4H), 3.95-3.97 (m, 2H), 3.77 (t, 3H, J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H), 2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1 .76 (m, 2H), 1 .04 (t, 3H, J=6.8 Hz).

…………………………………………

PATENT

WO2013155317

http://www.google.com/patents/WO2013155317A1?cl=en

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl] -3-carboxamide hydrobromide:

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl

(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3- carboxamide hydrobromide:

As used herein, “Compound I” refers to N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxamide. The hydrobromide of Compound I can be used to inhibit the histone methyltransferase activity of EZH2, either in a subject or in vitro. The hydrobromide of Compound I can also be used to treat cancer in a subject in need thereof.

Scheme 1

……………………………………..Compound I Compound I – HBr

HPLC

HPLC was conducted on an Agilent 1200 HPLC quaternary pump, low pressure mixing, with an in-line degasser. Analytical method conditions: 8 μΐ_^ sample (20 mg of ER-581982-06 diluted with 50 mL of a methanol to provide approximately 0.4 mg/mL solution) was injected onto a Agilent Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 3.5 um), Chromatography conditions: mobile phase A, water with 5mM ammonium formate; mobile phase B, 5 mM ammonium formate in 50/45/5 acetonitrile/methanol/water; flow rate, 1.5 ml/min.; gradient: isocratic at 10% B from 0 to 3 min; linear increase to 70% B from 3 to 7 min; isocratic at 70% B from 7 to 12 min; linear increase to 100% B from 12 to 15 min isocratic at 100% B from 15 to 20 min;

column temperature, 35 °C; detection, UV 230 nm. Approximate retention time of Compound I = 10.7 min.

Synthesis of Polymorph A

5-bromo-2-methyl-3-nitrobenzoic acid stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in cone. H₂S0₄ (400 mL), l,3-dibromo-5,5-dimethyl-2,4- imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid (140 g, 98%). The isolated compound was taken directly into the next step. 1H NMR (DMSO-J₆, 400 MHz) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate To a stirred solution of 5-bromo-2- methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate (1L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. 1H NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate (1) To a stirred solution of methyl 5- bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in- vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. 1H

NMR (CDCI₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (2) A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate (455.8 g, 1.87 mol), 1,2- Dichloroethane (4.56 L), and acetic acid (535 ml, 9.34 mol). To the mixture were added dihydro-2H-pyran-4(3H)-one (280 g, 2.80 mol) and sodium triacetoxyborohydride (594 g, 2.80 mol) maintaining the internal temperature below 40 °C. The mixture was stirred at 25 °C for 2.5 h and then the reaction was quenched with a solution of sodium hydroxide (448 g, 11.20 mol) in water (5.61 L). After stirring for 20 minutes at ambient temperature, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (3.65 L). The organic layers were combined, washed with brine (1.5 L), and concentrated under vacuum.

The residue was treated with ethyl acetate (1.8 L) and heated to 65-70 °C. The mixture was stirred at 65-70 °C for 15 minutes to give a clear solution and then treated with n-heptane (7.3 L) maintaining the temperature between 60-70 °C. Once the heptane was completely added to the solution, the mixture was held at 65-70 °C for 15 minutes and then allowed to cool to 18- 22 °C over 3 h. The resulting suspension was stirred at 18-22 °C for 4 h, cooled to 0-5 °C over 1 h, and held at 0-5 °C for 2 h. The precipitate was filtered, washed twice with n-heptane (1.4 L), and dried under vacuum to give the title compound (540 g, 88%). The XRPD pattern of this compound is shown in Figure 17.

Methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate (3)

To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 °C and sodium triacetoxyborohydride (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). 1H NMR DMSO-d₆, 400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-

[l,l’-biphenyl]-3-carboxylate (4): A mixture of methyl 5-bromo-3-(ethyl(tetrahydro-2H-pyran- 4-yl)amino)-2-methylbenzoate (580 g, 1.63 mol), 4-(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2-yl)benzyl)morpholine (592 g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 mol), and water (771 ml) was degassed by bubbling nitrogen through the mixture at 20 °C for 20 minutes and treated with tetrakis(triphenylphosphine)palladium(0) (14.11 g, 12.21 mmol). The resulting mixture was degassed for an additional 20 minutes and then heated to 87-89 °C for 17 h. After cooling to 20 °C, the mixture was diluted with ethyl acetate (5.80 L) and a solution of (R)-2-Amino-3-mercaptopropionic acid (232 g) in water (2.320 L). After stirring for 1 h at 20 °C, the organic layer was separated and washed again with a solution of (R)-2-Amino-3- mercaptopropionic acid (232 g) in water (2.320 L). The aqueous layers were combined and extracted with ethyl acetate (5.80 L). The organic layers were combined, washed with a solution of sodium hydroxide (93 g) in water (2.32 L), and concentrated under vacuum at 35 °C to give the title compound as an orange oil (1.21 kg, 164% yield).

5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxylic acid (5): Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylate (69.0 g, 152.5 mmol) (based on the theoretical yield from the previous step) was suspended in ethanol (380 mL) and treated with a solution of sodium hydroxide (24.84 g, 621.0 mmol) in water (207 mL). The mixture was stirred at 40°C for 18 h. After cooling to 0-5 °C, the mixture was neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL) maintaining the temperature below 25 °C. Then, the mixture was extracted twice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL). The organic layers were combined and concentrated under vacuum to give a crude product as a yellow solid (127g).

The crude product was dissolved in 2-methyltetrahydrofuran (656 mL) at 70 °C and then treated with IPA (828 mL). The mixture was allowed to cool to rt over 3-4 h and then stirred overnight at rt. The precipitate was filtered, washed twice with IPA (207 mL), and dried under vacuum to give the title compound as an off white solid (53.54 g, 80%). The XRPD pattern of this compound is shown in Figure 9.

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3-carboxamide

(Compound I): A mixture of 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylic acid (540 g, 1.23 mol) and 3-(aminomethyl)- 4,6-dimethyl-dihydro-pyridin-2(lH)-one hydrochloride (279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated with triethylamine (223 ml, 1.60 mol). The mixture was stirred at 25 °C for 30 min and treated with EDC-HC1 (354 g, 1.85 mol) and HOBT hydrate (283 g, 1.85 mol). The reaction mixture was stirred at rt for 16 h. After addition of triethylamine (292 ml, 2.09 mol), the mixture was cooled to 15 °C, diluted with water (10.1 L) maintaining the temperature below 30 °C, and stirred at 19-25 °C for 4 h. The resulting precipitate was filtered, washed twice with water (2.70 L), and dried under vacuum to give a crude product (695 g, wt-wt analysis = 78%).

For the further purification of the product, recrystallization was conducted. A crude product (20.00 g, 34.92 mmol) was suspended in a mixture of ethanol (190 ml) and water (10.00 ml) and heated to 75°C until a clear solution was obtained. The solution was allowed to cool to rt overnight. The precipitate was filtered, washed twice with a mixture of ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35 °C to give the title compound as an off white solid (14.0 g, 70% recovery from the crude and 90% yield based on wt-wt assay).

4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4-yl)methyl)morpholin- 4-ium bromide (Polymorph A): A crude N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)am

biphenyl]-3-carboxamide (595 g, 464 g based on wt-wt assay, 810.3 mmol) was suspended in ethanol (3.33 L). After heating to 70 °C, the mixture was treated with 48% aqueous HBr (97 ml, 850.8 mmol) and stirred at 70 °C for 30 min. The resulting orange-red solution was treated with ethyl acetate (3.33 L) maintaining the temperature above 60 °C. The mixture was slowly cooled to rt over 18 h. The mixture was cooled to 0 °C over 1 h and stirred at that temperature for 5.5 h. The resulting precipitate was filtered, washed twice with ethyl acetate (1.39 L), and dried under vacuum to give the title compound as an off white solid (515 g, 97% yield).

Recrystallization of Polymorph A: 4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4- yl)methyl)morpholin-4-ium bromide (0.50 g, 0.77 mmol; 95.6% pure by HPLC) was suspended in ethanol (3.0 mL) and heated to 80 °C until a clear solution was obtained. To the solution was added MTBE (5.0 mL) slowly. The resulting solution was allowed to cool to 18-22 °C over 3 h and stirred at 18-22 °C for 15 h. The precipitate was filtered, washed twice with MTBE (2 mL) and dried under vacuum to give 0.45 g of the title compound (89% recovery, 96.6% pure by HPLC).

Compound I is protonated at the nitrogen of the morpholino substituent, providing a monohydrobromide of Compound I having the following structure:

This particular monohydrobromide can be referred to as “4-((3′-(((4,6-dimethyl-2-oxo- l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′- methyl-[l, -biphenyl]-4-yl)methyl)morpholin-4-ium bromide.” Figure 11 depicts the X-ray crystal structure of this particular salt form.

…………………………………………………………..

see

WO-2015057859

Eisai Research Institute; Epizyme Inc

Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

…………………

Synthesis

Trade Names

Country	Trade Name	Vendor	Annotation
USA	Tazverik	Epizyme, 2020

Formulations

oral tabs. and suspension

References

- Knutson, S. K. et al., Proc. Natl. Acad. Sci USA, (2013) 110(19), 7922-7927.
- WO 2012 142504 (Epizyme/Eisai Co; 18.10.2012; appl. 13.4.2012; USA-prior. 13.4.2011).
- WO 2013 155317 (Epizyme/Eisai Co; 17.10.2013; appl. 11.4.2013).
- WO 2015 057859 (Epizyme/Eisai Co; 23.4.2015; appl. 15.10.2014; USA-prior. 16.10.2013).

EZH2 inhibitors for treating lymphona:
- WO 2015 195848 (Epizyme; 23.12.2015; appl. 17.6.2015; USA-prior. 17.6.2014).

////////

PAPER

RSC Advances (2015), 5(33), 25967-25978

http://pubs.rsc.org/en/content/articlelanding/2015/ra/c5ra02365c#!divAbstract

RSC Adv., 2015,5, 25967-25978,

DOI: 10.1039/C5RA02365C

The histone lysine methyltransferase EZH2 has been implicated as a key component in cancer aggressiveness, metastasis and poor prognosis. This study discovered a new class of hexahydroisoquinolin derivatives as EZH2 inhibitors. A structure–activity relationship study showed that the steric hindrance was important to the activity for EZH2. A preliminary optimization study led to the discovery of several potent compounds with low nanomolar to sub-nanomolar potency for EZH2. Biological evaluation indicated that SKLB1049 was a highly potent with improved solubility compared to EPZ6438, SAM-competitive, and cell-active EZH2 inhibitor that decreased global H3K27me3 in SU-DHL-6 and Pfeiffer lymphoma cells in a concentration- and time-dependent manner. Further study indicated that SKLB1049 caused cell arrest in G₀/G₁ phase. These compounds would be useful as chemical tools to further explore the biology of EZH2 and provided us with a start point to develop new EZH2 inhibitors.

Graphical abstract: Design, synthesis and biological evaluation of novel 1-methyl-3-oxo-2,3,5,6,7,8-hexahydroisoquinolins as potential EZH2 inhibitors

In vitro protocol:	Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7.
In vivo protocol:	Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7.

References

1: Knutson SK, Warholic NM, Johnston LD, Klaus CR, Wigle TJ, Iwanowicz D, Littlefield BA, Porter-Scott M, Smith JJ, Moyer MP, Copeland RA, Pollock RM, Kuntz KW, Raimondi A, Keilhack H. Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas. PLoS One. 2014 Dec 10;9(12):e111840. doi: 10.1371/journal.pone.0111840. eCollection 2014. PubMed PMID: 25493630; PubMed Central PMCID: PMC4262195.

2: Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, Kadowaki T, Uesugi M, Kuznetsov G, Kumar N, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Waters NJ, Smith JJ, Porter-Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Uenaka T, Pollock RM, Kuntz KW, Yokoi A, Keilhack H. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Cancer Ther. 2014 Apr;13(4):842-54. doi: 10.1158/1535-7163.MCT-13-0773. Epub 2014 Feb 21. PubMed PMID: 24563539

3. Inhibitors of human histone methyltransferase EZH2, and methods of use thereof for treating cancer. By Kuntz, Kevin W.; Knutson, Sarah K.; Wigle, Timothy James Nelson . From U.S. Pat. Appl. Publ. (2013), US 20130040906 A1 20130214.

4. Aryl-or heteroaryl-substituted benzamide compounds as anticancer agents and their preparation By Kuntz, Kevin Wayne; Chesworth, Richard; Duncan, Kenneth William; Keilhack, Heike; Warholic, Natalie; Klaus, Christine; Zheng, Wanjun; Seki, Masashi; Shirotori, Syuji; Kawano, Satoshi From PCT Int. Appl. (2012), WO 2012142504 A1 20121018.

5: Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM, Kuntz KW, Keilhack H. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7. doi: 10.1073/pnas.1303800110. Epub 2013 Apr 25. PubMed PMID: 23620515; PubMed Central PMCID: PMC3651445.

WO2013155317A1 *	Apr 11, 2013	Oct 17, 2013	Epizyme, Inc.	Salt form of a human hi stone methyltransf erase ezh2 inhibitor
WO2013155464A1 *	Apr 12, 2013	Oct 17, 2013	Epizyme, Inc.	Combination therapy for treating cancer
WO2014049488A1 *	Sep 16, 2013	Apr 3, 2014	Pfizer Inc.	Benzamide and heterobenzamide compounds
WO2014062732A1 *	Oct 15, 2013	Apr 24, 2014	Epizyme, Inc.	Substituted benzene compounds
WO2014062733A2 *	Oct 15, 2013	Apr 24, 2014	Epizyme, Inc.	Substituted benzene compounds
WO2014172044A1 *	Mar 14, 2014	Oct 23, 2014	Epizyme, Inc.	Substituted benzene compounds
WO2015004618A1 *	Jul 9, 2014	Jan 15, 2015	Glaxosmithkline Intellectual Property (No.2) Limited	Enhancer of zeste homolog 2 inhibitors
WO2015010049A1 *	Jul 18, 2014	Jan 22, 2015	Epizyme, Inc.	Substituted benzene compounds
WO2015010078A2	Jul 18, 2014	Jan 22, 2015	Epizyme, Inc.	Substituted 6,5-fused bicyclic heteroaryl compounds

Citing Patent	Filing date	Publication date	Applicant	Title
WO2011140325A1 *	May 5, 2011	Nov 10, 2011	Glaxosmithkline Llc	Indazoles
WO2012142504A1 *	Apr 13, 2012	Oct 18, 2012	Eisai Co., Ltd.	Aryl-or heteroaryl-substituted benzene compounds
WO2014062720A2 *	Oct 15, 2013	Apr 24, 2014	Epizyme, Inc.	Methods of treating cancer

WO2011140324A1 *	May 5, 2011	Nov 10, 2011	Glaxosmithkline Llc	Indoles
WO2011140325A1 *	May 5, 2011	Nov 10, 2011	Glaxosmithkline Llc	Indazoles
WO2012005805A1 *	May 5, 2011	Jan 12, 2012	Glaxosmithkline Llc	Azaindazoles
US4522811	Jul 8, 1982	Jun 11, 1985	Syntex (U.S.A.) Inc.	Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US5763263	Jul 24, 1996	Jun 9, 1998	Dehlinger; Peter J.	Method and apparatus for producing position addressable combinatorial libraries
US7563589	May 27, 2005	Jul 21, 2009	The University Of North Carolina At Chapel Hill	Including EED, EZH2 and SUZ12 wherein the reconstituted complex has histone methyltransferase (HMTase) activity for lysine 27 of histone H3 (H3-K27); cancer

References

^ Jump up to:^a ^b ^c ^d ^e ^f ^g ^h ⁱ ^j ^k ^l ^m ⁿ ^o ^p ^q ^r “FDA approves first treatment option specifically for patients with epithelioid sarcoma, a rare soft tissue cancer”. U.S. Food and Drug Administration (FDA) (Press release). 23 January 2020. Retrieved 23 January 2020. This article incorporates text from this source, which is in the public domain.
^ Lue JK, Amengual JE (October 2018). “Emerging EZH2 Inhibitors and Their Application in Lymphoma”. Curr Hematol Malig Rep. 13 (5): 369–382. doi:10.1007/s11899-018-0466-6. PMID 30112706. S2CID 52010283.
^ “Tazemetostat”. NCI Drug Dictionary. National Cancer Institute.
^ Jump up to:^a ^b ^c “Drug Trials Snapshots: Tazverik”. U.S. Food and Drug Administration (FDA). 23 January 2020. Retrieved 22 February 2020. This article incorporates text from this source, which is in the public domain.

External links

“Tazemetostat”. Drug Information Portal. U.S. National Library of Medicine.

Tazemetostat
Clinical data

Trade names	Tazverik
Other names	EPZ-6438
AHFS/Drugs.com	Monograph
MedlinePlus	a620018
License data	US DailyMed: Tazemetostat US FDA: Tazverik
Legal status
Legal status	US: ℞-only
Identifiers
IUPAC name [show]
CAS Number	1403254-99-8
PubChem CID	66558664
DrugBank	DB12887
ChemSpider	30208713
UNII	Q40W93WPE1
KEGG	D11485 D11444
ChEMBL	ChEMBL3414621
Chemical and physical data
Formula	C₃₄H₄₄N₄O₄
Molar mass	572.750 g·mol⁻¹
3D model (JSmol)	Interactive image
SMILES [hide] CCN(C1CCOCC1)C2=CC(=CC(=C2C)C(=O)NCC3=C(C=C(NC3=O)C)C)C4=CC=C(C=C4)CN5CCOCC5
InChI [hide] InChI=1S/C34H44N4O4/c1-5-38(29-10-14-41-15-11-29)32-20-28(27-8-6-26(7-9-27)22-37-12-16-42-17-13-37)19-30(25(32)4)33(39)35-21-31-23(2)18-24(3)36-34(31)40/h6-9,18-20,29H,5,10-17,21-22H2,1-4H3,(H,35,39)(H,36,40) Key:NSQSAUGJQHDYNO-UHFFFAOYSA-N

About EPZ-‐6438 Epizyme is developing EPZ-‐6438, a small molecule inhibitor of EZH2 created with our

proprietary product platform, for the treatment of non-‐Hodgkin lymphoma patients and patients with INI1-‐deficient solid tumors. In many human cancers, misregulated EZH2 enzyme activity results in misregulation of genes that control cell proliferation—without these control mechanisms, cancer cells are free to grow

About Epizyme, Inc.

Epizyme, Inc. is a clinical stage biopharmaceutical company creating novel epigenetic therapeutics for cancer patients.

Epizyme has built a proprietary product platform that the company uses to create small molecule inhibitors of a 96 member class of enzymes known as histone methyltransferases, or HMTs. HMTs are part of the system of gene regulation, referred

to as epigenetics, that controls gene expression. Genetic alterations can result in changes to

the activity of HMTs, making them oncogenic (cancer -‐causing). By focusing on the genetic drivers of cancers, Epizyme’s targeted science seeks to match the right medicines with the right patients.

Epizyme^®, Inc.
400 Technology Square, 4th Floor
Cambridge, MA 02139

Phone: (617) 229-5872
Fax: (617) 349-0707
contact@Epizyme.com

Victoria Richon, vice president of biological sciences, Epizyme Inc.

Jason Rhodes (left) has been appointed to president of Epizyme Inc.,

100 Technology Square

Central Square – The square – Cambridge, MA, United States

/////////TAZVERIK, EPIZYME, FDA 2020, APPROVALS 2020, Tazemetostat, EPZ-6438, EPZ 6438

K 912, NC 6300, Epirubicin nano

March 29, 2015 12:33 pm / Leave a comment

Epirubicin

Cancer Research UK

Epirubicin

Irish Cancer Society

Epirubicin

Macmillan Cancer Support

Epirubicin

NHS Evidence

Epirubicin – Substance Summary

PubChem

EpirubicinEmail

MedlinePlus

– See more at: http://www.cancerindex.org/Epirubicin#sthash.BBioCC6K.dpuf

PHASE 1 JAPAN SOLID TUMOURS

DNA/RNA Synthesis Inhibitor

WITH Nano Carrier Co.,Ltdhttp://pdf.irpocket.com/C4571/qnwX/eFou/vG1J.pdf

KOWA COMPANY LTD

CAS FREE FORM. 56420-45-2

Smiles

O=C2c1c(O)c5c(c(O)c1C(=O)c3cccc(OC)c23)C[C@@](O)(C(=O)CO)C[C@@H]5O[C@@H]4O[C@H]([C@H](O)[C@@H](N)C4)C
NMR
http://file.selleckchem.com/downloads/nmr/S122302-Epirubicin-Hydrochloride-NMR-Selleck.pdf
http://www.medkoo.com/Product-Data/Epirubicin/Epirubicin-QC-TZC20130604web.pdf

Epirubicin
CAS No.:	56420-45-2
Synonyms:	4′-Epi-DX; Epirubicina; WP 697; Pidorubicin; 4′-epiadriamycin; Adriblastin; epi-dx; Epiadriamycin; farmorubicin; imi28; Farmarubicin;
Formula:	C27H29NO11
Exact Mass:	543.17400

NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin.

Epirubicin is widely used to treat various human tumors. However, it is difficult to achieve a sufficient antitumor effect because of dosage limitation to prevent cardiotoxicity. We hypothesized that epirubicin-incorporating micelle would reduce cardiotoxicity and improve the antitumor effect. NC-6300 comprises epirubicin covalently bound to PEG polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure of 40-80 nm in diameter in an aqueous milieu. NC-6300 (10, 15 mg/kg) and epirubicin (10 mg/kg) were given i.v. three times to mice bearing s.c. or liver xenograft of human hepatocellular carcinoma Hep3B cells. Cardiotoxicity was evaluated by echocardiography in C57BL/6 mice that were given NC-6300 (10 mg/kg) or epirubicin (10 mg/kg) in nine doses over 12 weeks. NC-6300 showed a significantly potent antitumor effect against Hep3B s.c. tumors compared with epirubicin. Moreover, NC-6300 also produced a significantly longer survival rate than epirubicin against the liver orthotopic tumor of Hep3B. With respect to cardiotoxicity, epirubicin-treated mice showed significant deteriorations in fractional shortening and ejection fraction. In contrast, cardiac functions of NC-6300 treated mice were no less well maintained than in control mice. This study warrants a clinical evaluation of NC-6300 in patients with hepatocellular carcinoma or other cancers.

K-912（NC-6300）の概要 K-912（NC-6300）は、世界的に幅広く使用されているアントラサイクリン系の抗がん剤の一つであるエピルビシンを内包したミセル化ナノ粒子製剤で、その特性により、エピルビシンの有する心毒性の軽減が期待できます。さらに、pH 応答性システムを採用することで、腫瘍細胞内でのエピルビシンの放出量を高め、既存のエピルビシンに比べより強力な抗腫瘍効果が期待できます。

Epirubicin is an anthracycline drug used for chemotherapy. It can be used in combination with other medications to treat breast cancer in patients who have had surgery to remove the tumor. It is marketed by Pfizer under the trade name Ellence in the US andPharmorubicin or Epirubicin Ebewe elsewhere.

Similarly to other anthracyclines, epirubicin acts by intercalating DNA strands. Intercalation results in complex formation which inhibits DNA and RNA synthesis. It also triggers DNA cleavage by topoisomerase II, resulting in mechanisms that lead to cell death. Binding to cell membranes and plasma proteins may be involved in the compound’s cytotoxic effects. Epirubicin also generates free radicalsthat cause cell and DNA damage.

Epirubicin is favoured over doxorubicin, the most popular anthracycline, in some chemotherapy regimens as it appears to cause fewer side-effects. Epirubicin has a different spatial orientation of the hydroxyl group at the 4′ carbon of the sugar – it has the opposite chirality – which may account for its faster elimination and reduced toxicity. Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas.

Development history

The first trial of epirubicin in humans was published in 1980.^[1] Upjohn applied for approval by the U.S. Food and Drug Administration(FDA) in node-positive breast cancer in 1984, but was turned down because of lack of data.^[2] It appears to have been licensed for use in Europe from around this time however.^[3] In 1999 Pharmacia (who had by then merged with Upjohn) received FDA approval for the use of epirubicin as a component of adjuvant therapy in node-positive patients.

Patent protection for epirubicin expired in August 2007.

References

Bonfante, V; Bonadonna, G; Villani, F; Martini, A (1980). “Preliminary clinical experience with 4-epidoxorubicin in advanced human neoplasia”. Recent results in cancer research 74: 192–9. PMID 6934564. PM6934564.
“On Target”.
According to the proprietary database iddb.com

External links

1H NMR PREDICT

Epirubicin NMR spectra analysis, Chemical CAS NO. 56420-45-2 NMR spectral analysis, Epirubicin H-NMR spectrum

13C NMR PREDICT

Epirubicin NMR spectra analysis, Chemical CAS NO. 56420-45-2 NMR spectral analysis, Epirubicin C-NMR spectrum

COSY

1H NMR

Epirubicin
Systematic (IUPAC) name


(8R,10S)-10-((2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
Clinical data
Trade names	Ellence
AHFS/Drugs.com	monograph
MedlinePlus	a603003
Pregnancy category	D (Au, U.S.)
Legal status	℞-only (U.S.), POM (UK)
Routes of administration	Intravenous
Pharmacokinetic data
Bioavailability	NA
Protein binding	77%
Metabolism	Hepatic glucuronidationand oxidation
Excretion	Biliary and renal
Identifiers
CAS Registry Number	56420-45-2
ATC code	L01DB03
PubChem	CID 41867
DrugBank	DB00445
ChemSpider	38201
UNII	3Z8479ZZ5X
KEGG	D07901
ChEBI	CHEBI:47898
ChEMBL	CHEMBL417
Chemical data
Formula	C₂₇H₂₉N O₁₁
Molecular mass	543.519 g/mol

KOWA COMPANY LTD

Map for KOWA COMPANY LTD

Nano Carrier Co

P.S. : The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent.

Dabrafenib mesylate, GSK 2118436, ダブラフェニブ达拉菲尼, An antineoplastic agent that inhibits BRAF kinase

March 19, 2015 7:15 am / Leave a comment

DABRAFENIB

ダブラフェニブ

达拉菲尼,

1195765-45-7 BASE

1195768-06-9 cas of mesylate

Benzenesulfonamide, N-[3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-4-thiazolyl]-2-fluorophenyl]-2,6-difluoro-

N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

MW 519.56 BASE

MF C₂₃ H₂₀ F₃ N₅ O₂ S_{2 BASE}

Dabarefenib
Dabrafenib
GSK 2118436
Tafinlar
UNII-QGP4HA4G1B

US FDA APPROVAL….Date of Approval: May 29, 2013

update

Product details
Name	Tafinlar
Agency product number	EMEA/H/C/002604
Active substance	dabrafenib mesilate
International non-proprietary name (INN) or common name	dabrafenib
Therapeutic area (MeSH)	Melanoma
Anatomical therapeutic chemical (ATC) code	L01EC02

Publication details
Marketing-authorisation holder	Novartis Europharm Limited

Date of issue of marketing authorisation valid throughout the European Union

26/08/2013

An orally bioavailable inhibitor of B-raf (BRAF) protein with potential antineoplastic activity. Dabrafenib selectively binds to and inhibits the activity of B-raf, which may inhibit the proliferation of tumor cells which contain a mutated BRAF gene. B-raf belongs to the the raf/mil family of serine/threonine protein kinases and plays a role in regulating the MAP kinase/ERKs signaling pathway, which may be constitutively activated due to BRAF gene mutations

Dabrafenib (trade name Tafinlar) is a drug for the treatment of cancers associated with a mutated version of the gene BRAF. Dabrafenib acts as an inhibitor of the associated enzyme B-Raf, which plays a role in the regulation of cell growth. Dabrafenib has clinical activity with a manageable safety profile in clinical trials of phase 1 and 2 in patients with BRAF(V600)-mutated metastatic melanoma.^[1]^[2]

The Food and Drug Administration approved dabrafenib as a single agent treatment for patients with BRAF V600E mutation-positive advanced melanoma on May 30, 2013.^[3] Clinical trial data demonstrated that resistance to dabrafinib and other BRAF inhibitors occurs within 6 to 7 months.^[4] To overcome this resistance, the BRAF inhibitor dabrafenib was combined with the MEK inhibitor trametinib.^[4] As a result of this research, on January 8, 2014, the FDA approved the combination of dabrafenib and trametinib for the treatment of patients with BRAF V600E/K-mutant metastatic melanoma.^[5]

Inhibitor of BRAF(V600) mutants

Active Ingredient:	DABRAFENIB MESYLATE
Dosage Form;Route:	CAPSULE;ORAL
Proprietary Name:	TAFINLAR
Applicant:	GLAXOSMITHKLINE
Strength:	EQ 75MG BASE
NDA Application Number:	N202806
Product Number:	002
Approval Date:	May 29, 2013
Reference Listed Drug	Yes
RX/OTC/DISCN:	RX

Patent Data

Appl No	Prod No	Patent No	Patent Expiration	Drug Substance Claim	Drug Product Claim	Patent Use Code	Delist Requested
N202806	002	7994185	Jan 20, 2030	Y	Y	U – 1406
N202806	002	8415345	Jan 20, 2030	Y	Y	U – 1406

Exclusivity Data

NDA Appl No	Prod No	Exclusivity Code	Exclusivity Expiration
N202806	002	I – 678	Jan 8, 2017
N202806	002	ODE	Jan 9, 2021
N202806	002	NCE	May 29, 2018
N202806	002	ODE	May 29, 2020

PDF……http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/202806s000lbl.pdf

TERMS

I 678, TRAMETINIB, IN COMBINATION WITH DABRAFENIB, FOR THE TREATMENT OF PATIENTS WITH UNRESECTABLE OR METASTATIC MELANOMA WITH BRAF V600E OR V600K MUTATIONS AS DETECTED BY AN FDA-APPROVED TEST

ODE ORPHAN DRUG EXCLUSIVITY

NCE NEW CHEMICAL ENTITY

Analogs described herein were generally prepared according to Scheme S1. When the desired benzoic acid pre- cursors were unknown, the synthetic scheme began wi th esterification of bromo-acids 15a. Subsequent palladi-um-catalyzed amination witht-butyl carbamate afforded anilino esters15b. After esterification of benzoic acids15, or amination of bromo-esters leading to 15b, the anilino esters were reacted with an arylsulfonyl chloride toform the sulfonamide headgroup. Ester16was then condensed with the lithium anion of 2-chl

oro-4-methylpyrimidine to generate ketone intermediate17. Bromination of17with NBS followed by cyclization withisopropyl ort

-butyl thioamide afforded the desired thiazole core18. The tail was then installed by SNAr dis-placement at the chloropyrimidine in18

with either methanolic ammonia or a primary basicamine to generatethe desired analogues19

DABRAFENIB SYNTHESIS

WILL BE UPDATED

Inline image 1

—

http://www.google.com/patents/WO2011047238A1?cl=en

Method 1 : Compound B (first crystal form) – A/-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1 ,1 dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide

A suspension of A/-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]- 2-fluorophenyl}-2,6-difluorobenzenesulfonamide (196 mg, 0.364 mmol) and ammonia in methanol 7M (8 ml, 56.0 mmol) was heated in a sealed tube to 90 °C for 24 h. The reaction was diluted with DCM and added silica gel and concentrated. The crude product was chromatographed on silica gel eluting with 100% DCM to 1 :1 [DCM:(9:1 EtOAc:MeOH)]. The clean fractions were concentrated to yield the crude product. The crude product was repurified by reverse phase HPLC (a gradient of acetonitrile:water with 0.1 %TFA in both). The combined clean fractions were concentrated then partitioned between DCM and saturated NaHCO3. The DCM layer was separated and dried over Na₂SO₄. The title compound, /V-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1 – dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide was obtained (94 mg, 47% yield). ¹ H NMR (400 MHz, DMSO-c/6) δ ppm 10.83 (s, 1 H), 7.93 (d, J=5.2 Hz, 1 H), 7.55 – 7.70 (m, 1 H), 7.35 – 7.43 (m, 1 H), 7.31 (t, J=6.3 Hz, 1 H), 7.14 – 7.27 (m, 3 H), 6.70 (s, 2 H), 5.79 (d, J=5.13 Hz, 1 H), 1 .35 (s, 9 H). MS (ESI): 519.9 [M+H]⁺.

Method 2: Compound B (alternative crystal form) – A/-{3-[5-(2-Amino-4-pyrimidinyl)-2- (1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide 19.6 mg of A/-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (may be prepared in accordance with example 58a) was combined with 500 L of ethyl acetate in a 2-mL vial at room temperature. The slurry was temperature-cycled between 0-40°C for 48 hrs. The resulting slurry was allowed to cool to room temperature and the solids were collected by vacuum filtration. The solids were analyzed by Raman, PXRD, DSC/TGA analyses, which indicated a crystal form different from the crystal form resulting from Example 58a, above. Method 3: Compound B (alternative crystal form, large batch) – A/-{3-[5-(2-amino-4- pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide

tep A: methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate

Methyl 3-amino-2-fluorobenzoate (50 g, 1 eq) was charged to reactor followed by dichloromethane (250 mL, 5 vol). The contents were stirred and cooled to ~15°C and pyridine (26.2 mL, 1 .1 eq) was added. After addition of the pyridine, the reactor contents were adjusted to ~15°C and the addition of 2,6-diflurorobenzenesulfonyl chloride (39.7 mL, 1 .0 eq) was started via addition funnel. The temperature during addition was kept <25°C. After complete addition, the reactor contents were warmed to 20-25°C and held overnight. Ethyl acetate (150 mL) was added and dichloromethane was removed by distillation. Once distillation was complete, the reaction mixture was then diluted once more with ethyl acetate (5 vol) and concentrated. The reaction mixture was diluted with ethyl acetate (10 vol) and water (4 vol) and the contents heated to 50-55°C with stirring until all solids dissolve. The layers were settled and separated. The organic layer was diluted with water (4 vol) and the contents heated to 50-55° for 20-30 min. The layers were settled and then separated and the ethyl acetate layer was evaporated under reduced pressure to ~3 volumes. Ethyl Acetate (5 vol.) was added and again evaporated under reduced pressure to ~3 volumes.

Cyclohexane (9 vol) was then added to the reactor and the contents were heated to reflux for 30 min then cooled to 0 °C. The solids were filtered and rinsed with cyclohexane (2 x 100 mL). The solids were air dried overnight to obtain methyl 3-{[(2,6- difluorophenyl)sulfonyl]amino}-2-fluorobenzoate (94.1 g, 91 %).

Step B: A/-{3-[(2-chloro-4-pyhmidinyl)acetyl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide

Methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate (490 g, 1 equiv.), prepared generally in accordance with Step A, above, was dissolved in THF (2.45 L, 5 vols) and stirred and cooled to 0-3 °C. 1 M lithium bis(trimethylsilyl)amide in THF (5.25 L, 3.7 equiv.) solution was charged to the reaction mixture followed addition of 2- chloro-4-methylpyrimidine (238 g, 1 .3 equiv.) in THF (2.45 L, 5 vols). The reaction was then stirred for 1 hr. The reaction was quenched with 4.5M HCI (3.92 L, 8 vols). The aqueous layer (bootom layer) was removed and discarded. The organic layer was concentrated under reduced pressure to ~2L. IPAC (isopropyl acetate) (2.45L) was added to the reaction mixture which was then concentrated to ~2L. IPAC (0.5L) and MTBE (2.45 L) was added and stirred overnight under N₂. The solids were filtered. The solids and mother filtrate added back together and stirred for several hours. The solids were filtered and washed with MTBE (~5 vol). The solids were placed in vacuum oven at 50 °C overnight. The solids were dried in vacuum oven at 30 °C over weekend to obtain A/-{3-[(2-chloro-4-pyhmidinyl)acetyl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide (479 g, 72%).

Step C: A/-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

To a reactor vessel was charged /V-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}- 2,6-difluorobenzenesulfonamide (30 g, 1 eq) followed by dichloromethane (300 mL). The reaction slurry was cooled to ~10°C and N-bromosuccinimide (“NBS”) (12.09 g, 1 eq) was added in 3 approximately equal portions, stirring for 10-15 minutes between each addition. After the final addition of NBS, the reaction mixture was warmed to ~20°C and stirred for 45 min . Water (5 vol) was then added to the reaction vessel and the mixture was stirred and then the layers separated. Water (5 vol) was again added to the dichloromethane layer and the mixture was stirred and the layers separated. The dichloromethane layers were concentrated to -120 mL. Ethyl acetate (7 vol) was added to the reaction mixture and concentrated to -120 mL. Dimethylacetamide (270 mL) was then added to the reaction mixture and cooled to ~10°C. 2,2- Dimethylpropanethioamide (1 .3 g, 0.5 eq) in 2 equal portions was added to the reactor contents with stirring for ~5 minutes between additions. The reaction was warmed to 20-25 °C. After 45 min, the vessel contents were heated to 75°C and held for 1 .75 hours . The reaction mixture was then cooled to 5°C and water (270 ml) was slowly charged keeping the temperature below 30°C. Ethyl acetate (4 vol) was then charged and the mixture was stirred and layers separated. Ethyl acetate (7 vol) was again charged to the aqueous layer and the contents were stirred and separated. Ethyl acetate (7 vol) was charged again to the aqueous layer and the contents were stirred and separated. The organic layers were combined and washed with water (4 vol) 4 times and stirred overnight at 20-25°C. The organic layers were then concentrated under heat and vacuum to 120 mL. The vessel contents were then heated to 50°C and heptanes (120 mL) were added slowly. After addition of heptanes, the vessel contents were heated to reflux then cooled to 0°C and held for ~2 hrs. The solids were filtered and rinsed with heptanes (2 x 2 vol). The solid product was then dried under vacuum at 30°C to obtain /V-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonannide (28.8 g, 80%).

Step D: A/-{3-[5-(2-amino-4-pyhmidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonannide

In 1 gal pressure reactor, a mixture of A/-{3-[5-(2-chloro-4-pyrinnidinyl)-2-(1 ,1 – dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (120 g) prepared in accordance with Step C, above, and ammonium hydroxide (28-30%, 2.4 L, 20 vol) was heated in the sealed pressure reactor to 98-103 °C and stirred at this temperature for 2 hours. The reaction was cooled slowly to room temperature (20 °C) and stirred overnight. The solids were filtered and washed with minimum amount of the mother liquor and dried under vacuum. The solids were added to a mixture of EtOAc (15 vol)/ water (2 vol) and heated to complete dissolution at 60-70 °C and the aqueous layer was removed and discarded. The EtOAC layer was charged with water (1 vol) and neutralized with aq. HCI to ~pH 5.4-5.5. and added water (1 vol). The aqueous layer was removed and discarded at 60-70 °C. The organic layer was washed with water (1 vol) at 60-70 °C and the aqueous layer was removed and discarded. The organic layer was filtered at 60 °C and concentrated to 3 volumes. EtOAc (6 vol) was charged into the mixture and heated and stirred at 72 °C for 10 min , then cooled to 20°C and stirred overnight. EtOAc was removed via vacuum distillation to concentrate the reaction mixture to ~3 volumes. The reaction mixture was maintained at ~65-70°C for ~30mins. Product crystals having the same crystal form as those prepared in Example 58b (and preparable by the procedure of Example 58b), above, in heptanes slurry were charged. Heptane (9 vol) was slowly added at 65-70 °C. The slurry was stirred at 65-70 °C for 2- 3 hours and then cooled slowly to 0-5°C. The product was filtered, washed with

EtOAc/heptane (3/1 v/v, 4 vol) and dried at 45°C under vacuum to obtain A/-{3-[5-(2- amino-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide (102.3 g, 88%).

Method 4: Compound B (mesylate salt) – A/-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1 – dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide methanesulfonate

To a solution of /V-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonannide (204 mg, 0.393 mmol) in isopropanol (2 ml_), methanesulfonic acid (0.131 ml_, 0.393 mmol) was added and the solution was allowed to stir at room temperature for 3 hours. A white precipitate formed and the slurry was filtered and rinsed with diethyl ether to give the title product as a white crystalline solid (210 mg, 83% yield). ¹ H NMR (400 MHz, DMSO-c/6) δ ppm 10.85 (s, 1 H) 7.92 – 8.05 (m, 1 H) 7.56 – 7.72 (m, 1 H) 6.91 – 7.50 (m, 7 H) 5.83 – 5.98 (m, 1 H) 2.18 – 2.32 (m, 3 H) 1 .36 (s, 9 H). MS (ESI): 520.0 [M+H]⁺.

Method 5: Compound B (alternative mesylate salt embodiment) – A/-{3-[5-(2-amino-4- pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide methanesulfonate

A/-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}- 2,6-difluorobenzenesulfonamide (as may be prepared according to example 58a) (2.37g, 4.56 mmol) was combined with pre-filtered acetonitrile (5.25 vol, 12.4 ml_). A pre-filtered solution of mesic acid (1 .1 eq., 5.02 mmol, 0.48 g) in H₂O (0.75 eq., 1 .78 ml_) was added at 20°C. The temperature of the resulting mixture was raised to 50- 60°C while maintaining a low agitation speed. Once the mixture temperature reached to 50-60°C, a seed slurry of A/-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1 -dimethylethyl)-1 ,3- thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide methanesulfonate (1 .0 %w/w slurried in 0.2 vol of pre-filtered acetonitrile) was added, and the mixture was aged while agitating at a speed fast enough to keep solids from settling at 50-60°C for 2 hr. The mixture was then cooled to 0-5°C at 0.25°C/min and held at 0-5°C for at 6 hr. The mixture was filtered and the wet cake was washed twice with pre-filtered

acetonitrile. The first wash consisted of 14.2 ml (6 vol) pre-filtered acetonitrile and the second wash consisted of 9.5 ml (4 vol) pre-filtered acetonitrile. The wet solid was dried at 50°C under vacuum, yielding 2.39 g (85.1 % yield) of product. Typically, the salts of the present invention are pharmaceutically acceptable salts.

May 29, 2013 — GlaxoSmithKline plc announced today that the U.S. Food and Drug Administration (FDA) has approved Tafinlar (dabrafenib). Tafinlar is indicated as a single-agent oral treatment for unresectable melanoma (melanoma that cannot be removed by surgery) or metastatic melanoma (melanoma which has spread to other parts of the body) in adult patients with BRAF V600E mutation. Tafinlar is not indicated for the treatment of patients with wild-type BRAF melanoma. The mutation must be detected by an FDA-approved test, such as the companion diagnostic assay from bioMérieux S.A., THxID™-BRAF.

Among those with metastatic melanoma, approximately half have a BRAF mutation, which is an abnormal change in a gene that can enable some melanoma tumours to grow and spread

Tafinlar is approved for patients with the BRAF V600E mutation, which accounts for approximately 85 percent of all BRAF V600 mutations in metastatic melanoma.

GSK will be making Tafinlar available for prescription no later than in the early third quarter of 2013.

In 2010, GSK entered a collaboration with bioMérieux to develop a companion diagnostic test to detect BRAF V600 (V600E and V600K) gene mutations found in several cancers, including melanoma. bioMérieux has received FDA pre-market approval of THxID™-BRAF. Currently, it is the only FDA-approved test that detects the V600K mutation.

The primary outcome measure was the estimation of the overall intracranial response rate (OIRR) in each cohort. The OIRR for Cohort A was 18 percent (95% CI: 9.7, 28.2). For Cohort B, the OIRR was also 18 percent (95% CI: 9.9, 30.0). The median duration of response was 4.6 months (95% CI: 2.8, Not Reached) and 4.6 months (95% CI: 1.9, 4.6) in Cohort A and Cohort B, respectively.

Melanoma is the most serious and deadly form of skin cancer. According to statistics from the National Cancer Institute, in 2013 there will be an estimated 9,480 deaths resulting from melanoma in the United States. When melanoma spreads in the body, the disease is called metastatic melanoma.Approximately half of all people with metastatic melanoma have a BRAF mutation, which is an abnormal change in a gene that can enable some melanoma tumours to grow and spread.

One in two patients worldwide with metastatic melanoma is expected to survive for a year after diagnosis, while in the U.S., the five-year survival rate was 16 percent (2003-2009).The median age of a newly diagnosed metastatic melanoma patient is almost a decade younger than other cancers.

Tafinlar (dabrafenib) is now approved for the treatment of adult patients with unresectable or metastatic melanoma with BRAF V600E mutation as detected by an FDA-approved test. Limitation of use: Tafinlar is not recommended for use in patients with wild-type BRAF melanoma.

Tafinlar is not approved or licensed in Europe and may not be approved in other parts of the world for the treatment of patients with BRAF V600 mutation-positive unresectable melanoma or metastatic melanoma.

Dabrafenib mesylate is a kinase inhibitor. The chemical name for dabrafenib mesylate is N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzene sulfonamide, methanesulfonate salt. It has the molecular formula C₂₃H₂₀F₃N₅O₂S₂•CH₄O₃S and a molecular weight of 615.68. Dabrafenib mesylate has the following chemical structure:

TAFINLAR (dabrafenib) Structural Formula Illustration

Dabrafenib mesylate is a white to slightly colored solid with three pKas: 6.6, 2.2, and -1.5. It is very slightly soluble at pH 1 and practically insoluble above pH 4 in aqueous media.

TAFINLAR (dabrafenib) capsules are supplied as 50-mg and 75-mg capsules for oral administration. Each 50-mg capsule contains 59.25 mg dabrafenib mesylate equivalent to 50 mg of dabrafenib free base. Each 75-mg capsule contains 88.88 mg dabrafenib mesylate equivalent to 75 mg of dabrafenib free base.

The inactive ingredients of TAFINLAR are colloidal silicon dioxide, magnesium stearate, and microcrystalline cellulose. Capsule shells contain hypromellose, red iron oxide (E172), and titanium dioxide (E171).

Dabrafenib mesylate

1195768-06-9 cas of mesylate

N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide;methanesulfonic acid

Chemical structure

………………….

WO2015003571

达拉菲尼甲磺酸盐的新晶型及其制备方法

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=A1F1236472ED5D758B64CA11358EBB6C.wapp1nC?docId=WO2015003571&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Deficiencies of the prior art W, the main object of the present invention is to provide as follows has a better stability in an aqueous or aqueous systems Dara Feeney 曱

For purposes of this invention, the present invention provides Dallas Phoenix mesylate Form IV (hereinafter referred to as “Form IV”) and its preparation method. The type IV crystal is a hydrate; preferably each 摩尔达拉菲 Nepal mesylate contains about 1.5 moles of water.

Using Cu- Κα radiation, the crystalline form IV of X-ray powder diffraction pattern at diffraction angles 2Θ of 4.7 ± 0.2 °, 9.2 ± 0.2. , 12.8 ± 0.2. , 13.8 ± 0.2. 15.0 0.2 soil. And 16.3 ± 0.2 ° of the characteristic peaks.

Preferably, the crystalline form IV of the X-ray powder diffraction pattern at diffraction angles 2Θ of 4.7 ± 0.2. , 9.2 ± 0.2. , 12.8 ± 0 · 2. , 13 · 8 ± 0 · 2 ^o , 15.0 ± 0.2. , 16.3 ± 0 · 2. , 18.0 ± 0.2. , 18 · 6 ± 0.2. , 20 · 6 ± 0.2. , 22.9 ± 0.2 °, 23.8 ± 0.2. And 24.3 ± 0.2. Department characteristic peaks

Form IV of FIG differential scanning calorimetry (DSC) show: sample 151~ 105 ° C there is a large endothermic peak (solvent peak), the sample after dehydration melting range of 132 ~ 148 ° C, then at 200 ° C ~ 245 ° C with a heat transfer crystal peak at 249 ° C and finally melted.

The crystalline form IV has the following advantageous properties:

1) left at room temperature for one month and stable, stable for 1 month at room temperature for -97% RH;

2) known Form I in water suspension was stirred for 15 minutes into the free base monohydrate; and Form IV in water suspension was stirred for 15 minutes remain for 曱 salt Form IV, After stirring overnight converted to the free base monohydrate Form IV described more conducive to maintaining the solubility of the sample is larger than the free base state 曱 sulfonates, Form IV has a better stability in water / aqueous system or sex.

3) 0 to 22 hours compared to the elution amount, any detection points of Form IV of elution volume than the known polymorph I of the elution amount. Description Form IV has a better solubility and bioavailability.

4) 0 to 120 minutes elution amount compared to any detection points of Form IV gum Nang elution volume than the known polymorph I of the dissolution of glue Nang. Description Form IV gum Nang has better dissolution.

The Form IV was prepared using any one of the following methods:

1) The Dallas Feeney known mesylate polymorph I was dissolved in a mixed solution of tetrahydrofuran Yue alcohol, volatile crystallization, and then the precipitated crystals were separated and dried to obtain the Form IV;

The Yue alcohol and tetrahydrofuran in a volume ratio of 0.1 to 100: 1, preferably 0.5~50: 1, more preferably 0.5~5: 1;

2) The Dallas Phoenix Yue sulfonates known polymorph I was dissolved in acetone, volatile crystallization, and the precipitated crystals

Separated, dried, to give the Form IV;

3) The Dallas Phoenix 曱 known polymorph I salt is dissolved in isopropanol, after the addition of polyacrylic acid, volatile crystallization, and then the precipitated crystals were separated and dried to obtain the Form IV;

The polyacrylic acid in an amount of polymorph I of the known amount of 0.1% wt~10% wt, preferably

0.5% wt ~ 10% wt, more preferably 2% wt ~ 5% wt; an average molecular weight of the polyacrylic acid is 2000-5000.

Preparation of the above three methods, the known Dara Feeney 曱 sulfonate polymorph I at room temperature in an amount corresponding to its solubility in a solution of 0.1 to 1 times, preferably 0.5 to 1 times, more preferably 0.8 to 1 times;

The crystallization temperature of room temperature ~ 40 ° C, preferably at room temperature; the crystallization time is 1~14 days, preferably for two days; the dry, you can not vacuum or pressure, the pressure is preferably less than 0.09Mpa; temperature of 30 ° C ~ 120 ° C, preferably 4 (TC ~ 80 ° C, more preferably 40 ° C ~ 60 ° C; for 10 to 72 hours, preferably 10~48 hours, more preferably from 10- 24 hours;

4) The Dallas Phoenix Yue sulfonate polymorph Form II or V is placed to give the Form IV;

The placement of room temperature ~ 40 ° C, preferably room temperature; placement time from 15 minutes to 7 days, preferably

One day;

5) The temperature rise Dara Feeney 曱 sulfonate polymorph II to 120 ° C and then spontaneously cooled to room temperature to obtain the crystalline form

IV；

The preparation of Form I of Preparation Example 1 known

Methods Patent Document WO2009 / 137391 or CN200980126781.6 Example 58a and 58d known polymorph I. Preparation Specifically:

The N- {3- [5- (2- chloro-4-pyrimidinyl) -2- (1,1-Yue-yl-ethyl) -1,3-thiazol-4-yl] – 2-fluorophenyl 2,6-difluorophenyl sulfonamide (196 mg, 0.364mmol) and 7M ammonia in methanol (8ml, 56mmol) was added to a 25 ml autoclave, heated to 90 ° C for 24 hours, TLC showed the starting material the reaction was complete, The reaction system was cooled to room temperature, the solvent was concentrated and the residue was dry column chromatography to obtain N- {3- [5- (2- amino-4-pyrimidinyl) -2- (1,1-dimethylethyl ) -1,3-thiazol-4-yl] -2-fluorophenyl} -2,6-difluorobenzenesulfonamide 90 mg, yield: 45%.

The N- {3- [5- (2- amino-4-pyrimidinyl) -2- (1,1-Yue-yl-ethyl) -1,3-thiazol-4-yl] -2-fluorophenyl 2,6-difluorophenyl sulfonamide (204 mg, 0.393mmol) in isopropanol (2 mL) was added 曱 acid (0.131 ml, 0.393mmol) and the solution was stirred at room temperature for 3 hours. A white precipitate formed and the slurry was filtered and washed with diethyl ether to give N- [3- [5- (2- amino-4-pyrimidinyl) -2- (t-butyl) -4-thiazol-yl] -2-fluoro phenyl] -2,6-difluorobenzenesulfonamide 曱 sulphonates crystalline solid (221 mg, 87% yield) as a white.

1HNM (400MHz, DMSO-d6)5 ppm 10.85(s, lH)7.92-8.05(m, 1H), 7.56-7.72(m, 1H), 6.91-7.50(m, 7H)， 5.83-5.98(m, 1H) , 2.18-2.32(m, 3H) , 1.36(s, 9H)。

Preparation of crystal form obtained X-ray powder diffraction pattern shown in Figure 10. Report is consistent with the patent document WO2009 / 137391 or CN200980126781.6.

DSC chart is shown in Fig. Show: Known polymorphs I melt away as 247 ° C~250 ° C.

TGA spectrum shown in Figure 12. Show: decomposition temperature of 261 ° C.

Example 1

Take 10.02 mg polymorph IV (Example 7 Preparation) in 5 ml glass vial, add 0.5 ml of water, ultrasonic resulting suspension stirred at room temperature for 15 minutes, after centrifugation without drying, the present invention is to obtain crystalline form II. The yield was 10.00 mg; 99% yield.

X-ray powder diffraction pattern shown in Figure 6.

TGA pattern shown in Figure 7. Show: Form II at 50 ° C before the weight loss of about 4.6% (about 1.5 water), 50 ° C ~ 155 ° C 1.4% weight loss (about 0.5 water), the decomposition temperature of 287 ° C.

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PATENT

http://www.google.com/patents/WO2009137391A2?cl=en

WO 2009137391

Example 58a: Λ/-{3-r5-(2-Amino-4-pyrimidinylV2-(1.1-dimethylethylV1.3-thiazol-4-yll-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

Following a procedure analogous to the procedure described in Example 51, Step B using Λ/-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (196 mg, 0.364 mmol) and ammonia in methanol 7M (8 ml, 56.0 mmol) and heating to 90 ⁰C for 24 h, the title compound, Λ/-{3- [5-(2-amino-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide was obtained (94 mg, 47% yield). ¹H NMR (400 MHz, DMSO-d6) δ ppm 10.83 (s, 1 H), 7.93 (d, J=5.2 Hz, 1 H), 7.55 – 7.70 (m, 1 H), 7.35 –

7.43 (m, 1 H), 7.31 (t, J=6.3 Hz, 1 H), 7.14 – 7.27 (m, 3 H), 6.70 (s, 2 H), 5.79 (d, J=5.13 Hz, 1 H), 1.35 (s, 9 H). MS (ESI): 519.9 [M+H]⁺.

Example 58b: Λ/-{3-r5-(2-Amino-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yll-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

19.6 mg of Λ/-{3-[5-(2-Amino-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (may be prepared in accordance with example 58a) was combined with 500 μl_ of ethyl acetate in a 2-mL vial at room temperature. The slurry was temperature-cycled between 0-40⁰C for 48 hrs. The resulting slurry was allowed to cool to room temperature and the solids were collected by vacuum filtration. The solids were analyzed by Raman, PXRD, DSC/TGA analyses, which indicated a crystal form different from the crystal form resulting from Example 58a, above. Example 58c: Λ/-{3-r5-(2-amino-4-pyrimidinylV2-(1.1-dimethylethylV1.3-thiazol-4-yll-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

Step A: methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate

Methyl 3-amino-2-fluorobenzoate (50 g, 1 eq) was charged to reactor followed by dichloromethane (250 ml_, 5 vol). The contents were stirred and cooled to ~15°C and pyridine (26.2 ml_, 1.1 eq) was added. After addition of the pyridine, the reactor contents were adjusted to ~15°C and the addition of 2,6-diflurorobenzenesulfonyl chloride (39.7 ml_, 1.0 eq) was started via addition funnel. The temperature during addition was kept <25°C. After complete addition, the reactor contents were warmed to 20-25⁰C and held overnight. Ethyl acetate (150 ml.) was added and dichloromethane was removed by distillation. Once distillation was complete, the reaction mixture was then diluted once more with ethyl acetate (5 vol) and concentrated. The reaction mixture was diluted with ethyl acetate (10 vol) and water (4 vol) and the contents heated to 50- 55°C with stirring until all solids dissolve. The layers were settled and separated.

The organic layer was diluted with water (4 vol) and the contents heated to 50-55° for 20-30 min. The layers were settled and then separated and the ethyl acetate layer was evaporated under reduced pressure to ~3 volumes. Ethyl Acetate (5 vol.) was added and again evaporated under reduced pressure to ~3 volumes. Cyclohexane (9 vol) was then added to the reactor and the contents were heated to reflux for 30 min then cooled to 0 ⁰C. The solids were filtered and rinsed with cyclohexane (2 x 100 ml_). The solids were air dried overnight to obtain methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2- fluorobenzoate (94.1 g, 91 %).

Step B: Λ/-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide

The organic layer was concentrated under reduced pressure to ~2L. IPAC (isopropyl acetate) (2.45L) was added to the reaction mixture which was then concentrated to ~2L. IPAC (0.5L) and MTBE (2.45 L) was added and stirred overnight under N₂. The solids were filtered. The solids and mother filtrate added back together and stirred for several hours. The solids were filtered and washed with MTBE (~5 vol). The solids were placed in vacuum oven at 50 ⁰C overnight. The solids were dried in vacuum oven at 30 ⁰C over weekend to obtain Λ/-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide (479 g, 72%).

Step C: Λ/-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

To a reactor vessel was charged Λ/-{3-[(2-chloro-4-pyrimidinyl)acetyl]-2-fluorophenyl}- 2,6-difluorobenzenesulfonamide (30 g, 1 eq) followed by dichloromethane (300 ml_). The reaction slurry was cooled to ~10°C and N-bromosuccinimide (“NBS”) (12.09 g, 1 eq) was added in 3 approximately equal portions, stirring for 10-15 minutes between each addition. After the final addition of NBS, the reaction mixture was warmed to ~20°C and stirred for 45 min . Water (5 vol) was then added to the reaction vessel and the mixture was stirred and then the layers separated. Water (5 vol) was again added to the dichloromethane layer and the mixture was stirred and the layers separated.

The dichloromethane layers were concentrated to -120 ml_. Ethyl acetate (7 vol) was added to the reaction mixture and concentrated to -120 ml_. Dimethylacetamide (270 ml.) was then added to the reaction mixture and cooled to -1O⁰C. 2,2-Dimethylpropanethioamide (1.3 g, 0.5 eq) in 2 equal portions was added to the reactor contents with stirring for -5 minutes between additions. The reaction was warmed to 20-25 ⁰C. After 45 min, the vessel contents were heated to 75°C and held for 1.75 hours . The reaction mixture was then cooled to 5°C and water (270 ml) was slowly charged keeping the temperature below 30⁰C. Ethyl acetate (4 vol) was then charged and the mixture was stirred and layers separated. Ethyl acetate (7 vol) was again charged to the aqueous layer and the contents were stirred and separated.

Ethyl acetate (7 vol) was charged again to the aqueous layer and the contents were stirred and separated. The organic layers were combined and washed with water (4 vol) 4 times and stirred overnight at 20-25⁰C. The organic layers were then concentrated under heat and vacuum to 120 ml_. The vessel contents were then heated to 50⁰C and heptanes (120 ml.) were added slowly. After addition of heptanes, the vessel contents were heated to reflux then cooled to 0°C and held for -2 hrs. The solids were filtered and rinsed with heptanes (2 x 2 vol). The solid product was then dried under vacuum at 30⁰C to obtain Λ/-{3-[5-(2-chloro-4-pyrimidinyl)- 2-(1 , 1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (28.8 g, 80%).

Step D:

Λ/-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide

In 1 gal pressure reactor, a mixture of Λ/-{3-[5-(2-chloro-4-pyrimidinyl)-2-(1 ,1- dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (120 g) prepared in accordance with Step C, above, and ammonium hydroxide (28-30%, 2.4 L, 20 vol) was heated in the sealed pressure reactor to 98-103 ⁰C and stirred at this temperature for 2 hours. The reaction was cooled slowly to room temperature (20 ⁰C) and stirred overnight. The solids were filtered and washed with minimum amount of the mother liquor and dried under vacuum. The solids were added to a mixture of EtOAc (15 vol)/ water (2 vol) and heated to complete dissolution at 60-70 ⁰C and the aqueous layer was removed and discarded. The EtOAC layer was charged with water (1 vol) and neutralized with aq. HCI to ~pH 5.4-5.5. and added water (1vol). The aqueous layer was removed and discarded at 60-70 ⁰C.

The organic layer was washed with water (1 vol) at 60-70 ⁰C and the aqueous layer was removed and discarded. The organic layer was filtered at 60 ⁰C and concentrated to 3 volumes. EtOAc (6 vol) was charged into the mixture and heated and stirred at 72 ⁰C for 10 min , then cooled to 2O⁰C and stirred overnight. EtOAc was removed via vacuum distillation to concentrate the reaction mixture to ~3 volumes.

The reaction mixture was maintained at -65-7O⁰C for ~30mins. Product crystals having the same crystal form as those prepared in Example 58b (and preparable by the procedure of Example 58b), above, in heptanes slurry were charged. Heptane (9 vol) was slowly added at 65-70 ⁰C. The slurry was stirred at 65-70 ⁰C for 2-3 hours and then cooled slowly to 0-5⁰C. The product was filtered, washed with EtOAc/heptane (3/1 v/v, 4 vol) and dried at 45°C under vacuum to obtain Λ/-{3-[5-(2- amino-4-pyrimidinyl)-2-(1 , 1 -dimethylethyl)-1 ,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide (102.3 g, 88%).

Example 58d:

Λ/-{3-r5-(2-amino-4-pyrimidinvn-2-(1.1-dimethylethylV1.3-thiazol-4-yll-2- fluorophenyl}-2,6-difluorobenzenesulfonamide methanesulfonate

MESYLATE

To a solution of Λ/-{3-[5-(2-amino-4-pyrimidinyl)-2-(1 ,1-dimethylethyl)-1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (204 mg, 0.393 mmol) in isopropanol (2 ml_), methanesulfonic acid (0.131 ml_, 0.393 mmol) was added and the solution was allowed to stir at room temperature for 3 hours. A white precipitate formed and the slurry was filtered and rinsed with diethyl ether to give the title product as a white crystalline solid (210 mg, 83% yield).

¹H NMR (400 MHz, DMSO-d6) δ ppm 10.85 (s, 1 H) 7.92 – 8.05 (m, 1 H) 7.56 – 7.72 (m, 1 H) 6.91 – 7.50 (m, 7 H) 5.83 – 5.98 (m, 1 H) 2.18 – 2.32 (m, 3 H) 1.36 (s, 9 H). MS (ESI): 520.0 [M+H]⁺.WO2009137391

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PAPER

ACS Medicinal Chemistry Letters (2013), 4(3), 358-362.

ACS Med. Chem. Lett., 2013, 4 (3), pp 358–362

DOI: 10.1021/ml4000063

http://pubs.acs.org/doi/abs/10.1021/ml4000063

The title compound,N-{3-[5-(2-amino-4-pyrimidinyl)-2-(1,1-dimethylethyl)-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenze

nesulfonamide was obtained (94 mg, 47% yield).

Dabrafenib base

1H NMR

(400 MHz, DMSO-d6) δ ppm 10.83 (s, 1 H), 7.93 (d,J=5.2 Hz, 1 H), 7.55 – 7.70 (m, 1 H), 7.35 – 7.43 (m, 1 H), 7.31(t,J=6.3 Hz, 1 H), 7.14 – 7.27 (m, 3 H), 6.70 (s, 2 H),5.79 (d,J=5.13 Hz, 1 H), 1.35 (s, 9 H).

MS (ESI): 519.9 [M+H]+.

13C NMR (100 MHz, DMSO-d6) δ ppm 182.1, 164.0, 160.6, 159.4, 158.0, 154.9,

152.4, 145.8, 136.6, 135.1, 130.0,

128.4, 125.6, 124.7, 114.1, 113.9, 105.7, 38.3, 31.0.

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Patent

http://www.google.com/patents/WO2014158467A1?cl=en

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WO 2014066606

http://www.google.com/patents/WO2014066606A2?cl=en

Step C : N- {3-[5-(2-chloro-4-pyrimidinyl)-2-(l , 1 -dimethylethyl)-l ,3-thiazol-4-yl]- 2-fluorophenyl}-2,6-difluorobenzenesulfonamide

To a reactor vessel was charged N- {3-[(2-chloro-4-pyrimidinyl)acetyl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (30 g, 1 eq) followed by dichloromethane (300 mL). The reaction slurry was cooled to ~10°C and N-bromosuccinimide (“NBS”) (12.09 g, 1 eq) was added in 3 approximately equal portions, stirring for 10-15 minutes between each addition. After the final addition of NBS, the reaction mixture was warmed to ~20°C and stirred for 45 min . Water (5 vol) was then added to the reaction vessel and the mixture was stirred and then the layers separated. Water (5 vol) was again added to the dichloromethane layer and the mixture was stirred and the layers separated. The dichloromethane layers were concentrated to -120 mL. Ethyl acetate (7 vol) was added to the reaction mixture and concentrated to -120 mL. Dimethylacetamide (270 mL) was then added to the reaction mixture and cooled to ~10°C. 2,2-Dimethylpropanethioamide (1.3 g, 0.5 eq) in 2 equal portions was added to the reactor contents with stirring for ~5 minutes between additions. The reaction was warmed to 20-25 °C. After 45 min, the vessel contents were heated to 75°C and held for 1.75 hours . The reaction mixture was then cooled to 5°C and water (270 ml) was slowly charged keeping the temperature below 30°C. Ethyl acetate (4 vol) was then charged and the mixture was stirred and layers separated. Ethyl acetate (7 vol) was again charged to the aqueous layer and the contents were stirred and separated. Ethyl acetate (7 vol) was charged again to the aqueous layer and the contents were stirred and separated. The organic layers were combined and washed with water (4 vol) 4 times and stirred overnight at 20-25°C. The organic layers were then concentrated under heat and vacuum to 120 mL. The vessel contents were then heated to 50°C and heptanes (120 mL) were added slowly. After addition of heptanes, the vessel contents were heated to reflux then cooled to 0°C and held for ~2 hrs. The solids were filtered and rinsed with heptanes (2 x 2 vol). The solid product was then dried under vacuum at 30°C to obtain N-{3-[5-(2-chloro-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3- thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (28.8 g, 80%).

Compound B is disclosed and claimed, along with pharmaceutically acceptable salts thereof, as being useful as an inhibitor of BRaf activity, particularly in the treatment of cancer, in PCT patent application PCT/US09/42682. Compound B is embodied by Examples 58a through 58e of the application. The PCT application was published on 12 November 2009 as publication WO2009/137391, and is hereby incorporated by reference.

Suitably, Compound B may be prepared according to the methods below:

Method 1 : Compound B (first crystal form) – N-{3-[5-(2-Amino-4-pyrimidinyl)-2- (1,1 -dimethylethyl)- 1 ,3-thiazol-4-yl]- -fluorophenyl} -2,6-difluorobenzenesulfonamide

A suspension of N-{3-[5-(2-chloro-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3- thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (196 mg, 0.364 mmol) and ammonia in methanol 7M (8 ml, 56.0 mmol) was heated in a sealed tube to 90 °C for 24 h. The reaction was diluted with DCM and added silica gel and concentrated. The crude product was chromatographed on silica gel eluting with 100% DCM to 1 : 1 [DCM: (9: 1 EtOAc:MeOH)]. The clean fractions were concentrated to yield the crude product. The crude product was repurified by reverse phase HPLC (a gradient of acetonitrile: water with 0.1%TFA in both). The combined clean fractions were concentrated then partitioned between DCM and saturated NaHC0₃. The DCM layer was separated and dried over Na₂S0₄. The title compound, N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)- l,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide was obtained (94 mg, 47% yield). 1H NMR (400 MHz, DMSO- 6) δ ppm 10.83 (s, 1 H), 7.93 (d, J=5.2 Hz, 1 H), 7.55 – 7.70 (m, 1 H), 7.35 – 7.43 (m, 1 H), 7.31 (t, J=6.3 Hz, 1 H), 7.14 – 7.27 (m, 3 H), 6.70 (s, 2 H), 5.79 (d, J=5.13 Hz, 1 H), 1.35 (s, 9 H). MS (ESI): 519.9 [M+H]⁺.

Method 2: Compound B (alternative crystal form) – N-{3-[5-(2-Amino-4- pyrimidinyl)-2-(l,l-dimethylethyl)-l,3-thiazol-4-yl]-2-fluorophenyl}-2,6- difluorobenzenesulfonamide 19.6 mg of N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(l,l- dimethylethyl)- 1 ,3-thiazol-4-yl]-2-fluorophenyl} -2,6-difluorobenzenesulfonamide (may be prepared in accordance with example 58a) was combined with 500 L of ethyl acetate in a 2-mL vial at room temperature. The slurry was temperature-cycled between 0-40°C for 48 hrs. The resulting slurry was allowed to cool to room temperature and the solids were collected by vacuum filtration. The solids were analyzed by Raman, PXRD, DSC/TGA analyses, which indicated a crystal form different from the crystal form resulting from Example 58a, above.

Method 3: Compound B (alternative crystal form, large batch) – N-{3-[5-(2-amino- 4-pyrimidinyl)-2-(l , 1 -dimethylethyl)- 1 ,3-thiazol-4-yl]-2-fluorophenyl} -2,6- difluorobenzenesulfonamide

Step D : N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3-thiazol-4-yl]-

2-fluorophenyl}-2,6-difluorobenzenesulfonamide

In 1 gal pressure reactor, a mixture of N-{3-[5-(2-chloro-4-pyrimidinyl)-2-(l,l- dimethylethyl)- 1 ,3-thiazol-4-yl]-2-fluorophenyl} -2,6-difluorobenzenesulfonamide ( 120 g) prepared in accordance with Step C, above, and ammonium hydroxide (28-30%, 2.4 L, 20 vol) was heated in the sealed pressure reactor to 98-103 °C and stirred at this temperature for 2 hours. The reaction was cooled slowly to room temperature (20 °C) and stirred overnight. The solids were filtered and washed with minimum amount of the mother liquor and dried under vacuum. The solids were added to a mixture of EtOAc (15 vol)/ water (2 vol) and heated to complete dissolution at 60-70 °C and the aqueous layer was removed and discarded. The EtOAC layer was charged with water (1 vol) and neutralized with aq. HC1 to ~pH 5.4-5.5. and added water (lvol). The aqueous layer was removed and discarded at 60-70 °C. The organic layer was washed with water (1 vol) at 60-70 °C and the aqueous layer was removed and discarded. The organic layer was filtered at 60 °C and concentrated to 3 volumes. EtOAc (6 vol) was charged into the mixture and heated and stirred at 72 °C for 10 min , then cooled to 20°C and stirred overnight. EtOAc was removed via vacuum distillation to concentrate the reaction mixture to ~3 volumes. The reaction mixture was maintained at ~65-70°C for ~30mins. Product crystals having the same crystal form as those prepared in Example 58b (and preparable by the procedure of Example 58b), above, in heptanes slurry were charged. Heptane (9 vol) was slowly added at 65-70 °C. The slurry was stirred at 65-70 °C for 2-3 hours and then cooled slowly to 0- 5°C. The product was filtered, washed with EtO Ac/heptane (3/1 v/v, 4 vol) and dried at 45°C under vacuum to obtain N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3- thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (102.3 g, 88%>).

MESYLATE

Method 4: Compound B (mesylate salt) – N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l- dimethylethyl)- 1 ,3-thiazol-4-yl]-2-fluorophenyl} -2,6-difluorobenzenesulfonamide methanesulfonate

To a solution of N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3- thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide (204 mg, 0.393 mmol) in isopropanol (2 mL), methanesulfonic acid (0.131 mL, 0.393 mmol) was added and the solution was allowed to stir at room temperature for 3 hours. A white precipitate formed and the slurry was filtered and rinsed with diethyl ether to give the title product as a white crystalline solid (210 mg, 83% yield). 1H NMR (400 MHz, DMSO- 6) δ ppm 10.85 (s, 1 H) 7.92 – 8.05 (m, 1 H) 7.56 – 7.72 (m, 1 H) 6.91 – 7.50 (m, 7 H) 5.83 – 5.98 (m, 1 H) 2.18 – 2.32 (m, 3 H) 1.36 (s, 9 H). MS (ESI): 520.0 [M+H]⁺.

Method 5: Compound B (alternative mesylate salt embodiment) – N-{3-[5-(2- amino-4-pyrimidinyl)-2-(l , 1 -dimethylethyl)-l ,3-thiazol-4-yl]-2-fluorophenyl} -2,6- difluorobenzenesulfonamide methanesulfonate

N- {3-[5-(2-amino-4-pyrimidinyl)-2-(l , 1 -dimethylethyl)- 1 ,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide (as may be prepared according to example 58a) (2.37g, 4.56 mmol) was combined with pre-filtered acetonitrile (5.25 vol, 12.4 mL). A pre-filtered solution of mesic acid (1.1 eq., 5.02 mmol, 0.48 g) in H₂0 (0.75 eq., 1.78 mL) was added at 20°C. The temperature of the resulting mixture was raised to 50-60°C while maintaining a low agitation speed. Once the mixture temperature reached to 50- 60°C, a seed slurry of N-{3-[5-(2-amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3-thiazol- 4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide methanesulfonate (1.0 %w/w slurried in 0.2 vol of pre-filtered acetonitrile) was added, and the mixture was aged while agitating at a speed fast enough to keep solids from settling at 50-60°C for 2 hr. The mixture was then cooled to 0-5°C at 0.25°C/min and held at 0-5°C for at 6 hr. The mixture was filtered and the wet cake was washed twice with pre-filtered acetonitrile. The first wash consisted of 14.2 ml (6 vol) pre-filtered acetonitrile and the second wash consisted of 9.5 ml (4 vol) pre-filtered acetonitrile. The wet solid was dried at 50°C under vacuum, yielding 2.39 g (85.1% yield) of product

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WO 2014195852

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014195852&recNum=9&maxRec=777&office=&prevFilter=&sortOption=&queryString=EN_ALL%3Anmr+AND+PA%3Aglaxosmithkline&tab=PCTDescription

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WO 2014169770

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CN 104109159

http://www.google.co.in/patents/CN1597899A?cl=en

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CN 103588767

http://www.google.com/patents/CN103588767A?cl=en

Dara Phoenix (Dabrafenib) by the British GlaxoSmithKline (GSK) has developed Sisu threonine protein kinase (BRAF) inhibitor, as monotherapy ro ー kinds of clothes capsules for carrying BRAF V600E mutation surgical unresectable melanoma or metastatic melanoma treatment of adult patients, Dara Phoenix mesylate in May 2013 was approved by the US Food and Drug Administration (FDA), and is listed on the United States, the trade name Tafinlar (Da Feina). Since the European Medicines Agency (EMA) Committee for Medicinal Products for human use (CHMP) positive evaluation of Tafinlar, making the drug is expected to become after Roche’s Weiluofeini (Vemurafinib) to enter the European market, following a second BRAF inhibitors.

The chemical name Phoenix Dallas: N- [3- [5- (2- amino-4-pyrimidinyl) -2_ (tert-butyl) ~ ~ thiazol-4-yl] _2_ fluorophenyl] – 2,6_-difluorobenzenesulfonamide.

World Patent No. W02009137391, No. W02011047238 and W02012148588 number reported Dallas and Phoenix and its medicinal value synthesis method of the composition. According to the structural characteristics of Dara Phoenix and its analogues, the synthesis of such substances currently have A, B and C are three routes.

A more common route is the synthetic route, by reaction of 3-amino-2-fluorobenzoate (IX) first and 2,6_-difluorobenzene sulfonyl chloride (III) to amidation reaction occurs sulfonamide intermediate ( X); intermediate (X) with 2-chloro-4-methyl pyrimidine (XI) The condensation reaction occurs under the action of a strong base to give the intermediate (XII); intermediate (XII) to give the intermediate bromo

(XIII); intermediate (XIII) with 2,2_ dimethyl thiopropionamide (VI) to give the cyclized intermediate (XIV); and finally, the intermediate (XIV) by ammonolysis to afford the title compound Dallas Phoenix (I).

Different [0009] B is the first route by reaction of 3-amino-2-fluorobenzoate (IX) amino group protection, and thus condensation, cyclization, and bromo; then be obtained by deprotection of the amino group and the sulfonamide Intermediate (XIV); similarly, the intermediate

(XIV) obtained by ammonolysis target compound Dara Phoenix (I).

c route design features that first aminolysis reaction, and then give the desired product by deprotection and amino sulfonamide reaction. Clearly, this design is suitable for the route of these substituted amino ー aminolysis reaction, and for compounds such as Dallas Phoenix having pyrimidinylamino structure is not applicable. The reason is that if there are two aromatic amino groups will make the final sulfonamide ー reaction step to lose selectivity.

Example IV: the reaction flask was added N- [3- (5- formyl-2-t-butyl-ko -4_ thiazolyl) -2_ fluorophenyl] -2,6_ difluoro benzenesulfonamide (VIII) (5.4g, 11.5mmol), N, N- dimethylformamide dimethyl acetal (DMF-DMA) (2.74g, 23mmol) and xylene 50mL, heated to 140 ° C. About every four hours methanol was distilled out of the resulting reaction system, the reaction takes about 24 hours in total, the end of the reaction was detected by TLC. Cool, add hexane 40mL, have produced a yellow solid, filtered, and dried solids obtained after January nitrate melon (1.36,11.5mmol), sodium hydroxide (0.46g, 11.5mmol) and n-Ding enjoy 5OmL, warmed to 120 ° C, The reaction for 12 inches, TLC the reaction was complete. Cooling, with a crystal precipitated crystallized slowly for 3 inches, and filtered. The filter cake starched water, filtered and dried to yield an off-white solid Dara Phoenix (I) 3.58g, yield 60%.

………………………………………………….

WO 2014193898

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=04EC351C32F86C79CF575B3B4B7F46C3.wapp1nA?docId=WO2014193898&recNum=63&maxRec=11870&office=&prevFilter=&sortOption=&queryString=EN_ALL%3Anmr+AND+PA%3Amerck&tab=PCTDescription

References

Gibney, G. T.; Zager, J. S. (2013). “Clinical development of dabrafenib in BRAF mutant melanoma and other malignancies”. Expert Opinion on Drug Metabolism & Toxicology 9 (7): 1. doi:10.1517/17425255.2013.794220. PMID 23621583. edit
Huang, T.; Karsy, M.; Zhuge, J.; Zhong, M.; Liu, D. (2013). “B-Raf and the inhibitors: From bench to bedside”. Journal of Hematology & Oncology 6: 30. doi:10.1186/1756-8722-6-30. PMC 3646677. PMID 23617957. edit
“GSK melanoma drugs add to tally of U.S. drug approvals”. Reuters. May 30, 2013.
“Combined BRAF and MEK Inhibition in Melanoma with BRAF V600 Mutations” 367 (18). New England Journal of Medicine. November 1, 2012. pp. 1694–703. doi:10.1056/NEJMoa1210093. PMC 3549295. PMID 23020132.

“Dabrafenib/Trametinib Combination Approved for Advanced Melanoma”. OncLive. January 9, 2013.

Updates

Dabrafenib prediction
1H NMR PREDICT

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N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide NMR spectra analysis, Chemical CAS NO. 1195765-45-7 NMR spectral analysis, N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide H-NMR spectrum

13C NM PREDICT

logo
N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide NMR spectra analysis, Chemical CAS NO. 1195765-45-7 NMR spectral analysis, N-[3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide C-NMR spectrum

logo

COSY NMR PREDICT

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HMBC, HSBC NMR PREDICT

INTERMEDIATES

INT 1
Methyl 3-{[(2,6-difluorophenyl)sulfonyl]amino}-2-fluorobenzoate;Methyl 3-(tert-butoxycarbonylamino)-2-fluorobenzoate, 1195768-23-0

INT2

methyl 3-bromo-2-fluorobenzylate; PC3663; fluorobromobenzoic acid methyl ester;

3-bromo-2-fluorobenzoic acid methyl ester; 206551-41-9

INT3

methyl 3-amino-2-fluorobenzoate, CAS No. 1195768-18-3

INT4

Methyl 3-(tert-butoxycarbonylamino)-2-fluorobenzoate, CAS No. 1195768-19-4

INT5

CAS No. 1042055-86-6 Methyl 3-(tert-butoxycarbonylamino)-2-fluorobenzoate

INT6

2-fluoro-3-bromobenzoic acid;3-Bromo-2-fluoro-benzoic acid, CAS No. 161957-56-8

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SYNTHESIS

SYN 1

DABARAFENIB

GLAXOSMITHKLINE LLC; HOOS, Axel; GRESHOCK, Joel Patent: WO2014/66606 A2, 2014 ; Location in patent: Page/Page column 20; 24 ;

SYN 2

WO2011/47238 A1, ;

SYN 3

ACS Medicinal Chemistry Letters, , vol. 4, # 3 p. 358 – 362

SYN 4

ACS Medicinal Chemistry Letters, , vol. 4, # 3 p. 358 – 362

SYN 5

ACS Medicinal Chemistry Letters, , vol. 4, # 3 p. 358 – 362

SYN 6
WO2011/47238 A1, ;

SYN 7

methyl 3-amino-2-fluorobenzoate, CAS No. 1195768-18-3WO2011/47238

UPDATES WATCH REGULARLY

WO2015003571

达拉菲尼甲磺酸盐的新晶型及其制备方法

Brief Description

Figure 1 Form IV of the present invention an X-ray powder diffraction pattern.

Figure 2 is a schematic diagram Form IV of DSC language.

Figure 3 Form IV of the present invention TGA profiles.

Figure 4 is a dynamic water adsorption of Form IV of the invention, FIG.

Figure 5 Form IV of the present invention 1HNMR spectrum.

Figure 6 of the present invention, Form II X-ray powder diffraction pattern.

Figure 7 of the present invention, Form II TGA profiles.

Figure 8 Form III of the present invention an X-ray powder diffraction pattern.

Figure 9 Form V of the present invention, an X-ray powder diffraction pattern.

Figure 58a and in Example 10 in accordance with Patent Document WO2009 / 137391 or in CN200980126781.6

58d method described for the preparation of polymorph I of the known X-ray powder diffraction pattern.

Figure 11 is in accordance with Patent Document WO2009 / 137391 or CN200980126781.6 in the method of Example 58a and 58d described for the preparation of polymorph I of the known DSC pattern.

12 is in accordance with Patent Document WO2009 / 137391 or CN200980126781.6 in the method of Example 58a and 58d described for the preparation of polymorph I of the known TGA profiles.

Figure 13 is a known polymorph I in Comparative Example 1 in various stages XRPD comparison chart with the sample from top to bottom in the order of: Dara Phoenix free base hydrate, a known polymorph I in water was stirred for 15 After minutes to obtain a sample, and a known polymorph I.

Figure 14 Form IV in the present invention Comparative Example 1 each stage XRPD comparison chart with the sample from top to bottom in the order of: Form IV, Form IV in water with stirring for 15 minutes to obtain a sample, Form IV After stirring overnight in water to obtain a sample, as well as the free base of the hydrate Dara Phoenix. Figure 15 is a Comparative Form IV polymorph I of the known elution compared to the situation in Figure 1 (A to Form IV, ■ known Form 1).

Figure 16 is known in the polymorph I of Comparative Example 2 in various stages XRPD comparison chart (figure from top to bottom as follows: Form I is known API by “wet granulation” process of granulation (excluding section 3-step tablet) obtained by the sample, the known polymorphs I and amount of excipients formulated physically mixed formulation obtained sample, lactose monohydrate and microcrystalline cellulose according to Formulation physical sample after mixing, Dara Feeney free base hydrate, as well as known Form 1).

17 is a crystalline form IV according to the present invention in Comparative Example 2 in various stages XRPD comparison chart (from top to bottom as follows: In Form IV according to API “wet granulation” process of granulation (not included in Step 3 tableting) after the sample obtained, Form IV and excipients Formulation amount by physically mixing the obtained sample, the sample lactose monohydrate and microcrystalline cellulose according to Formulation after physical mixing, and Form IV).

FIG 5

Dabrafenib
Systematic (IUPAC) name

N-{3-[5-(2-aminopyrimidin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl}-2,6-difluorobenzenesulfonamide
Clinical data
Trade names	Tafinlar
Pregnancy category	US: D
Legal status	US: ℞-only
Identifiers
CAS number	1195765-45-7
ATC code	L01XE23
PubChem	CID 44462760
ChemSpider	25948204
ChEBI	CHEBI:75045
ChEMBL	CHEMBL2028663
Chemical data
Formula	C₂₃H₂₀F₃N₅O₂S₂
Molecular mass	519.56 g/mol

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