OA19483A - Substituted indoline derivatives as dengue viral replication inhibitors. - Google Patents
Substituted indoline derivatives as dengue viral replication inhibitors. Download PDFInfo
- Publication number
- OA19483A OA19483A OA1201900377 OA19483A OA 19483 A OA19483 A OA 19483A OA 1201900377 OA1201900377 OA 1201900377 OA 19483 A OA19483 A OA 19483A
- Authority
- OA
- OAPI
- Prior art keywords
- mmol
- compound
- methoxy
- phenyl
- reduced pressure
- Prior art date
Links
- 208000001490 Dengue Diseases 0.000 title claims abstract description 8
- 206010012310 Dengue fever Diseases 0.000 title claims abstract description 8
- 125000003387 indolinyl group Chemical class N1(CCC2=CC=CC=C12)* 0.000 title abstract 2
- 230000002401 inhibitory effect Effects 0.000 title description 4
- 239000003112 inhibitor Substances 0.000 title 1
- 230000029812 viral genome replication Effects 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 174
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 6
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 14
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 7
- 201000009910 diseases by infectious agent Diseases 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000000546 pharmaceutic aid Substances 0.000 claims description 2
- 125000001153 fluoro group Chemical group F* 0.000 claims 2
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 claims 2
- 239000003443 antiviral agent Substances 0.000 claims 1
- 125000001309 chloro group Chemical group Cl* 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 claims 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 114
- 238000000034 method Methods 0.000 abstract description 3
- 206010047461 Viral infection Diseases 0.000 abstract 3
- 208000001756 Virus Disease Diseases 0.000 abstract 3
- 238000002360 preparation method Methods 0.000 abstract 3
- 230000017613 viral reproduction Effects 0.000 abstract 3
- 230000002265 prevention Effects 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 178
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 141
- 230000002829 reduced Effects 0.000 description 124
- 239000000243 solution Substances 0.000 description 102
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 92
- 235000019439 ethyl acetate Nutrition 0.000 description 89
- 230000015572 biosynthetic process Effects 0.000 description 70
- 238000003786 synthesis reaction Methods 0.000 description 70
- 230000002194 synthesizing Effects 0.000 description 70
- 239000002904 solvent Substances 0.000 description 67
- 239000000543 intermediate Substances 0.000 description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 64
- 239000011541 reaction mixture Substances 0.000 description 64
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 62
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 62
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 62
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 61
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 54
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Substances C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 54
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 50
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 47
- 235000019341 magnesium sulphate Nutrition 0.000 description 46
- 238000003818 flash chromatography Methods 0.000 description 45
- 239000000741 silica gel Substances 0.000 description 45
- 229910002027 silica gel Inorganic materials 0.000 description 45
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 42
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 40
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 39
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 39
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 38
- CURLTUGMZLYLDI-UHFFFAOYSA-N carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 36
- 239000001569 carbon dioxide Substances 0.000 description 36
- 229910002092 carbon dioxide Inorganic materials 0.000 description 36
- 239000012044 organic layer Substances 0.000 description 36
- TUCNEACPLKLKNU-UHFFFAOYSA-N ethanone Chemical compound C[C]=O TUCNEACPLKLKNU-UHFFFAOYSA-N 0.000 description 34
- JNWBBCNCSMBKNE-UHFFFAOYSA-N HATU Chemical compound F[P-](F)(F)(F)(F)F.C1=CN=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 JNWBBCNCSMBKNE-UHFFFAOYSA-N 0.000 description 33
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 33
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 33
- 238000006243 chemical reaction Methods 0.000 description 32
- IMNFDUFMRHMDMM-UHFFFAOYSA-N n-heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 31
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 28
- 239000012071 phase Substances 0.000 description 28
- 210000004027 cells Anatomy 0.000 description 27
- 238000005160 1H NMR spectroscopy Methods 0.000 description 26
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 26
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 26
- 239000012074 organic phase Substances 0.000 description 24
- 239000002244 precipitate Substances 0.000 description 22
- 230000005526 G1 to G0 transition Effects 0.000 description 21
- 229910000027 potassium carbonate Inorganic materials 0.000 description 19
- 238000000746 purification Methods 0.000 description 19
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N DMSO-d6 Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 18
- 235000015320 potassium carbonate Nutrition 0.000 description 17
- 239000012267 brine Substances 0.000 description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 16
- -1 hydrohalic acids Chemical class 0.000 description 16
- 238000001665 trituration Methods 0.000 description 16
- 238000003756 stirring Methods 0.000 description 15
- 241000700605 Viruses Species 0.000 description 14
- 239000002253 acid Substances 0.000 description 14
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 14
- 229910052796 boron Inorganic materials 0.000 description 13
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 13
- 239000007787 solid Substances 0.000 description 13
- 239000007864 aqueous solution Substances 0.000 description 12
- 238000002844 melting Methods 0.000 description 11
- 238000007792 addition Methods 0.000 description 10
- FERIUCNNQQJTOY-UHFFFAOYSA-M butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 10
- 238000001816 cooling Methods 0.000 description 10
- GLXDVVHUTZTUQK-UHFFFAOYSA-M lithium;hydroxide;hydrate Chemical compound [Li+].O.[OH-] GLXDVVHUTZTUQK-UHFFFAOYSA-M 0.000 description 10
- 230000003287 optical Effects 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- 239000000843 powder Substances 0.000 description 10
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 10
- IJOOHPMOJXWVHK-UHFFFAOYSA-N Trimethylsilyl chloride Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 238000004808 supercritical fluid chromatography Methods 0.000 description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 9
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 8
- 239000003643 water by type Substances 0.000 description 8
- BPACUYIQSQIXCZ-UHFFFAOYSA-N 3-methoxy-5-(1,2,4-triazol-1-yl)aniline Chemical compound COC1=CC(N)=CC(N2N=CN=C2)=C1 BPACUYIQSQIXCZ-UHFFFAOYSA-N 0.000 description 7
- 101700033661 ACTB Proteins 0.000 description 7
- 102100011550 ACTB Human genes 0.000 description 7
- 101710032514 ACTI Proteins 0.000 description 7
- YNESATAKKCNGOF-UHFFFAOYSA-N Lithium bis(trimethylsilyl)amide Chemical compound [Li+].C[Si](C)(C)[N-][Si](C)(C)C YNESATAKKCNGOF-UHFFFAOYSA-N 0.000 description 7
- 239000007832 Na2SO4 Substances 0.000 description 7
- 229920000401 Three prime untranslated region Polymers 0.000 description 7
- 238000004166 bioassay Methods 0.000 description 7
- 239000002552 dosage form Substances 0.000 description 7
- 239000001963 growth media Substances 0.000 description 7
- 239000002609 media Substances 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- CZFUAUSGMISVFX-UHFFFAOYSA-N 6-(trifluoromethoxy)-2,3-dihydro-1H-indole Chemical compound FC(F)(F)OC1=CC=C2CCNC2=C1 CZFUAUSGMISVFX-UHFFFAOYSA-N 0.000 description 6
- WWBVZCBWSHFMLP-UHFFFAOYSA-N 6-(trifluoromethyl)-2,3-dihydro-1H-indole Chemical compound FC(F)(F)C1=CC=C2CCNC2=C1 WWBVZCBWSHFMLP-UHFFFAOYSA-N 0.000 description 6
- VTNHMFNIBCWZCZ-UHFFFAOYSA-N FC1=CC(=C(C=C1)C(C(=O)N1CCC2=CC=C(C=C12)C(F)(F)F)NC1=CC(=CC(=C1)N1N=CN=C1)OC)OCCO Chemical compound FC1=CC(=C(C=C1)C(C(=O)N1CCC2=CC=C(C=C12)C(F)(F)F)NC1=CC(=CC(=C1)N1N=CN=C1)OC)OCCO VTNHMFNIBCWZCZ-UHFFFAOYSA-N 0.000 description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N Triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-M acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 6
- 238000002425 crystallisation Methods 0.000 description 6
- 230000005712 crystallization Effects 0.000 description 6
- HBFXVTVOSLPOEY-UHFFFAOYSA-N ethoxyethane;2-propan-2-yloxypropane Chemical compound CCOCC.CC(C)OC(C)C HBFXVTVOSLPOEY-UHFFFAOYSA-N 0.000 description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 6
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- 238000004448 titration Methods 0.000 description 6
- 229940040692 Lithium Hydroxide Monohydrate Drugs 0.000 description 5
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium on carbon Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 5
- 230000000840 anti-viral Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
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- 125000003626 1,2,4-triazol-1-yl group Chemical group [*]N1N=C([H])N=C1[H] 0.000 description 4
- ILAHWRKJUDSMFH-UHFFFAOYSA-N Boron tribromide Chemical compound BrB(Br)Br ILAHWRKJUDSMFH-UHFFFAOYSA-N 0.000 description 4
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butanoic acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate dianion Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- ZXLHFPMPCDUKET-UHFFFAOYSA-N ClC1=CC(=C(C=C1)CC(=O)OCC)O Chemical compound ClC1=CC(=C(C=C1)CC(=O)OCC)O ZXLHFPMPCDUKET-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- QAEDZJGFFMLHHQ-UHFFFAOYSA-N Trifluoroacetic anhydride Chemical compound FC(F)(F)C(=O)OC(=O)C(F)(F)F QAEDZJGFFMLHHQ-UHFFFAOYSA-N 0.000 description 4
- 210000003501 Vero Cells Anatomy 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
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- 150000002500 ions Chemical class 0.000 description 4
- 239000010410 layer Substances 0.000 description 4
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
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- 229920002676 Complementary DNA Polymers 0.000 description 3
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- 230000000875 corresponding Effects 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- 108010037444 diisopropylglutathione ester Proteins 0.000 description 1
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide DMF Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 description 1
- UBHZUDXTHNMNLD-UHFFFAOYSA-N dimethylsilane Chemical compound C[SiH2]C UBHZUDXTHNMNLD-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 108091005938 enhanced green fluorescent protein Proteins 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 125000004464 hydroxyphenyl group Chemical group 0.000 description 1
- 230000000155 isotopic Effects 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 230000002934 lysing Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- FLXDYQAAHLOBCM-UHFFFAOYSA-N methyl 2-(4-chloro-2-hydroxyphenyl)acetate Chemical compound COC(=O)CC1=CC=C(Cl)C=C1O FLXDYQAAHLOBCM-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- QDHHCQZDFGDHMP-UHFFFAOYSA-N monochloramine Chemical compound ClN QDHHCQZDFGDHMP-UHFFFAOYSA-N 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- WSFCGNCBLRNXCO-UHFFFAOYSA-N oxolane;sulfuric acid Chemical compound C1CCOC1.OS(O)(=O)=O WSFCGNCBLRNXCO-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000001184 potassium carbonate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 239000001187 sodium carbonate Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- VDZXAOBGHPKGCS-UHFFFAOYSA-M sodium;1,4-dioxane;hydroxide Chemical compound [OH-].[Na+].C1COCCO1 VDZXAOBGHPKGCS-UHFFFAOYSA-M 0.000 description 1
- ODGROJYWQXFQOZ-UHFFFAOYSA-N sodium;boron(1-) Chemical compound [B-].[Na+] ODGROJYWQXFQOZ-UHFFFAOYSA-N 0.000 description 1
- MHSYOMOOZBKVFH-UHFFFAOYSA-M sodium;hydrogen carbonate;sodium Chemical compound [Na].[Na+].OC([O-])=O MHSYOMOOZBKVFH-UHFFFAOYSA-M 0.000 description 1
- WGRULTCAYDOGQK-UHFFFAOYSA-M sodium;sodium;hydroxide Chemical compound [OH-].[Na].[Na+] WGRULTCAYDOGQK-UHFFFAOYSA-M 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000000707 stereoselective Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 229960001367 tartaric acid Drugs 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- PHCBRBWANGJMHS-UHFFFAOYSA-J tetrasodium;disulfate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O PHCBRBWANGJMHS-UHFFFAOYSA-J 0.000 description 1
- 230000001225 therapeutic Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
Abstract
The present invention relates to substituted indoline derivatives, methods to prevent or treat dengue viral infections by using said compounds and also relates to said compounds for use as a medicine, more preferably for use as a medicine to treat or prevent dengue viral infections. The present invention furthermore relates to pharmaceutical compositions or combination preparations of the compounds, to the compositions or preparations for use as a medicine, more preferably for the prevention or treatment of dengue viral infections. The invention also relates to processes for preparation of the compounds.
Description
Pharmaceutically acceptable salts of said compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Suitable base salts are formed from bases which form nonîo toxic salts.
The pharmaceutically acceptable acid salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid addition sait forms that the compounds of formula (I) are able to form. These pharmaceutically acceptable 15 acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic {i.e. butane-dioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-amino5 salicylic, pamoic acid and the like acids.
The compounds of the invention may also exist in un-solvated and solvated forms.
The term “solvaté” is used herein to describe a molecular complex comprising the compound ofthe invention and one or more pharmaceutically acceptable solvent îo molécules, for example, éthanol.
The term “polymorph” refers to the ability ofthe compound ofthe invention to exist in more than one form or crystal structure.
The compounds of the présent invention may be administered as crystalline or amorphous products. They may be obtained for example as solid plugs, powders, or films by methods such as précipitation, crystallization, freeze drying, spray drying, or evaporative drying. They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term “excipient” is used herein to describe any ingrédient other than the compound(s) of the invention. The choice of excipient dépends largely on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
The compounds of the présent invention or any subgroup thereof may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited ail compositions usually employed for 30 systemically administering drugs. To préparé the pharmaceutical compositions of this invention, an effective amount ofthe particular compound, optionally in addition sait form, as the active ingrédient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of préparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral or rectal administration. For example, in preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid préparations such as suspensions, syrups, élixirs, émulsions,
-7and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case 5 solid pharmaceutical carriers are obviously employed. Also included are solid form préparations that can be converted, shortly before use, to liquid forms.
It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of îo dosage. Unit dosage form as used herein refers to physically discrète units suitable as unitary dosages, each unit containing a predetermined quantity of active ingrédient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, 15 wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.
Those of skill in the treatment of infectious diseases will be able to détermine the effective amount from the test results presented hereinafter. In general it is contemplated that an effective daily amount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 25 mg of active ingrédient per unit dosage form.
The exact dosage and frequency of administration dépends on the particular compound of the invention used, the particular condition being treated, the severity ofthe condition being treated, the âge, weight and general physical condition of 30 the particular patient as well as other médication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is évident that the effective amount may be lowered or increased depending on the response ofthe treated subject and/ordepending on the évaluation ofthe physician prescribing the compounds ofthe instant invention. The effective amount ranges mentioned above are therefore only guidelines and are not intended to limit the scope or use ofthe invention to any extent.
The présent disclosure is also intended to include any isotopes of atoms présent in the compounds of the invention. For example, isotopes of hydrogen include tritium
and deuterium and isotopes of carbon include C-13 and C-14.
The présent compounds used in the current invention may also exist in their stereochemically isomeric form, defining ail possible compounds made up of the 5 same atoms bonded by the same sequence of bonds but having different threedimensional structures, which are not interchangeable. Unless otherwise mentioned or indicated, the Chemical désignation of compounds encompasses the mixture of ail possible stereochemically isomeric forms, which said compounds might possess.
Said mixture may contain ail diastereomers and/or enantiomers of the basic molecular structure of said compound. Ail stereochemically isomeric forms of the compounds used in the présent invention either in pure form or in admixture with each other are intended to be embraced within the scope of the présent invention 15 including any racemic mixtures or racemates.
Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or 20 intermediates. In particular, the term 'stereoisomerically pure' concems compounds or intermediates having a stereoisomeric excess of at least 80% (i. e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric 25 excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms 'enantiomerically pure' and 'diastereomerically pure' should be understood in a similar way, but then having regard to the enantiomeric excess, respectively the diastereomeric excess of the mixture in 30 question.
Pure stereoisomeric forms of compounds and intermediates used in this invention may be obtained by the application of art-known procedures. For instance, enantiomers may be separated from each other by the sélective crystallization of 35 their diastereomeric salts with optically active acids or bases. Examples thereof are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphosulfonic acid. Altematively, enantiomers may be separated by chromatographie techniques using chiral stationary phases. Said pure stereochemically isomeric forms may
-9also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically. Preferably, if a spécifie stereoisomer is desired, said compound will be synthesized by stereospecific methods of préparation. These methods will advantageously employ enantiomerically pure starting materials.
The compounds of formula (I) of the présent invention ail hâve at least one chiral carbon atom as indicated in the figure below by the carbon atom Iabelled with * :
Due to the presence of said chiral carbon atom, a “compound of formula (I)” can be the (R)-enantiomer, the (S)-enantiomer, the racemic form, or any possible combination ofthe two individual enantiomers in any ratio. When the absolute (R)or (S)-configuration of an enantiomer is not known, this enantiomer can also be identified by indicating whether the enantiomer is dextrorotatory (+)- or levorotatory (-)- after measuring the spécifie optical rotation of said particular enantiomer.
In an aspect the présent invention relates to a first group of compound of formula (I) wherein the compounds of formula (I) hâve the (+) spécifie rotation.
In a further aspect the présent invention relates to a second ground of compounds of formula (I) wherein the compounds of formula (I) hâve the (-) spécifie rotation.
Examples
LC/MS methods
The High Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).
-10Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time...) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software.
Compounds are described by their experimental rétention times (Rt) and ions. If not specified differently in the table of data, the reported molecular ion îo corresponds to the [M+H]+ (protonated molécule) and/or [M-Hp (deprotonated molécule). In case the compound was not directly ionizable the type of adduct is specified (i.e. [Μ+ΝΗ4Γ, [M+HCOO]', etc...). For molécules with multiple isotopic patterns (Br, Cl), the reported value is the one obtained for the lowest isotope mass. Ail results were obtained with experimental uncertainties that are commonly 15 associated with the method used.
Hereinafter, “SQD” means Single Quadrupole Detector, “MSD” Mass Sélective Detector, “RT” room température, “BEH” bridged ethylsiloxane/silica hybrid, “DAD” Diode Array Detector, ”HSS” High Strength silica.
LC/MS Method codes (Flow expressed in mL/min; column température (T) in °C; Run time in minutes).
| Method code | Instrument | Column | Mobile phase | Gradient | Flow Col T | Run time (min) |
| LC-A | Waters: Acquity® UPLC®-DADQuattro Micro™ | Waters: BEH® C18 (1.7 pm, 2.1 x100 mm) | A: 95% CH3COONH4 7mM / 5% CH3CN, B: CH3CN | 84.2% A for 0.49 min, to 10.5% A in 2.18 min, held for 1.94 min, back to 84.2% A in 0.73 min, held for 0.73 min. | 0.343 mL/min 40°C | 6.2 |
| LC-B | Waters: Acquity® H-Class - DAD and SQD2TM | Waters: BEH® C18 (1.7 pm, 2.1 x 100 mm) | A: 95% CH3COONH4 7mM / 5% CH3CN, B: CH3CN | 84.2% A/15.8% B to 10.5% A in 2.18 min, held for 1.96 min, back to 84.2% A/15.8% B in 0.73 min, held for 0.49 min. | 0.343 mL/min 40°C | 6.1 |
| LC-C | Waters: Acquity® UPLC®-DADSQD | Waters: BEH C18(1.7pm, 2.1 x50 mm) | A: 10mM CH3COONH4 in 95% H2O + 5% | From 95% A to 5% A in 1.3 min, held for 0.7 min. | 0.8 mL/min | 2 |
| Method code | Instrument | Column | Mobile phase | Gradient | Flow Col T | Run time (min) |
| CH3CN B: CH3CN | 55°C | |||||
| LC-D | Waters: Acquity® UPLC®-DADSQD | Waters: HSS T3(1.8pm, 2.1 x 100 mm) | A: 10mM CH3COONH4 in 95% H2O + 5% CH3CN B: CH3CN | From 100% A to 5% A in 2.10 min, to 0% A in 0.90 min, to 5% A in 0.5 min | 0.7 mL/min 55°C | 3.5 |
SFC/MS methods
The SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO2) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS). It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time...) in order to obtain ions allowing the identification of the compound’s nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software.
Analytical SFC/MS Methods (Flow expressed in mL/min; column température (T) in °C; Run time in minutes, Backpressure (BPR) in bars.
| Method code | column | mobile phase | gradient | Flow Col T | Run time |
| BPR | |||||
| SFC-A | Daicel Chiralpak® IA column (5 pm, 150 x 4.6 mm) | A:CO2 B: EtOH (+0.3% iPrNH2) | 30% B hold 7 min, | 3 35 | 7 100 |
| SFC-B | Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) | A:CO2 B: EtOH (+0.3% iPrNH2) | 30% B hold 7 min, | 3 35 | 7 100 |
| SFC-C | Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) | A:CO2 B: EtOH (+0.3% iPrNH2) | 40% B hold 7 min, | 3 35 | 7 100 |
| Method | column | mobile phase | gradient | Flow | Run time |
| code | Col T | BPR | |||
| SFC-D | Daicel Chiralpak® IC column (5 pm, 150 x 4.6 mm) | A:CO2 B: IPrOH (+0.3% iPrNH2) | 25% B hold 7 min, | 3 35 | 7 100 |
| SFC-E | Daicel Chiralpak® IC column (5 pm, 150 x 4.6 mm) | A:CO2 B: iPrOH (+0.3% iPrNH2) | 30% B hold 7 min, | 3 35 | 7 100 |
| SFC-F | Daicel Chiralpak® AD-3 column (3 pm, 100 x 4.6 mm) | A:CO2 B: IPrOH (+0.3% iPrNH2) | 30% B hold 3 min, | 3.5 35 | 3 103 |
| SFC-G | Daicel Chiralpak® AD-H column (5 pm, 150 x 4.6 mm) | A:CO2 B: EtOH | 30% B hold 7 min, | 3 35 | 7 100 |
| SFC-H | Daicel Chiralpak® AS-3 column (3 pm, 100 x 4.6 mm) | A:CO2 B: iPrOH (+0.3% iPrNH2) | 20% B hold 10 min, | 3.5 35 | 10 103 |
| SFC-I | Daicel Chiralpak® AS-3 column (3 pm, 100 x 4.6 mm) | A:CO2 B: EtOH (+0.3% iPrNH2) | 10% B hold 10 min, | 3.5 35 | 10 103 |
| SFC-J | Daicel Chiralcel® OD-3 column (3 pm, 100 x 4.6 mm) | A:CO2 B:MeOH (+0.3% IPrNH2) | 20% B hold 6 min, | 3.5 35 | 6 103 |
| SFC-K | Daicel Chiralpak® AS3 column (3.0 pm, 150 x 4.6 mm) | A:CO2 B: EtOH (+0.2% IPrNH2 +3% H2O) | 10%-50% B in 6 min, hold 3.5 min | 2.5 40 | 9.5 110 |
Meltinq Points
Values are either peak values or melt ranges, and are obtained with experimental uncertainties that are commonly associated with this analytical method.
DSC823e (indicated as DSC)
For a number of compounds, melting points were determined with a DSC823e
-13(Mettler-Toledo). Melting points were measured with a température gradient of 10°C/minute. Maximum température was 300°C.
Optical Rotations:
Optical rotations were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and reported as follows: [α]° (λ, c g/10C)ml, solvent, T°C).
[α]λτ = (100a) / (/x c) : where I is the path length in dm and c is the concentration in g/100 ml for a sample at a température T (°C) and a wavelength λ (in nm). If the wavelength of light used is 589 nm (the sodium D line), then the symbol D might be used instead. The sign of the rotation (+ or -) should always be given. When using this équation the concentration and solvent are always provided in parenthèses after the rotation. The rotation is reported using degrees and no units of concentration are given (it is assumed to be g/100 ml).
Abbreviations used in experimental part
| (M+H)+ | protonated molecular ion | HCl | hydrochloric acid |
| aq. | aqueous | HPLC | high performance liquid chromatography |
| Boc | fert-butyloxycarbonyl | iPrNH2 | isopropylamine |
| BoczO | di-tert-butyl dicarbonate | iPrOH | 2-propanol |
| br | broad | K2CO3 | potassium carbonate |
| CH3CN | acetonitrile | UAIH4 | lithium aluminium hydride |
| CHCI3 | chloroform | m/z | mass-to-charge ratio |
| CH2CI2 | dichloromethane | Me | methyl |
| CH3OH | methanol | MeOH | methanol |
| CO2 | carbon dioxide | MgSO4 | magnésium sulfate |
| d DCM__ DIEA | jdoublet _____ _ _ _ dichloromethane_____ _ diisopropylethylamine | min n2___________________ Na2CO3 | _minute(s)_________ nitrogen________ sodium carbonate |
| DIPE | diisopropyl ether | Na2SO4 | sodium sulfate |
| DMA | dimethylacetamide | NaBH4 | sodium borohydride |
| DMAP | 4-dimethylaminopyridine | NaHCO3 | sodium bicarbonate |
| DME | 1,2-dimethoxyethane | NaOH | sodium hydroxide |
| DMF | dimethylformamide | NH4CI | ammonium chloride |
| DMSO | dimethyl sulfoxide | q | quartet |
| eq. | équivalent | rt or RT | room température |
| Et2O | diethyl ether | s | singlet |
| Et3N | triethylamine | t | triplet |
| EtOAc | ethyl acetate | tBuOK | potassium terf-butanolaat |
| EtOH | éthanol | ΤΙΞΑ | triethylamine |
| H2O | water | TFA | trifluoroacetic acid |
| H2SO4 | sulfuric acid | THF | tetrahydrofuran |
| HATU | O-(7-aza-1 H-benzotriazol-1 yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate -CAS [148893-10-1] | TMSCI | trimethylsilyl chloride |
Example 1 : synthesis of 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1H-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 1) and chiral séparation into enantiomers 1A and 1B.
HCl (4M in dioxane)
MeOH rt, 1h
Enantiomers 1A and 1B
Synthesis of intermediate 1a:
A solution of 4-fluoro-2-methoxyphenylacetic acid [CAS 886498-61 -9] (10 g, 54.3 mmol) in EtOH (200 mL) and H2SO4 (2 mL) was heated under reflux for 12 h. Water was added and the mixture was concentrated under reduced pressure to half of the original volume. Ice was added. The solution was basified with K2CO3
-15and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give ethyl 2-(4-fluoro-2-methoxyphenyl)acetate 1a (11.6 g). The compound was used directly in the next step.
Synthesis of intermediate 1b:
A 1M solution of boron tribromide in CH2CI2 (109.3 mL, 109.3 mmol) was added dropwise to a solution of ethyl 2-(4-fluoro-2-methoxyphenyl)acetate 1a (11.6 g, 54.7 mmol) in CH2CI2 (300 mL) at -30°C. The reaction was stirred at -20°C for 1 h, 10 and then quenched with CH3OH. The pH was adjusted to 8 by adding a saturated water solution of NaHCOs. The solution was extracted with CH2CI2 and the combined organic layers were dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give ethyl 2-(4-fluoro-2hydroxyphenyl)acetate 1b (10.8 g). The compound was used directly in the next 15 step without further purification.
Synthesis of intermediate 1c:
To a mixture of ethyl 2-(4-fluoro-2-hydroxyphenyl)acetate 1b (10.6 g, 53.5 mmol) and césium carbonate (34.8 g, 106.9 mmol) in DMF (200 mL) at 10°C was added 20 (2-bromoethoxy)(tert-butyl)dimethylsilane [CAS 86864-60-0] (13.8 mL, 64.2 mmol).
The reaction mixture was stirred at room température overnight. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μΜ, 40 g, heptane/EtOAc 25 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4fluorophenyl)acetate 1c (17.7 g).
Synthesis of intermediate 1d:
To a 1M lithium bis(trimethylsilyl)amide solution in THF (28.05 mL, 28.05 mmol), cooled at -78°C, was added a solution of ethyl 2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 1c (5 g, 14.03 mmol) in THF (30 mL). After stirring for 1 h at -78°C, chlorotrimethylsilane (2.85 mL, 22.4 mmol) was added. The reaction mixture was stirred at -78°C for 15 min. N35 Bromosuccinimide (3 g, 16.8 mmol) in THF (30 mL) was added and stirring was continued at -55°C for 2 h. The reaction mixture was poured out into H2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to give ethyl 2-bromo-219483
-16(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 1d (6.57 g) which was used in the next step without further purification.
Synthesis of intermediate 1e:
A mixture of ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4fluorophenyl)acetate 1d (3.1 g, 7.12 mmol), 3-methoxy-5-(1/7-1,2,4-triazol-1yl)aniline [CAS 1220630-56-7] (2.03 g, 10.7 mmol) and diisopropylethylamine (2.45 mL, 14.2 mmol) in CH3CN (60 mL) was stirred at 50°C for 18 h. The reaction mixture was concentrated under reduced pressure. The residue was taken up with EtOAc and washed with 0.5N HCl, water and brine. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc gradient 80/20 to 60/40) to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4fluorophenyl)-2-((3-methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)acetate 1e (2.5 g).
Synthesis of intermediate 1f:
A solution of lithium hydroxide monohydrate (226 mg, 5.397 mmol) in water (25 mL) was added portionwise to a solution of ethyl 2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 yl)phenyl)amino)acetate 1e (2.45 g, 4.498 mmol) in a solvent mixture of THF/CH3OH (1/1) (50 mL) at 10°C. The reaction was stirred at room température for 6 h, diluted with water and cooled to 0°C. The solution was slowly acidified with 0.5N HCl to pH 6, and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(1/71,2,4-triazol-1-yl)phenyl)amino)acetic acid 1f (2.05 g). The compound was used directly in the next step without further purification.
Synthesis of intermediate 1g:
To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)acetic acid 1f (1.58 g, 3.06 mmol) in DMF (20 mL) were added HATU (1.74 g, 4.60 mmol), diisopropylethylamine (1.5 mL, 9.17 mmol) and 6-(trifluoromethyl)indoline [CAS 181513-29-1] (572 mg, 3.06 mmol). The reaction mixture was stirred at room température for 2 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert19483
-17butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-tnazol-1 yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 1g (2.1 g). The crude compound was used directly in the next step.
Synthesis of Compound 1 and chiral séparation into Enantiomers 1A and 1 B: Under a N2 flow, at 5°C, 4M HCl in dioxane (7.65 mL, 30.6 mmol) was added dropwise to a solution of 2-(2-(2-((fert-butyldimethylsilyl)oxy)ethoxy)-4fluorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(6(trifluoromethyl)indolin-1-yl)ethanone 1g (2.1 g, 3.06 mmol) in MeOH (40 mL).
The reaction was stirred at room teperature for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was crystallized from CHsCN/diisopropyl ether to give 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1yl)ethanone 1 (800 mg) as a racemate.
The enantiomers of Compound 1 (720 mg) were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® IA 5 pm 250 x 20 mm, Mobile phase: 70% CO2, 30% EtOH (+0.3% iPrNH2)). The first eluted enantiomer (303 mg) was crystallized from Et2O to give Enantiomer 1A (270 mg). The second eluted enantiomer (320 mg) was crystallized from Et2Û to give Enantiomer 1B (274 mg).
Compound 1:
1H NMR (500 MHz, DMSO-d6) δ ppm 3.15 - 3.30 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.85 (m, 2 H) 4.06 - 4.17 (m, 3 H) 4.45 (td, 7=10.3, 6.1 Hz, 1 H) 4.98 (t, 7=5.4 Hz, 1 H) 5.83 (d, 7=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (t, 7=1.9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, 7=11.2, 2.4 Hz, 1 H) 7.37 - 7.42 (m, 2 H) 7.44 - 7.49 (m, 1 H) 8.16 (s, 1 H) 8.39 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-A): Rt 3.06 min, MH+ 572 Melting point: 151°C
Enantiomer 1A:
1H NMR (500 MHz, DMSO-de) δ ppm 3.15 - 3.29 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.06 - 4.19 (m, 3 H) 4.45 (td, 7=10.2, 6.3 Hz, 1 H) 4.97 (t, 7=5.5 Hz, 1 H) 5.83 (d, 7=8.8 Hz, 1 H) 6.36 (s, 1 H) 6.66 (t, 7=1.9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, 7=11.3, 2.5 Hz, 1 H) 7.37 - 7.42 (m, 2 H) 7.46 (d, 7=7.9 Hz, 1 H) 8.16 (S, 1 H) 8.39 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-A): Rt 3.06 min, MH+572
-18[a]D 20: .44.3° (C 0.2525, DMF)
Chiral SFC (method SFC-A): Rt 2.59 min, MH+ 572, chiral purity 100%.
Melting point: 163°C
Enantiomer 1 B:
1H NMR (500 MHz, DMSO-cfe) δ ppm 3.15 - 3.30 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.85 (m, 2 H) 4.06 - 4.19 (m, 3 H) 4.45 (td, J=10.3, 6.1 Hz, 1 H) 4.97 (t, J=5.5 Hz, 1 H) 5.83 (d, J=8.5 Hz, 1 H) 6.36 (s, 1 H) 6.66 (t, J=) .9 Hz, 1 H) 6.76 - 6.82 (m, 2 H) 6.84 (s, 1 H) 6.98 (dd, J=11.2, 2.4 Hz, 1 H) 7.36 - 7.43 (m, 2 H) 7.46 (d, J=7.9 Hz, 10 1 H) 8.16 (s, 1 H) 8.39 (s, 1 H) 9.13 (s, 1 H)
LC/MS (method LC-A): Rt 3.06 min, MH+ 572
[a]D 20: +36.2° (c 0.2567, DMF)
Chiral SFC (method SFC-A): Rt 3.15 min, MH+572, chiral purity 98.07%.
Melting point: 162°C 15
Example 2 : synthesis of 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1H-1,2,4-triazol-1-yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1yl)ethanone (Compound 2) and chiral séparation into Enantiomers 2A and 2B.
MeOH rt, 1h
HCl (4M in dioxane)
enantiomers 2A and 2B
Synthesis of intermediate 2a:
A mixture of 1-methoxy-4-nitro-2-(trifluoromethyl)benzene [CAS 654-76-2] (24.5 g, 110.8 mmol) and 4-chlorophenoxyacetonitrile [CAS 3598-13-8] (20.4 g, 121.9 mmol) in DMF (100 mL) was added dropwise over 30 min to a stirred solution of tBuOK (27.35 g, 243.7 mmol) in DMF (100 mL) at -10°C. After addition, the purple
-19solution was maintained at -10°C for 1 h. 500 ml. of ice-water and 500 mL of 6N HCl were added and the precipitate was filtered off, washed with water and dried under reduced pressure to afford 40.4 g of 2-(5-methoxy-2-nitro-4(trifluoromethyl)phenyl)acetonitrile 2a (used as such in the next step).
Synthesis of intermediate 2b:
A solution of 2-(5-methoxy-2-nitro-4-(trifluoromethyl)phenyl)acetonitrile 2a (26 g , 99.9 mmol) in ethanol/water (9/1) (500 mL) and AcOH (5.2 mL) was hydrogenated for 1 h at a pressure of 3.5 Bar with 10% Pd/C (15.3 g) as the catalyst. The îo reaction mixture was filtered through a pad of celite® and the filter cake was washed with a solvent mixture of CH2CI2 and CH3OH. The filtrate was concentrated under reduced pressure. The residue was filtered through a glass filter charged with silica 60-200 pm using heptane/EtOAc 80/20 as the eluent. The fractions containing the expected compound were combined and the solvent was 15 concentrated under reduced pressure to give 5-methoxy-6-(trifluoromethyl)-1 Hindole 2b (15.6 g).
Synthesis of intermediate 2c:
At 0°C, BHs-Pyridine (23.5 mL, 232.4 mmol) was added dropwise to a solution of 20 5-methoxy-6-(trifluoromethyl)-1/-/-indole 2b (10 g, 46.5 mmol) in EtOH (60 mL). 6N
HCl (140 mL) was slowly added while maintaining the température below 10°C.
The mixture was stirred at 0°C for 2 h. Water (200 mL) was added and the mixture was basified to pH 8-9 with a concentrated aqueous solution of NaOH (the reaction température was kept below 20°C). The precipitate was filtered off, 25 washed with water (twice) and co-evaporated under reduced pressure with toluene to give 5-methoxy-6-(trifluoromethyl)indoline 2c (9 g).
Synthesis of intermediate 2d:
To a solution of 2-(2-(2-((fert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((330 methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)acetic acid 1f (1.02 g, 1.97 mmol) in DMF (10 mL) were added HATU (1.13 g, 2.96 mmol), diisopropylethylamine (979 pL, 5.92 mmol) and 5-methoxy-6-(trifluoromethyl)indoline 2c (429 mg, 1.97 mmol). The reaction mixture was stirred at room température for 2 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water 35 and taken up with EtOAc. The organic layer was washed with a 10% solution of
K2CO3 in water, water, dried over MgSOq, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 19483
-20yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 2d (1.36 g).
The compound was used as such in the next reaction step.
Synthesis of Compound 2 and chiral séparation into Enantiomers 2A and 2B: Under a N2 flow, at 5°C, 4M HCl in dioxane (4.75 mL, 18.99 mmol) was added dropwise to a solution of 2-(2-(2-((ter(-butyldimethylsilyl)oxy)ethoxy)-4fluorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(5-methoxy6-(trifluoromethyl)indolin-1-yl)ethanone 2d (1.36 g, 1.9 mmol) in MeOH (25 mL). The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc. The organic phase was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was crystallized from MeOH to give 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 2 (850 mg) as a racemate.
The enantiomers of Compound 2 (800 mg) were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® IA 5 pm 250 x 20 mm, Mobile phase: 6% CH2CI2, 70% CO2, 24% EtOH (+0.3% iPrNH2)). The, first eluted enantiomer (370 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 2A (329 mg). The second eluted enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CH2CI2/MeOH 99/1). The pure fractions were combined and the solvent was concentrated under reduced pressure. The residue (320 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 2B (262 mg).
Compound 2:
Ή NMR (500 MHz, DMSO-de) δ ppm 3.15 - 3.30' (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.04 - 4.18 (m, 3 H) 4.43 (td, J=10.4, 6.3 Hz, 1 H) 4.99 (t, J=5.7 Hz, 1 H) 5.81 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.65 (t, J=1.9 Hz, 1 H) 6.75 6.81 (m, 2 H) 6.83 (s, 1 H) 6.98 (dd, 1.2, 2.4 Hz, 1 H) 7.24 (s, 1 H) 7.39 (dd, J=8.5, 6.9 Hz, 1 H) 8.16 (s, 1 H) 8.35 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-A): Rt 2.99 min, MH+ 602 Melting point: 192°C
Enantiomer 2A:
1H NMR (400 MHz, DMSO-de) δ ppm 3.16 - 3.28 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.03 - 4.18 (m, 3 H) 4.37 - 4.49 (m, 1 H) 4.97 (t, J=5.Q Hz, 1
-21H) 5.81 (d, J=8.1 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.73 - 6.81 (m, 2 H) 6.83 (s, 1
H) 6.97 (dd, J=11.1, 2.0 Hz, 1 H) 7.23 (s, 1 H) 7.39 (t, J=7.6 Hz, 1 H) 8.15 (s, 1 H) 8.35 (s, 1 H) 9.12 (s, 1 H)
LC/MS (method LC-A): Rt 2.97 min, MH+602
[a]D 20: -45.0° (c 0.2425, DMF)
Chiral SFC (method SFC-A): Rt4.14 min, MH+ 602, chiral purity 100%.
Enantiomer 2B:
1H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 - 3.28 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 10 (m, 2 H) 3.84 (s, 3 H) 4.02 - 4.20 (m, 3 H) 4.42 (td, J=10.2, 6.3 Hz, 1 H) 4.97 (t,
J=5.6 Hz, 1 H) 5.81 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.73 - 6.81 (m, 2 H) 6.83 (s, 1 H) 6.97 (dd, J=11.4, 2.3 Hz, 1 H) 7.23 (s, 1 H) 7.36 - 7.43 (m, 1 H) 8.15 (s, 1 H) 8.35 (s, 1 H) 9.12 (s, 1 H) LC/MS (method LC-A): Rt2.97 min, MH+ 602
[a]D 20: +43.4° (c 0.2007, DMF)
Chiral SFC (method SFC-A): Rt 5.08 min, MH+602, chiral purity 100%.
Example 3 : synthesis of 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1 /-/-1,2,4-triazol-1 -yl)phenyl)amino)-1 -(6-(trifluoromethoxy)indolin-1 -yl)ethanone (Compound 3) and chiral séparation into Enantiomers 3A and 3B.
HATU, iPr2NEt DMF, rt2h
MeOH rt, 1h
HCl (4M in dioxane)
Chiral séparation
Enantiomers 3A and 3B
Synthesis of intermediate 3a:
To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3methoxy-5-(1/-/-1,2,4-triazol-1-yl)phenyl)amino)acetic acid 1f (1.02 g, 1.974 mmol)
-22in DMF (10 mL) were added HATU (1.13 g, 2.96 mmol), diisopropylethylamine (979 pL, 5.92 mmol) and 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (401 mg, 1.97 mmol). The reaction mixture was stirred at room température for 2 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed 5 with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3a (1.34 g). The crude compound was 10 used directly in the next reaction step.
Synthesis of Compound 3 and chiral séparation into Enantiomers 3A and 3B: Under aN2 flow, at 5°C, 4M HCl in dioxane (4.27 mL, 17.1 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-415 fluorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)-1 -(6(trifluoromethoxy)indolin-1-yl)ethanone 3a (1.2 g, 1.71 mmol) in MeOH (25 mL). The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
The organic phase was separated, dried over MgSO4, filtered and the solvent was 20 concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2Cl2/MeOH gradient 99.5/0.5 to 99/1). The pure fractions were combined and concentrated to dryness under reduced pressure to give 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3 25 (550 mg) as a racemate. Part of this fraction was crystallized from MeOH to provide Compound 3 (36 mg).
The remaining material was used to separate the enantiomers of Compound 3 via Préparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 20 mm, 30 Mobile phase: 65% CO2, 35% EtOH (+0.3% iPrNH2)). The first eluted enantiomer (210 mg) was solidified by trituration with diisopropyl ether/heptane to give Enantiomer 3A (182 mg). The second eluted enantiomer (230 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CH2Cl2/MeOH 99/1 ). The pure fractions were combined and the solvent was concentrated under 35 reduced pressure. The residue (180 mg) was solidified by trituration with diisopropyl ether/heptane to give Enantiomer 3B (137 mg).
-23Compound 3:
1H NMR (500 MHz, DMSO-c/6) δ ppm 3.06 - 3.25 (m, 2 H) 3.73 (s, 3 H) 3.75 - 3.86 (m, 2 H) 4.06 - 4.17 (m, 3 H) 4.38 - 4.50 (m, 1 H) 4.95 (br s, 1 H) 5.81 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.75 - 6.81 (m, 2 H) 6.83 (s, 1 H) 6.94 - 7.04 (m, 2 5 H) 7.33 (d, J=8.6 Hz, 1 H) 7.36 - 7.43 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.11 (s, 1
H)
LC/MS (method LC-A): Rt 3.13 min, MH+ 588
Melting point: 178°C
Enantiomer 3A:
1H NMR (400 MHz, DMSO-ds) δ ppm 3.06 - 3.26 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.05 - 4.18 (m, 3 H) 4.38 - 4.50 (m, 1 H) 4.97 (t, J=5.3 Hz, 1 H) 5.82 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.74 - 6.86 (m, 3 H) 6.94 - 7.04 (m, 2 H) 7.34 (d, J=8.1 Hz, 1 H) 7.36 - 7.42 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H)
LC/MS (method LC-A): Rt 3.11 min, MH+588
[a]D 20: -38.2° (c 0.28, DMF)
Chiral SFC (method SFC-B): Rt3.38 min, MH+ 588, chiral purity 100%.
Enantiomer 3B:
1H NMR (400 MHz, DMSO-de) δ ppm 3.07 - 3.26 (m, 2H) 3.72 (s, 3 H) 3.74 - 3.86 (m, 2 H) 4.04 - 4.20 (m, 3 H) 4.38 - 4.50 (m, 1 H) 4.97 (t, J=5.6 Hz, 1 H) 5.81 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.74 - 6.87 (m, 3 H) 6.94 - 7.03 (m, 2 H) 7.34 (d, J=8.1 Hz, 1 H) 7.36 - 7.42 (m, 1 H) 8.04 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H) LC/MS (method LC-A): Rt 3.11 min, MH+ 588
[a]D 20: +40.9° (c 0.23, DMF)
Chiral SFC (method SFC-B): Rt5.31 min, MH+588, chiral purity 100%.
Example 4 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 4) and chiral séparation into Enantiomers 4A and 4B.
Synthesis of intermediate 4a:
A solution of 2-(4-chloro-2-methoxyphenyl)acetic acid [CAS 170737-95-8] (20 g, 5 101 mmol) in dry THF (300 mL) was cooled at 0°C. Oxalyl chloride (18 mL, 202 mmol) and two drops of DMF were added. The reaction mixture was stirred at room température for 30 min. The solvent was evaporated under reduced pressure. The residue was dissolved in éthanol (300 mL) and the reaction mixture was stirred at room température for 1 h. The reaction mixture was concentrated îo under reduced pressure to give ethyl 2-(4-chloro-2-methoxyphenyl)acetate 4a (23 g), which was used in the next step without further purification.
Synthesis of intermediate 4b:
To a solution of ethyl 2-(4-chloro-2-methoxyphenyl)acetate 4a (10 g, 44 mmol) in 15 CH2CI2 (350 mL), cooled at -30°C, was added dropwise a 1M BBr3 solution in
CH2CI2 (87.5 mL, 87.5 mmol) while maintaining the température below -20 °C. The reaction mixture was stirred at -30°C for 1 h before quenching with methanol. The pH was adjusted to 8 by addition of an aqueous saturated solution of NaHCOs.
The phases were separated. The aqueous phase was extracted with CH2CI2. The 20 organic phases were combined, dried over MgSO4, filtered and concentrated
-25under reduced pressure to afford ethyl 2-(4-chloro-2-hydroxyphenyl)acetate 4b (9.5 g), which was used in the next step without further purification.
Synthesis of intermediate 4c:
To a mixture of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate 4b [CAS 1261826-30-5] (2.82 g, 13.1 mmol) and césium carbonate (8.56 g, 26.3 mmol) in DMF (50 mL) was added benzyl 2-bromoethyl ether [CAS 1462-37-9] (2.29 g, 14.5 mmol). The reaction mixture was stirred at room température for 24 h. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (2% to 20%) in heptane to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetate 4c (4.17 g).
Synthesis of intermediate 4d:
To a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetate 4c (4.17 g, 12.0 mmol) in a mixture of EtOH (80 mL) and THF (40 mL) was added 0.5N NaOH (72 mL, 36.0 mmol). The reaction mixture was stirred at room température for 3 h. The reaction mixture was partially concentrated under reduced pressure to remove 20 the organic solvents. The residue was acidified to pH 2-3 with 1N HCl and the mixture was extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure to give 2-(2-(2(benzyloxy)ethoxy)-4-chlorophenyl)acetic acid 4d (3.83 g).
Synthesis of intermediate 4e:
A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetic acid 4d (7.12 g, 22.2 mmol) in thionyl chloride (50 mL, 689 mmol) was stirred at room température for 18 h. The reaction mixture was concentrated under reduced pressure and coevaporated with toluene to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetyl 30 chloride 4e (7.53 g) which was used in the next step without further purification.
Synthesis of intermediate 4f:
A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetyl chloride 4e (5.29 g, 15.6 mmol) in CH3CN (50 mL) was added dropwise under N2-atm to a stirring 35 mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (2.92 g, 15.6 mmol) and sodium bicarbonate (1.44 g, 17.1 mmol) in CH3CN (50 mL). The reaction mixture was stirred at room température for 65 h and poured out into water (500 mL). The product was extracted (2x) with Et20. The combined organic layers were washed
-26with brine, dried over MgSO4, filtered and evaporated under reduced pressure. The residue solidified upon standing. The product was stirred up in diisopropyl ether (25 mL), filtered off, washed (3x) with diisopropyl ether, and dried under vacuum at 45°C to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-1-(65 (trifluoromethyl)indolin-l-yl)ethanone 4f (6.97 g).
Synthesis of intermediate 4g:
A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-1-(6(trifluoromethyl)indolin-l-yl)ethanone 4f (1.5 g, 3.06 mmol) in 2-Me-THF (125 mL) îo was stirred under N2-flow and cooled to -78°C. A solution of 1M lithium bis(trimethylsilyl)amide in THF (6.12 mL, 6.12 mmol) was added dropwise and the resulting mixture was stirred at -78°C for 20 minutes. Chlorotrimethylsilane (626 pL, 4.90 mmol) was added dropwise and the mixture was stirred at -78°C for 25 min. A solution of A/-bromosuccinimide (599 mg, 3.37 mmol) in 2-Me-THF (50 mL) was added dropwise and the reaction mixture was stirred at -78°C for 1 h. An aqueous saturated solution of NH4CI (60 mL) was added at once, and the resulting mixture was stirred without cooling until the température reached 0°C. Water (20 mL) was added and, after stirring for 30 min, the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated under reduced pressure, and co20 evaporated with CH3CN to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2bromo-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4g (1.16 g). The product was used without further purification in the next step.
Synthesis of intermediate 4h:
To a stirred solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromo-1-(6(trifluoromethyl)indolin-1-yl)ethanone 4g (1.74 g, 3.06 mmol) in CH3CN (100 mL) under N2-atm were added 3-methoxy-5-(1/7-1,2,4-triazol-1-yl)aniline [CAS 1220630-56-7] (874 mg, 4.59 mmol), and diisopropylethylamine (1.06 mL, 6.12 mmol) and the reaction mixture was stirred at room température for 20 h and then at 50°C for 7 h. The mixture was cooled to room température and poured out into stirring H2O (400 mL). The product was extracted (2x) with Et20. The combined organic layers were dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a gradient of heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure and co-evaporated with toluene. The residue was dried under vacuum at 50°C to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4h (1.43 g).
-27Synthesis of Compound 4 and chiral séparation into Enantiomers 4A and 4B:
A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 H1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4h (1.43 g, 2.11 mmol) in a solvent mixture of THF (15 mL) in EtOAc (75 mL) was hydrogenated for 5 h at room température under atmospheric pressure of H2 using Pd/C (0.5 g) as the catalyst. The catalyst was removed by filtration over dicalite®. The filter cake was washed several times with THF, and the combined filtrâtes were evaporated under reduced pressure. The solid residue was stirred up in a solvent mixture CH2Cl2/EtOAc/MeOH 1/2/1. The precipitate was filtered off, washed (2x) with EtOAc, and dried under vacuum at 45°C to provide racemic 2-(4chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-( 1/7-1,2,4-triazol-1 yl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 4, 600 mg).
The enantiomers of Compound 4 (700 mg) were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® Diacel AD 20 x 250 mm, Mobile phase: CO2, MeOH/iPrOH (50/50) + 0.4% iPrNH2). The product fractions were combined and evaporated under reduced pressure to provide Enantiomer 4A as the first eluted product and Enantiomer 4B as the second eluted product. Both enantiomers were further purified by flash chromatography on silica gel (4 g) using a gradient heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and evaporated under reduced pressure. The residue was stirred in water (3 mL) and MeOH (0.75 mL). The solids were filtered off, washed (3x) with water, and dried under vacuum at 50°C to provide Enantiomer 4A (81 mg) and Enantiomer 4B (132 mg).
Enantiomer 4A:
1H NMR (400 MHz, DMSO-d6) δ ppm 3.18 - 3.28 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.84 30 (m, 2 H) 4.08 - 4.19 (m, 3 H) 4.44 (td, 7=10.2, 6.7 Hz, 1 H) 4.96 (t, 7=5.6 Hz, 1 H)
5.84 (d, J=8.Q Hz, 1 H) 6.35 (t, 7=2.1 Hz, 1 H) 6.66 (t, 7=1.7 Hz, 1 H) 6.79 - 6.87 (m, 2 H) 7.02 (dd, 7=8.3, 1.9 Hz, 1 H) 7.15 (d, 7=1.8 Hz, 1 H) 7.34 - 7.43 (m, 2 H) 7.43 - 7.51 (m, 1 H) 8.15 (s, 1 H) 8.38 (br s, 1 H) 9.12 (s, 1 H) LC/MS (method LC-C): Rt 1.16 min, MH+ 588
[a]D 20: -42.9° (c 0.515, DMF)
Chiral SFC (method SFC-K): Rt 2.91 min, MH+ 588, chiral purity 100%.
-28Enantiomer 4B:
1H NMR (400 MHz, DMSO-cfe) δ ppm 3.15 - 3.28 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.84 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.44 (td, J=10.1,6.6 Hz, 1 H) 4.96 (t, J=5.5 Hz, 1 H) 5.84 (d, J=8.6 Hz, 1 H) 6.35 (t, J=2.0 Hz, 1 H) 6.67 (t, J=) .9 Hz, 1 H) 6.79 - 6.87 (m, 2 H) 7.03 (dd, J=8.1,2.0 Hz, 1 H) 7.15 (d, J=2.0 Hz, 1 H) 7.34 - 7.42 (m, 2 H)
7.43 - 7.49 (m, 1 H) 8.16 (s, 1 H) 8.38 (br s, 1 H) 9.13 (s, 1 H)
LC/MS (method LC-C): Rt 1.15 min, MH+ 588
[a]D 20: +39.5° (c 0.595, DMF)
Chiral SFC (method SFC-K): Rt2.78 min, MH+ 588, chiral purity 100%.
Example 5 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5(1H-1,2,4-triazol-1-yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1yl)ethanone (Compound 5) and chiral séparation into Enantiomers 5A and 5B.
HCl (4M in dioxane)
MeOH rt, 1h
chiral séparation enantiomers 5Aand 5B
-29Synthesis of intermediate 5a:
To a mixture of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate 4b (5.2 g, 24.2 mmol) and césium carbonate (15.8 g, 48.5 mmol) in DMF (90 mL) at 10°C was added (2bromoethoxy)(fe/ï-butyl)dimethylsilane [CAS 86864-60-0] (6.26 mL, 29.1 mmol). 5 The reaction mixture was stirred at room température ovemight. H2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, heptane/EtOAc 80/20). The pure fractions were combined and the solvent was removed under 10 reduced pressure to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)acetate 5a (7.8 g).
Synthesis of intermediate 5b:
To a 1M lithium bis(trimethylsilyl)amide solution in THF (41.8 mL,41.8 mmol), 15 cooled to -70°C was added a solution of ethyl 2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5a (7.8 g, 20.9 mmol) in THF (45 mL). After 1 h at -70°C, chlorotrimethylsilane (4.24 mL, 33.5 mmol) was added. The reaction mixture was stirred at -70°C for 15 min. A/-Bromosuccinimide (4.46 g, 25.1 mmol) in THF (45 mL) was added and stirring was continued at 20 55°C for 2 h. The reaction mixture was poured out into H2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure to give ethyl 2-bromo-2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5b (10.1 g) which was used in the next step without further purification. 25
Synthesis of intermediate 5c:
A mixture of ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)acetate 5b (4.75 g, 10.5 mmol), 3-methoxy-5-(1/-/-1,2,4-triazol-1yl)aniline [CAS 1220630-56-7] (3 g, 15.8 mmol) and diisopropylethylamine (3.62 30 mL, 21.0 mmol) in CH3CN (90 mL) was stirred at 50°C for 24 h. The reaction mixture was concentrated under reduced pressure. The residue was taken up with EtOAc and washed with 0.5N HCl, water and brine. The organic phase was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc 35 gradient 80/20 to 70/30) to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)acetate 5c (3.7 g)19483
-30Synthesis of intermediate 5d:
Lithium hydroxide monohydrate (523 mg, 12.5 mmol) in water (25 mL) was added portionwise to a solution of ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1H-1,2,4-triazol-1-yl)phenyl)amino)acetate 5c (3.5 5 g, 6.24 mmol) in THF/CH3OH (1/1) (50 mL) at 10°C. The reaction was stirred at room température for 2 h, diluted with water and cooled down to 0°C. The solution was slowly acidified with 0.5N HCl to pH 6 and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-410 chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)acetic acid 5d (3.1 g). The compound was used as such in the next step.
Synthesis of intermediate 5e:
A mixture of 5-methoxy-6-(trifluoromethyl)indoline 2c (400 mg, 1.84 mmol), 2-(215 (2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4triazol-1-yl)phenyl)amino)acetic acid 5d (982 mg, 1.84 mmol), HATU (1.05 g, 2.76 mmol) and diisopropylethylamine (913 pL, 5.53 mmol) in DMF (10 mL) was stirred at room temperaure for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, 20 washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-{2-{{tertbutyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 5e (1.35 g). The compound was used as such in the next reaction step.
Synthesis of Compound 5 and chiral séparation into Enantiomers 5A and 5B:
Under a N2 flow, at 5°C, 4M HCl in dioxane (4.6 mL, 18.4 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(5-methoxy30 6-(trifluoromethyl)indolin-1-yl)ethanone 5e (1.35 g, 1.84 mmol) in MeOH (25 mL).
The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
The organic phase was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2CI2/MeOH 98.5/1.5). The pure fractions were combined and concentrated to dryness under reduced pressure. The residue (980 mg) was crystallized from MeOH to afford 2-(4-chloro-2-(219483
-31hydroxyethoxy)phenyl)-2-((3-methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(5methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 5 (805 mg) as a racemate.
The enantiomers of Compound 5 (771 mg) were separated via Préparative Chiral
SFC (Stationary phase: Chiralpak® IA 5 pm 250x20 mm, Mobile phase: 6% CH2CI2, 70% CO2, 24% EtOH (+0.3% iPrNH2)). The first eluted enantiomer (375 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 5A (308 mg). The second eluted enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CH2Cl2/MeOH 99/1 ). The pure fractions were combined and the solvent was concentrated under reduced pressure. The residue (340 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 5B (291 mg).
Compound 5:
1H NMR (500 MHz, DMSO-d6) δ ppm 3.15 - 3.30 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.85 (s, 3 H) 4.06 - 4.20 (m, 3 H) 4.41 (td, J=10.2, 6.3 Hz, 1 H) 4.99 (t, J=5.5 Hz, 1 H) 5.82 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.65 (t, J=) .9 Hz, 1 H) 6.80 6.85 (m, 2 H) 7.02 (dd, J=8.2, 1.9 Hz, 1 H) 7.15 (d, J=2.2 Hz, 1 H) 7.24 (s, 1 H) 7.37 (d, J=8.5 Hz, 1 H) 8.16 (s, 1 H) 8.34 (s, 1 H) 9.13 (s, 1 H)
LC/MS (method LC-A): Rt 3.13 min, MH+ 618
Melting point: 228°C
Enantiomer 5A:
1H NMR (400 MHz, DMSO-de) δ ppm 3.16 - 3.28 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.06 - 4.21 (m, 3 H) 4.35 - 4.46 (m, 1 H) 4.97 (t, J=5.6 Hz, 1
H) 5.81 (d, J=8.Q Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 11 H) 6.77 - 6.86 (m, 2 H) 7.02 (dd, J=8.3, 1.8 Hz, 1 H) 7.14 (d, J=1.5 Hz, 1 H) 7.24 (s, 1 H) 7.38 (d, J=8.6 Hz, 1 H) 8.15 (s, 1 H) 8.34 (s, 1 H) 9.12 (s, 1 H) LC/MS (method LC-A): Rt 3.11 min, MH+618
[a]D 20: -40.3° (c 0.2383, DMF)
Chiral SFC (method SFC-C): Rt 2.75 min, MH+ 618, chiral purity 100%.
Enantiomer 5B:
1H NMR (400 MHz, DMSO-ofe) δ ppm 3.16 - 3.28 (m, 2 H) 3.72 (s, 3 H) 3.74 - 3.83 (m, 2 H) 3.84 (s, 3 H) 4.06 - 4.20 (m, 3 H) 4.36 - 4.46 (m, 1 H) 4.97 (t, J=5.6 Hz, 1
H) 5.81 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.65 (s, 1 H) 6.78 - 6.85 (m, 2 H) 7.01 (dd, J=8.3, 1.8 Hz, 1 H) 7.14 (d, J=ï .5 Hz, 1 H) 7.24 (s, 1 H) 7.38 (d, J=8.1 Hz, 1 H) 8.15 (s, 1 H) 8.34 (s, 1 H) 9.12 (s, 1 H)
LC/MS (method LC-A): Rt3.11 min, MH+618
-32[a]D 20: +40.0° (c 0.22, DMF)
Chiral SFC (method SFC-C): Rt3.60 min, MH+618, chiral purity 98.47%.
Example 6 (Method 1) : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-25 ((3-methoxy-5-( 1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin1-yl)ethanone (Compound 6) and chiral séparation into Enantiomers 6A and 6B.
Chiral séparation
Enantiomers 6B and 6B io Synthesis of intermediate 6a:
A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (837 mg, 4.12 mmol), 2-(2-(2-((terf-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1/71,2,4-triazol-1-yl)phenyl)amino)acetic acid 5d (2.196 g, 4.12 mmol), HATU (2.35 g, 6.18 mmol) and diisopropylethylamine (2 mL, 12.36 mmol) in DMF (20 mL) was stirred at room température for 2 h. The mixture was diluted with water. The resulting gummy material was taken up with EtOAc, washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, heptane/EtOAc gradient 70/30 to
60/40). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)-1 -(6(trifluoromethoxy)indolin-1-yl)ethanone 6a (1.65 g).
Synthesis of Compound 6 and chiral séparation into Enantiomers 6A and 6B:
Under a N2 flow, at 5°C, 4M HCl in dioxane (6.5 mL, 21.6 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-419483
-33chlorophenyl)-2-((3-methoxy-5-(1 H-1,2,4-triazol-1 -yl)phenyl)amino)-1 -(6(trifluoromethoxy)indolin-1-yl)ethanone 6a (1.85 g, 2.58 mmol) in MeOH (40 mL). The reaction was stirred at room temperaure for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
The organic phase was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The compound was crystallized from CH2CI2 to afford 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(1/-/1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6 (1.47 g) as a racemate.
The enantiomers of Compound 6 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, Mobile phase: 70% CO2, 30% iPrOH (+0.3% iPrNH2)). The first eluted enantiomer (585 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CH2Cl2/MeOH 99/1 ) to give, 15 after solidification in MeOH/diisopropyl ether/heptane, Enantiomer 6A (491 mg).
The second eluted enantiomer (400 mg) was further purified by flash chromatography on silica gel (15-40 pm, 24 g, CH2Cl2/MeOH 99/1) to give, after solidification in MeOH/diisopropyl ether/heptane, Enantiomer 6B (467 mg).
Compound 6:
1H NMR (400 MHz, DMSO-d6) δ ppm 3.08 - 3.23 (m, 2 H) 3.73 (s, 3 H) 3.75 - 3.84 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.37 - 4.49 (m, 1 H) 4.94 (br s, 1 H) 5.82 (d, J=8.6 Hz, 1 H) 6.35 (s, 1 H) 6.64 - 6.68 (m, 1 H) 6.79 - 6.86 (m, 2 H) 6.98 - 7.05 (m, 2 H) 7.15 (d, J=2.Q Hz, 1 H) 7.34 (d, J=8.6 Hz, 1 H) 7.37 (d, J=8A Hz, 1 H) 8.04 (s, 1 H) 25 8.15 (s, 1 H) 9.11 (s, 1 H)
LC/MS (method LC-A): Rt 3.27 min, MH+ 604
Melting point: 161°C
Enantiomer 6A:
Ή NMR (500 MHz, DMSO-dô) δ ppm 3.10 - 3.25 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.84 (m, 2 H) 4.06 - 4.23 (m, 3 H) 4.39 - 4.48 (m, 1 H) 4.99 (br t, J=5.4 Hz, 1 H) 5.83 (br d, J=8.5 Hz, 1 H) 6.36 (br s, 1 H) 6.67 (s, 1 H) 6.84 (s, 1 H) 6.88 (br d, J=8.5 Hz, 1 H) 7.03 (brt, J=7.6 Hz, 2 H) 7.16 (s, 1 H) 7.36 (dd, J=11.8, 8.4 Hz, 2 H) 8.04 (brs, 1 H) 8.17 (s, 1 H) 9.14 (s, 1 H)
LC/MS (method LC-A): Rt 3.25 min, MH+604
[a]D 20: +45.9° (c 0.29, DMF)
Chiral SFC (method SFC-D): Rt4.20 min, MH+ 604, chiral purity 100%.
-34Enantiomer 6B:
1H NMR (500 MHz, DMSO-cfe) δ ppm 3.09 - 3.25 (m, 2 H) 3.73 (s, 3 H) 3.74 - 3.83 (m, 2 H) 4.06 - 4.21 (m, 3 H) 4.43 (td, 0.2, 6.6 Hz, 1 H) 4.98 (t, J=5.4 Hz, 1 H) 5.83 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (s, 1 H) 6.83 (s, 1 H) 6.88 (d, J=8.8 Hz, 1 5 H) 6.99 - 7.06 (m, 2 H) 7.15 (d, J=) .6 Hz, 1 H) 7.36 (dd, J=12.6, 8.2 Hz, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.14 (s, 1 H)
LC/MS (method LC-A): Rt 3.31 min, MH+ 604
[a]D 20: -46.3° (c 0.3, DMF)
Chiral SFC (method SFC-D): Rt 5.30 min, MH+604, chiral purity 100%.
Example 6 (Method 2) : synthesis of2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2((3-methoxy-5-(1H-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin1-yl)ethanone (Compound 6).
Cl
2) CISiMe3, -70’C 15 min
3) N BS, -70°C 2h
1) 1.5 M LiHMDS in THF THF, -70°C 1h
CH3CN, 50°C 24h
6b
HATU, iPr2NEt DMF, rt 2h
Synthesis of intermediate 6b:
To a 1.5M lithium bis(trimethylsilyl)amide solution in THF (23 mL, 34.4 mmol) cooled at -70°C under a N2 flow was added a solution of ethyl 2-(4-chloro-220 hydroxyphenyl)acetate 4c (6 g, 17.2 mmol) in THF (35 mL). After 1 h at -70°C, chlorotrimethylsilane (3.5 mL, 27.5 mmol) was added. The reaction mixture was stirred at -70°C for 15 min. A/-Bromosuccinimide (3.7 g, 20.6 mmol) in THF (35 mL) was added and stirring was continued at -70°C for 2 h. The reaction mixture was poured out into H2O and extracted with EtOAc. The organic phases were
-35combined, dried over MgSO4, filtered and concentrated under reduced pressure to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 6b (8.2 g) which was used in the next step without further purification.
Synthesis of intermediate 6c:
A mixture of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 6b (6.5 g, 15.2 mmol), 3-methoxy-5-( 1/7-1,2,4-triazol-1-yl)aniline [CAS 1220630-56-7] (4.6 g, 24.1 mmol) and diisopropylethylamine (5.3 mL, 30.4 mmol) in CH3CN (130 mL) was stirred at 50°C for 24 h. The solvent was concentrated under reduced pressure. The residue was diluted with EtOAc. The solution was filtered to remove solid particles (residual aniline). The organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc gradient 80/20 to 70/30). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl
2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol -1 yl)phenyl)amino)acetate 6c (4.8 g).
Synthesis of intermediate 6d:
At 10°C, Lithium hydroxide monohydrate (500 mg, 11.9 mmol) was added to a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 H1,2,4-triazol-1-yl)phenyl)amino)acetate 6c (3.2 g, 5.96 mmol) in MeOH/THF/water (1/1/1 ) (50 mL). The mixture was stirred at room température for 2 h. The mixture was diluted with ice water and cooled to 0°C. The resulting mixture was acidified up to pH 6-7 with 0.5N HCl and extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)acetic acid 6d (2.75 g). The compound was used in the next reaction step without further purification.
Synthesis of intermediate 6e:
A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (1.2 g, 5.89 mmol), 2(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 yl)phenyl)amino)acetic acid 6d (2.5 g, 4.91 mmol), HATU (2.29 g, 6.01 mmol) and diisopropylethylamine (1.99 mL, 12.0 mmol) in DMF (18 mL) was stirred at room température for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The compound was
-36purified by flash chromatography on silica gel (15-40 pm, 220 g, heptane/EtOAc 50/50). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3methoxy-5-(1/-/-1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1yl)ethanone 6e (2.5 g).
Synthesis of Compound 6:
A mixture of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1/-/1,2,4-triazol-1-yl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6e (2 g, 2.88 mmol) in EtOAc/MeOH/THF (1/1/1) (100 mL) was hydrogenated for 50 min under atmospheric pressure of H2 with Pd/C (10%) (3.07 g, 2.88 mmol) as the catalyst. The reaction was diluted with MeOH and filtered through a pad of celite®. The filtrate was concentrated under reduced pressure. The residue (1.42 g) was combined with another batch (total amount: 1.65 g) and purified via achiral SFC (stationary phase: NH2 5 pm 150 x 30 mm, mobile phase: 70% CO2, 30% iPrOH (+0.3% IPrNH2)). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(4-chloro-2-(2hydroxyethoxy)phenyl)-2-((3-methoxy-5-(1H-1,2,4-triazol-1-yl)phenyl)amino)-1-(6(trifluoromethoxy)indolin-1-yl)ethanone 6 (1.36 g) as a racemate.
Example 7 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-1-(5-fluoro-6(trifluoromethyl)indolin-l -yl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 yl)phenyl)amino)ethanone (Compound 7) and chiral séparation into Enantiomers 7Aand 7B.
Synthesis of intermediate 7a:
At 0°C, BHs-Pyhdine (10.45 mL, 103.4 mmol) was added dropwise to a solution of 5-fluoro-6-(trifluoromethyl)-1/-/-indole [CAS 1493800-10-4] (7 g, 34.5 mmol) in
EtOH (45 mL). 6N HCl (105 mL) was slowly added while maintaining the température below 10°C. The mixture was stirred at 0°C for 2 h. Water (210 mL) was added and the mixture was basified to pH 8-9 with a concentrated aqueous solution of NaOH (the reaction température was kept below 20°C). EtOAc was added. The organic layer was separated, washed with water, dried over MgSO4, îo filtered and the solvent was concentrated under reduced pressure. The residue was co-evaporated under reduced pressure with toluene. The crude was purified by flash chromatography on silica gel (20-45 pm, 120 g, CH2Cl2/MeOH 98.5/1.5). The pure fractions were combined and the solvent was removed under reduced pressure to give 5-fluoro-6-(trifluoromethyl)indoline 7a (3.5 g).
Synthesis of intermediate 7b:
A mixture of 5-fluoro-6-(trifluoromethyl)indoline 7a (385 mg, 1.88 mmol), 2-(2-(2((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1H-1,2,4triazol-1-yl)phenyl)amino)acetic acid 5d (1 g, 1.88 mmol), HATU (1.07 g, 2.814 mmol) and diisopropylethylamine (930 pL, 5.63 mmol) in DMF (10 mL) was stirred at room température for 2 h. The mixture was diluted with water. The resulting gummy material was taken up with EtOAc. The organic layer was washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent
-38was concentrated under reduced pressure to give 2-(2-(2-((tertbutyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin1-yl)-2-((3-methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)ethanone 7b (1.4 g).
Synthesis of Compound 7 and chiral séparation into Enantiomers 7A and 7B: Under a N2 flow, at 5°C, 4M HCl in dioxane (4.9 mL, 19.4 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-((3-methoxy-5-(1/7-1,2,4triazol-1-yl)phenyl)amino)ethanone 7b (1.4 g, 1.94 mmol) in MeOH (25 mL). The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2Cl2/MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-1-(5-fluoro-6(trifluoromethyl)indolin-l -yl)-2-((3-methoxy-5-(1 Η-Ί ,2,4-triazol-1 yl)phenyl)amino)ethanone 7 (737 mg) as a racemate.
The enantiomers of Compound 7 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 60% CO2, 40% EtOH (+0.3% iPrNH2)). The first eluted enantiomer (325 mg) was crystallized from diisopropyl ether/petroleum ether to give Enantiomer 7A (244 mg). The second eluted enantiomer (310 mg) was crystallized from diisopropyl ether/petroleum ether to give Enantiomer 7B (220 mg).
Compound 7:
1 H NMR (500 MHz, DMSO-d6) δ ppm 3.19 - 3.30 (m, 2 H) 3.63 - 3.87 (m, 5 H) .05 - 4.24 (m, 3 H) 4.40 - 4.49 (m, 1 H) 4.97 (br s, 1 H) 5.83 (br d, 7=6.9 Hz, 1 H) 30 6.35 (br s, 1 H) 6.67 (br s, 1 H) 6.80 - 6.89 (m, 2 H) 7.03 (br d, 7=7.3 Hz, 1 H) 7.15 (br s, 1 H) 7.37 (br d, 7=7.6 Hz, 1 H) 7.47 (br d, J=9.5 Hz, 1 H) 8.16 (br s, 1 H) .39 (br d, J=4A Hz, 1 H) 9.14 (br s, 1 H) LC/MS (method LC-B): Rt3.14 min, MH+ 606 Melting point: 140°C 35
Enantiomer 7A:
1H NMR (500 MHz, DMSO-de) δ ppm 3.20 - 3.30 (m, 2 H) 3.69 - 3.86 (m, 5 H) 4.06 - 4.23 (m, 3 H) 4.40 - 4.50 (m, 1 H) 4.98 (br t, J=5.2 Hz, 1 H) 5.83 (br d, J=8.8 Hz, 1 H) 6.35 (br s, 1 H) 6.67 (s, 1 H) 6.82 - 6.89 (m, 2 H) 7.03 (br d, 7=8.2 Hz, 1 H)
-397.16 (s, 1 H) 7.37 (d, 7=8.2 Hz, 1 H) 7.47 (brd, 7=10.1 Hz, 1 H) 8.17 (s, 1 H) 8.39 (brd, 7=6.3 Hz, 1 H) 9.14 (s, 1 H)
LC/MS (method LC-A): Rt 3.27 min, MH+606
[a]D 20: -44.3° (c 0.282, DMF)
Chiral SFC (method SFC-B): Rt2.89 min, MH+ 606, chiral purity 100%.
Melting point: 166°C
Enantiomer 7B:
1H NMR (500 MHz, DMSO-de) δ ppm 3.18 - 3.30 (m, 2 H) 3.69 - 3.86 (m, 5 H) 10 4.07 - 4.22 (m, 3 H) 4.40 - 4.50 (m, 1 H) 4.97 (br t, 7=5.2 Hz, 1 H) 5.83 (br d, 7=8.8
Hz, 1 H) 6.35 (br s, 1 H) 6.67 (s, 1 H) 6.81 - 6.89 (m, 2 H) 7.03 (br d, 7=8.2 Hz, 1 H)
7.16 (s, 1 H) 7.37 (brd, 7=8.2 Hz, 1 H) 7.47 (brd, 7=10.1 Hz, 1 H) 8.17 (s, 1 H)
8.39 (br d, 7=6.3 Hz, 1 H) 9.14 (s, 1 H)
LC/MS (method LC-A): Rt 3.27 min, MH+ 606
[a]D 20:+35.6° (c 0.281, DMF)
Chiral SFC (method SFC-B): Rt4.92 min, MH+606, chiral purity 100%.
Melting point: 100°C
Example 8 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-520 (1/7-1,2,4-triazol-1-yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethoxy)indolin-1yl)ethanone (Compound 8) and chiral séparation into Enantiomers 8Aand 8B.
| F NBS F FT -l ’ F O toluene, 5°C 2h I tBuOK NMP F,0^ ----- fT X 80°C 4h Ο - Ι Cl /A / ' ο°Me ^A, ' 3N A\ f HO h ^A N-N 5d | trimethylsilylacetylene Cul,PdCI2(PPh3)2 f XX ’ fT xx O Br 70°C ovemight (y 8a 8b H c n H :: r-N BH3-Pyridine ' A? ---- A KJA EtOH, 0°C 3h ° 8c 8d Cl aX0'—aX / \ OMe 8d °χ x h’ A HATU,iPr2NEt F4 T || ) N-N DMF, rt2h F Α^^ΧΑA Λ > ? 8e N |
enantiomers 8A and 8B
Synthesis of intermediate 8a:
A solution of 4-methoxy-3-(trifluoromethoxy)aniline [CAS 647855-21-8] (3.1 g, 15.0 mmol) in toluene (50 mL) was treated with /V-bromosuccinimide (2.8 g, 15.7 mmol) at 5°C and the resulting mixture was stirred at 5-10°C for 2 h. The mixture was diluted with water and extracted with EtOAc. The combined extracts were dried over MgSO4, filtered and evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 pm, 24 g, heptane/EtOAc gradient 95/5 to 90/10) The pure fractions were combined and evaporated to dryness to give 2-bromo-4-methoxy-5-(trifluoromethoxy)aniline 8a (2.5 g).
Synthesis of intermediate 8b:
A solution of 2-bromo-4-methoxy-5-(trifluoromethoxy)aniline 8a (2.72 g, 9.51 mmol) in DMF (30 mL) was degassed with N2for 15 min.
Dichlorobis(triphenylphosphine)palladium(ll) (667 mg, 0.95 mmol), copper(l) iodide (362 mg, 1.90 mmol), triethylamine (3.96 mL, 28.53 mmol) and trimethylsilylacetylene (3.95 mL, 28.5 mmol) were added. The reaction mixture was heated at 70°C for 12 h under a N2 flow. After cooling to room température, the reaction mixture was diluted with H2O and extracted with EtOAc. The organic phases were combined, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 80 g, heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 4-methoxy-5-(trifluoromethoxy)-2((trimethylsilyl)ethynyl)aniline 8b (1.4 g).
Synthesis of intermediate 8c:
To a solution of 4-methoxy-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 8b (1.2 g, 3.96 mmol) in NMP (11 mL) under a N2 flow, was added tBuOK (1.33 g, 11.9 mmol) in one portion. The reaction mixture was heated at 80°C for 4 h, poured out into ice/water and acidified with 3N HCl to pH 4-5. The reaction mixture was extracted with EtOAc. The organic phases were combined, washed with H2O, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g,
-41heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 5-methoxy-6-(trifluoromethoxy)-1/7-indole 8c (490 mg).
Synthesis of intermediate 8d:
At 0°C, BHs-Pyridine (10.5 mL, 103.8 mmol) was added dropwise to a solution of 5-methoxy-6-(trifluoromethoxy)-1/7-indole 8c (8 g, 34.6 mmol) in EtOH (45 mL). 6N HCl (6 mL) was added dropwise while maintaining the température below 10°C. The mixture was stirred at 0°C for 3 h. Water (210 mL) was added and the mixture was basified to pH 8-9 with a concentrated solution of NaOH in water (the reaction îo température was kept below 20°C). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 5-methoxy-6(trifluoromethoxy)indoline 8d (7.5 g).
Synthesis of intermediate 8e:
A mixture of 5-methoxy-6-(trifluoromethoxy)indoline 8d (437 mg, 1.88 mmol), 2-(2(2-((fert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1/7-1,2,4triazol-1-yl)phenyl)amino)acetic acid 5d (1 g, 1.88 mmol), HATU (1.07 g, 2.81 20 mmol) and diisopropylethylamine (930 pL, 5.63 mmol) in DMF (10 mL) was stirred at room température for 2 h. The mixture was diluted with water. The resulting gummy material was taken up with EtOAc. The organic solution was washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert25 butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 H-1,2,4-triazol-1 yl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethoxy)indolin-1-yl)ethanone 8e (1.5 g). The compound was used as such in the next reaction step.
Synthesis of Compound 8 and chiral séparation into Enantiomers 8A and 8B:
Under a N2 flow, at 5°C, 4M HCl in dioxane (4.9 mL, 19.4 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 -yl)phenyl)amino)-1 -(5-methoxy6-(trifluoromethoxy)indolin-1-yl)ethanone 8e (1.4 g, 1.94 mmol) in MeOH (25 mL). The reaction was stirred at room température for 1 h. The mixture was cooled to
0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2Cl2/MeOH/NH4OH 98.4/1.5/0.1).
-42The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after crystallization from CH2CI2, 2-(4-chloro-2-(2hydroxyethoxy)phenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(5methoxy-6-(trifluoromethoxy)indolin-1-yl)ethanone 8 (850 mg) as a racemate.
The enantiomers of Compound 8 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® IC 5 pm 250 x 30 mm, Mobile phase: 60% CO2, 40% iPrOH). The first eluted enantiomer (410 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 8A (314 mg). The second eluted enantiomer (388 mg) was solidified by trituration with diisopropyl ether to give Enantiomer 8B (300 mg).
Compound 8:
1H NMR (400 MHz, DMSO-d6) δ ppm 3.07 - 3.27 (m, 2 H) 3.70 - 3.85 (m, 8 H) 4.07
- 4.19 (m, 3 H) 4.35 - 4.45 (m, 1 H) 4.97 (t, J=5.6 Hz, 1 H) 5.80 (d, J=8.6 Hz, 1 H)
6.34 (s, 1 H) 6.63 - 6.67 (m, 1 H) 6.80 - 6.87 (m, 2 H) 7.02 (dd, J=8.1,2.0 Hz, 1 H) 7.14 (d, J=2.0 Hz, 1 H) 7.20 (s, 1 H) 7.37 (d, J=8.6 Hz, 1 H) 8.06 (s, 1 H) 8.15 (s, 1 H) 9.12 (s, 1 H) LC/MS (method LC-A): Rt 3.20 min, MH+ 634
Melting point: 181°C
Enantiomer 8A:
1H NMR (500 MHz, DMSO-ds) δ ppm 3.09 - 3.26 (m, 2 H) 3.70 - 3.85 (m, 8 H)
4.08 - 4.20 (m, 3 H) 4.40 (td, J=10.3, 6.5 Hz, 1 H) 4.99 (br t, J=5A Hz, 1 H) 5.81 (d, 25 J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (s, 1 H) 6.83 (s, 1 H) 6.86 (d, J=8.8 Hz, 1 H) 7.03 (dd, J=8.2, 1.3 Hz, 1 H) 7.15 (d, J=1.6 Hz, 1 H) 7.21 (s, 1 H) 7.38 (d, J=8.2 Hz, 1 H) 8.07 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-B): Rt 3.09 min, MH+634 [a]D 20: +39.3° (c 0.3, DMF)
Chiral SFC (method SFC-E): Rt 3.39 min, MH+ 634, chiral purity 100%.
Enantiomer 8B:
1H NMR (500 MHz, DMSO-de) δ ppm 3.09 - 3.27 (m, 2 H) 3.70 - 3.85 (m, 8 H) 4.06 -4.19 (m, 3 H) 4.40 (td, J=10.2, 6.6 Hz, 1 H) 4.99 (brt, J=5.2 Hz, 1 H) 5.81 (d,
J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (s, 1 H) 6.83 (s, 1 H) 6.86 (d, J=8.8 Hz, 1 H) 7.03 (dd, J=8.2,1.6 Hz, 1 H) 7.15 (d, J=1.6 Hz, 1 H) 7.21 (s, 1 H) 7.38 (d, J=8.2 Hz, 1 H) 8.07 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-A): Rt 3.07 min, MH+ 634 [a]D20:.44 4° (C 0.295, DMF)
-43Chiral SFC (method SFC-E): Rt 5.69 min, MH+634, chiral purity 100%.
Example 9 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-1-(5-fluoro-6(trifluoromethoxy)indolin-l -yl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 5 yl)phenyl)amino)ethanone (Compound 9) and chiral séparation into Enantiomers 9A and 9B.
kno3, H2SO4
Fe, NH4CI
9b
Pd(PPh3)CI2
Cul, Et3N 90°C,16h
0Cto25°C, 16h iPrOH, water
60°C 16h
HCl (4M in dioxane)
MeOH, rt 1h
Cl
Chiral séparation
Enantiomers 9A and 9B
Synthesis of intermediate 9a:
A solution of 4-bromo-2-fluoro-1-(trifluoromethoxy)benzene [CAS 105529-58-6] (98.7 g, 381.1 mmol) in concentrated H2SO4 (98%, 200 mL), was cooled to 0°C with an ice-bath. KNO3 (43.0 g, 425.3 mmol) was added portionwise. After addition, the ice-bath was removed and the mixture was stirred at room température for 16
h. The reaction mixture was poured out into ice-water (2 L) while stirring. The mixture was extracted with CH2CI2 (3x 500 mL). The combined organic layers were washed with a saturated aqueous NaHCOs solution (2x 500 mL), brine (500 mL), dried over MgSO4, filtered and concentrated under reduced pressure to afford 1-bromo-5-fluoro-2-nitro-4-(trifluoromethoxy)benzene 9a (117.2 g), which was used in the next step without further purification.
-44Synthesis of intermediate 9b:
To a stirred suspension of 1-bromo-5-fluoro-2-nitro-4-(trifluoromethoxy)benzene
9a (70.0 g, 230 mmol) and NH4CI (123.2 g, 2.30 mol) in iPrOH (1 L) and water (330 mL) was added reductive iron powder (64.3 g, 1.15 mol) under N2atmosphere. The reaction mixture was stirred at 60°C for 16 h. The reaction mixture was diluted with EtOAc (1 L) and filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was partitioned between EtOAc (1 L) and water (800 mL). The layers were separated and the organic 10 phase was washed with brine (1 L), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by distillation under reduced pressure (oil pump, b.p. 60~64°C). 2-Bromo-4-fluoro-5-(trifluoromethoxy)aniline 9b (47.3 g) was obtained as a yellow oil.
Synthesis of intermediate 9c:
To a mixture of 2-bromo-4-fluoro-5-(trifluoromethoxy)aniline 9b (18.4 g, 67.2 mmol), ethynyl(trimethyl)silane (19.9 g, 202.4 mmol, 28.00 mL) in Et3N (300 mL) was added Cul (1.28 g, 6.72 mmol) and Pd(PPh3)2Cl2 (2.40 g, 3.42 mmol). The reaction mixture was heated under N2-atmosphere at 90°C for 16 h. After cooling 20 to room température, the mixture was diluted with MTBE (300 mL) and filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (ISCO®, 220 g SepaFlash® Silica Flash Column, eluent: gradient of 0 to 5% EtOAc in Petroleum ether @1 OOmL/min). 4-Fluoro-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 25 9c (16.1 g, 90% purity) was obtained as a brown oil.
Synthesis of intermediate 9d:
A mixture of 4-fluoro-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 9c (16.1 g, 55.3 mmol) and tBuOK (18.6 g, 165.8 mmol) in NMP (220.00 mL) was heated at 30 90°C for 16 h under N2-atmosphere. After cooling to room température, the reaction mixture was poured out into ice-water (1 L) and extracted with MTBE (3x 300mL). The combined organic phases were washed with water (2x 200 mL), brine (300 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (ISCO®, 35 120 g SepaFlash® Silica Flash Column, eluent: gradient of 0 to 5% EtOAc in
Petroleum ether @ 85 mL/min) to afford 5-fluoro-6-(trifluoromethoxy)-1/7-indole 9d (11 g) product as a dark-green oil. The residue was combined with another fraction (total amount = 17.2 g) and further purified by distillation under reduced pressure
-45(oil pump, b.p. 60~64°C) to provide 5-fluoro-6-(trifluoromethoxy)-1H-indole 9d (14.7 g, 95% purity) as a colorless oil.
Synthesis of intermediate 9e:
At 0°C, BHs-Pyridine (13.8 mL, 136.9 mmol) was added dropwise to a solution of 5-fluoro-6-(trifluoromethoxy)-1H-indole 9d (6 g, 27.4 mmol) in EtOH (40 mL). 6N HCl (90 mL) was added dropwise while maintaining the température below 10°C. The mixture was stirred at 0°C for 2 h. Water (100 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the îo reaction température was kept below 20°C). The mixture was extracted with CH2CI2. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 5.52 g of 5-fluoro-6(trifluoromethoxy)indoline 9e. The compound was used in the next reaction step 15 without further purification.
Synthesis of intermediate 9f:
A mixture of 5-fluoro-6-(trifluoromethoxy)indoline 9e (169 mg, 0.76 mmol), 2-(2-(2((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1/7-1,2,420 triazol-1-yl)phenyl)amino)acetic acid 5d (407 mg, 0.76 mmol), HATU (435 mg, 1.15 mmol) and diisopropylethylamine (379 pL, 2.29 mmol) in DMF (3.9 mL) was stirred at room température for 2 h. The mixture was diluted with water. The resulting gummy material was taken up with EtOAc. The organic solution was washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 24 g, heptane/EtOAc 70/30). The pure fractions were combined and the solvent concentrated under reduced pressure to give 2-(2-(2-((fert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-1 -(5-fluoro-6-(trifluoromethoxy)indolin-1 -yl)-2-((3-methoxy-5-(1 H30 1,2,4-triazol-1-yl)phenyl)amino)ethanone 9f (257 mg).
Synthesis of Compound 9 and chiral séparation into Enantiomers 9A and 9B: Under a N2 flow, at 5°C, 4M HCl in dioxane (873 pL, 3.49 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-435 chlorophenyl)-1-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-2-((3-methoxy-5-(1H1,2,4-triazol-1-yl)phenyl)amino)ethanone 9f (257 mg, 0.35 mmol) in MeOH (4 mL). The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
-46The organic layer was separated, washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 12 g, CH2Cl2/MeOH 98.5/1.5). The pure fractions were combined and the solvent was concentrated 5 under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-1 -(5fluoro-6-(trifluoromethoxy)indolin-1 -yl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 yl)phenyl)amino)ethanone 9 (210 mg) as a racemate. A small fraction (17 mg) was further purified via reverse phase chromatography (Stationary phase: YMC-actus Triart-C18 10 pm 30 x 150mm, Mobile phase: Gradient from 50% NH4HCO3 0.2%, 10 50% CH3CN to 0% NH4HCO30.2%, 100% CH3CN) to afford 7 mg. The residue was solidified by lyophilization from a solvent mixture of CH3CN (1 ml) and water (4 mL).
The enantiomers of Compound 9 (190 mg) were separated via Préparative Chiral 15 SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 65%
CO2, 35% EtOH (+0.3% iPrNH2)). The first eluted enantiomer (58 mg) was dissolved in CH3CN (2 ml), water (8 mL) was added and the mixture was lyophilized to give Enantiomer 9A (58 mg) as a powder. The second eluted enantiomer (59 mg) was dissolved in CH3CN (2 ml), water (8 mL) was added and 20 the mixture was lyophilized to give Enantiomer 9B (59 mg) as a powder.
Compound 9:
1H NMR (500 MHz, DMSO-d6) δ ppm 3.13 - 3.30 (m, 2 H) 3.72 (s, 3H) 3.70 - 3.80 (m, 2 H) 4.06 - 4.22 (m, 3 H) 4.42 (td, J=10.4, 6.3 Hz, 1 H) 4.97 (br s, 1 H) 5.82 (d, 25 J=8.5 Hz, 1 H) 6.34 (s, 1 H) 6.66 (t, J=1.9 Hz, 1 H) 6.82 (s, 1 H) 6.87 (d, J=8.2 Hz,
H) 7.03 (dd, J=8.2, 1.9 Hz, 1 H) 7.15 (d, J=2.2 Hz, 1 H) 7.36 (d, J=8.2 Hz, 1 H) 7.45 (d, J=9.8 Hz, 1 H) 8.14 - 8.18 (m, 2 H) LC/MS (method LC-A): Rt 3.32 min, MH+ 622
Enantiomer 9A:
1H NMR (400 MHz, DMSO-de) δ ppm 3.12 - 3.28 (m, 2 H) 3.72 (s, 3H) 3.69 - 3.82 (m, 2 H) 3.99 - 4.22 (m, 3 H) 4.42 (td, J=10.0, 6.8 Hz, 1 H) 4.96 (t, J=5.3 Hz, 1 H) 5.81 (d, J=9.1 Hz, 1 H) 6.34 (s, 1 H) 6.66 (t, J=1.8 Hz, 1 H) 6.80 - 6.88 (m, 2 H) 7.02 (dd, J=8A, 2.0 Hz, 1 H) 7.15 (d, J=2.Q Hz, 1 H) 7.36 (d, J=8.6 Hz, 1 H) 7.45 35 (d, J=10.1 Hz, 1 H) 8.13 - 8.18 (m, 2 H) 9.13 (s, 1 H)
LC/MS (method LC-B): Rt3.17 min, MH+622
[a]D 20: -35.1° (c 0.276, DMF)
Chiral SFC (method SFC-B): Rt2.75 min, MH+ 622, chiral purity 100%.
-47Enantiomer 9B:
1H NMR (400 MHz, DMSO-cfe) δ ppm 3.12 - 3.28 (m, 2 H) 3.72 (s, 3H) 3.69 - 3.82 (m, 2 H) 3.99 - 4.22 (m, 3 H) 4.42 (td, J=10.0, 6.8 Hz, 1 H) 4.96 (t, J=5.3 Hz, 1 H) 5.81 (d, J=9.1 Hz, 1 H) 6.34 (s, 1 H) 6.66 (t, J=) .8 Hz, 1 H) 6.80 - 6.88 (m, 2 H)
7.02 (dd, J=8.1,2.0 Hz, 1 H) 7.15 (d, J=2.0 Hz, 1 H) 7.36 (d, J=8.6 Hz, 1 H) 7.45 (d, J=10.1 Hz, 1 H) 8.13 - 8.18 (m, 2 H) 9.13 (s, 1 H) LC/MS (method LC-B): Rt 3.17 min, MH+ 622 [a]D 20: +32.3° (c 0.254, DMF)
Chiral SFC (method SFC-B): Rt 3.75 min, MH+622, chiral purity 100%.
.0
Example 10 : synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy5-(1/-/-1,2,4-triazol-1-yl)phenyl)amino)-1-(4-methyl-6-(trïfluoromethoxy)indolin-1yl)ethanone (Compound 10) and chiral séparation into Enantiomers 10Aand 10B.
rt1 h
1) NaNO2,H2SO4t H2O
CH3COOH, rt 30 min
TFAA
Dioxane
HNO3. Ac2O
55°C ovemight
2) urea, H2O, rt 10 min
3) Kl, H2O, rt 30 min
80°C 4 h
reflux, 40 min tSuOK NMP
BH3-Pyridine
2M K2COs MeOH
70°C ovemight
Fe, NH4CI
EtOH, H2O
T rimethylsilylacetylene
Cul, PdCI2(PPh3)2
10e
EtOH, 0°C 3h
HCl (4M in dioxane)
MeOH, rt 1h
Cl
Chiral séparation
Et3N
65°C ovemight
Enantiomers 10A and 10B
Synthesis of intermediate 10a:
To a solution of 2-methyl-4-(trifluoromethoxy)aniline [CAS 86256-59-9] (10.0 g, 52.3 mmol) in dioxane (20 ml.) was added trifluoroacetic anhydride (8 mL, 57.2 mmol). The reaction mixture was stirred at room température for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc and 1N HCl. The phases were separated. The organic phase was washed with a saturated solution of NaHCOs in water, H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure to afford 14.7 g of 2,2,2-trifluoro-A/-(2-methyl-4-(trifluoromethoxy)phenyl)acetamide 10a as a white powder. The compound was used in the next step without further purification.
Synthesis of intermediate 10c:
To acetic anhydride (11.4 mL, 61.1 mmol), cooled at 0°C was added dropwise 70% nitric acid (3.9 mL). 2,2,2-Trifluoro-N-(2-methyl-4-(trifluoromethoxy)phenyl)15 acetamide 10a (5 g, 17.4 mmol) was added portionwise and the reaction mixture was heated at 55°C for 12 h. After cooling to room température, the reaction mixture was diluted with EtOAc and washed with H2O. The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in methanol (46 mL). 2M K2CO3 (23 mL, 46 mmol) was added and the reaction mixture was heated at 70°C for 4 h. More 2M K2CO3 (10 mL, 20 mmol) was added and the reaction mixture was heated at 70°C for 12 h. The reaction mixture was partially concentrated under reduced pressure to remove methanol. The residue was extracted with EtOAc. The organic phase was washed with H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (20% to 50%) in heptane to afford 3.6 g of 2methyl-6-nitro-4-(trifluoromethoxy)aniline 10c as a yellow solid.
Synthesis of intermediate 10d:
To a solution of 2-methyl-6-nitro-4-(trifluoromethoxy)aniline 10c (1.8 g, 7.69 mmol) in acetic acid (10.9 mL) was added dropwise a solution of sodium nitrite (0.806 g, 11.7 mmol) in Η28Ο4/Η2Ο (2 mL, 1/1). The reaction mixture was stirred at room température for 30 min. H2O (22 mL) and urea (0.802 g, 13.4 mmol) were added. After 10 min at room température, a solution of potassium iodide (1.7 g, 10.2 mmol) in H2O (11 mL) was added dropwise. The reaction mixture was stirred at room température for 30 min. The yellow solid was filtered off, washed with H2O and dried to give 2.4 g of 2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 10d.
-49Synthesis of intermediate 10e:
To a solution of 2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 10d (3.5 g, 10.0 mmol) in EtOH (30 mL) was added a solution of NH4CI (2.7 g, 49.9 mmol) in H2O (30 mL). The reaction mixture was heated at 50°C. Iron (2.6 g, 46.9 mmol) 5 was added and the reaction mixture was heated under reflux for 40 min. After cooling to room température, the reaction mixture was filtered through celite®. The solids were washed with EtOH. The filtrate was partially concentrated under reduced pressure to remove EtOH. The residue was partitioned between EtOAc and a saturated solution of NaHCOs in water. The phases were separated. The 10 organic phase was washed with H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25%) in heptane to afford 2.9 g of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 10e as a yellow oil.
Synthesis of intermediate 10f:
A solution of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 10e (2.9 g, 9.1 mmol) in triethylamine (23 mL) was degassed with argon for 15 min.
Dichlorobis(triphenylphosphine)palladium(ll) (0.327 g, 0.47 mmol), copper(l) iodide (0.164 g, 0.86 mmol) and trimethylsilylacetylene (1.8 mL, 13.1 mmol) were added. 20 The reaction mixture was heated at 65°C for 12 h. After cooling to room température, the reaction mixture was diluted with H2O and extracted with EtOAc (3x). The organic phases were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25 20%) in heptane to afford 2.6 g of 3-methyl-5-(trifluoromethoxy)-2((trimethylsilyl)ethynyl)aniline 10f as an orange oil.
Synthesis of intermediate 10g:
To a solution of 3-methyl-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 10f 30 (2.7 g, 9.3 mmol) in NMP (27 mL) was added tBuOK (3.1 g, 27.8 mmol). The reaction mixture was heated at 80°C for 4 h. After cooling to room température, the reaction mixture was diluted with H2O and extracted with EtOAc (2x). The organic phases were combined, washed with H2O and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by 35 flash chromatography on silica gel using a gradient of EtOAc (0% to 20%) in heptane to afford 1.7 g of 4-methyl-6-(trifluoromethoxy)-1/7-indole 10g as an orange oil.
-50Synthesis of intermediate 10h:
At 0°C, BH3-Pyridine (1.2 mL, 11.6 mmol) was added dropwise to a solution of 4methyl-6-(trifluoromethoxy)-1H-indole 10g (0.5 g, 2.32 mmol) in EtOH (3 mL). 6N HCl (6 mL) was slowly added dropwise while maintaining the reaction température 5 below 10°C. The mixture was stirred at 0°C for 3 h. Water (12 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction température was kept below 20°C). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and îo the solution was concentrated under reduced pressure to give 450 mg of 4-methyl6-(trifluoromethoxy)indoline 10h.
Synthesis of intermediate 10i:
A mixture of 4-methyl-6-(trifluoromethoxy)indoline 10h (163 mg, 0.75 mmol), 2-(215 (2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA,2,4triazol-1-yl)phenyl)amino)acetic acid 5d (400 mg, 0.75 mmol), HATU (428 mg, 1.13 mmol) and diisopropylethylamine (372 pL, 2.25 mmol) in DMF (3.8 mL) was stirred at room température for 2 h. The mixture was diluted with water. The resulting gummy material was taken up with EtOAc. The organic layer was 20 washed with a solution of K2CO310% in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 pm, 24 g, heptane/EtOAc gradient 80/20 to 70/30). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert25 butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4-triazol-1 yl)phenyl)amino)-1-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)ethanone 10i (226 mg).
Synthesis of Compound 10 and chiral séparation into Enantiomers 10A and 30 10B:
Under a N2 flow, at 5°C, 4M HCl in dioxane (772 pL, 3.1 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(4-methyl-6(trifluoromethoxy)indolin-1-yl)ethanone 10i (226 mg, 0.31 mmol) in MeOH (4 mL). 35 The reaction was stirred at room température for 1 h. The mixture was cooled to 0°C, basified with a 10% aqueous solution of K2CO3 and extracted with EtOAc.
The organic layer was separated, washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 12 g, CH2Cl2/MeOH 98.5/1.5). A second purification was performed via reverse phase chromatography (Stationary phase: YMC-actus Triart-C18 10 pm 30 x 150mm, Mobile phase: Gradient from 55% NH4HCO3 0.2%, 45% CH3CN to 0% NH4HCO30.2% , 100%
CH3CN). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl )-2-((3methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-1 -(4-methyl-6(trifluoromethoxy)indolin-1-yl)ethanone 10 (78 mg) as a racemate. A small fraction was solidified by trituration with CH3CN/diisopropyl ether to provide Compound 10 (9 mg). The remaining amount was used to separate the Enantiomers of
Compound 10 via Préparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 60% CO2, 40% iPrOH). The first eluted enantiomer (27 mg) was dissolved in CH3CN (2 ml), water (8 mL) was added and the mixture was lyophilized to give Enantiomer 10A (25 mg) as a powder. The 15 second eluted enantiomer (28 mg) was dissolved in CH3CN (2 ml), water (8 mL) was added and the mixture was lyophilized to give Enantiomer 10B (22 mg) as a powder.
Compound 10:
Ή NMR (500 MHz, DMSO-d6) δ ppm 2.21 (s, 3 H) 3.01 - 3.13 (m, 2 H) 3.72 (s, 3H) 3.70 - 3.82 (m, 2 H) 4.08 - 4.22 (m, 3 H) 4.40 - 4.48 (m, 1 H) 4.98 (t, J=5.4 Hz, 1 H) 5.82 (d, 7=8.5 Hz, 1 H) 6.35 (br s, 1 H) 6.66 (s, 1 H) 6.82 - 6.90 (m, 3 H) 7.02 (dd, J=8.4, 1.7 Hz, 1 H) 7.15 (d, J=1.6 Hz, 1 H) 7.36 (d, 7=8.2 Hz, 1 H) 7.89 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H)
LC/MS (method LC-B): Rt 3.26 min, MH+ 618
Enantiomer 10A:
1H NMR (500 MHz, DMSO-c/6) δ ppm 2.21 (s, 3 H) 3.01 - 3.14 (m, 2 H) 3.72 (s, 3H) 3.71 - 3.83 (m, 2 H) 4.07 - 4.22 (m, 3 H) 4.44 (td, J=10.2, 6.5 Hz, 1 H) 4.98 (t, 30 J=5.5 Hz, 1 H) 5.82 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (t, 7=1.9 Hz, 1 H) 6.81 6.90 (m, 3 H) 7.02 (dd, 7=8.4, 2.1 Hz, 1 H) 7.15 (d, J=1.9 Hz, 1 H) 7.36 (d, J=8.2 Hz, 1 H) 7.89 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H) LC/MS (method LC-B): Rt 3.26 min, MH+618 [a]D20: -38.4° (c 0.279, DMF)
Chiral SFC (method SFC-F): Rt 1.41 min, MH+ 618, chiral purity 100%.
-52Enantiomer 10B:
1H NMR (500 MHz, DMSO-cfe) δ ppm 2.21 (s, 3 H) 3.00 - 3.14 (m, 2 H) 3.72 (s, 3H) 3.71 - 3.82 (m, 2 H) 4.06 - 4.23 (m, 3 H) 4.44 (td, J=) 0.1,6.9 Hz, 1 H) 4.95 - 5.02 (m, 1 H) 5.82 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.66 (t, J=1.7 Hz, 1 H) 6.82 - 6.90 (m, 5 3 H) 7.02 (dd, J=8.5,1.9 Hz, 1 H) 7.15 (d, J=1.9 Hz, 1 H) 7.36 (d, J=8.2 Hz, 1 H)
7.89 (s, 1 H) 8.16 (s, 1 H) 9.13 (s, 1 H)
LC/MS (method LC-B): Rt3.26 min, MH+ 618
[a]D 20: +37.5° (c 0.299, DMF)
Chiral SFC (method SFC-F): Rt 1.82 min, MH+618, chiral purity 100%.
.0
Example 11 : synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(1H-1,2,4-triazol-1yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 11) and chiral séparation into Enantiomers 11Aand 11 B.
1) 1.5 M LiHMDS in THF THF, -78°C 15 min
OMe
2) CISiMe3, -78°C 15 min
3) NBS, -70°C 1h
CH3CN/iPr2NEt
65°C 24h
Chiral séparation
Enantiomers 11A and 11B
Synthesis of intermediate 11a:
To a suspension of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate 4b (8.5 g, 39.6 mmol) and CS2CO3 (25.8 g, 79.2 mmol) in DMF (130 mL) at 10°C was added dropwise tert-butyl 4-bromobutanoate [CAS 110611-91-1] (7 mL, 39.6 mmol). The mixture was stirred at room température overnight. The mixture was diluted with
-53EtOAc and water. The layers were decanted. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc 90/10). The pure fractions were combined and concentrated to dryness to give tert-butyl 4-(5-chloro-2-(2-ethoxy-2oxoethyl)phenoxy)butanoate 11a (12.7 g).
Synthesis of intermediate 11b:
A flask was charged with LiHMDS 1.5 M in THF (23.5 mL, 35.3 mmol) under a N2 ίο flow and the solution was cooled to -78°C. A solution of tert-butyl 4-(5-chloro-2-(2ethoxy-2-oxoethyl)phenoxy)butanoate 11a (6.3 g, 17.6 mmol) in THF (60 mL) was added dropwise and the mixture was stirred at -78°C for 15 min.
Chlorotrimethylsilane (3.6 mL, 28.3 mmol) was added. After 15 min at -78°C, NBromosuccinimide (3.77 g, 21.2 mmol) in THF (40 mL) was added and the mixture 15 was stirred at -70°C for 1 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was separated, washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to yield tert-butyl 4-(2-(1 -bromo-2-ethoxy-2-oxoethyl)-5-chlorophenoxy)butanoate 11b (7.6 g). The compound was used directly in the next reaction step without further 20 purification.
Synthesis of intermediate 11c:
To a solution of tert-butyl 4-(2-(1 -bromo-2-ethoxy-2-oxoethyl)-5chlorophenoxy)butanoate 11b (7.6 g, 17.4 mmol) in CH3CN (140 mL) at room 25 température, was added diisopropylethylamine (4.8 mL, 27.9 mmol) and 3methoxy-5-(1H-1,2,4-triazol-1-yl)aniline [CAS 1220630-56-7] (4 g, 20.9 mmol). The mixture was stirred at 65°C for 24 h. The mixture was diluted with EtOAc, washed with 0.5N HCl (twice) and water. The organic layer was dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc 85/15 to 70/30). The pure fractions were combined and concentrated to dryness to give tert-butyl 4-(5-chloro-2-(2-ethoxy-1-((3-methoxy-5-(1/7-1,2,4-triazol-1 yl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate 11c (6.6 g).
Synthesis of intermediate 11 d:
A mixture of tert-butyl 4-(5-chloro-2-(2-ethoxy-1-((3-methoxy-5-(1H-1,2,4-triazol-1yl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate 11c (6.6 g, 12.1 mmol) and lithium hydroxide monohydrate (1.52 g, 36.3 mmol) in THF/water (1/1) (160 mL)
-54was stirred at room température for 1 h. The mixture was diluted with water. The aqueous solution was slowly acidified with 3N HCl and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-45 chlorophenyl)-2-((3-methoxy-5-(1H-1,2,4-triazol-1-yl)phenyl)amino)acetic acid 11d (6.2 g). The crude product was used without further purification in the next step.
Synthesis of intermediate 11e:
A mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (290 mg, 1.55 mmol), 10 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 H-1,2,4triazol-1-yl)phenyl)amino)acetic acid 11d (800 mg, 1.55 mmol), HATU (880 mg, 2.32 mmol) and diisopropylethylamine (770 pL, 4.64 mmol) in DMF (30 mL) was stirred at room température for 2 h. The mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The 15 organic layer was washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was crystallized from diisopropyl ether to give tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/7-1,2,4-triazol1 -yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1 yl)ethyl)phenoxy)butanoate 11e (500 mg).
Synthesis of Compound 11 and chiral séparation into Enantiomers 11A and 11B:
A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/7-1,2,4-triazol-1yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoate 25 11e (500 mg, 0.729 mmol) in 4M HCl in dioxane (5 mL) was stirred at 5°C for 3 h and at room température for 8 h. The precipitate was filtered off, washed with dioxane/diisopropyl ether and dried to give 4-(5-chloro-2-(1-((3-methoxy-5-(1H1,2,4-triazol-1-yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1yl)ethyl)phenoxy)butanoic acid 11 (430 mg, 0.4 H2O (determined by titration)) as a 30 racemate.
The eEnantiomers of Compound 11 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 65% CO2, 35% EtOH). The first eluted enantiomer (80 mg) was solidified by titration with 35 petroleum ether/diisopropyl ether to give Enantiomer 11A (65 mg). The second eluted enantiomer (126 mg) was solidified by titration with petroleum ether/diisopropyl ether to give Enantiomer 11B (110 mg).
-55Compound 11:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.94 - 2.02 (m, 2 H) 2.31 - 2.42 (m, 2 H) 3.15 - 3.35 (m, 2 H) 3.73 (s, 3 H) 4.01 - 4.24 (m, 3 H) 4.22 - 4.49 (m, 1 H) 5.73 (s, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.82 (s, 1 H) 7.02 (dd, J=8A, 2.0 Hz, 1 H) 7.13 (d, J=2.0
Hz, 1 H) 7.33 (d, J=8.0 Hz, 1 H) 7.38 (d, J=7.6 Hz, 1 H) 7.45 (d, J=7.6 Hz, 1 H)
8.16 (s, 1 H) 8.38 (s, 1 H) 9.15 (s, 1 H) LC/MS (method LC-A): Rt 2.70 min, MH+ 630
Enantiomer 11 A:
1H NMR (500 MHz, DMSO-ofe) δ ppm 1.98 (br s, 2 H) 2.28 - 2.45 (m, 2 H) 3.13 3.29 (m, 2 H) 3.74 (s, 3 H) 4.02 - 4.17 (m, 3 H) 4.36 - 4.44 (m, 1 H) 5.74 (br d, J=8.5 Hz, 1 H) 6.35 (br s, 1 H) 6.68 (s, 1 H) 6.80 - 6.88 (m, 2 H) 7.03 (br d, J=7.9 Hz, 1 H) 7.14 (s, 1 H) 7.33 (d, J=7.8 Hz, 1 H) 7.37 - 7.40 (m, 1 H) 7.46 (brd, J=7.6 Hz, 1 H) 8.16 (s, 1 H) 8.39 (s, 1 H) 9.16 (s, 1 H) 12.13 (brs, 1 H)
LC/MS (method LC-A): Rt 2.79 min, MH+ 630
[a]D 20: -28.9° (c0.26, DMF)
Chiral SFC (method SFC-G): Rt 3.31 min, MH+ 630, chiral purity 100%.
Enantiomer 11 B:
1H NMR (500 MHz, DMSO-cfe) δ ppm 1.91 - 2.04 (m, 2 H) 2.28 - 2.46 (m, 2 H) 3.16
- 3.30 (m, 2 H) 3.73 (s, 3 H) 4.02 - 4.18 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.73 (br d, J=8.5 Hz, 1 H) 6.34 (br s, 1 H) 6.68 (s, 1 H) 6.80 - 6.87 (m, 2 H) 7.02 (br d, J=7.9 Hz, 1 H) 7.13 (s, 1 H) 7.31 - 7.41 (m, 2 H) 7.45 (br d, J=7.9 Hz, 1 H) 8.16 (s, 1 H) 8.38 (s, 1 H) 9.16 (s, 1 H) 12.13 (brs, 1 H)
LC/MS (method LC-A): Rt 2.79 min, MH+ 630
[a]D 20: +23.8° (C 0.29, DMF)
Chiral SFC (method SFC-G): Rt 4.32 min, MH+630, chiral purity 100%.
Example 12 : synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(1H-1,2,4-triazol-130 yl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2oxoethyl)phenoxy)butanoic acid (Compound 12) and chiral séparation into Enantiomers 12Aand 12B.
Synthesis of intermediate 12a:
A mixture of 5-methoxy-6-(trifluoromethyl)indoline 2c (630 mg, 2.9 mmol), 2-(2-(4(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 5 yl)phenyl)amino)acetic acid Tld (1.5 g, 2.9 mmol), HATU (1.65 g, 4.35 mmol) and diisopropylethylamine (1.45 mL, 8.7 mmol) in DMF (30 mL) was stirred at room température for 2 h. The mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic solution was washed with water, dried over MgSO4, filtered and the solvent was concentrated îo under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc 60/40). The pure fractions were combined and concentrated to dryness to give, after crystallization from ether/diisopropyl ether, tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/-/-1,2,4-triazol1-yl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-215 oxoethyl)phenoxy)butanoate 112a (1.45 g).
Synthesis of Compound 12 and chiral séparation into Enantiomers 12A and 12B:
A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/7-1,2,4-triazol-1 20 yl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2oxoethyl)phenoxy)butanoate 12a (1.45 g, 2.03 mmol) in 4M HCl in dioxane (12 mL) was stirred at 5°C for 3 h and at room température for 12 h. The precipitate was filtered off, washed with dioxane/diisopropyl ether and dried to provide crude Compound 12 (1.02 g). A small part (90 mg) was further purified by achiral SFC (Stationary phase: 2-Ethylpyridine 6 pm 150 x 21.2 mm, Mobile phase: 60% CO2, 40% iPrOH) to give, after solidification by trituration with CHsCN/diisopropyl ether,
-574-(5-chloro-2-(1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2-(5-methoxy6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)phenoxy)butanoic acid 12 (70 mg,) as a racemate. The remaining amount was used to separate the enantiomers.
The enantiomers of Compound 12 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® AS-H 5 pm 250 x 20 mm, Mobile phase: 63% CO2, 37% iPrOH). The first eluted enantiomer (458 mg) was stirred in a mixture of 1N HCl and EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was solidified îo by trituration with diisopropyl ether/petroleum ether to give Enantiomer 12A (270 mg). The second eluted enantiomer (405 mg) was stirred in a mixture of 1N HCl and EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was solidified by trituration with diisopropyl ether/petroleum ether to give Enantiomer 12B (272 mg).
Compound 12:
Ή NMR (400 MHz, DMSO-d6) δ ppm 1.95 - 2.04 (m, 2 H) 2.31 - 2.45 (m, 2 H) 3.15 - 3.28 (m, 2 H) 3.73 (s, 3 H) 3.84 (s, 3 H) 3.98 - 4.17 (m, 3 H) 4.33 - 4.41 (m, 1 H) 5.70 (br d, 7=8.6 Hz, 1 H) 6.33 (s, 1 H) 6.66 (s, 1 H) 6.77 - 6.83 (m, 2 H) 7.01 (br d, 20 7=8.6 Hz, 1 H) 7.12 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, 7=8.6 Hz, 1 H) 8.15 (s, 1 H) 8.34 (s, 1 H) 9.13 (s, 1 H) 12.07 (br s, 1 H) LC/MS (method LC-A): Rt 2.72 min, MH+ 660
Enantiomer 12A:
1H NMR (500 MHz, DMSO-ds) δ ppm 1.94 - 2.05 (m, 2 H) 2.31 - 2.46 (m, 2 H) 3.16 - 3.31 (m, 2 H) 3.73 (s, 3 H) 3.85 (s, 3 H) 3.99 - 4.18 (m, 3 H) 4.38 (td, 7=10.2, 6.5 Hz, 1 H) 5.71 (d, 7=8.5 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.82 (s, 1 H) 6.83 (d, 7=9.5 Hz, 1 H) 7.02 (dd, 7=8.2, 1.6 Hz, 1 H) 7.13 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, 7=8.2 Hz, 1 H) 8.16 (s, 1 H) 8.34 (s, 1 H) 9.15 (s, 1 H) 12.11 (br s, 1 H)
LC/MS (method LC-A): Rt 2.70 min, MH+660
[a]D 20: +30.4° (c 0.257, DMF)
Chiral SFC (method SFC-H): Rt 3.71 min, MH+ 660, chiral purity 100%.
Enantiomer 12B:
Ή NMR (500 MHz, DMSO-de) δ ppm 1.91 - 2.08 (m, 2 H) 2.32 - 2.44 (m, 2 H) 3.16 - 3.31 (m, 2 H) 3.73 (s, 3 H) 3.85 (s, 3 H) 3.99 - 4.17 (m, 3 H) 4.38 (td, 7=10.3, 6.6 Hz, 1 H) 5.71 (d, 7=8.5 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.79 - 6.85 (m, 2 H) 7.02 (d, 7=8.1 Hz, 1 H) 7.13 (s, 1 H) 7.23 (s, 1 H) 7.33 (d, 7=8.2 Hz, 1 H) 8.16 (s, 1 H) 8.34 (s, 1 H) 9.15 (s, 1 H) 12.12 (br s, 1 H)
-58LC/MS (method LC-A): Rt 2.70 min, MIT 630
[a]D 10 * * * * 15 * * * * 20 * * * * 25: -36.9° (c 0.287, DMF)
Chiral SFC (method SFC-H): Rt 5.91 min, MH+660, chiral purity 100%.
Example 13 : synthesis of 4-(5-chloro-2-(1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 y|)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 13) and chiral séparation into Enantiomers 13A and 13B.
Synthesis of intermediate 13a:
A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (590 mg, 2.9 mmol),
2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 /7-1,2,4triazol-1-yl)phenyl)amino)acetic acid 11d (1.5 g, 2.9 mmol), HATU (1.65 g, 4.35 mmol) and diisopropylethylamine (1.45 mL, 8.7 mmol) in DMF (60 mL) was stirred at room température for 12 h. The mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 120 g, heptane/EtOAc 60/40). The pure fractions were combined and concentrated to dryness to give, after crystallization from ether/diisopropyl ether, tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/7-1,2,4triazol-1-yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoate 13a (1.05 g).
-59Synthesis of Compound 13 and chiral séparation into Enantiomers 13A and 13B:
A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1H-1,2,4-triazol-1 yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1yl)ethyl)phenoxy)butanoate 13a (1.05 g ,1.50 mmol) in 4M HCl in dioxane (9.5 mL) was stirred at 5°C for 3 h and at room temperaure for 12 h. The precipitate was filtered off, washed with dioxane/diisopropyl ether and dried to give 4-(5-chloro-2(1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2-oxo-2-(6(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoic acid 13 (965 mg, 0.23 H2O (determined by titration)) as a racemate.
The enantiomers of Compound 13 were separated via Préparative Chiral SFC (Stationary phase: Chiralpak® AS-H 5 pm 250 x 20 mm, Mobile phase: 80% CO2, 20% EtOH). The first eluted enantiomer (390 mg) was solidified by trituration with Petroleum ether/diisopropyl ether to give Enantiomer 13A (260 mg). The second eluted enantiomer (350 mg) was solidified by trituration with petroleum ether/diisopropyl ether to give Enantiomer 13B (188 mg).
Compound 13:
1H NMR (500 MHz, DMSO-cfe) δ ppm 1.94 - 2.03 (m, 2 H) 2.33 - 2.41 (m, 2 H) 3.10 - 3.24 (m, 2 H) 3.73 (s, 3 H) 4.04 - 4.26 (m, 3 H) 4.39 (td, J=10.2, 6.5 Hz, 1 H) 5.71 (s, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.81 (s, 1 H) 6.99 - 7.07 (m, 2 H) 7.13 (d, J=A .6 Hz, 1 H) 7.27-7.39 (m, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.15 (s, 1 H) 12.08 (br s, 1 H) LC/MS (method LC-B): Rt 2.73 min, MH+ 646
Enantiomer 13A:
1H NMR (500 MHz, DMSO-cfe) δ ppm 1.93 - 2.03 (m, 2 H) 2.34 - 2.47 (m, 2 H) 3.13 - 3.21 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.17 (m, 3 H) 4.39 (td, J=10.1, 6.6 Hz, 1 H) 5.72 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.68 (s, 1 H) 6.82 (s, 1 H) 6.87 (d, J=8.2 Hz, 1 H) 7.02 (t, J=8.4 Hz, 1 H) 7.13 (s, 1 H) 7.27 - 7.39 (m, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.15 (s, 1 H) 12.10 (br s, 1 H)
LC/MS (method LC-A): Rt2.83 min, MH+646
[ct]D 20: +38.4° (c 0.276, DMF)
Chiral SFC (method SFC-I): Rt4.96 min, MH+ 646, chiral purity 100%.
Enantiomer 13B:
1H NMR (500 MHz, DMSO-cfe) δ ppm 1.91 - 2.02 (m, 2 H) 2.33 -2.41 (m, 2 H) 3.10 - 3.24 (m, 2 H) 3.73 (s, 3 H) 4.04 - 4.17 (m, 3 H) 4.39 (td, J=î0.1, 6.6 Hz, 1 H) 5.72 (d, J=8.5 Hz, 1 H) 6.35 (s, 1 H) 6.68 (s, 1 H) 6.81 - 6.89 (m, 2 H) 6.99 - 7.05 (m, 2
H) 7.12 - 7.15 (m, 1 H) 7.27-7.40 (m, 2 H) 8.04 (s, 1 H) 8.16 (s, 1 H) 9.16 (s, 1 H)
12.11 (brs, 1 H)
LC/MS (method LC-A): Rt 2.83 min, MH+ 646
[a]D 20: -43.2° (c 0.273, DMF)
Chiral SFC (method SFC-I): Rt 6.56 min, MIT 646, chiral purity 100%.
Example 14 : synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(1/7-1,2,4-triazol-1yl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)-2,2dimethylbutanoic acid (Compound 14) and chiral séparation into Enantiomers 14A and 14B.
CS2CO3
DMF, rt overnight
1M NaOH
MeOH, dioxane 0°C 2h, rt 14h
HATU, DIPEA
DMF, rt 5h
1) LiHMDS, 1M in THF 2-Me-THF, -78°C, 30 min
2) TMSCI, -78°C, 25 min
3) NBS, -78°C, 45 min
14d
OMe
DIPEA
CH3CN, 50°C18h, 70°C6h
1M NaOH dioxane rt 40h
Chiral séparation
Enantiomer 14A + Enantiomer 14B
Synthesis of intermediate 14a:
To a mixture of methyl 2-(4-chloro-2-hydroxyphenyl)acetate [CAS 518979-09-4] (1 g, 4.99 mmol) and césium carbonate (3.25 g, 9.97 mmol) in DMF (30 mL) was added methyl 4-bromo-2,2-dimethylbutanoate [CAS 4833-99-2] (1.09 g, 5.23 mmol). The reaction mixture was stirred at room température for 20 h. the reaction mixture was poured out into stirring water (150 mL) and the product was extracted (2x) with Et2O. The combined organic layers were washed with brine, dried over
MgSO4, filtered, and evaporated under reduced pressure. The product crystallized upon standing at room température. The solid residue was stirred up in 5 mL diisopropyl ether. The precipitate was filtered off, washed (3x) with diisopropyl ether, and dried under vacuum at 45°C to provide methyl 4-(5-chloro-2-(225 methoxy-2-oxoethyl)phenoxy)-2,2-dimethylbutanoate 14a (0.897 g).
-61Synthesis of intermediate 14b:
A solution of methyl 4-(5-chloro-2-(2-methoxy-2-oxoethyl)phenoxy)-2,2dimethylbutanoate 14a (0.897 g, 2.73 mmol) in a solvent mixture of MeOH (10 mL) 5 and dioxane (5 mL) was cooled on an ice-bath. At 0°C, 1M NaOH (2.73 mL, 2.73 mmol) was added carefully. The reaction mixture was stirred at 0°C for 2 h, and at room température for 14 h. The reaction mixture was poured out into water (50 mL), stirred for 15 minutes and left standing for 30 minutes. The solid fraction (unreacted intermediate 14a) was filtered off, and washed (3x) with water. The îo combined filtrâtes were acidified by dropwise addition of 1N HCl (2.8 mL) while stirring. After 10 min, the precipitate was filtered off, washed (3x) with water, and dried under vacuum at 45°C to provide 2-(4-chloro-2-(4-methoxy-3,3-dimethyl-4oxobutoxy)phenyl)acetic acid 14b (0.576 g).
Synthesis of intermediate 14c:
To a stirring solution of 2-(4-chloro-2-(4-methoxy-3,3-dimethyl-4oxobutoxy)phenyl)acetic acid 14b (576 mg, 1.83 mmol), 6(trifluoromethoxy)indoline [CAS 959235-95-1] (409 mg, 2.01 mmol) and diisopropylethylamine (907 pL, 5.49 mmol) in DMF (7.5 mL) under N2-atm was 20 added HATU (1.07 g, 2.75 mmol), and the reaction mixture was stirred at room température for 5 h. Water (30 mL) was added, and the product was extracted (2x) with Et2O. The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a gradient of heptane/EtOAc 25 100/0 to 0/100. The desired fractions were combined and evaporated under reduced pressure, and co-evaporated with toluene. The residue was dried under vacuum at 45°C to provide methyl 4-(5-chloro-2-(2-oxo-2-(6(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)-2,2-dimethylbutanoate 14c (790 mg) as a powder.
Synthesis of intermediate 14d:
A solution of methyl 4-(5-chloiO-2-(2-oxo-2-(6-(trifluoromethoxy)indolin-1yl)ethyl)phenoxy)-2,2-dimethylbutanoate 14c (790 mg, 1.58 mmol) in 2-Me-THF (30 mL) was stirred under N2-flow and cooled to -78°C. A solution of 1M lithium 35 bis(trimethylsilyl)amide in THF (3.16 mL, 3.16 mmol) was added dropwise and the resulting mixture was stirred at -78°C for 30 minutes. Chlorotrimethylsilane (323 pL, 2.53 mmol) was added dropwise and the mixture was stirred at -78°C for 25 min. A solution of /V-bromosuccinimide (352 mg, 1.98 mmol) in 2-Me-THF (7.5 mL) and
-62THF (2.5 mL) was added dropwise and the reaction mixture was stirred at -78°C for 45 min. An aqueous saturated solution of NH4CI (50 mL) was added slowly, and the resulting mixture was stirred without cooling until the température reached 0°C. Water (10 mL) was added and, after stirring for 30 min, the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated under reduced pressure, and co-evaporated with CH3CN to provide methyl 4-(2-(1bromo-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)-5-chlorophenoxy)-2,2dimethylbutanoate 14d (915 mg). The product was used without further purification in the next step.
Synthesis of intermediate 14e:
To a stirred solution of methyl 4-(2-(1 -bromo-2-oxo-2-(6-(trifluoromethoxy)indolin1-yl)ethyl)-5-chlorophenoxy)-2,2-dimethylbutanoate 14d (915 mg, 1.58 mmol) in CH3CN (40 mL), under N2-atm, were added 3-methoxy-5-(1H-1,2,4-triazol-115 yl)aniline [CAS 1220630-56-7] (301 mg, 1.58 mmol), and diisopropylethylamine (545 pL, 3.16 mmol) and the reaction mixture was stirred at 50°C for 18 h and 70°C for 6 h. The mixture was cooled to room température and poured out into stirring H2O (200 mL). The product was extracted (2x) with Et2O. The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (40 g) using a gradient of heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure and co-evaporated with dioxane to provide methyl 4-(5-chloro-2-(1-((3methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2-oxo-2-(625 (trifluoromethoxy)indolin-l -yl)ethyl)phenoxy)-2,2-dimethylbutanoate 14e (1.09 g). The product was used in the next step without purification.
Synthesis of Compound 14 and chiral séparation into Enantiomers 14A and 14B:
1M NaOH (3.95 mL, 3.95 mmol) was added to a stirring solution of methyl 4-(5chloro-2-(1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2-oxo-2-(6(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)-2,2-dimethylbutanoate 14e (1.09 g, 1.58 mmol) in dioxane (6.5 mL The reaction mixture was stirred at room température for 40 h. Water (21 mL) and 1N HCl (4.1 mL) were added and after stirring for 10 minutes, the precipitate was filtered off, and washed (3x) with water. The solid residue (0.9 g) was stirred up in CH2CI2 (7.5 mL) for 45 minutes, filtered off, washed (3x) with CH2CI2, and dried under vacuum at 45°C to provide racemic 4-(5-chloro-2-(1 -((3-methoxy-5-(1 H-1,2,4-triazol-1 -yl)phenyl)amino)-2-oxo-2-(619483
-63(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)-2,2-dimethylbutanoic acid (Compound 14, 590 mg).
The enantiomers of Compound 14 (557 mg) were separated via Préparative Chiral 5 SFC (Stationary phase: Chiralpak® Diacel AD 20 x 250 mm, Mobile phase: CO2, EtOH + 0.4% iPrNH2). The fractions containing the first eluted product were combined, evaporated under reduced pressure and co-evaporated with CH3CN. The residue was crystallized from Et2O/heptane 3/1, filtered off, washed (3x) with Et2O, and dried under vacuum at 50°C to provide Enantiomer 14A (96 mg). The 10 fractions containing the second eluted product were combined, evaporated under reduced pressure and co-evaporated with CH3CN. The residue was crystallized from Et2O, filtered off, washed (3x) with Et2O, and dried under vacuum at 50°C to provide Enantiomer 14B (35 mg + 106 mg (second crop)).
Compound 14:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.11 (d, J=5.1 Hz, 6 H) 1.86 - 2.03 (m, 2 H) 3.09 - 3.28 (m, 2 H) 3.73 (s, 3 H) 4.01 - 4.18 (m, 3 H) 4.38 (td, J=10.2, 6.6 Hz, 1 H) 5.69 (d, J=8.Q Hz, 1 H) 6.33 (t, J=2.0 Hz, 1 H) 6.67 (t, J=1.9 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.97 - 7.05 (m, 2 H) 7.18 (d, J=1 .8 Hz, 1 H) 7.31 (d, J=8A Hz, 1 H) 7.34 20 (d, J=8.1 Hz, 1 H) 8.03 (br s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (br s, 1 H)
LC/MS (method LC-C): Rt 1.12 min, MH+ 674
Enantiomer 14A:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.11 (d, J=5.1 Hz, 6 H) 1.87 - 2.02 (m, 2 H) 25 3.09 - 3.26 (m, 2 H) 3.73 (s, 3 H) 4.02 - 4.18 (m, 3 H) 4.37 (td, J=10.2, 6.6 Hz, 1 H)
5.69 (d, J=8.8 Hz, 1 H) 6.33 (t, J=2.2 Hz, 1 H) 6.67 (t, J=).8 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.99 - 7.04 (m, 2 H) 7.18 (d, J=2.0 Hz, 1 H) 7.31 (d, J=8A Hz, 1 H) 7.34 (d, J=8.4 Hz, 1 H) 8.03 (br s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (brs, 1 H) LC/MS (method LC-D): Rt 2.09 min, MH+ 674
[a]D 20: -32.8° (c 0.528, DMF)
Chiral SFC (method SFC-K): Rt 3.63 min, MH+ 674, chiral purity 100%.
Melting point: 111°C
Enantiomer 14B:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.11 (d, J=5.1 Hz, 6 H) 1.87 - 2.02 (m, 2 H) 3.10 - 3.27 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.18 (m, 3 H) 4.38 (td, J=10.2, 6.5 Hz, 1 H) 5.69 (d, J=8.6 Hz, 1 H) 6.33 (t, J=1.9 Hz, 1 H) 6.67 (t, J=1.9 Hz, 1 H) 6.79 - 6.86 (m, 2 H) 6.98 - 7.05 (m, 2 H) 7.18 (d, J=1.8 Hz, 1 H) 7.31 (d, J=8A Hz, 1 H) 7.34 (d, J=8.1 Hz, 1 H) 8.03 (brs, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.25 (brs, 1 H)
-64LC/MS (method LC-D): Rt 2.08 min, MH+ 674
[a]D20: +32.8° (c 0.515, DMF)
Chiral SFC (method SFC-K): Rt4.22 min, MH+ 674, chiral purity 100%.
Melting point: 177°C
Example 15 : synthesis of 4-(5-chloro-2-(2-(5-fluoro-6-(trifluoromethoxy)indolin-1yl)-1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2oxoethyl)phenoxy)butanoic acid (Compound 15) and chiral séparation into Enantiomers 15Aand 15B.
ci
Synthesis of intermediate 15a:
A mixture of 5-fluoro-6-(trifluoromethoxy)indoline 9e (321 mg, 1.45 mmol), 2-(2-(415 (tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazol-1 yl)phenyl)amino)acetic acid 11d (750 mg, 1.45 mmol), HATU (827 mg, 2.18 mmol) and diisopropylethylamine (719 pL, 4.35 mmol) in DMF (30 mL) was stirred at room température for 12 h. The mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic solution 20 was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 pm, 40 g, CH2Cl2/MeOH 98/2). The pure fractions were combined and concentrated to dryness to give tertbutyl 4-(5-chloro-2-(2-(5-fluoro-6-(trifluoromethoxy)indolin-1 -yl)-1 -((3-methoxy-525 (1/-7-1,2,4-triazol-1-yl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate 15a (1.05 g).
-65Synthesis of Compound 15 and chiral séparation into Enantiomers 15A and 15B:
A solution of tert-butyl 4-(5-chloro-2-(2-(5-fluoro-6-(trifluoromethoxy)indolin-1-yl)-1((3-methoxy-5-(1/7-1,2,4-triazol-1-yl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate
15a (1.05 g , 1.46 mmol) in 4M HCl in dioxane (8 mL) was stirred at 5°C for 3 h and at room température for 12 h. The precipitate was filtered off, washed with dioxane/diisopropyl ether and dried. The residue (580 mg) was dissolved in THF (2.5 mL) and a solution of lithium hydroxide monohydrate (180 mg, 4.277 mmol) in water (2.5 mL) was added dropwise. The mixture was stirred at room température ίο for 18 h. The reaction was cooled to 0°C and water and ice were added. The pH was adjusted to 6 by the addition of 3N HCl. The product was extracted with EtOAc. The organic layer was separated, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. A small fraction of the residue was crystallized from CH2CI2 to give 4-(5-chloro-2-(2-(5-fluoro-615 (trifluoromethoxy)indolin-l -yl)-1 -((3-methoxy-5-(1 /7-1,2,4-triazol-1 yl)phenyl)amino)-2-oxoethyl)phenoxy)butanoic acid 15 (24 mg) as a racemate. The remaining amount was further purified by flash chromatography on silica gel (20-45 pm, 24 g, CH2Cl2/MeOH gradient 99.5/0.5 to 90/10). The pure fractions were combined and concentrated to dryness to provide a second fraction of
Compound 15 (382 mg).
The enantiomers of Compound 15 (382 mg) were separated via Préparative Chiral SFC (Stationary phase: Chiralcel® OD-H 5 pm 250 x 30 mm, Mobile phase: 75% CO2, 25% MeOH (+0.3% ÎPrNH2)). The first eluted enantiomer (178 mg) was solidified by titration with heptane/diisopropyl ether to give Enantiomer 15A (140 mg). The second eluted enantiomer (187 mg) was solidified by titration with heptane/diisopropyl ether to give Enantiomer 15B (136 mg).
Compound 15:
1H NMR (500 MHz, DMSO-cfe) δ ppm 1.90 - 2.04 (m, 2 H) 2.28 - 2.46 (m, 2 H) 3.11 - 3.28 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.18 (m, 3 H) 4.33 - 4.42 (m, 1 H) 5.71 (d, J=8.8 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.81 (s, 1 H) 6.87 (br d, J=8.8 Hz, 1 H) 7.02 (dd, 7=8.2, 1.9 Hz, 1 H) 7.12 (s, 1 H) 7.31 (d, 7=8.2 Hz, 1 H) 7.43 (d, 7=9.8 Hz, 1 H) 8.12-8.20 (m, 2 H) 9.16 (s, 1 H) 12.12 (br s, 1 H)
LC/MS (method LC-B): Rt 2.72 min, MH+ 664 Melting point: 188°C
-66Enantiomer 15A:
1H NMR (400 MHz, DMSO-de) δ ppm 1.88 - 2.12 (m, 2 H) 2.19 -2.42 (m, 2 H) 2.96 - 3.24 (m, 2 H) 3.73 (s, 3 H) 3.89 - 4.16 (m, 3 H) 4.25 - 4.43 (m, 1 H) 5.72 (d, 7=8.6 Hz, 1 H) 6.34 (s, 1 H) 6.66 (s, 1 H) 6.79 - 6.86 (m, 2 H) 7.01 (dd, 7=8.3, 1.8 Hz, 1
H) 7.09 - 7.14 (m, 1 H) 7.32 (d, 7=8.1 Hz, 1 H) 7.41 (d, 7=9.6 Hz, 1 H) 8.12 - 8.17 (m, 2 H) 9.16 (s, 1 H)
LC/MS (method LC-B): Rt2.75 min, MH+664
[a]D 20: -31.5° (c 0.267, DMF)
Chiral SFC (method SFC-J): Rt 2.66 min, MH+ 664, chiral purity 99.69%.
Enantiomer 15B:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.88 - 2.04 (m, 2 H) 2.22 -2.43 (m, 2 H) 3.12 - 3.40 (m, 2 H) 3.72 (s, 3H) 4.03 - 4.17 (m, 3 H) 4.33 - 4.43 (m, 1 H) 5.72 (d, 7=8.6 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.78 - 6.87 (m, 2 H) 7.01 (dd, 7=8.3, 1.8 Hz, 1
H) 7.09 - 7.13 (m, 1 H) 7.32 (d, 7=8.1 Hz, 1 H) 7.41 (d, 7=9.6 Hz, 1 H) 8.12 - 8.18 (m, 2 H) 9.16 (s, 1 H)
LC/MS (method LC-B): Rt 2.73 min, MH+ 664
[a]D 20: +28.2° (c 0.262, DMF)
Chiral SFC (method SFC-J): Rt 3.53 min, MH+664, chiral purity 98.93%.
Exemple 16 : synthesis of4-(5-chloro-2-(1-((3-methoxy-5-(1H-1,2,4-triazol-1yl)phenyl)amino)-2-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)-2oxoethyl)phenoxy)butanoic acid (Compound 16) and chiral séparation into Enantiomers 16Aand 16B.
5°C 2h, rt 12h
HCl (4 M in dioxane)
Chiral séparation
Enantiomers 16A and 16B
-67Synthesis of intermediate 16a:
A mixture of 4-methyl-6-(trifluoromethoxy)indoline 10h (336 mg, 1.55 mmol), 2-(2(4-(terf-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(1 HA ,2,4-triazoÎ-1 yl)phenyl)amino)acetic acid 11d (800 mg, 1.55 mmol), HATU (883 mg, 2.32 mmol) 5 and diisopropylethylamine (767 pL, 4.64 mmol) in DMF (30 mL) was stirred at room température for 12 h. The mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic solution was washed with a 10% solution of K2CO3 in water, water, dried over MgSO4, filtered and the solvent was concentrated under reduced pressure. The residue 10 was purified by flash chromatography on silica gel (15-40 pm, 40 g, heptane/EtAOc gradient 90/10 to 70/30). The pure fractions were combined and concentrated to dryness to give tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1/-/1,2,4-triazol-1 -yl)phenyl)amino)-2-(4-methyl-6-(trifluoromethoxy)indolin-1 -yl )-2oxoethyl)phenoxy)butanoate 16a (816 mg).
Synthesis of Compound 16 and chiral séparation into Enantiomers 16A and 16B:
A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(1H-1,2,4-triazol-1yl)phenyl)amino)-2-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)-220 oxoethyl)phenoxy)butanoate 16a (816 mg ,1.14 mmol) in 4M HCl in dioxane (7 mL) was stirred at 5°C for 2 h and at room température for 12 h. The precipitate was filtered off, washed with dioxane/diisopropyl ether and dried. The residue was purified by flash chromatography on silica gel (20-45 pm, 40 g, CH2Cl2/MeOH gradient 100/0 to 95/5). Pure fractions were combined and solvent was evaporated under reduced pressure to give, after crystallization from CHsCN/diisopropyl ether, 4-(5-chloro-2-(1 -((3-methoxy-5-(1 HA ,2,4-triazol-1 -yl)phenyl)amino)-2-(4-methyl-6(trifluoromethoxy)indolin-1-yl)-2-oxoethyl)phenoxy)butanoic acid 16 (495 mg).
The enantiomers of Compound 16 were separated via Préparative Chiral SFC 30 (Stationary phase: Chiralpak® AD-H 5 pm 250 x 30 mm, Mobile phase: 65% CO2, 35% iPrOH (+0.3% iPrNH2)). The first eluted enantiomer (145 mg) was further purified by flash chromatography on silica gel (10-40 pm, 24 g, CH2Cl2/MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after solidification by trituration with diisopropyl ether/pentane, Enantiomer 16A (99 mg). The second eluted enantiomer (149 mg) was further purified by flash chromatography on silica gel (10-40 pm, 24 g,
-68CH2Cl2/MeOH 98/2). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after solidification by trituration with diisopropyl ether/pentane, Enantiomer 16B (95 mg).
Compound 16:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.91 - 2.05 (m, 2 H) 2.20 (s, 3 H) 2.29 - 2.43 (m, 2 H) 3.01 - 3.27 (m, 2 H) 3.72 (s, 3 H) 4.02 - 4.18 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (br d, J=8.6 Hz, 1 H) 6.34 (br s, 1 H) 6.67 (s, 1 H) 6.78 - 6.90 (m, 3 H) 7.01 (brd, J=7.1 Hz, 1 H) 7.12 (s, 1 H) 7.31 (d, J=8.1 Hz, 1 H) 7.88 (br s, 1 H) 8.15 (s, 1
H) 9.14 (s, 1 H) 12.07 (br s, 1 H)
LC/MS (method LC-A): Rt 2.93 min, MH+ 660
Melting point: 214°C
Enantiomer 16A:
1H NMR (400 MHz, DMSO-cfe) δ ppm 1.92 - 2.04 (m, 2 H) 2.20 (s, 3 H) 2.30 - 2.44 (m, 2 H) 2.99 - 3.16 (m, 2 H) 3.73 (s, 3 H) 4.04 - 4.17 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (br d, J=8.6 Hz, 1 H) 6.34 (br s, 1 H) 6.67 (s, 1 H) 6.79 - 6.89 (m, 3 H) 7.02 (brd, J=8.6 Hz, 1 H) 7.12 (s, 1 H) 7.32 (d, J=8A Hz, 1 H) 7.89 (s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.09 (br s, 1 H)
LC/MS (method LC-A): Rt 2.94 min, MH+660
[a]D 20: -35.4° (c 0.263, DMF)
Chiral SFC (method SFC-F): Rt 1.61 min, MH+ 660, chiral purity 100%.
Enantiomer 16B:
1H NMR (400 MHz, DMSO-ofe) δ ppm 1.91 - 2.04 (m, 2 H) 2.20 (s, 3 H) 2.30 - 2.44 (m, 2 H) 3.00 - 3.15 (m, 2 H) 3.73 (s, 3 H) 4.03 - 4.17 (m, 3 H) 4.35 - 4.44 (m, 1 H) 5.71 (d, J=8.6 Hz, 1 H) 6.34 (s, 1 H) 6.67 (s, 1 H) 6.79 - 6.89 (m, 3 H) 7.02 (dd, J=8A, 1.5 Hz, 1 H) 7.10-7.14 (m, 1 H) 7.32 (d, J=8.59 Hz, 1 H) 7.89 (s, 1 H) 8.15 (s, 1 H) 9.14 (s, 1 H) 12.10 (brs, 1 H)
LC/MS (method LC-A): Rt 2.94 min, MH+ 660
[a]D 20: +34.3° (c 0.274, DMF)
Chiral SFC (method SFC-F): Rt 2.22 min, MH+660, chiral purity 99.47%.
-69Table : compounds prepared as described above
| Compound | Structure | Optical rotation |
| 1 | F och3 W o | racemic |
| 1A | Αί FC . YAU 1 Γ N~N Ua o N | [a]D 20 = -44.8° |
| 1B | F Au..... N | [a]D 20 = +36.2° |
| 2 | F OCH3 FC. XTh M* j y s n-n CHaO^^7 A | racemic |
| 2A | F och3 1 L > N-14 ch3o'A>^/ V | [a]D 20 = -45.0° |
| 2B | F och3 «Xl/A F H\s / 1 1 ) N-n ch3oAA-/ Qn> | [a]D 20 = +43.4° |
| Compound | Structure | Optical rotation |
| 3 | F CO Il I ) N-N Μ-/ o N | racemic |
| 3A | F Η0-γθ P=H, TT) N—N U? v | [a]D 20 = -38.2° |
| 3B | F HC'^^0TP OCH3 .AA Tl) N-N Υ^ΑΎ f i | [a]D 20 = +40.9° |
| 4 | Cl __OCH3 F C _a kl Fl ΐ h 3 y^x- N \ T [ > n-n AA-/ L 5 N | racemic |
| 4A | Cl och3 Am FC. T 1 T \ N~N AAv Γ i XNX | [a]D 20 = -42.9° |
| 4B | Cl râcT 1 T 5 n-n M'Y o N | [a]D 20 = +39.5° |
| 5 | Cl och3 F C. -.‘Ti 1 t y n-n CHaO··^^7 4>n> | racemic |
| Compound | Structure | Optical rotation |
| 5A | CI och3 f œ κι H / 1 T / N-N CHaO-^^^7 JJ | [ab20 = -40.3° |
| 5B | Cl OCH3 o-J^L/K F r ~ Il / TT) N-N Jl/Uy l \\ ch3c<^ | [a]D 20 =+40.0° |
| 6 | Cl PCH3 M IX> n | racemic |
| 6A | Cl HO·—'^'cr^r PCHs %AJ/=S FsGQ^/^Jn H~\— T 1 > N-N N | [a]D 20 = +45.9° |
| 6B | Ci hon/-No7\J 0Ch3 f,» °t7Q uo n XN | [a]D 20 = -46.3° |
| 7 | Cl l0/x,U,) och3 °i «“Q r 3 N \ Il 1 ) n~n v | racemic |
| 7A | Cl ho^Q T Γ ) N-N fAA2 v | [a]D 20 = -44.3° |
| Compound | Structure | Optical rotation |
| 7B | Cl ,och3 CK JL /==\ N—£ ? F C Λ 1 H V_/ N \ T T 2 N-N F.VA2 v | [a]D 20 = +35.6° |
| 8 | Cl och3 IL/ 81-14 CHacA^/ V | racemic |
| 8A | Cl ΝΟ-χ/^ΧΧ/ OCH3 α^Λ+>_ FaCOx-a^XL »^Α—ζ Il Γ 2 n-n ch3c<^ %> | [a]D 20 = +39.3° |
| 8B | Cl HCL -ÎJ HCx/l/K/ OCH3 FaûO-^Lf ”~V^ 1 Γ } N-N /L/A/ 1 w CHaO-^^ | [a]D 20 = -44.4° |
| 9 | C! __och3 F CCI «Γ v y 11) N-N fXA7 y | racemic |
| 9A | Ci HC< LLX och Ûl_H FaCO^J Il T } N~N F^> v | [a]D 20 = -35.10 |
| 9B | C! HCk LLX nru FaCO^J HM II) n-n fAA2 v | [a]D 20 = +32.3° |
| Compound | Structure | Optical rotation |
| 10 | Cl .och3 f ca [ΤΟ Il I > N—N V^7 o I N ch3 | racemic |
| 10A | Cl •Af Il I > N—N 'CA-/ f 5 T XN ch3 | [a]D 20 = -38.4° |
| 10B | Cl /-Γ3 f ca ; rQ I Γ 5 n-n •CÿAV £ T XN CH3 | [a]D 20 = +37.5° |
| 11 | Cl __OCH3 FsC^^^ZT Tl; n-n | racemic |
| 11A | C! __OCH3 FaCx^sjTrT —\ Il T > n-n Μ-/ o N | [a]D 20 = -28.9° |
| 11B | C! HOX''^^xo'x^r Pch3 ï F —\ Il I > N-N | [a]D 20 = +23.8° |
| Compound | Structure | Optical rotation |
| 12 | Cl ,och3 T T \ N-N | racemic |
| 12A | Cl “M T T Y N-N KAj i \\ ch3o^^ %> | [a]D 20 = +30.4° |
| 12B | Cl JLX OCH3 T I > N-N AA? / \\ CH3O^-^ | [a]D 20 = -36.9° |
| 13 | Cl ΗΟγ^^θΧ^ PCH3 F 3 ejUs>^γ- N \ 1 Τ'} N-N Μ-/ o N | racemic |
| 13A | 1 û ΖΛΪ/1 >=ο ï o I | [a]D 20 = +38.4° |
| 13B | Cl Hœ^^Xb T 0^/=( f3co^^_Jj hV_^ Μ-/ o N | [a]D 20 = -43.2° |
| 14 | Cl __0CH3 0 °Xn7) T 1 ? N-N Μ-/ O N | racemic |
| Compound | Structure | Optical rotation |
| 14A | Cl OCH3 Il T > N-N | [a]D 20 = -32.8° |
| 14B | Cl och3 T ojM f3co^^XY h\_^ IX> Π XNX | [a]D 20 =+32.8° |
| 15 | Cl ΗΟγ-^^Q/Cj OCH3 0 0;V^N—Z) F3co.^7r -\ Il I > N-N fa>? y | racemic |
| 15A | ci HO. _ïj ï T Γ N-N FJUY v | [a]D 20 =-31.5° |
| 15B | Cl ΗΟ<χ-\^\ nrw Y^ ° >) A 3 F3CO^^3f H\_ II) N-N V | [a]D 20 = +28.2° |
| 16 | Cl __PCH3 AA Il I > N-N v^7 o I N ch3 | racemic |
| 16A | Cl OCH3 o c ™ I H V / 1 Γ ) N—N w O 1 N ch3 | [a]D 20 = -35.4° |
| Compound | Structure | Optical rotation |
| 16B | Cl HŒ _ _ Îj OCH3 Tl I n-n S A7 o I N ch3 | [a]D 20 = +34.3° |
ANTIVIRAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION
DENV-2 antiviral assay
The antiviral activity of ail the compounds ofthe invention was tested against the 5 DENV-2 16681 strain which was labeled with enhanced green fluorescent protein (eGPF). The culture medium consists of minimal essential medium supplemented with 2% of heat-inactivated fêtai calf sérum, 0.04% gentamycin (50mg/mL) and 2mM of L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 25μL was added to 384-well plates (2500 cells/well), which already îo contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (200nL). In addition, each compound concentration is tested in quadruplicate (final concentration range: 25μΜ - 0.000064μΜ or 2.5μΜ - 0.0000064μΜ for the most active compounds). Finally, each plate contains wells which are assigned as virus contrais (containing cells and virus in the absence of compound), cell contrais (containing cells in the absence of virus and compound) and medium contrais (containing medium in the absence of cells, virus and compounds). To the wells assigned as medium control, 25pL of culture medium was added instead of Vero cells. Once the cells were added to the plates, 20 the plates were incubated for 30 minutes at room température to allow the cells to distribute evenly within the wells. Next, the plates were incubated in a fully humidified incubator (37°C, 5% CO2) until the next day. Then, DENV-2 strain 16681, labeled with eGFP, was added at a multiplicity of infection (MOI) of 0.5. Therefore, 15pL of virus suspension was added to ail the wells containing test compound and to the wells assigned as virus control. In parallel, 15pL of culture medium was added to the medium and cell contrais. Next, the plates were incubated for 3 days in a fully humidified incubator (37°C, 5% CO2). At the day of the read out, the eGFP fluorescence was measured using an automated fluorescence microscope at 488 nm (blue laser). Using an in-house LIMS system, 30 inhibition dose response curves for each compound were calculated and the half maximal effective concentration (EC50) was determined. Therefore, the percent inhibition (I) for every test concentration is calculated using the following formula: I
-77= 100*(St-Scc)/(Svc-Scc); St, Scc and Svc are the amount of eGFP signal in the test compound, cell control and virus control wells, respectively. The ECso represents the concentration of a compound at which the virus réplication is inhibited with 50%, as measured by a 50% réduction of the eGFP fluorescent intensity compared to the virus control. The ECso is calculated using linear interpolation (Table 1).
In parallel, the toxicity ofthe compounds was assessed on the same plates. Once the read-out for the eGFP signal was done, 40pL of ATPIite, a cell viability stain, was added to ail wells ofthe 384-well plates. ATP is présent in ail metabolically 10 active cells and the concentration déclinés very rapidly when the cells undergo necrosis or apoptosis. The ATPLite assay System is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. The plates were incubated for 10 minutes at room température. Next, the plates were measured on a ViewLux. The half maximal cytotoxic concentration (CCso) was also determined, defined as the concentration required to reduce the luminescent signal by 50% compared to that of the cell control wells. Finally, the selectivity index (SI) was determined for the compounds, which was calculated as followed: SI = CCso/ECso.
Table 1 : ECso, CCso, and SI for the compounds ofthe invention in the DENV-2 antiviral assay
| compound# | EC50(pM) | N | CC50 (μΜ) | N | SI | N |
| 1 | 0.0031 | 4 | 12 | 4 | 3350 | 4 |
| 1A | 1.00 | 3 | 7.7 | 3 | 7.8 | 3 |
| 1B | 0.0028 | 4 | 9.8 | 2 | 3530 | 2 |
| 2 | 0.0028 | 4 | 13 | 4 | 4000 | 4 |
| 2A | 0.27 | 3 | 12 | 3 | 43 | 3 |
| 2B | 0.0018 | 3 | 10 | 3 | 4180 | 3 |
| 3 | 0.0019 | 4 | 8.5 | 4 | 5210 | 4 |
| 3A | 0.52 | 3 | 11 | 3 | 22 | 3 |
| 3B | 0.00099 | 3 | 8.7 | 3 | 11400 | 3 |
| 4A | 1.2 | 4 | 7.8 | 4 | 6.4 | 4 |
| 4B | 0.0010 | 3 | 11 | 3 | 16100 | 3 |
| 5 | 0.00082 | 3 | 11 | 3 | 11200 | 3 |
| 5A | 0.042 | 3 | 11 | 3 | 275 | 3 |
| 5B | 0.00062 | 3 | 9.2 | 3 | 14400 | 3 |
| 6 | 0.00097 | 3 | 3.3 | 3 | 3370 | 3 |
| compound# | ΕΟδο(μΜ) | N | CC50 (μΜ) | N | SI | N |
| 6A | 0.00059 | 9 | 8.5 | 9 | 14900 | 9 |
| 6B | 0.092 | 7 | 7.3 | 7 | 79 | 7 |
| 7 | 0.0030 | 3 | 10 | 3 | 3410 | 3 |
| 7A | 0.43 | 3 | 11 | 3 | 26 | 3 |
| 7B | 0.0018 | 3 | 7.6 | 3 | 4180 | 3 |
| 8 | 0.0021 | 3 | 9.5 | 3 | 4410 | 3 |
| 8A | 0.0015 | 3 | 12 | 3 | 7360 | 3 |
| 8B | 0.25 | 3 | 12 | 3 | 47 | 3 |
| 9 | 0.0016 | 4 | 3.9 | 3 | 2850 | 3 |
| 9A | 0.25 | 3 | 4.2 | 4 | 17 | 3 |
| 9B | 0.00060 | 3 | 10 | 3 | 13800 | 3 |
| 10 | 0.00035 | 3 | 10 | 3 | 26600 | 3 |
| 10A | 0.063 | 3 | 10 | 3 | 165 | 3 |
| 10B | 0.00025 | 3 | 11 | 3 | 51400 | 3 |
| 11 | 0.00031 | 3 | 15 | 3 | 53500 | 3 |
| 11A | 0.045 | 3 | 11 | 3 | 246 | 3 |
| 11B | 0.00020 | 3 | 12 | 3 | 45200 | 3 |
| 12 | 0.0022 | 3 | 13 | 3 | 5660 | 3 |
| 12A | 0.0012 | 3 | 13 | 3 | 11000 | 3 |
| 12B | 0.32 | 3 | 12 | 3 | 37 | 3 |
| 13 | 0.00023 | 3 | 11 | 3 | 59500 | 3 |
| 13A | 0.00012 | 4 | 12 | 4 | 103601 | 4 |
| 13B | 0.012 | 3 | 12 | 3 | 972 | 3 |
| 14 | 0.00041 | 3 | 10 | 3 | 24900 | 3 |
| 14A | 0.42 | 3 | 9.1 | 3 | 22 | 3 |
| 14B | 0.00027 | 4 | 9.4 | 4 | 35500 | 4 |
| 15 | 0.00022 | 3 | 11 | 3 | 58700 | 3 |
| 15A | 0.0053 | 3 | 10 | 3 | 1970 | 3 |
| 15B | 0.00013 | 3 | 11 | 4 | 94800 | 3 |
| 16 | 0.00011 | 3 | 11 | 3 | 95800 | 3 |
| 16A | 0.014 | 3 | 11 | 3 | 800 | 3 |
| 16B | 0.000069 | 5 | 6.2 | 5 | >174673 | 5 |
N= the number of independent experiments in which the compounds were tested.
Tetravalent reverse transcriptase quantitative-PCR (RT-qPCR) assay: Protocol A.
The antiviral activity ofthe compounds of the invention was tested against DENV-1 5 strain TC974#666 (NCPV), DENV-2 strain 16681, DENV-3 strain H87 (NCPV) and
DENV-4 strain H241 (NCPV) in a RT-qPCR assay. Therefore, Vero cells were infected with either DENV-1, or -2, or -3, or -4 in the presence or absence of test compounds. At day 3 post-infection, the cells were lysed and cell lysâtes were used to préparé cDNA of both a viral target (the 3’UTR of DENV; Table 2) and a 5 cellular reference gene (β-actin, Table 2). Subsequently, a duplex real time PCR was performed on a Lightcycler480 instrument. The generated Cp value is inversely proportional to the amount of RNA expression of these targets. Inhibition of DENV réplication by a test compound results in a shift of Cp’s for the 3’UTR gene. On the other hand, if a test compound is toxic to the cells, a similar effect on îo β-actin expression will be observed. The comparative ΔΔΟρ method is used to calculate ECso, which is based on the relative gene expression ofthe target gene (3’UTR) normalized with the cellular housekeeping gene (β-actin). In addition, CC50 values are determined based on the Cp values acquired for the housekeeping gene β-actin.
Table 2: Primers and probes used for the real-time, quantitative RT-PCR .
| Primer/probe | Target | Sequenceab |
| F3utr258 | DENV 3’- UTR | 5'-CGGTTAGAGGAGACCCCTC-3' |
| R3utr425 | DENV 3’- UTR | 5'-GAGACAGCAGGATCTCTGGTC-3' |
| P3utr343 | DENV 3’- UTR | FAM-5'-AAGGACTAG-ZEN- AGGTTAGAGGAGACCCCCC-3'-/ABkFQ |
| Factin743 | β-actin | 5'-GGCCAGGTCATCACCATT-3' |
| Ractin876 | β-actin | 5'-ATGTCCACGTCACACTTCATG-3' |
| Pactin773 | β-actin | HEX-5'-TTCCGCTGC-ZEA/-CCTGAGGCTCTC-3'lABkFQ |
a Reporter dyes (FAM, HEX) and quenchers (ZEN and lABkFQ) éléments are indicated in bold and italics.
b The nucléotide sequence of the primers and probes were selected from the conserved région in the 3’UTR région of the dengue virus genome, based on the alignment of 300 nucléotide sequences ofthe four dengue serotypes deposited in Genbank (Gong et al., 2013, Methods Mol Biol, Chapter 16).
The culture medium consisted of minimal essential medium supplemented with 2% of heat-inactivated fêtai calf sérum, 0.04% gentamycin (50mg/mL) and 2mM of
-80L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 75pL/well was added in 96-well plates (10000 cells/well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (500nL; final concentration range: 25μΜ 0.000064μΜ or 2.5μΜ - 0.0000064μΜ for the most active compounds). In addition, each plate contains wells which are assigned as virus Controls (containing cells and virus in the absence of compound) and cell Controls (containing cells in the absence of virus and compound). Once the cells were ίο added in the plates, the plates were incubated in a fully humidified incubator (37°C, 5% CO2) until the next day. Dengue viruses serotype-1,2, 3 and4 were diluted in order to obtain a Cp of -22-24 in the assay. Therefore, 25pL of virus suspension was added to ail the wells containing test compound and to the wells assigned as virus control. In parallel, 25pL of culture medium was added to the cell 15 Controls. Next, the plates were incubated for 3 days in a fully humidified incubator (37°C, 5% CO2). After 3 days, the supernatant was removed from the wells and the cells were washed twice with ice-cold PBS (~100pL). The cell pellets within the 96-well plates were stored at -80°C for at least 1 day. Next, RNA was extracted using the Cells-to-CT™ lysis kit, according to the manufacturer’s guideline (Life
Technologies). The cell lysâtes can be stored at -80°C or immediately used in the reverse transcription step.
In préparation of the reverse transcription step, mix A (table 3A) was prepared and 7.57pL/well was dispensed in a 96-well plate. After addition of 5pL of the cell lysâtes, a five minute dénaturation step at 75°C was performed (table 3B).
Afterwards, 7.43pL of mix B was added (table 3C) and the reverse transcription step was initiated (table 3D) to generate cDNA.
Finally, a RT-qPCR mix was prepared, mix C (table 4A), and 22.02 pL/well was dispensed in 96-well LightCycler qPCR plates to which 3pL of cDNA was added and the qPCR was performed according to the conditions in table 4B on a 30 LightCycler 480.
Using the LightCycler software and an in-house LIMS system, dose response curves for each compound were calculated and the half maximal effective concentration (EC50) and the half maximal cytotoxic concentration (CC50) were determined (Tables 5-8).
-81Table 3: cDNA synthesis usina Mix A, dénaturation, Mix B and reverse transcription.
Mix A
| Plates | 8 | ||||
| Samples | 828 | Reaction Vol. Je!)____________ | 20 | ||
| Mix item | Concentration | Volume for (pl) | |||
| Unit | Stock | Final | 1 sample | x samples | |
| Milli-Q H2O | 7.27 | 6019.56 | |||
| R3utr425 | μΜ | 20 | 0.27 | 0.15 | 124.20 |
| Ractin876 | μΜ | 20 | 0.27 | 0.15 | 124.20 |
| Volume mix/well (pl) | 7.57 | ||||
| Cell lysâtes | 5.00 |
B Dénaturation step:
| Step | Temp | Time |
| Dénaturation | 75°C | 5' |
| Hold | 4°C | hold |
C Mix B
| Samples | 864 | ||||
| Mix Item | Concentration | Volume for (pl) | |||
| Unit | Stock Final | 1 sample | x samples | ||
| Expand HIFI buffer 2 | X | 10.00 | 1.00 | 2.00 | 1728.0 |
| MgCI2 | mM | 25.00 | 3.50 | 2.80 | 2419.2 |
| dNTPs | mM | 10.00 | 1.00 | 2.00 | 1728.0 |
| Rnase inhibitor | U/μΙ | 40.00 | 1.00 | 0.50 | 432.0 |
| Expand RT | U/μΙ | 50.00 | 0.33 | 0.13 | 112.3 |
| Total Volume Mix (U·) | 7.43 |
D Protocol cDNA synthesis
| Step | Temp | Time |
| Rev transe | 42°C | 30’ |
| Dénaturation | 99°C | 5' |
| Hold | 4°C | hold |
-82Table 4: qPCR mix and protocol.
A Mix C
| Samples | 833 | Reaction Vol. (PD | 25 | ||
| Mix Item | Concentration | Volume for (μΙ) | |||
| Unit | Stock | Final | 1 sample | x samples | |
| HjO PCR grade Roche | 7.74 | 6447.42 | |||
| Roche 2xMM mix | X | 2 | 1 | 12.50 | 10412.50 |
| F3utr258 | μΜ | 20 | 0.3 | 0.38 | 316.54 |
| R3utr425 | μΜ | 20 | 0.3 | 0.38 | 316.54 |
| P3utr343 | μΜ | 20 | 0.1 | 0.13 | 108.29 |
| Factin743 | μΜ | 20 | 0.3 | 0.38 | 316.54 |
| Ractin876 | μΜ | 20 | 0.3 | 0.38 | 316.54 |
| Pactin773 | μΜ | 20 | 0.1 | 0.13 | 108.29 |
| Volume Mix / Tube (μΙ) | 22.02 | ||||
| cDMA | 3.00 |
| B | Protocol qPCR3 | ||||
| Step | Temp | Time | Ramp rate | ||
| preincub/denat | 95°C | 10 min | 4.4 | ||
| Dénaturation | 95°C | 10 sec | 4.4 | 40 cycles | |
| annealing | 58°C | 1 min | 2.2 | ||
| Elongation | 72°C | 1 sec | 4.4 | ||
| Cooling | 40°C | 10 sec | 1.5 |
Table 5: EC5q, CC50, and SI for the compounds against serotype 1 in the RT-qPCR
| assays | ||||||
| Protocol A | ||||||
| RT-qPCR serotype 1 T | C974 | l#666 | ||||
| compound# | ECso (pM) | N | CC50 (pM) | N | SI | N |
| 1B | 0.0015 | 4 | >2.5 | 4 | >2290 | 4 |
| 2B | 0.0060 | 5 | >2.5 | 5 | >744 | 5 |
| 3B | 0.0024 | 3 | >2.5 | 2 | >1550 | 2 |
| 4B | 0.00057 | 4 | >2.5 | 4 | >8060 | 4 |
| 5B | 0.0020 | 4 | >2.5 | 4 | >981 | 4 |
| 6A | 0.00064 | 4 | >2.5 | 4 | >10900 | 4 |
| 7B | 0.00088 | 3 | >2.5 | 3 | >6750 | 3 |
| Protocol A | ||||||
| RT-qPCR serotype 1 TC972 | t#666 | |||||
| compound# | ËC50 (μΜ) | N | CC50 (μΜ) | N | SI | N |
| 8A | 0.0020 | 3 | >2.5 | 3 | >1570 | 3 |
| 9B | 0.00099 | 3 | >2.5 | 3 | >2860 | 3 |
| 10B | 0.00036 | 3 | >2.5 | 3 | >8670 | 3 |
| 11B | 0.000095 | 3 | >2.5 | 3 | >41800 | 3 |
| 12A | 0.0021 | 3 | 11 | 3 | 3850 | 3 |
| 13A | 0.00012 | 3 | >2.5 | 3 | >32500 | 3 |
| 14B | 0.00022 | 3 | 2.3 | 3 | 8720 | 3 |
| 15B | 0.00013 | 3 | 4.9 | 3 | 46500 | 3 |
| 16B | 0.000092 | 3 | >1.0 | 3 | >23100 | 3 |
N= the number of independent experiments in which the compounds were tested.
Table 6: ECso, CC50, and SI for the compounds against serotype 2 in the RT-qPCR assays
| Protocol A | ||||||
| RT-qPCR serotype 2 16681 | ||||||
| compound# | EC50 (pM) | N | CC50 (pM) | N | SI | N |
| 1B | 0.0024 | 3 | >2.5 | 1 | >1480 | 1 |
| 2B | 0.0021 | 3 | 4.3 | 3 | 2070 | 3 |
| 3B | 0.0014 | 3 | 13 | 3 | 5680 | 3 |
| 4B | 0.00045 | 3 | 2.4 | 3 | 6270 | 3 |
| 5B | 0.00052 | 3 | 2.8 | 3 | 6730 | 3 |
| 6A | 0.00049 | 5 | 11 | 5 | 18700 | 5 |
| 7B | 0.0019 | 3 | >2.5 | 2 | >9150 | 2 |
| 8A | 0.00038 | 3 | >0.99 | 3 | 3620 | 3 |
| 9B | 0.00047 | 3 | >2.5 | 2 | >4250 | 2 |
| 10B | 0.00015 | 3 | >1.0 | 3 | >10700 | 3 |
| 11B | 0.000059 | 3 | 5.0 | 3 | 64900 | 3 |
| 12A | 0.00042 | 3 | 14 | 3 | 19800 | 3 |
| 13A | 0.000057 | 3 | >2.5 | 3 | >53100 | 3 |
| 14B | 0.00016 | 4 | 2.6 | 3 | >9320 | 3 |
| 15B | 0.000100 | 3 | 6.3 | 4 | >22600 | 3 |
| 16B | 0.000055 | 3 | >1.0 | 3 | >44200 | 3 |
N= the number of independent experiments in which the compounds were tested.
-84Table 7: ECso, CCso, and SI for the compounds against serotype 3 in the RT-qPCR
| Protocol A | ||||||
| RT-qPCR serotype 3 | H 87 | |||||
| compound# | ECso (pM) | N | CC50 (pM) | N | SI | N |
| 1B | 0.025 | 3 | >2.5 | 3 | >123 | 3 |
| 2B | 0.038 | 4 | >2.5 | 4 | >118 | 4 |
| 3B | 0.023 | 3 | >2.5 | 3 | >136 | 3 |
| 4B | 0.011 | 3 | 2.3 | 3 | 228 | 3 |
| 5B | 0.015 | 4 | >2.2 | 4 | >116 | 4 |
| 6A | 0.0081 | 4 | >2.5 | 4 | >227 | 4 |
| 7B | 0.013 | 3 | >2.5 | 3 | >259 | 3 |
| 8A | 0.015 | 3 | >2.5 | 3 | >203 | 3 |
| 9B | 0.0064 | 4 | >1.0 | 1 | >103 | 1 |
| 10B | 0.0059 | 3 | >2.5 | 3 | >414 | 3 |
| 11B | 0.0065 | 3 | >2.5 | 3 | >991 | 3 |
| 12A | 0.036 | 3 | 12 | 3 | 384 | 3 |
| 13A | 0.0018 | 3 | >2.5 | 3 | >1580 | 3 |
| 14B | 0.0055 | 3 | >2.5 | 3 | >644 | 3 |
| 15B | 0.0028 | 3 | 5.8 | 3 | 3130 | 3 |
| 16B | 0.00087 | 3 | >1.0 | 3 | >1780 | 3 |
N= the number of independent experiments in which the compounds were tested.
Table 8: EC5q, CCso, and SI for the compounds against serotype 4 in the RT-qPCR assays
| Protocol A | ||||||
| RT-q | PCR serotype 4 | H241 | ||||
| compound# | EC50 (pM) | N | CC50 (pM) | N | SI | N |
| 1B | 0.18 | 3 | 2.2 | 2 | 10 | 2 |
| 2B | 0.15 | 3 | >2.5 | 3 | >11 | 3 |
| 3B | 0.098 | 3 | >2.5 | 2 | >25 | 2 |
| 4B | 0.076 | 3 | 1.8 | 3 | 23 | 3 |
| 5B | 0.090 | 3 | >2.5 | 2 | >33 | 2 |
| 6A | 0.060 | 6 | >2.5 | 4 | >53 | 4 |
| 7B | 0.12 | 3 | 1.1 | 3 | 10 | 3 |
| 8A | 0.056 | 3 | 1.1 | 2 | 21 | 2 |
| 9B | 0.087 | 3 | 2.5 | 3 | 27 | 3 |
| Protocol A | ||||||
| RT-q | PCR serotype 4 | H241 | ||||
| compound# | EC50 (μΜ) | N | CCso (μΜ) | N | SI | N |
| 10B | 0.032 | 3 | 1.1 | 2 | 27 | 2 |
| 11B | 0.020 | 3 | 3.3 | 3 | 184 | 3 |
| 12A | 0.10 | 3 | 5.5 | 3 | 96 | 3 |
| 13A | 0.010 | 3 | 2.6 | 3 | 251 | 3 |
| 14B | 0.023 | 3 | 1.4 | 3 | 61 | 3 |
| 15B | 0.015 | 3 | 2.3 | 3 | 174 | 3 |
| 16B | 0.0053 | 3 | 1.2 | 2 | 181 | 2 |
N= the number of independent experiments in which the compounds were tested.
Claims (7)
1. A compound having formula (I)
wherein
R1 is chloro or fluoro,
R2 is -CH2CH2OH, or C3_5alkylCOOH;
îo R3 is trifluoromethyl, or trifluoromethoxy;
R4 is hydrogen, fluoro, or methoxy; and
R5 is hydrogen or methyl;
or a pharmaceutically acceptable sait, solvaté or polymorph thereof.
15
2. A compound or its stereoisomeric form, a pharmaceutically acceptable sait, solvaté or polymorph thereof according to claim 1 wherein said compound is selected from the group of
3. The compound according to claim 1 wherein said compound has the (+) spécifie rotation.
5
4. The compound according to claim 1 wherein said compound is selected from :
5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 together with one or more pharmaceutically acceptable excipients, diluents or carriers.
6. The pharmaceutical composition according to claim 5 which comprises a second or further active ingrédient.
7. The pharmaceutical composition according to claim 6 wherein the second or îo further active ingrédient is an antiviral agent.
8 A compound of formula (I) according to any one of claims 1 to 4 for use as a medicine.
15 9. A compound of formula (I) according to any one of claims 1 to 4 for use in the treatment of Dengue infection and for the prévention or treatment of disease associated with Dengue infection.
-8910. A compound of formula (I) for use according to claim 9 wherein the Dengue infection is infection by viruses of the DENV-1, DENV-2, DENV-3 or DENV-4 strain.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP17164048.5 | 2017-03-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA19483A true OA19483A (en) | 2020-10-23 |
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