CA2869945C

Modulators of atp-binding cassette transporters

Abstract

Compounds of the present invention and pharmaceutically acceptable compositions thereof, are useful as modulators of ATP-Binding Cassette ('ABC') transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator ('CFTR'). The present invention also relates to methods of treating ABC transporter mediated diseases using compounds of the present invention.

CA2869945C, drawing sheet 1 — Sheet 1 of 6

Term

0.5 yearsleft in the term

Expires 9 April 2027.

3 claims: 1 independent, 2 dependent

1
CLAIMS:1. A process of preparing compounds of the following formula Ic: wherein, Ri is -Z a R4, wherein each Z A is independently a bond or an optionally substituted branched or straight Ci_6 aliphatic chain wherein up to two carbon units of Z A are optionally and independently replaced by -CO-, -CS-, -CONR A -, -CONR A NR A -, -CO2-, -OCO-, -nr a co2-, -o-, -nr a conr a -, -oconr a -, -nr a nr a -, -nr a co-, -S-, -so-, -so2-, -NR a -, -SO2NR a -, -NR a SO2-, or -NR A SO2NR A -, Each R4 is independently R A , halo, -OH, -NH 2 , -NO 2 , -CN, or -OCF3, Each R a is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl;Each R 2 is independently -Z B Rs, wherein each Z B is independently a bond or an optionally substituted branched or straight Cj.6 aliphatic chain wherein up to two carbon units of Z B are optionally and independently replaced by -CO-, -CS-, -CONR B -, -CONR b NR b -, -CO2-, -OCO-, -NR b CO2-, -O-, -NR b C0NR b -, OCONR b -, -NR b NR b -, -NR b CO-, -S-, SO-, -SO2-, -NR b -, -SO2NR b -, -NR b SO2-, or -NR B SO2NR B -, Each R5 is independently R B , halo, -OH, -NH 2 , -NO 2 , -CN, -CF3, or -OCF3, Each R B is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl, Or, any two adjacent R 2 groups together with the atoms to which they are attached form an optionally substituted carbocycle or an optionally substituted heterocycle;Ring A is an optionally substituted 3-7 membered monocyclic ring having 0-3 heteroatoms selected from N, O, and S;Ring B is a group having formula la: CA 02869945 2016-04-13 66822-1083D1 or a pharmaceutically acceptable salt thereof, wherein p is 0-2, Each R 3 and R’ 3 is independently -Z Rô, where each Z is independently a bond or an 5 optionally substituted branched or straight Ci-6 aliphatic chain wherein up to two carbon units of Z are optionally and independently replaced by -CO-, -CS-, - CONR -, -C0NR c NR c -, -CO2-, -OCO-, -NR c CO2-, -O-, -NR c C0NR c -, -0C0NR c -, - NR C NR C -, -NR c CO-, -S-, -SO-, -SO2-, -NR C -, -SO2NR c -, -NR c SO2-, or -NR C SO 2 NR C -, Each Rô is independently R c , halo, -OH, -NH 2 , -NO 2 , -CN, or -OCF 3 , p 10 Each R is independently hydrogen, an optionally substituted aliphatic, an optionally substituted cycloaliphatic, an optionally substituted heterocycloaliphatic, an optionally substituted aryl, or an optionally substituted heteroaryl, Or, any two adjacent R 3 groups together with the atoms to which they are attached form an optionally substituted heterocycle;and 15 n is 1-3;comprising the steps of converting the acid of the following formula: to the corresponding acid chloride of the following formula: O CA 02869945 2016-04-13 66822-1083D1 266 wherein R2, n, and ring A are as defined above, and coupling the acid chloride with an amine of the following formula: wherein Ri and Ring B are as defined above or alternatively, reacting the acid with a coupling reagent to provide an active ester and coupling the active ester with an amine of the aforementioned formula.
2
The process of claim 1, wherein n is 2 and two adjacent R2 groups together with the atoms to which they are attached form an optionally substituted heterocycle.

Independent claims2

410 paragraphs in 59 sections, as filed

(57) Abrégé/Abstract:

Compounds of the present invention and pharmaceutically acceptable compositions thereof, are useful as modulators of ATPBinding Cassette ('ABCj transporters or fragments thereof, including Cystic Fibrosis Transmembrane Conductance Regulator ('CFTRj. The present invention also relates to methods of treating ABC transporter mediated diseases using compounds of the present invention.

Canada http://opic.gc.ca Ottawa-Hull KIA 0C9 · http://cipo.gc.ca

O PI c

C I P o

OPIC-CIPO 191

CA 2869945 C 2018/01/23 (11)(21) 2 869 945 (13) C (72) lnventeurs(suite)/lnventors(continued): MILLER, MARK T., US; MCCARTNEY, JASON, US;

NUMA, MEHDI MICHEL JAMEL, US; YANG, XIAOQING, US (73) Propriétaires(suite)/Owners(continued):VERTEX PHARMACEUTICALS INCORPORATED, US (74) Agent: SMART & BIGGAR

-2CA 02869945 2014-11-04

ABSTRACT

DEMANDES OU BREVETS VOLUMINEUX

LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVETS COMPREND PLUS D’UN TOME.

CECI EST LE TOME _1__DE _2__

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE THAN ONE VOLUME.

THIS IS VOLUME 2 OF 2

NOTE: For additional volumes please contact the Canadian Patent Office.

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6.89 (m, 4H), 6.08 (d, J = 1.5 Hz, 1H), 6.03 (s, 2H), 3.31 (d, J = 6.2 Hz, 2H), 1.80 (s, 3H),

1.41 - 1.38 (m, 2H), 1.26 (s, 6H), 1.04 - 1.01 (m, 2H).

[00999] Example 116: l-iBenzoMU^ldioxoI-S-ylJ-A^Z-^-methyl-J-aÆ-tetrazol-SyI)butan-2-yl)-lZT-indol-5-yl)cyclopropanecarboxamide [001000] l-(Benzo[</j[l,3]dioxol-5-yl)-2V-(2-(4-cyano-2-methyibutan-2-yl)-127-indol-5yl)cyclopropanecarboxamide (83 mg, 0.20 mmol) was dissolved in XJV-dimethylformamide (1 mL) containing ammonium chloride (128 mg, 2.41 mmol), sodium azide (156 mg, 2.40 mmol); and a magnetic stir bar. The reaction mixture was heated at 110 °C for 40 minutes in a microwave reactor. The crude product was filtered and then purified by preparative HPLC using a gradient of 0-99% acetonitrile in water containing 0.05% trifluoroacetic acid to yield l-(benzo[i/][l,3]dioxol-5-yl)-7V-(2-(2-methyl-4-(12f-tetrazol-5-yl)butan-2-yl)-lH-indol-5 yl)cyclopropanecarboxamide. ESI-MS m/z calc. 458.2, found 459.2 (M+l)+. Retention time of 1.53 minutes. ’H NMR (400 MHz, CD3CN) 9.23 (s, 1H), 7.51 - 7.48 (m, 2H), 7.19 (d, J= 8.6 Hz, 1H), 7.06 - 7.03 (m, 2H), 6.95 - 6.89 (m, 2H), 6.17 (dd, J= 0.7, 2.2 Hz, 1H), 6.02 (s, 2H), 2.61 - 2.57 (m, 2H), 2.07 - 2.03 (m, 2H), 1.55-1.51 (m, 2H), 1.39 (s, 6H), 1.12-1.09 (m, 2H).

[001001] Example 117: l-(Benzo[rf][l,3]dioxol-5-yI)-JV-(2-(piperidiii-2-yl)-lH-indoI-5yl)cyclopropanecarboxamide [001002] tert-Butyl 2-(5-(l-(benzo[d][l,3]dioxol-5-yl)cyclo-propanecarboxamido)-lf7indol-2-yl)piperidine-l-carboxylate (55 mg, 0.11 mmol) was dissolved in dichloromethane (2.5 mL) containing trifluoroacetic acid (1 mL). The reaction mixture was stirred for 6 h at room temperature. The crude product was purified by preparative HPLC using a gradient of 0-99% acetonitrile in water containing 0.05% trifluoroacetic acid to yield 1(benzo[c/][l,3]dioxol-5-yl)-7V-(2-(piperidin-2-yl)-l//-indol-5-yl)cyclopropanecarboxamide. ESI-MS m/z calc. 403.2, found 404.4 (M+l)+. Retention time of 0.95 minutes.

[001003] Example 118: S-teri-Butyl-lH-indoï-ô-ylamine

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[001004] 2-Bromo-4-fôrr-butyI-phenylamin® [001005] To a solution of 4-ierf-Butyl-phenylamine (447 g, 3.00 mol) in DMF (500 mL) was added dropwise NBS (531 g, 3.00 mol) in DMF (500 mL) at room temperature. Upon completion, the reaction mixture was diluted with water and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4 and concentrated. The crude product was directly used in the next step without further purification.

[001006] 2-Bromo-4-tert‘-butyl-5-nitro-phenylamine [001007] 2-Bromo-4-ier/-butyl-phenylamine (160 g, 0.71 mol) was added dropwise to H2SO4 (410 mL) at room temperature to yield-a clear solution. This clear solution was then cooled down to —5 to —10 °C. A solution of KNO3 (83 g, 0.82 mol) in H2SO4 (410 mL) was added dropwise while the temperature was maintained between —5 to -10 °C. Upon completion, the reaction mixture was poured into ice / water and extracted with EtOAc. The combined organic layers were washed with 5% NajCOj and brine, dried over Na2SO4 and concentrated. The residue was purified by a column chromatography (ethyl acetate/petroleum ether 1:10) to give 2-bromo-4-reri-butyl-5-nitro-phenylamine as a yellow solid (150 g, 78%).

HCCSiMej Br Pd(PPh3)2CI2

---------->

NH, Cut·

Tol, H2O

[001008] 4-ter/-Butyl-5-nitro-2-trimethylsilanylethynyI-phenylamine [001009] To a mixture of 2-bromo-4-/èrf-butyl-5-nitro-phenylamine (27.3 g, 100 mmol) in toluene (200 mL) and water (100 mL) was added EtjN (27.9 mL, 200 mmol), Pd(PPh3)2Cl2 (2.11 g, 3.00 mmol), Cui (950 mg, 0.500 mmol) and trimethylsilyl acetylene (21.2 mL, 150 mmol) under a nitrogen atmosphere. The reaction mixture was heated at 70 °C in a sealed pressure flask for 2.5 h., cooled down to room temperature and filtered through a short plug

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248 of Celite. The filter cake was washed with EtOAc. The combined filtrate was washed with 5% NH4OH solution and water, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography (0 - 10 % ethyl acetate/petroleum ether) to provide 4tert-butyl-5-nitro-2-trimethylsilanylethynyl-phenylamine as a brown viscous liquid (25 g, 81 %).

[001010] 5-iert-Butyl-6-nitro-lH-indoIe [001011] To a solution of 4-ier/-butyl-5-nitro-2-trimethylsilanylethynyl-phenylamine (25 g, 86 mmol) in DMF (100 mL) was added Cui (8.2 g, 43 mmol) under a nitrogen atmosphere.

The mixture was heated at 135 °C in a sealed pressure flask overnight, cooled down to room temperature and filtered through a short plug of Celite. The filter cake was washed with EtOAc. The combined filtrate was washed with water, dried over Na2SO4 and concentrated.

The crude product was purified by column chromatography (10 — 20 % ethyl aetate/hexane) to provide 5-teri-butyl-6-nitro-lH-indole as a yellow solid (13 g, 69 %).

[001012] 5-fôri-Butyl-lH-indol-6-yIamine [001013] Raney Nickel (3 g) was added to 5-?er/-butyl-6-nitro-lH-indole (15 g, 67 mmol) in methanol (100 mL). The mixture was stirred under hydrogen (1 atm) at 30 °C for 3 h. The catalyst was filtered off. The filtrate was dried over Na2SO4 and concentrated. The crude dark brown viscous oil was purified by column chromatography (10 - 20 % ethyl acetate/petroleum ether) to give 5-ter/-butyI-lH-indoI-6-ylamine as a gray solid (11 g, 87 %). 'H NMR (300 MHz, DMSO-d6) δ 10.3 (br s, 1H), 7.2 (s, 1H), 6.9 (m, 1H), 6.6 (s, IH), 6.1 (m, IH), 4.4 (br s, 2H), 1.3 (s, 9H).

[001014] A person skilled in the chemical arts can use the examples and schemes along with known synthetic methodologies to synthesize compounds of the present invention, including the compounds in Table 3, below.

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Table 3 : Physical data of exemplary compounds.

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> 9</td><td> 487.3</td><td> 2.12</td><td> H NMR (400 MHz, DMSOd6) 8.65 (s, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 1.4 Hz, 1H), 7.38 (d, J = 8.3 Ηζ,ΙΗ), 7307.25 (m, 2H), 7.08 (dd, J =8.8, 1.9 Hz, 1H), 6.11 (s, 1H), 4.31 (t,J = 7.4 Hz, 2H), 3.64(1, J = 7.3 Hz, 2H), 3.20 (t, J = 7.6 Hz, 2H), 1.92 (t, J = 7.6 Hz, 2H), 1.45 (m, 2H), 1.39 (s, 6H), 1.10 (m, 2H)</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> 42</td><td> 505.3</td><td> 2.23</td><td> H NMR (400 MHz, DMSOd6) 8.33 (s, IH), 7.52 (s, IH), 7.42-7.39 (m, 2H), 7.337.25 (m, 2H), 6.14(s, IH), 4.99 (s„lH), 4.31-4.27 (m, 3H), 3.64 (t, J = 7.0 Hz, 2H), 3.20 (t, J = 7.6 Hz,2H), 1.91 (t, J = 7.6 Hz, 2H), 1.46 (m, 2H), 1.39 (s, 6H), 1.13 (m, 2H)</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMK</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> 99</td><td> 393.1</td><td> 3.18</td><td> H NMR (400 MHz, DMSOd6) 11.80 (s, 1H), 8.64 (s, 1H), 7.83 (m, 1H), 7.33-7.26 (m, 2H), 7.07 (m, 1H), 7.02 (m, 1H), 6.966.89 (m, 2H), 6.02 (s, 2H), 4.33 (q, J = 7.1 Hz, 2H), 1.421.39 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.061.03 (m, 2H)</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> 116</td><td> 397.3</td><td> 3.52</td><td> H NMR (400 MHz, DMSOd6) U.27(d,J “ 1.9 Hz, 1H), 8.66 (s, IH), 8.08 (d, J =1.6 Hz, IH), 7.657.61 (m, 3H), 7.46-7.40 (m, 2H), 7.31 (d, J = 8.7 Hz, 1H), 7.25-7.17 (m. 2H), 7.03 (d, J = 1.6 Hz, IH), 6.98-6.87 (m, 2H), 6.02 (s, 2H), 1.43-1.39 (m, 2H), 1.061.02 (m,2H)</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

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<td> Compound No.</td><td> LC/MS 1 M+l</td><td> LCZRT Min</td><td> NMR</td>

<td> 184</td><td> 461.3</td><td> 2.4</td><td> H NMR (400 MHz, DMSOd6) 10.89 (s, IH), 8.29 (s, IH), 7.52 (s, IH), 7.42-7.37 (m, 2H}> 7.32 (dd, J =8.3,1.4 Hz, 1H), 7.01 (d, J = 10.9 Hz, IH), 6.05 (d, J = 1.7 Hz, IH), 4.29 (t, J = 5.0 Hz, IH), 3.23 (m, 2H), 1.81 (t, J = 7.7 Hz, 2H), 1.46 (m, 2H), 1.29 (s, 6H), 1.13 (m, 2H)</td>

<td> Compound No.</td><td> Lt;MS M+l</td><td> LC/Kl Min</td><td> PUYXW.</td>

<td> 226</td><td> 377.5</td><td> 3.47</td><td> H NMR (400 MHz, DMSOd6) 10.76 (s, IH), 8.39 (s, IH), 7.55 (s, IH), 7.15-7.13 (m, IH), 7.036.89 (m, 4H), 6.03 (m, 3H), 1.41-1.38 (m, 2H), 1.32 (s, 9H), 1.04-1.01 (m, 2H)</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> Compound No.</td><td> LC/MS M+I</td><td> LC/RT Min</td><td> «MK</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

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<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

<td> 294</td><td> 443.4</td><td> 1.98</td><td> H NMR (400 MHz, DMSO) 8.62 (s, 1H), 7.55 (d,J=1.8 Hz, 1H), 7.50 (d, J =1.5 Hz, IH), 7.38 (d, J = 8.3 Hz, IH), 7.30 - 7.24 (m, 2H), 7.05 (dd, J = 2.0,8.8 Hz, IH), 6.13 (s, 1H), 4.88 (t, J = 5.5 Hz, IH), 4.14 (t, J = 6.1 Hz, 2H), 3.61 (m,2H), 3.21 (septet, J = 6.8 Hz, 1H), 1.471.44 (m, 2H), 1.26 (d, J = 6.8 Hz, 6H), 1.11 1.08 (m, 2H)</td>

<td> Compound No.</td><td> LC/MS M+l</td><td> LC/RT Min</td><td> NMR</td>

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257 [001015] VII. ASSAYS FOR DETECTING AND MEASURING AF508-CFTR CORRECTION PROPERTIES OF COMPOUNDS [001016] Membrane potential optical methods for assaying AF508-CFTR· modulation properties of compounds [001017] The optical membrane potential assay utilized voltage-sensitive FRET sensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y. Tsien (1995) “Voltage sensing by fluorescence resonance energy transfer in single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y. Tsien (1997) “Improved indicators of cell membrane potential that use fluorescence resonance energy transfer” Chem Biol 4(4): 269-77) in combination with instrumentation for measuring fluorescence changes such as the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades, et al. (1999) “Cell-based assays and instrumentation for screening ion-channel targets” Drug Discov Today 4(9): 431-439).

[001018] These voltage sensitive assays are based on the change in fluorescence resonant energy transfer (FRET) between the membrane-soluble, voltage-sensitive dye, DiSBAC2(3), and a fluorescent phospholipid, CC2-DMPE, which is attached to the outer leaflet of the plasma membrane and acts as a FRET donor. Changes in membrane potential (Vm) cause the negatively charged DiSBAC2<3) to redistribute across the plasma membrane and the amount of energy transfer from CC2-DMPE changes accordingly. The changes in fluorescence emission were monitored using VIPR™ II, which is an integrated liquid handler and fluorescent detector designed to conduct cell-based screens in 96- or 384-well microtiter plates.

[001019] Identification of Correction Compounds [001020] To identify small molecules that correct the trafficking defect associated with AF508-CFTR; a single-addition HTS assay format was developed. The cells were incubated in serum-free medium for 16 hrs at 37 °C in the presence or absence (negative control) of test compound. As a positive control, cells plated in 384-well plates were incubated for 16 hrs at 27 °C to “temperature-correct” AF5O8-CFTR. The cells were subsequently rinsed 3X with

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Krebs Ringers solution and loaded with the voltage-sensitive dyes. To activate AF5O8CFTR, 10 μΜ forskolin and the CFTR potentiator, genistein (20 μΜ), were added along with Cl’-free medium to each well. The addition of Cl’-free medium promoted CF efflux in response to AF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRET-based voltage-sensor dyes.

[001021] Identification of Potentiator Compounds [001023] To identify potentiators of AF508-CFTR, a double-addition HTS assay format was developed. During the first addition, a Cl’-free medium with or without test compound was added to each well. After 22 sec, a second addition of Cl’-free medium containing 2-10 μΜ forskolin was added to activate AF508-CFTR. The extracellular Cl' concentration following both additions was 28 mM, which promoted Cl’ efflux in response to AF508-CFTR activation and the resulting membrane depolarization was optically monitored using the FRET-based voltage-sensor dyes.SolutionsBath Solution #1 : (in mM) NaCl 160, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, pH 7.4 with NaOH.

[001026] Chloride-free bath solution: Chloride salts in Bath Solution #1 are substituted with gluconate salts.

[001027] CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at 20°C.

[001028] DiSBAC2(3): Prepared as a 10 mM stock in DMSO and stored at -20°C.

[001029] Cell Culture [001030] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for optical measurements of membrane potential. The cells are maintained at 37 °C in 5% CO2 and 90 % humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm2 culture flasks. For all optical assays, the cells were seeded at 30,000/well in 384-well matrigel-coated plates and cultured for 2 hrs at 37 °C before culturing at 27 °C for 24 hrs. for the potentiator assay. For the correction assays, the cells are cultured at 27 °C or 37 °C with and without compounds for 16 — 24 hoursElectrophysioIogical Assays for assaying AF5O8CFTR modulation properties of compoundsUssing Chamber AssayUssing chamber experiments were performed on polarized epithelial cells expressing AF5O8-CFTR to further characterize the AF508-CFTR modulators identified in the optical assays. FRT‘4F5<,8‘cftr

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259 epithelial cells grown on Costar Snapwell cell culture inserts were mounted in an Ussing chamber (Physiologic Instruments, Inc., San Diego, CA), and the monolayers were continuously short-circuited using a Voltage-clamp System (Department of Bioengineering, University of Iowa, IA, and, Physiologic Instruments, Inc., San Diego, CA). Transepithelial resistance was measured by applying a 2-mV pulse. Under these conditions, the FRT epithelia demonstrated resistances of 4 Κ.Ω/ cm2 or more. The solutions were maintained at 27 °C and bubbled with air. The electrode offset potential and fluid resistance were corrected using a cell-free insert. Under these conditions, the current reflects the flow of CF through AF508-CFTR expressed in the apical membrane. The Isc was digitally acquired using an MP100A-CE interface and AcqKnowledge software (v3.2.6; BIOP AC Systems, Santa Barbara, CA).

[001034] Identification of Correction Compounds [001035] Typical protocol utilized a basolateral to apical membrane CF concentration gradient. To set up this gradient, normal ringer was used on the basolateral membrane, whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large CF concentration gradient across the epithelium. All experiments were performed with intact monolayers. To fully activate AF508-CFTR, forskolin (10 μΜ) and the PDE inhibitor, IBMX (100 μΜ), were applied followed by the addition of the CFTR potentiator, genistein (50 μΜ).

[001036] As observed in other cell types, incubation at low temperatures of FRT cells stably expressing AF508-CFTR increases the functional density of CFTR in the plasma membrane. To determine the activity of correction compounds, the cells were incubated with 10 μΜ of the test compound for 24 hours at 37°C and were subsequently washed 3X prior to recording. The cAMP- and genistein-mediated Isc in compound-treated cells was normalized to the 27°C and 37°C controls and expressed as percentage activity. Pieincubation of the cells with the correction compound significantly increased the cAMP- and genistein-mediated Isc compared to the 37°C controls.

[001037] Identification of Potentiator Com-pounds [001038] Typical protocol utilized a basolateral to apical membrane Cl concentration gradient. To set up this gradient, normal ringers was used on the basolateral membrane and was permeabilized with nystatin (360 pg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give a large Cl concentration gradient across the epithelium. All experiments were performed 30 min after nystatin permeabilization. Forskolin (10 μΜ) and all test compounds were added to both sides of the

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260 cell culture inserts, Lne efficacy ot tne putative Atcub-ut·TK potentiators was compared to that of the known potentiator, genistein.

[001039] Solutions [001040] Basolateral solution (in mM): NaCl (135), CaCh (1.2), MgCty (1.2), K2HPO4 (2.4), KHPO4 (0.6), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (10), and dextrose (10). The solution was titrated to pH 7.4 with NaOH.

[001041J Apical solution (in mM): Same as basolateral solution with NaCl replaced with Na Gluconate (135).

[001042] Cell Culture [001043] Fisher rat epithelial (FRT) cells expressing AF508-CFTR (pRT<iF5O8-CFirR) were used for Ussing chamber experiments for the putative AF508-CFTR modulators identified from our optical assays. The cells were cultured on Costar Snapwell cell culture inserts and cultured for five days at 37 °C and 5% CO2 in Coon’s modified Ham’s F-12 medium supplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100 pg/ml streptomycin. Prior to use for characterizing the potentiator activity of compounds, the cells were incubated at 27 °C for 16-48 hrs to correct for the AF508-CFTR. To determine the activity of corrections compounds, the cells were incubated at 27 °C or 37 °C with and without the compounds for 24 hours.

[001044] The macroscopic AF508-CFTR current (Iafsos) in temperature- and test compoundcorrected NIH3T3 cells stably expressing AF508-CFTR were monitored using the perforatedpatch, whole-cell recording. Briefly, voltage-clamp recordings of Iafsos were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City, CA). All recordings were acquired at a sampling frequency of 10 kHz and lowpass filtered at 1 kHz. Pipettes had a resistance of 5 - 6 ΜΩ when filled with the intracellular solution. Under these recording conditions, the calculated reversal potential for CT (Eci) at room temperature was -28 mV. All recordings had a seal resistance > 20 GQ and a series resistance <15 ΜΩ. Pulse generation, data acquisition, and analysis were performed using a PC equipped with a Digidata 1320 A/D interface in conjunction with Clampex 8 (Axon Instruments Inc.). The bath- contained < -250 μΐ of saline and was continuously perifosed at a rate of 2 ml/min using a gravity-driven perfusion system.

[001045] Identification of Correction Compounds [001046] To determine the activity of correction compounds for increasing the density of functional AF508-CFTR in the plasma membrane, we used the above-described perforatedCA 02869945 2014-11-04

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261 patch-recording techniques to measure the current density tollowing 24-hr treatment with the correction compounds. To fully activate AF508-CFTR, 10 μΜ forskolin and 20 μΜ genistein were added to the cells. Under our recording conditions, the current density following 24-hr incubation at 27°C was higher than that observed following 24-hr incubation at 37 °C. These results are consistent with the known effects of low-temperature incubation on the density of AF508-CFTR in the plasma membrane. To determine the effects of correction compounds on CFTR current density, the cells were incubated with 10 pM of the test compound for 24 hours at 37°C and the current density was compared to the 27°C and 37°C controls (% activity). Prior to recording, the cells were washed 3X with extracellular recording medium to remove any remaining test compound. Preincubation with 10 pM of correction compounds significantly increased the cAMP- and genistein-dependent current compared to the 37°C controls.

(001047] Identification of Potentiator Compounds (001048] The ability of AF508-CFTR potentiators to increase the macroscopic AF508-CFTR Cf current (Iafsos) in N1H3T3 cells stably expressing AF508-CFTR was also investigated using perforated-patch-recording techniques. The potentiators identified from the optical assays evoked a dose-dependent increase in Iafscs with similar potency and efficacy observed in the optical assays. In all cells examined, the reversal potential before and during potentiator application was around -30 mV, which is the calculated Eci (-28 mV).

[001049] Solutions [001050] Intracellular solution (in mM): Cs-aspartate (90), CsCl (50), MgCl2 (1), HEPES (10), and 240 pg/ml amphotericin-B (pH adjusted to 7.35 with CsOH).

[001051] Extracellular solution (in mM): ZV-methyl-D-glucamine (NMDG)-Cl (150), MgCl2 (2), CaCl2 (2), HEPES (10) (pH adjusted to 7.35 with HC1).

[001052] Cell Culture [001053] NIH3T3 mouse fibroblasts stably expressing AF508-CFTR are used for whole-cell recordings. The cells are maintained at 37 °C in 5% CO2 and 90 % humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, I X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm2 culture flasks. For whole-cell recordings, 2,500 - 5,000 cells were seeded on poly-L-lysine-coated glass coverslips and cultured for 24 - 48 hrs at 27 °C before use to test the activity of potentiators; and incubated with or without the correction compound at 37 °C for measuring the activity of correctors.

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262 [001054] The single-channel activities of temperature-corrected AF5U8-CFTK stably expressed in NÏH3T3 cells and activities of potentiator compounds were observed using excised inside-out membrane patch. Briefly, voltage-clamp recordings of single-channel activity were performed at room temperature with an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). All recordings were acquired at a sampling frequency of 10 kHz and low-pass filtered at 400 Hz. Patch pipettes were fabricated'from Coming Kovar Sealing #7052 glass (World Precision Instruments, Inc., Sarasota, FL) and had a resistance of 5 - 8 ΜΩ when filled with the extracellular solution. The AF508-CFTR was activated after excision, by adding 1 mM Mg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalytic subunit (PKA; Promega Corp. Madison, WI). After channel activity stabilized, the patch was perifused using a gravity-driven microperfusion system. The inflow was placed adjacent to the patch, resulting in complete solution exchange within 1-2 sec. To maintain AF508-CFTR activity during the rapid perifusion, the nonspecific· phosphatase inhibitor F' (10 mM NaF) was added to the bath solution. Under these recording conditions, channel activity remained constant throughout the duration of the patch recording (up to 60 min). Currents produced by positive charge moving from the intra- to extracellular solutions (anions moving in the opposite direction) are shown as positive currents. The pipette potential (Vp) was maintained at 80 mV.

[001055] Channel activity was analyzed from membrane patches containing < 2 active channels. The maximum number of simultaneous openings determined the number of active channels during the course of an experiment. To determine the single-channel current amplitude, the data recorded from 120 sec of AF508-CFTR activity was filtered “off-line” at 100 Hz and then used to construct all-point amplitude histograms that were fitted with multigaussian functions using Bio-Patch Analysis software (Bio-Logic Comp. France). The total microscopic current and open probability (Po) were determined from 120 sec of channel activity. The Po was determined using the Bio-Patch software or from the relationship Po = I7i(N), where I = mean current, i = single-channel current amplitude, and N = number of active channels in patch.

[001056] Solutions [001057] Extracellular solution (in mM): NMDG (150), aspartic acid (150), CaCL (5), MgCh (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).

[001058] Intracellular solution (in mM): NMDG-C1 (150), MgCh (2), EGTA (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with HC1).

[001059] Cell Culture

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263 [001060] NIH3T3 mouse fibroblasts stably expressing AF5O8-CFTR are used for excisedmembrane patch-clamp recordings. The cells are maintained at 37 °C in 5% CO2 and 90 % humidity in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine, 10 % fetal bovine serum, 1 X NEAA, β-ΜΕ, 1 X pen/strep, and 25 mM HEPES in 175 cm2 culture flasks. For single channel recordings, 2,500 - 5,000 cells were seeded on poly-L-lysinecoated glass coverslips and cultured for 24 - 48 hrs at 27 °C before use.

[001061] Compounds of the invention are useful as modulators of ATP binding cassette transporters. Using the procedures described above, the activities, i.e., EC50s, of compounds of the present invention have been measured to be from about 3.8 nM to about 13.5 μΜ. Furthermore, using those methods described above, the efficacies of compounds of the present invention have been measured to be from about 35 % to about 110%.

[001062] OTHER EMBODIMENTS [001063] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

DEMANDES OU BREVETS VOLUMINEUX

LA PRÉSENTE PARTIE DE CETTE DEMANDE OU CE BREVETS COMPREND PLUS D’UN TOME.

CECI EST LE TOME _1__DE _2__

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

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Contents59

104 members in 22 offices

This recordMember, by publication datePriority claim12 more offices are in the list below

Priority claims9

Priority claims
Document	Office	Kind	Date
60790459	United States of America	–	2006-04-07
79045906	United States of America	P	2006-04-07
79045906	United States of America	P	2006-04-07
2648719	Canada	A	2007-04-09
2648719	Canada	A	2007-04-09
2648719	–	–	–
60790459	–	–	–
CA20072648719	–	–	–
US20060790459P	–	–	–

Members104

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Document	Office	Kind
AU2007235260A1	Australia	A1	2007-10-18
CA2648719A1	Canada	A1	2007-10-18
CA2869945A1	Canada	A1	2007-10-18
US2007244159A1	United States of America	A1	2007-10-18
WO2007117715A2	World Intellectual Property Organization (WIPO)	A2	2007-10-18
WO2007117715A3	World Intellectual Property Organization (WIPO)	A3	2008-01-10
EP2007756A2	European Patent Office (EPO)	A2	2008-12-31
MX2008012945A	Mexico	A	2009-01-15
US2009131492A1	United States of America	A1	2009-05-21
CN101460489A	China	A	2009-06-17
IL194576D0	Israel	D0	2009-08-03
JP2009533351A	Japan	A	2009-09-17
ZA200809290B	South Africa	B	2009-10-28
US7645789B2	United States of America	B2	2010-01-12
US2010113555A1	United States of America	A1	2010-05-06
RU2008144124A	Russian Federation	A	2010-05-20
US7776905B2	United States of America	B2	2010-08-17
US2010331344A1	United States of America	A1	2010-12-30
US2011060024A1	United States of America	A1	2011-03-10
US2011071206A1	United States of America	A1	2011-03-24
NZ571803A	New Zealand	A	2011-12-22
BRPI0710965A2	Brazil	A2	2012-01-31
RU2451018C2	Russian Federation	C2	2012-05-20
US8415387B2	United States of America	B2	2013-04-09
AU2013205183A1	Australia	A1	2013-05-16
AU2007235260B2	Australia	B2	2013-05-30
NZ596889A	New Zealand	A	2013-06-28
US2013178471A1	United States of America	A1	2013-07-11
CN103254177A	China	A	2013-08-21
US8575209B2	United States of America	B2	2013-11-05
US8598181B2	United States of America	B2	2013-12-03
EP2674428A1	European Patent Office (EPO)	A1	2013-12-18
US8623905B2	United States of America	B2	2014-01-07
JP5420395B2	Japan	B2	2014-02-19
JP2014031384A	Japan	A	2014-02-20
US2014057906A1	United States of America	A1	2014-02-27
US2014080826A1	United States of America	A1	2014-03-20
NZ611485A	New Zealand	A	2014-09-26
US8912199B2	United States of America	B2	2014-12-16
US8952049B2	United States of America	B2	2015-02-10
US8952050B2	United States of America	B2	2015-02-10
CA2648719C	Canada	C	2015-02-24
US2015094304A1	United States of America	A1	2015-04-02
US2015119441A1	United States of America	A1	2015-04-30
IN2423KON2014A	India	A	2015-05-01
US2015174098A1	United States of America	A1	2015-06-25
JP2015120762A	Japan	A	2015-07-02
EP2007756B1	European Patent Office (EPO)	B1	2015-08-26
DK2007756T3	Denmark	T3	2015-10-05
PT2007756E	Portugal	E	2015-11-02
SI2007756T1	Slovenia	T1	2015-11-30
JP5827297B2	Japan	B2	2015-12-02
ES2554353T3	Spain	T3	2015-12-18
PL2007756T3	Poland	T3	2016-01-29
EP2674428B1	European Patent Office (EPO)	B1	2016-04-06
DK2674428T3	Denmark	T3	2016-04-18
HUE026145T2	Hungary	T2	2016-05-30
PT2674428T	Portugal	T	2016-07-14
AU2013205183B2	Australia	B2	2016-07-28
SI2674428T1	Slovenia	T1	2016-07-29
ES2580803T3	Spain	T3	2016-08-26
US2016271105A1	United States of America	A1	2016-09-22
HUE027630T2	Hungary	T2	2016-10-28
EP3091011A1	European Patent Office (EPO)	A1	2016-11-09
US2016332997A1	United States of America	A1	2016-11-17
IL194576A	Israel	A	2016-12-29
PL2674428T3	Poland	T3	2017-01-31
CY1116979T1	Cyprus	T1	2017-04-05
CY1117731T1	Cyprus	T1	2017-05-17
US2017189389A1	United States of America	A1	2017-07-06
US9758510B2	United States of America	B2	2017-09-12
EP3091011B1	European Patent Office (EPO)	B1	2017-12-27
CA2869945CThis record	Canada	C	2018-01-23
DK3091011T3	Denmark	T3	2018-02-26
PT3091011T	Portugal	T	2018-03-07
ES2659364T3	Spain	T3	2018-03-15
LT3091011T	Lithuania	T	2018-04-10
US9974781B2	United States of America	B2	2018-05-22
EP3327016A1	European Patent Office (EPO)	A1	2018-05-30
US2018162842A1	United States of America	A1	2018-06-14
HUE036165T2	Hungary	T2	2018-06-28
PL3091011T3	Poland	T3	2018-06-29
SI3091011T1	Slovenia	T1	2018-06-29
US10022352B2	United States of America	B2	2018-07-17
CY1120045T1	Cyprus	T1	2018-12-12
US2019076419A1	United States of America	A1	2019-03-14
US10239867B2	United States of America	B2	2019-03-26
US2020115366A1	United States of America	A1	2020-04-16
BRPI0710965B1	Brazil	B1	2020-08-18
US10975061B2	United States of America	B2	2021-04-13
US10987348B2	United States of America	B2	2021-04-27
BRPI0710965B8	Brazil	B8	2021-05-25
EP3327016B1	European Patent Office (EPO)	B1	2021-06-16
EP3882245A1	European Patent Office (EPO)	A1	2021-09-22
SI3327016T1	Slovenia	T1	2021-09-30
PL3327016T3	Poland	T3	2021-10-04
HUE055205T2	Hungary	T2	2021-11-29
ES2882684T3	Spain	T3	2021-12-02
US2022411410A1	United States of America	A1	2022-12-29
US2023100634A1	United States of America	A1	2023-03-30

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DEMANDES OU BREVETS VOLUMINEUX

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