MODULATORS OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

This disclosure provides modulators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), pharmaceutical compositions containing at least one such modulator, methods of treatment of cystic fibrosis using such modulators and pharmaceutical compositions, and processes for making such modulators.

The invention relates to modulators of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), pharmaceutical compositions containing the modulators, methods of treatment of cystic fibrosis and CFTR-mediated disorders using such modulators and pharmaceutical compositions, and processes for making such modulators.

Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 83,000 children and adults worldwide. Despite progress in the treatment of CF, there is no cure.

In patients with CF, mutations in CFTR endogenously expressed in respiratory epithelia lead to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to increased mucus accumulation in the lung and accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, result in death. In addition, the majority of males with cystic fibrosis are infertile, and fertility is reduced among females with cystic fibrosis.

Sequence analysis of the CFTR gene has revealed a variety of disease-causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B S. et al. (1989) Science 245:1073-1080; Kerem, B S. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 2000 mutations in the CF gene have been identified; currently, the CFTR2 database contains information on at least 322 of these identified mutations, with sufficient evidence to define at least 281 mutations as disease-causing. The most prevalent disease-causing mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence and is commonly referred to as the F508del mutation. This mutation occurs in many of the cases of cystic fibrosis and is associated with severe disease.

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelia cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of 1480 amino acids that encode a protein which is made up of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R)-domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.

Chloride transport takes place by the coordinated activity of ENaC (epithelial sodium channel) and CFTR present on the apical membrane and the Na+—K+-ATPase pump and Cl− channels expressed on the basolateral surface of the cell. Secondary active transport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell via Cl− channels, resulting in a vectoral transport. Arrangement of Na+/2Cl−/K+ co-transporter, Na+—K+-ATPase pump and the basolateral membrane K+ channels on the basolateral surface and CFTR on the luminal side coordinate the secretion of chloride. Because water is probably never actively transported itself, its flow across epithelia depends on tiny transepithelial osmotic gradients generated by the bulk flow of sodium and chloride.

A number of CFTR modulators have recently been identified. These modulators can be characterized as, for example, potentiators, correctors, potentiator enhancers/co-potentiators, amplifiers, readthrough agents, and nucleic acid therapies. CFTR modulators that increase the channel gating activity of mutant and wild-type CFTR at the epithelial cell surface are known as potentiators. Correctors improve faulty protein processing and resulting trafficking to the epithelial surface. Ghelani and Schneider-Futschik (2020) ACS Pharmacol. Transl. Sci. 3:4-10. There are three CFTR correctors approved by the U.S. FDA for treatment of cystic fibrosis. However, monotherapy with some CFTR correctors has not been found to be effective enough and as a result combination therapy with a potentiator is often needed to enhance CFTR activity. There is currently only one CFTR potentiator that is approved for the treatment of cystic fibrosis. Thus, although the treatment of cystic fibrosis has been transformed by these new small molecule CFTR modulators, new and better modulators are needed to prevent disease progression, reduce the severity of the cystic fibrosis and other CFTR-mediated diseases, and to treat the more severe forms of these diseases.

One aspect of the invention provides novel compounds, including compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof and pharmaceutically acceptable salts of any of the foregoing.

For example, compounds of Formula I can be depicted as:

and includes deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:

—Si(RZ3)2—, and

In some embodiments of Formula I, X is —C(RX1)2—. In some embodiments of Formula I, —C(RX1)2— is a group selected from

In some embodiments of Formula I, X is —CO—.

In some embodiments of Formula I, X is

In some embodiments of Formula I, X is a group selected from:

In some embodiments of Formula I, X is

In some embodiments of Formula I, Ring A is a cyclic group selected from phenyl, pyrazole, and oxadiazole.

In some embodiments of Formula I, X is selected from:

In some embodiments of Formula I, each RX1 is independently selected from H, C1-C6 alkyl, halogen, cyano, —ORX2, and C1-C6 fluoroalkyl. In some embodiments of Formula I, each RX2 is independently selected from H and C1-C4 alkyl. In some embodiments of Formula I, each RX1 is independently selected from H, F, —CF3, —CH3, —OH, —OCH3, and CN.

In some embodiments of Formula I, each Y is independently selected from —C(RY)2—, —CO—, —NRYN—, and

In some embodiments of Formula I, each RY is independently selected from hydrogen, hydroxy, halogen, and C1-C6 alkyl. In some embodiments of Formula I, each RY is independently selected from H, —OH, —F, and —CH3.

In some embodiments of Formula I, each RYN is independently selected from:

In some embodiments of Formula I, each RYN is independently selected from: H, —CH3,

In some embodiments of Formula I, Ring B is selected from:

In some embodiments of Formula I, Ring B is selected from:

In some embodiments of Formula I, each R2 is H.

In some embodiments of Formula I, each R1 is independently selected from C1-C6 fluoroalkyl, —N(R2)2, and —CN. In some embodiments of Formula I, each R1 is independently selected from —CF3, —NH2, and —CN.

In some embodiments of Formula I, Z is selected from

In some embodiments of Formula I, Z is selected from:

In some embodiments of Formula I, Z is

wherein Ring C is selected from C6-C10 aryl. In some embodiments of Formula I, the group:

is selected from:

In some embodiments of Formula I, the group:

In some embodiments of Formula I, Z is

In some embodiments of Formula I, Z is

In some embodiments of Formula I, Z is

In some embodiments of Formula I, Z is

wherein (R) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention. In some embodiments of Formula I, Z is

wherein (S) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention.

In some embodiments of Formula I, RZ1 is selected from C1-C6 fluoroalkyl.

In some embodiments of Formula I, RZ2 is selected from halogen and hydroxy.

In some embodiments of Formula I, n is selected from 5, 6, and 7.

In some embodiments of Formula I, m is selected from 1 and 2.

In some embodiments, compounds of Formula I can be depicted as Formula I′:

and includes deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:

—Si(RZ3)2—, and

In some embodiments of Formula I′, X is —C(RX1)2—. In some embodiments of Formula I′, —C(RX1)2— is a group selected from —CH2—,

In some embodiments of Formula I′, X is —CO—.

In some embodiments of Formula I′, X is

In some embodiments of Formula I′, X is a group selected from:

In some embodiments of Formula I′, X is

In some embodiments of Formula I′, Ring A is a cyclic group selected from phenyl, pyrazole, and oxadiazole.

In some embodiments of Formula I′, X is selected from: —CO—, —CH2—,

In some embodiments of Formula I′, each RX1 is independently selected from H, C1-C6 alkyl, halogen, cyano, —ORX2, and C1-C6 fluoroalkyl. In some embodiments of Formula I′, each RX2 is independently selected from H and C1-C4 alkyl. In some embodiments of Formula I′, each RX2 is independently selected from H, F, —CF3, —CH3, —OH, —OCH3, and CN.

In some embodiments of Formula I′, each Y is independently selected from —C(RY)2—, —CO—, —NRYN—, and

In some embodiments of Formula I′, each RY is independently selected from hydrogen, hydroxy, halogen, and C1-C6 alkyl. In some embodiments of Formula I′, each RY is independently selected from H, —OH, —F, and —CH3.

In some embodiments of Formula I′, each RYN is independently selected from:

In some embodiments of Formula I′, each RYN is independently selected from: H, —CH3,

In some embodiments of Formula I′, Ring B is selected from:

In some embodiments of Formula I′, Ring B is selected from:

In some embodiments of Formula I′, each R2 is H.

In some embodiments of Formula I′, each R1 is independently selected from C1-C6 fluoroalkyl, —N(R2)2, and —CN. In some embodiments of Formula I′, each R1 is independently selected from —CF3, —NH2, and —CN.

In some embodiments of Formula I′, Z is selected from

In some embodiments of Formula I′, Z is selected from:

In some embodiments of Formula I′, Z is

wherein Ring C is selected from C6-C10 aryl. In some embodiments of Formula I′, the group:

is selected from:

In some embodiments of Formula I′, the group:

In some embodiments of Formula I′, Z is

In some embodiments of Formula I′, Z is

In some embodiments of Formula I′, Z is

In some embodiments of Formula I′, Z is

wherein (R) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention. In some embodiments of Formula I′, Z is

wherein (S) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention.

In some embodiments of Formula I′, RZ1 is selected from C1-C6 fluoroalkyl.

In some embodiments of Formula I′, RZ2 is selected from halogen and hydroxy.

In some embodiments of Formula I′, n is selected from 5, 6, and 7.

In some embodiments of Formula I′, m is selected from 1 and 2.

The compounds of the invention also include compounds of Formulae Ia and Ib:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

In some embodiments of Formulae Ia and Ib, the portion of the compound represented by:

wherein (R) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention. In some embodiments of Formula I, the portion of the compound represented by

wherein (S) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention.

The compounds of the invention also include compounds of Formulae Ia′ and Ib′:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

In some embodiments of Formulae Ia′ and Ib′, the portion of the compound represented by:

wherein (R) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention. In some embodiments of Formula I, the portion of the compound represented by

wherein (S) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention.

The compounds of the invention also include compounds of Formulae IIa, IIb, IIc, IId, IIe, and IIf:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

In some embodiments of Formulae IIa, IIb, IIc, IId, IIe, and IIf, the portion of the compound represented by:

wherein (R) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention. In some embodiments of Formula I″, the portion of the compound represented by

wherein (S) refers to the stereochemical designation of the central carbon atom under the Cahn-Ingold-Prelog convention.

Another aspect of the invention provides pharmaceutical compositions comprising at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, and at least one pharmaceutically acceptable carrier, which compositions may further include at least one additional active pharmaceutical ingredient. Thus, another aspect of the invention provides methods of treating the CFTR-mediated disease cystic fibrosis comprising administering at least one of compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, and at least one pharmaceutically acceptable carrier, optionally as part of a pharmaceutical composition comprising at least one additional component, to a subject in need thereof.

In certain embodiments, the pharmaceutical compositions of the invention comprise at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing. In some embodiments, compositions comprising at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing may optionally further comprise at least one compound chosen from Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing.

Another aspect of the invention provides methods of treating the CFTR-mediated disease cystic fibrosis comprising administering to a patient in need thereof at least one compound chosen from the novel compounds disclosed herein, pharmaceutically acceptable salts thereof, and deuterated derivatives of any of the foregoing, and optionally further administering one or more additional CFTR modulating agents selected from (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide (Compound II), N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide (Compound III) or N-(2-(tert-butyl)-5-hydroxy-4-(2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Compound III-d), 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane carboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound IV), N-(1,3-dimethylpyrazol-4-yl)sulfonyl-6-[3-(3,3,3-trifluoro-2,2-dimethyl-propoxy)pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound V), N-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound VI), (14S)-8-[3-(2-{dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione (Compound VII), (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound VIII); N-(benzenesulfonyl)-6-(3-fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound IX), and N-[(6-amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound X).

Another aspect of the invention provides methods of treating the CFTR-mediated disease cystic fibrosis comprising administering to a patient in need thereof at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, and optionally further administering one or more additional CFTR modulating agents selected from:

disclosed in Journal of Cystic Fibrosis (2018), 17(5), 595-606, and:

disclosed in WO 2016/105485. In one embodiment, the additional CFTR modulating agent is ASP-11. In one embodiment, the additional CFTR modulating agent comprises PTI-428.

Another aspect of the invention provides methods of treating the CFTR-mediated disease cystic fibrosis comprising administering to a patient in need thereof at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, and optionally further administering one or more additional CFTR modulating agents selected from:

disclosed in United States Patent Application Publication No. 2016-0120841;

disclosed in WO 2018/065921;

disclosed in WO 2017/062581; ABBV-2851, disclosed in WO 2017/009804; GLPG2737, disclosed in United States Patent Application Publication No. 2017-0101405; ABBV-3748; ABBV-3903; and ABBV-119.

Definitions

“Compound II,” as used herein, refers to (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide, which can be depicted with the following structure:

Compound II may be in the form of a pharmaceutically acceptable salt. Compound II and methods of making and using Compound II are disclosed in WO 2010/053471, WO 2011/119984, WO 2011/133751, WO 2011/133951, and WO 2015/160787, each incorporated herein by reference.

“Compound III” as used throughout this disclosure refers to N-(5-hydroxy-2,4-di-tert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide (also known as N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide), which is depicted by the structure:

Compound III may also be in the form of a pharmaceutically acceptable salt. Compound III and methods of making and using Compound III are disclosed in WO 2006/002421, WO 2007/079139, WO 2010/108162, and WO 2010/019239, each incorporated herein by reference.

In some embodiments, a deuterated derivative of Compound III (Compound III-d) is employed in the compositions and methods disclosed herein. A chemical name for Compound III-d is N-(2-(tert-butyl)-5-hydroxy-4-(2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)phenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide, as depicted by the structure:

Compound III-d may be in the form of a pharmaceutically acceptable salt. Compound III-d and methods of making and using Compound III-d are disclosed in WO 2012/158885, WO 2014/078842, WO 2019/109021, and U.S. Pat. No. 8,865,902, incorporated herein by reference.

“Compound IV” as used herein, refers to 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid, which is depicted by the chemical structure:

Compound IV may be in the form of a pharmaceutically acceptable salt. Compound IV and methods of making and using Compound IV are disclosed in WO 2007/056341, WO 2009/073757, and WO 2009/076142, incorporated herein by reference.

“Compound V” as used herein, refers to N-(1,3-dimethylpyrazol-4-yl)sulfonyl-6-[3-(3,3,3-trifluoro-2,2-dimethyl-propoxy)pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide, which is depicted by the chemical structure:

Compound V may be in the form of a pharmaceutically acceptable salt. Compound V and methods of making and using Compound V are disclosed in WO 2018/107100 and WO 2019/113476, incorporated herein by reference.

“Compound VI” as used herein, refers to N-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide, which is depicted by the chemical structure:

Compound VI may be in the form of a pharmaceutically acceptable salt. Compound VI and methods of making and using Compound VI are disclosed in WO 2018/064632 and WO 2019/113476, incorporated herein by reference.

“Compound VII” as used herein, refers to (14S)-8-[3-(2-{dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione, which is depicted by the chemical structure:

Compound VII may be in the form of a pharmaceutically acceptable salt. Compound VII and methods of making and using Compound VII are disclosed in WO 2019/161078, WO 2020/102346, and PCT Application No. PCT/US2020/046116, incorporated herein by reference.

“Compound VIII” as used herein, refers to (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione, which is depicted by the chemical structure:

Compound VIII may be in the form of a pharmaceutically acceptable salt. Compound VIII and methods of making and using Compound VIII are disclosed in WO 2020/206080, incorporated herein by reference.

“Compound IX” as used herein, refers to N-(benzenesulfonyl)-6-(3-fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide, which is depicted by the chemical structure:

Compound IX may be in the form of a pharmaceutically acceptable salt. Compound IX and methods of making and using Compound IX are disclosed in WO 2016/057572, incorporated herein by reference.

“Compound X” as used herein, refers to N-[(6-amino-2-pyridyl)sulfonyl]-6-(3-fluoro-5-isobutoxy-phenyl)-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide, which is depicted by the chemical structure:

Compound X may be in the form of a pharmaceutically acceptable salt. Compound X and methods of making and using Compound X are disclosed in WO 2016/057572, incorporated herein by reference.

As used herein, the term “pi bond” refers to a covalent bond formed by the p orbitals of adjacent atoms. Pi bonds exist where there is a multiple bond, i.e., a double or triple bond, between two atoms. For example, a carbon-carbon double bond consists of one pi bond, and a carbon-carbon triple bond consists of two pi bonds.

As used herein, the term “haloalkyl group” refers to an alkyl group substituted with one or more halogen atoms.

As used herein, the term “fluoroalkyl” refers to an alkyl group substituted with one or more fluorine atoms. In some embodiments, a fluoroalkyl group is substituted by 1-6 fluorine atoms. In some embodiments, a fluoroalkyl group is perfluorinated.

The term “alkoxy” as used herein refers to an alkyl or cycloalkyl covalently bonded to an oxygen atom. Alkoxy groups may be substituted or unsubstituted.

As used herein, the term “haloalkoxy group” refers to an alkoxy group substituted with one or more halogen atoms.

As used herein, the term “fluoroalkoxy” refers to an alkoxy group substituted with one or more fluorine atoms. In some embodiments, a fluoroalkoxy group is substituted by 1-6 fluorine atoms. In some embodiments, a fluoroalkoxy group is perfluorinated.

The term “aryl,” as used herein, is a functional group or substituent derived from an aromatic ring and encompasses monocyclic aromatic rings and bicyclic, tricyclic, and fused ring systems wherein at least one ring in the system is aromatic. Non-limiting examples of aryl groups include phenyl, naphthyl, and 1,2,3,4-tetrahydronaphthalenyl.

The terms “heteroaryl ring” and “heteroaryl” as used herein refers to an aromatic ring comprising at least one ring atom that is a heteroatom, such as O, N, or S. Heteroaryl groups encompass monocyclic rings and bicyclic, tricyclic, bridged, fused, and spiro ring systems (including mono spiro and dispiro rings) wherein at least one ring in the system is aromatic. Non-limiting examples of heteroaryl rings include pyridine, quinoline, indole, and indoline.

As used herein, the terms “heterocyclyl ring” and “heterocyclyl” refer to a non-aromatic hydrocarbon containing 3 to 12 atoms in a ring (such as, for example 3-10 atoms) comprising at least one ring atom that is a heteroatom, such as O, N, S, or Si and may include one or more unsaturated bonds. “Heterocyclyl” rings encompass monocyclic, bicyclic, tricyclic, polycyclic, bridged, fused, and spiro rings, including mono spiro and dispiro rings.

“Substituted” indicates that at least one hydrogen of the “substituted” group is replaced by a substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent chosen from a specified group, the substituent may be either the same or different at each position.

As used herein, “deuterated derivative(s)” means the same chemical structure, with one or more hydrogen atoms replaced by a deuterium atom.

As used herein, “CFTR” means cystic fibrosis transmembrane conductance regulator.

As used herein, the terms “CFTR modulator,” “CFTR-modulating compound,” and “CFTR-modulating agent” interchangeably refer to a compound that directly or indirectly increases the activity of CFTR. The increase in activity resulting from a CFTR modulator includes but is not limited to compounds that correct, potentiate, stabilize and/or amplify CFTR.

As used herein, the term “CFTR corrector” refers to a compound that facilitates the processing and trafficking of CFTR to increase the amount of CFTR at the cell surface. Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, and Compound X disclosed herein are CFTR correctors.

As used herein, the term “CFTR potentiator” refers to a compound that increases the channel activity of CFTR protein located at the cell surface, resulting in enhanced ion transport. The novel compounds disclosed herein are CFTR potentiators. Compound III and Compound III-d disclosed herein are CFTR potentiators.

As used herein, the terms “CFTR potentiator enhancer”, CFTR potentiation enhancer”, and “CFTR co-potentiator” are used interchangeably and refer to a compound that enhances CFTR potentiation.

As used herein, the term “active pharmaceutical ingredient” (“API”) or “therapeutic agent” refers to a biologically active compound.

The terms “patient” and “subject” are used interchangeably and refer to an animal including humans.

The terms “effective dose” and “effective amount” are used interchangeably herein and refer to that amount of a compound that produces the desired effect for which it is administered (e.g., improvement in CF or a symptom of CF, or lessening the severity of CF or a symptom of CF). The exact amount of an effective dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the terms “treatment,” “treating,” and the like generally mean the improvement in one or more symptoms of CF or lessening the severity of CF or one or more symptoms of CF in a subject. “Treatment,” as used herein, includes, but is not limited to, the following: increased growth of the subject, increased weight gain, reduction of mucus in the lungs, improved pancreatic and/or liver function, reduction of chest infections, and/or reductions in coughing or shortness of breath. Improvements in or lessening the severity of any of these symptoms can be readily assessed according to standard methods and techniques known in the art.

As used herein, the term “in combination with,” when referring to two or more compounds, agents, or additional active pharmaceutical ingredients, means the administration of two or more compounds, agents, or active pharmaceutical ingredients to the patient prior to, concurrent with, or subsequent to each other.

The terms “about” and “approximately,” when used in connection with doses, amounts, or weight percentages of ingredients of a composition or a dosage form, include the value of a specified dose, amount, or weight percentage or a range of the dose, amount, or weight percentage that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent. The terms “about” and “approximately” may refer to an acceptable error for a particular value as determined by one of skill in the art, which depends in part on how the values is measured or determined. In some embodiments, the terms “about” and “approximately” mean within 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of a given value or range.

It will be appreciated that certain compounds of this invention may exist as separate stereoisomers or enantiomers and/or mixtures of those stereoisomers or enantiomers. As used in the chemical structures disclosed herein, a “wedge” () or “hash” () bond to a stereogenic atom indicates a chiral center of known absolute stereochemistry (i.e. one stereoisomer). As used in the chemical structures disclosed herein, a “wavy” bond () to a stereogenic atom indicates a chiral center of unknown absolute stereochemistry (i.e. one stereoisomer). As used in the chemical structures disclosed herein, a “wavy” bond () to a double-bonded carbon indicates a mixture of E/Z isomers. As used in the chemical structures disclosed herein, a  (“straight”) bond to a stereogenic atom indicates where there is a mixture (e.g., a racemate or enrichment). As used herein, two  (“straight”) bonds to a double-bonded carbon indicates that the double bond possesses the E/Z stereochemistry as drawn. As used in the chemical structures disclosed herein, a  (i.e., a “wavy” line perpendicular to a “straight” bond to group “A”) indicates that group “A” is a substituent whose point of attachment is at the end of the bond that terminates at the “wavy” line.

Certain compounds disclosed herein may exist as tautomers and both tautomeric forms are intended, even though only a single tautomeric structure is depicted. For example, a description of Compound A is understood to include its tautomer Compound B and vice versa, as well as mixtures thereof:

As used herein, “minimal function (MF) mutations” refer to CFTR gene mutations associated with minimal CFTR function (little-to-no functioning CFTR protein) and include, for example, mutations associated with severe defects in ability of the CFTR channel to open and close, known as defective channel gating or “gating mutations”; mutations associated with severe defects in the cellular processing of CFTR and its delivery to the cell surface; mutations associated with no (or minimal) CFTR synthesis; and mutations associated with severe defects in channel conductance.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt form of a compound of this disclosure wherein the salt is nontoxic. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. A “free base” form of a compound, for example, does not contain an ionically bonded salt.

The phrase “deuterated derivative or pharmaceutically acceptable salt thereof” is used interchangeably with “deuterated derivative thereof or pharmaceutically acceptable salt of any of the forgoing” in reference to one or more compounds or formulae of the invention. The phrase “pharmaceutically acceptable salt and deuterated derivative thereof” is used interchangeably with “pharmaceutically acceptable salt thereof and deuterated derivative of any of the forgoing” in reference to one or more compounds or formulae of the invention.

One of ordinary skill in the art would recognize that, when an amount of “a compound or a pharmaceutically acceptable salt thereof” is disclosed, the amount of the pharmaceutically acceptable salt form of the compound is the amount equivalent to the concentration of the free base of the compound. It is noted that the disclosed amounts of the compounds or their pharmaceutically acceptable salts thereof herein are based upon their free base form.

Suitable pharmaceutically acceptable salts are, for example, those disclosed in S. M. Berge, et al. J. Pharmaceutical Sciences, 1977, 66, 1-19. For example, Table 1 of that article provides the following pharmaceutically acceptable salts:

DETAILED DESCRIPTION OF EMBODIMENTS

In addition to compounds of Formula I, pharmaceutically acceptable salts thereof, and deuterated derivatives of those compounds and salts, the invention provides compounds of Formulae I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing.

For example, in some embodiments, the compound of Formula I is selected from compounds of any one of Formulae Ta and Tb:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

For example, in some embodiments, the compound of Formula I is selected from compounds of any one of Formulae Ia′ and Ib′:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

For example, in some embodiments, the compound of Formula I is selected from compounds of any one of Formulae IIa, IIb, IIc, IId, IIe, and IIf:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein all variables are as defined for Formula I.

Also disclosed herein are compounds having a formula chosen from any one of the formulae depicted in Table 7, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of those compounds and deuterated derivatives.

Methods of Treatment

Any of the novel compounds disclosed herein, such as for example, compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing can act as a CFTR modulator, i.e., it modulates CFTR activity in the body. Individuals suffering from a mutation in the gene encoding CFTR may benefit from receiving a CFTR modulator. A CFTR mutation may affect the CFTR quantity, i.e., the number of CFTR channels at the cell surface, or it may impact CFTR function, i.e., the functional ability of each channel to open and transport ions. Mutations affecting CFTR quantity include mutations that cause defective synthesis (Class I defect), mutations that cause defective processing and trafficking (Class II defect), mutations that cause reduced synthesis of CFTR (Class V defect), and mutations that reduce the surface stability of CFTR (Class VI defect). Mutations that affect CFTR function include mutations that cause defective gating (Class III defect) and mutations that cause defective conductance (Class IV defect). Some CFTR mutations exhibit characteristics of multiple classes. Certain mutations in the CFTR gene result in cystic fibrosis.

Thus, in some embodiments, the invention provides methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis in a patient comprising administering to the patient an effective amount of any of the novel compounds disclosed herein, such as for example, compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, alone or in combination with another active ingredient, such as another CFTR modulating agent. In some embodiments, the patient has an F508del/minimal function (MF) genotype, F508del/F508del genotype (homozygous for the F508del mutation), F508del/gating genotype, or F508del/residual function (RF) genotype. In some embodiments the patient is heterozygous and has one F508del mutation. In some embodiments, the patient is homozygous for the F508del mutation. In some embodiments the patient is homozygous for the N1303K mutation. In some embodiments, the patient has a G551D mutation.

In some embodiments, 1 mg to 1000 mg of a compound disclosed herein, a deuterated derivative thereof or a pharmaceutically acceptable salt of the compound or deuterated derivative are administered daily.

In some embodiments, the patient is heterozygous and has an F508del mutation on one allele and a mutation on the other allele selected from Table 2:

CFTR Mutations

Mutation

In some embodiments, the disclosure also is directed to methods of treatment using isotope-labelled compounds of the afore-mentioned compounds, or pharmaceutically acceptable salts thereof, wherein the formula and variables of such compounds and salts are each and independently as described above or any other embodiments described above, provided that one or more atoms therein have been replaced by an atom or atoms having an atomic mass or mass number which differs from the atomic mass or mass number of the atom which usually occurs naturally (isotope labelled). Examples of isotopes which are commercially available and suitable for the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively.

The isotope-labelled compounds and salts can be used in a number of beneficial ways. They can be suitable for medicaments and/or various types of assays, such as substrate tissue distribution assays. For example, tritium (3H)— and/or carbon-14 (14C)-labelled compounds are particularly useful for various types of assays, such as substrate tissue distribution assays, due to relatively simple preparation and excellent detectability. For example, deuterium (2H)-labelled ones are therapeutically useful with potential therapeutic advantages over the non-2H-labelled compounds. In general, deuterium (2H)-labelled compounds and salts can have higher metabolic stability as compared to those that are not isotope-labelled owing to the kinetic isotope effect described below. Higher metabolic stability translates directly into an increased in vivo half-life or lower dosages, which could be desired. The isotope-labelled compounds and salts can usually be prepared by carrying out the procedures disclosed in the synthesis schemes and the related description, in the example part and in the preparation part in the present text, replacing a non-isotope-labelled reactant by a readily available isotope-labelled reactant.

In some embodiments, the isotope-labelled compounds and salts are deuterium (2H)-labelled ones. In some specific embodiments, the isotope-labelled compounds and salts are deuterium (2H)-labelled, wherein one or more hydrogen atoms therein have been replaced by deuterium. In chemical structures, deuterium is represented as “2H” or “D.”

When discovering and developing therapeutic agents, the person skilled in the art attempts to optimize pharmacokinetic parameters while retaining desirable in vitro properties. It may be reasonable to assume that many compounds with poor pharmacokinetic profiles are susceptible to oxidative metabolism.

The deuterium (2H)-labelled compounds and salts can modulate the oxidative metabolism of the compound by way of the primary kinetic isotope effect. The primary kinetic isotope effect is a change of the rate for a chemical reaction that results from exchange of isotopic nuclei, which in turn is caused by the change in ground state energies necessary for covalent bond formation after this isotopic exchange. Exchange of a heavier isotope usually results in a lowering of the ground state energy for a chemical bond and thus causes a reduction in the rate-limiting bond breakage. If the bond breakage occurs in or in the vicinity of a saddle-point region along the coordinate of a multi-product reaction, the product distribution ratios can be altered substantially. For explanation: if deuterium is bonded to a carbon atom at a non-exchangeable position, rate differences of kM/kD=2-7 are typical. For a further discussion, see S. L. Harbeson and R. D. Tung, Deuterium In Drug Discovery and Development, Ann. Rep. Med. Chem. 2011, 46, 403-417, which is incorporated herein by reference.

The concentration of the isotope(s) (e.g., deuterium) incorporated into the isotope-labelled compounds and salt of the disclosure may be defined by the isotopic enrichment factor. The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. In some embodiments, if a substituent in a compound of the disclosure is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

Combination Therapies

One aspect disclosed herein provides methods of treating cystic fibrosis and other CFTR-mediated diseases using any of the novel compounds disclosed herein, such as for example, compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, in combination with at least one additional active pharmaceutical ingredient.

Thus, in some embodiments, the invention provides methods of treating, lessening the severity of, or symptomatically treating cystic fibrosis in a patient comprising administering to the patient an effective amount of any of the novel compounds disclosed herein, such as for example, compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of those compounds and deuterated derivatives, alone or in combination with at least one additional active pharmaceutical ingredient, such as, e.g., a CFTR modulating agent.

In some embodiments, at least one additional active pharmaceutical ingredient is selected from mucolytic agents, bronchodilators, antibiotics, anti-infective agents, and anti-inflammatory agents.

In some embodiments, the additional therapeutic agent is an antibiotic. Exemplary antibiotics useful herein include tobramycin, including tobramycin inhaled powder (TIP), azithromycin, aztreonam, including the aerosolized form of aztreonam, amikacin, including liposomal formulations thereof, ciprofloxacin, including formulations thereof suitable for administration by inhalation, levofloxacin, including aerosolized formulations thereof, and combinations of two antibiotics, e.g., fosfomycin and tobramycin.

In some embodiments, the additional agent is a mucolyte. Exemplary mucolytes useful herein includes Pulmozyme®.

In some embodiments, the additional agent is a bronchodilator. Exemplary bronchodilators include albuterol, metaproterenol sulfate, pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In some embodiments, the additional agent is an anti-inflammatory agent, i.e., an agent that can reduce the inflammation in the lungs. Exemplary such agents useful herein include ibuprofen, docosahexanoic acid (DHA), sildenafil, inhaled glutathione, pioglitazone, hydroxychloroquine, or simvastatin.

In some embodiments, the additional agent is a nutritional agent. Exemplary nutritional agents include pancrelipase (pancreating enzyme replacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®, Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation. In one embodiment, the additional nutritional agent is pancrelipase.

In some embodiments, at least one additional active pharmaceutical ingredient is selected from CFTR modulating agents. In some embodiments, the CFTR modulating agent is a CFTR corrector. In some embodiments, the CFTR modulating agent is a CFTR potentiator enhancer/co-potentiator (for example, ASP-11). In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR amplifier. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR readthrough agent. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR nucleic acid therapy.

In some embodiments, the at least one additional active pharmaceutical ingredient is a ENaC inhibitor. In some embodiments, the at least one additional active pharmaceutical ingredient is a TMEM16A modulator. In some embodiments, the at least one additional active pharmaceutical ingredient is a GPR39 agonist.

In some embodiments, the at least one additional active pharmaceutical ingredient is chosen from (a) Compound II and deuterated derivatives and pharmaceutically acceptable salts thereof; (b) Compound IV and deuterated derivatives and pharmaceutically acceptable salts thereof; (c) Compound V and deuterated derivatives and pharmaceutically acceptable salts thereof, (d) Compound VI and deuterated derivatives and pharmaceutically acceptable salts thereof; (e) Compound VII and deuterated derivatives and pharmaceutically acceptable salts thereof; and (f) Compound VIII and deuterated derivatives and pharmaceutically acceptable salts thereof. Thus, in some embodiments, the combination therapies provided herein comprise a compound selected from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives; and at least one compound chosen from Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, the combination therapies provided herein comprise (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives; (b) at least one compound chosen from Compound II, Compound IV, and pharmaceutically acceptable salts and deuterated derivatives thereof, and (c) at least one compound chosen from Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, the combination therapies provided herein comprise (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives; (b) at least one compound selected from Compound II and pharmaceutically acceptable salts and deuterated derivatives thereof; and (c) at least one compound chosen from Compound VII and deuterated derivatives and pharmaceutically acceptable salts thereof.

In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound II and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of those compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound IV and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, at least one compound chosen from compounds Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of those compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound V and deuterated derivatives and pharmaceutically acceptable slats thereof. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of those compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound VI and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound VII and deuterated derivatives and pharmaceutically acceptable salts thereof. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing compounds and deuterated derivatives, is administered in combination with at least one compound chosen from Compound VIII and deuterated derivatives and pharmaceutically acceptable salts thereof.

In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof; at least one compound chosen from Compound II, Compound IV, and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II, Compound IV, and pharmaceutically acceptable salts thereof; and at least one compound chosen from Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and pharmaceutically acceptable salts thereof are administered twice daily. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof; at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof are administered once daily. In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof are administered twice daily.

Compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and their deuterated derivatives and pharmaceutically acceptable salts thereof can be administered in a single pharmaceutical composition or separate pharmaceutical compositions. Such pharmaceutical compositions can be administered once daily or multiple times daily, such as twice daily. As used herein, the phrase that a given amount of API (e.g., Compound II, Compound VII, or pharmaceutically acceptable salts thereof) is administered once or twice daily or per day means that said given amount is administered per dosing, which may occur once or twice daily.

In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof is administered in a first pharmaceutical composition; and at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof is administered in a second pharmaceutical composition.

In some embodiments, at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof is administered in a first pharmaceutical composition; at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof is administered in a second pharmaceutical composition; and at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof is administered in a third pharmaceutical composition.

Any suitable pharmaceutical compositions known in the art can be used for compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and deuterated derivatives and pharmaceutically acceptable salts thereof. Some exemplary pharmaceutical compositions for Compound II and its pharmaceutically acceptable salts can be found in WO 2011/119984 and WO 2014/014841, incorporated herein by reference. Some exemplary pharmaceutical compositions for Compound III and its pharmaceutically acceptable salts can be found in WO 2007/134279, WO 2010/019239, WO 2011/019413, WO 2012/027731, and WO 2013/130669, and some exemplary pharmaceutical compositions for Compound III-d and its pharmaceutically acceptable salts can be found in U.S. Pat. Nos. 8,865,902, 9,181,192, 9,512,079, WO 2017/053455, and WO 2018/080591, all of which are incorporated herein by reference. Some exemplary pharmaceutical compositions for Compound IV and its pharmaceutically acceptable salts can be found in WO 2010/037066, WO 2011/127421, and WO 2014/071122, incorporated herein by reference. Some exemplary pharmaceutical compositions for Compound V and its pharmaceutically acceptable salts can be found in WO 2019/152940, incorporated herein by reference. Some exemplary pharmaceutical compositions for Compound VI and its pharmaceutically acceptable salts can be found in WO 2019/079760, incorporated herein by reference.

Pharmaceutical Compositions

Another aspect of the invention provides a pharmaceutical composition comprising at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, and at least one pharmaceutically acceptable carrier.

In some embodiments, the invention provides pharmaceutical compositions comprising at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof in combination with at least one additional active pharmaceutical ingredient. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR modulator. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR corrector. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR potentiator. In some embodiments, the at least one additional active pharmaceutical ingredient is a compound that enhances CFTR potentiation, i.e., a CFTR potentiator enhancer/co-potentiator. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR amplifier. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR readthrough agent. In some embodiments, the at least one additional active pharmaceutical ingredient is a CFTR nucleic acid therapy. In some embodiments, the at least one additional active pharmaceutical ingredient is a ENaC inhibitor. In some embodiments, the at least one additional active pharmaceutical ingredient is a TMEM16A modulator. In some embodiments, the at least one additional active pharmaceutical ingredient is a GPR39 agonist. In some embodiments, the pharmaceutical composition comprises at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof and at least two additional active pharmaceutical ingredients, each of which is a CFTR corrector. In some embodiments, the pharmaceutical composition comprises at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof and at least two additional active pharmaceutical ingredients, one of which is a CFTR corrector and one of which is a CFTR potentiator enhancer.

In some embodiments, the invention provides a pharmaceutical composition comprising (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, (b) at least one compound chosen from Compound II, Compound IV, Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and pharmaceutically acceptable salts thereof, and (c) at least one pharmaceutically acceptable carrier.

In some embodiments, the invention provides a pharmaceutical composition comprising (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, (b) at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, and (c) at least one pharmaceutically acceptable carrier.

In some embodiments, the invention provides a pharmaceutical composition comprising (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, (b) at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof, and (c) at least one pharmaceutically acceptable carrier.

In some embodiments, the disclosure provides a pharmaceutical composition comprising (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, (b) at least one compound chosen from Compound II, Compound IV, and pharmaceutically acceptable salts thereof, (c) at least one compound chosen from Compound V, Compound VI, Compound VII, Compound VIII, Compound IX, Compound X, and pharmaceutically acceptable salts thereof, and (d) at least one pharmaceutically acceptable carrier.

In some embodiments, the disclosure provides a pharmaceutical composition comprising (a) at least one compound chosen from compounds of Formulae I, I′, Ia, Ib, Ia′, Ib′, IIa, IIb, IIc, IId, IIe, and IIf, Compounds 1-271, and deuterated derivatives and pharmaceutically acceptable salts thereof, (b) at least one compound chosen from Compound II and pharmaceutically acceptable salts thereof, (c) at least one compound chosen from Compound VII and pharmaceutically acceptable salts thereof, and (d) at least one pharmaceutically acceptable carrier.

Any pharmaceutical composition disclosed herein may comprise at least one pharmaceutically acceptable carrier. In some embodiments, the at least one pharmaceutically acceptable carrier is chosen from pharmaceutically acceptable vehicles and pharmaceutically acceptable adjuvants. In some embodiments, the at least one pharmaceutically acceptable is chosen from pharmaceutically acceptable fillers, disintegrants, surfactants, binders, lubricants.

The pharmaceutical compositions described herein are useful for treating cystic fibrosis and other CFTR-mediated diseases.

As described above, pharmaceutical compositions disclosed herein may optionally further comprise at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be chosen from adjuvants and vehicles. The at least one pharmaceutically acceptable carrier, as used herein, includes any and all solvents, diluents, other liquid vehicles, dispersion aids, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier is incompatible with the compounds of this disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. Non-limiting examples of suitable pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates, glycine, sorbic acid, and potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts, and electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars (such as lactose, glucose and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (such as cocoa butter and suppository waxes), oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil), glycols (such as propylene glycol and polyethylene glycol), esters (such as ethyl oleate and ethyl laurate), agar, buffering agents (such as magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, non-toxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants.

1. A compound selected from compounds of Formula I:

—Si(RZ3)2—, and

7. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-6, wherein each Y is independently selected from —C(RY)2—, —CO—, —NRYN—, and

8. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-7, wherein each RY is independently selected from hydrogen, hydroxy, halogen, and C1-C6 alkyl.

9. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-8, wherein each RY is independently selected from H, —OH, —F, and —CH3.

10. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-9, wherein each RYN is independently selected from:

12. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-11, wherein Ring B is selected from:

14. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-13, wherein each R2 is H.

15. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-14, wherein each R1 is independently selected from C1-C6 fluoroalkyl, —N(R2)2, and —CN.

16. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-15, wherein each R1 is independently selected from —CF3, —NH2, and —CN.

17. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-16, wherein RZ1 is selected from C1-C6 fluoroalkyl.

18. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-17, wherein RZ2 is selected from halogen and hydroxy.

19. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-18, wherein Z is selected from

20. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-19, wherein Z is selected from:

21. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-20, wherein n is selected from 5, 6, and 7.

22. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiment 1-21, wherein

24. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-23, wherein each wherein each —NRYN— is independently selected from:

25. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-24, wherein Ring B is

26. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-25, wherein —(Y)n— is a group selected from:

27. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Embodiment 1, wherein the compound is selected from Formula IA:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:

and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:

36. A pharmaceutical composition comprising a compound, deuterated derivative, or pharmaceutically acceptable salt of any one of Embodiments 1-35 and a pharmaceutically acceptable carrier.

37. The pharmaceutical composition according to Embodiment 36, further comprising one or more additional therapeutic agent(s).

38. The pharmaceutical composition according to Embodiment 37, wherein the one or more additional therapeutic agent(s) comprise(s) a compound with CFTR modulating activity or a salt or deuterated derivative thereof.

39. The pharmaceutical composition according to Embodiment 37 or Embodiment 38, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR corrector.

40. The pharmaceutical composition according to any one of Embodiments 37-39, wherein the one or more additional therapeutic agent(s) comprise(s) (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide (Compound II):

41. The pharmaceutical composition according to any one of Embodiments 37-40, wherein the one or more additional therapeutic agent(s) comprise(s) 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound IV):

42. The pharmaceutical composition according to any one of Embodiments 37-41, wherein the one or more additional therapeutic agent(s) comprise(s) N-(1,3-dimethylpyrazol-4-yl)sulfonyl-6-[3-(3,3,3-trifluoro-2,2-dimethyl-propoxy)pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound V):

43. The pharmaceutical composition according to any one of Embodiments 37-42, wherein the one or more additional therapeutic agent(s) comprise(s) N-(benzenesulfonyl)-6-[3-[2-[1-(trifluoromethyl)cyclopropyl]ethoxy]pyrazol-1-yl]-2-[(4S)-2,2,4-trimethylpyrrolidin-1-yl]pyridine-3-carboxamide (Compound VI):

44. The pharmaceutical composition according to any one of Embodiments 37-43, wherein the one or more additional therapeutic agent(s) comprise(s) (14S)-8-[3-(2-{dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione (Compound VII):

45. The pharmaceutical composition according to any one of Embodiments 37-44, wherein the one or more additional therapeutic agent(s) comprise(s) (11R)-6-(2,6-dimethylphenyl)-11-(2-methylpropyl)-12-{spiro[2.3]hexan-5-yl}-9-oxa-2λ6-thia-3,5,12,19-tetraazatricyclo[12.3.1.14,8]nonadeca-1(17),4(19),5,7,14(18),15-hexaene-2,2,13-trione (Compound VIII):

46. The pharmaceutical composition according to any one of Embodiments 37-45, wherein the one or more additional therapeutic agent(s) comprise(s) at least one compound selected from PTI-428, ABBV-2222, ABBV-2851, GLPG2737, ABBV-3221, ABBV-3748, ABBV-3903, ABBV-119, FDL-169, ARN5562, ARN21586, ARN22081, ARN22652, ARN23765, ARN23766, and PTI-801.

47. The pharmaceutical composition according to any one of Embodiments 37-46, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR potentiator enhancer.

48. The pharmaceutical composition according to any one of Embodiments 37-47, wherein the one or more additional therapeutic agent(s) comprise(s) ASP-11.

49. The pharmaceutical composition according to any one of Embodiments 37-48, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR potentiator.

50. The pharmaceutical composition according to any one of Embodiments 37-49, wherein the one or more additional therapeutic agent(s) comprise(s) a compound selected from N-(5-hydroxy-2,4-di-tert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide (Compound III):

51. The pharmaceutical composition according to any one of Embodiments 37-50, wherein the one or more additional therapeutic agent(s) comprise(s) at least one compound selected from FDL-176, PTI-808, GLPG1837/ABBV-974, GLPG2451/ABBV-2451, QBW251 (Icenticaftor), GLPG3067/ABBV-3067 (Navocaftor), and ABBV-191.

52. The pharmaceutical composition according to any one of Embodiments 37-51, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR amplifier.

53. The pharmaceutical composition according to any one of Embodiments 37-52, wherein the one or more additional therapeutic agent(s) comprise(s) PTI-428.

54. The pharmaceutical composition according to any one of Embodiments 37-53, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR readthrough agent.

55. The pharmaceutical composition according to any one of Embodiments 37-54, wherein the one or more additional therapeutic agent(s) comprise(s) ELX-02.

56. The pharmaceutical composition according to any one of Embodiments 37-55, wherein the one or more additional therapeutic agent(s) comprise(s) a nucleic acid therapy.

57. The pharmaceutical composition according to any one of Embodiments 37-56, wherein the one or more additional therapeutic agent(s) comprise(s) at least one agent selected from MRT5005, Lunar-CF, and RCT223.

58. The pharmaceutical composition according to any one of Embodiments 37-57, wherein the one or more additional therapeutic agent(s) comprise(s) an ENaC inhibitor.

60. The pharmaceutical composition according to any one of Embodiments 37-59, wherein the one or more additional therapeutic agent(s) comprise(s) a TMEM16A modulator.

61. The pharmaceutical composition according to any one of Embodiments 37-60, wherein the one or more additional therapeutic agent(s) comprise(s) ETD002.

62. The pharmaceutical composition according to any one of Embodiments 37-61, wherein the one or more additional therapeutic agent(s) comprise(s) a GPR39 Agonist.

63. The pharmaceutical composition according to any one of Embodiments 37-62, wherein the one or more additional therapeutic agent(s) comprise(s) DS-1039.

64. A method of treating cystic fibrosis, comprising administering an effective amount of the compound, salt, or deuterated derivative according to any one of Embodiments 1-35 or the pharmaceutical composition according to any one of Embodiments 36-63 to a patient in need thereof.

65. The method according to Embodiment 64, further comprising administering one or more additional therapeutic agent(s).

66. The method according to Embodiment 65, wherein the one or more additional therapeutic agent(s) comprise(s) a compound with CFTR modulating activity or a salt or deuterated derivative thereof.

67. The method according to Embodiment 65 or Embodiment 66, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR corrector.

68. The method according to any one of Embodiments 65-67, wherein the one or more additional therapeutic agent(s) comprise(s) Compound II.

69. The method according to any one of Embodiments 65-68, wherein the one or more additional therapeutic agent(s) comprise(s) Compound IV.

70. The method according to any one of Embodiments 65-69, wherein the one or more additional therapeutic agent(s) comprise(s) Compound V.

71. The method according to any one of Embodiments 65-70, wherein the one or more additional therapeutic agent(s) comprise(s) Compound VI.

72. The method according to any one of Embodiments 65-71, wherein the one or more additional therapeutic agent(s) comprise(s) Compound VII.

73. The method according to any one of Embodiments 65-72, wherein the one or more additional therapeutic agent(s) comprise(s) Compound VIII.

74. The method according to any one of Embodiments 65-73, wherein the one or more additional therapeutic agent(s) comprise(s) at least one compound selected from PTI-428, ABBV-2222, ABBV-2851, GLPG2737, ABBV-3221, ABBV-3748, ABBV-3903, ABBV-119, ABBV-2851, FDL-169, ARN5562, ARN21586, ARN22081, ARN22652, ARN23765, ARN23766, and PTI-801.

75. The method according to any one of Embodiments 65-74, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR potentiator enhancer.

76. The method according to any one of Embodiments 65-75, wherein the one or more additional therapeutic agent(s) comprise(s) ASP-11.

77. The method according to any one of Embodiments 65-76, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR potentiator.

78. The method according to any one of Embodiments 65-77, wherein the one or more additional therapeutic agent(s) comprise(s) a compound selected from Compound III and Compound III-d.

79. The method according to any one of Embodiments 65-78, wherein the one or more additional therapeutic agent(s) comprise(s) at least one compound selected from FDL-176, PTI-808, GLPG1837/ABBV-974, GLPG2451/ABBV-2451, QBW251 (Icenticaftor), GLPG3067/ABBV-3067 (Navocaftor), and ABBV-191.

80. The method according to any one of Embodiments 65-79, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR amplifier.

81. The method according to any one of Embodiments 65-80, wherein the one or more additional therapeutic agent(s) comprise(s) PTI-428.

82. The method according to any one of Embodiments 65-81, wherein the one or more additional therapeutic agent(s) comprise(s) a CFTR readthrough agent.

83. The method according to any one of Embodiments 65-82, wherein the one or more additional therapeutic agent(s) comprise(s) ELX-02.

84. The method according to any one of Embodiments 65-83, wherein the one or more additional therapeutic agent(s) comprise(s) a nucleic acid therapy.

85. The method according to any one of Embodiments 65-84, wherein the one or more additional therapeutic agent(s) comprise(s) at least one agent selected from MRT5005, Lunar-CF, and RCT223.

86. The method according to any one of Embodiments 65-85, wherein the one or more additional therapeutic agent(s) comprise(s) an ENaC inhibitor.

88. The method according to any one of Embodiments 65-87, wherein the one or more additional therapeutic agent(s) comprise(s) a TMEM16A modulator.

89. The method according to any one of Embodiments 65-88, wherein the one or more additional therapeutic agent(s) comprise(s) ETD002.

90. The method according to any one of Embodiments 65-89, wherein the one or more additional therapeutic agent(s) comprise(s) a GPR39 Agonist.

91. The method according to any one of Embodiments 65-90, wherein the one or more additional therapeutic agent(s) comprise(s) DS-1039.

92. The compound, salt, or deuterated derivative of any one of Embodiments 1-35 or the pharmaceutical composition according to any one of Embodiments 36-63 for use in the treatment of cystic fibrosis.

93. Use of the compound, salt, or deuterated derivative of any one of Embodiments 1-35 or the pharmaceutical composition according to any one of Embodiments 36-63 in the manufacture of a medicament for the treatment of cystic fibrosis.

Examples

General Experimental Procedures

Abbreviations

General UPLC-MS/HPLC-MS/GC Analytical Methods

LC Method G: Analytical reverse phase HPLC-MS using a Merckmillipore Chromolith SpeedROD C18 column (50×4.6 mm) and a dual gradient run from 5% to 100% mobile phase B over 6 minutes. Mobile phase A=water (+0.1% trifluoroacetic acid). Mobile phase B=acetonitrile (+0.1% trifluoroacetic acid).

LC Method N: Analytical reverse phase UPLC-MS using an Acquity UPLC-MS BEH C18 column (30×2.1 mm, 1.7 m particle size) made by Waters (pn: 186002349), and a dual gradient run from 1% to 99% mobile phase B over 2.9 minutes. Mobile phase A=water (+5 mM ammonium hydroxide). Mobile phase B=acetonitrile. Flow rate=1.5 mL/min, injection volume=1.5 μL, and column temperature=60° C.

LC Method V: Analytical reverse phase HPLC-MS using a Kinetex EVO C18 column (2.1×50 mm 2.6 μm particle size), Temp: 45° C.; Flow: 1.0 mL/min; Run time: 1.5 min. Mobile phase: Initial 98% of mobile phase A (10 mM ammonium formate in water:acetonitrile, 95:5, pH 9) and 2% mobile phase B (acetonitrile) linear gradient to 98% acetonitrile for 1.15 min then hold at 98% acetonitrile for 0.2 min then return to 98% water and 10 mM ammonium formate for 0.05 min and hold for 0.1 min.

LC Method DD: Analytical reverse phase HPLC-MS using a Merckmillipore Chromolith SpeedROD C18 column (50×4.6 mm) and a dual gradient run from 5% to 100% mobile phase B over 12 minutes. Mobile phase A=water (+0.1% trifluoroacetic acid). Mobile phase B=acetonitrile (+0.1% trifluoroacetic acid).

PREPARATION OF COMMON INTERMEDIATES

Intermediate 1: Preparation of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-bromo-5-(trifluoromethyl)pyridine-2-carboxylate

A mixture of methyl 3-chloro-5-(trifluoromethyl)pyridine-2-carboxylate (47.3 g, 197.43 mmol), diphenylmethanimine (47 g, 259.33 mmol), Xantphos (9.07 g, 15.675 mmol), and cesium carbonate (131 g, 402.06 mmol) in dioxane (800 mL) was degassed by bubbling nitrogen for 30 minutes. Pd(OAc)2 (3.52 g, 15.679 mmol) was added and the system was purged with nitrogen three times. The reaction mixture was heated at 100° C. for 18 h. The reaction was cooled to room temperature and filtered on a pad of Celite. The cake was washed with ethyl acetate and solvents were evaporated under reduced pressure to give methyl 3-(benzhydrylideneamino)-5-(trifluoromethyl)pyridine-2-carboxylate (90 g, 84%) as yellow solid. ESI-MS m/z calc. 384.10855, found 385.1 (M+1)+; Retention time: 2.24 minutes (LC Method B).

To a suspension of methyl 3-(benzhydrylideneamino)-5-(trifluoromethyl)pyridine-2-carboxylate (65 g, 124.30 mmol) in methanol (200 mL) was added HCl (3 M in methanol) (146 mL of 3 M, 438 mmol). The mixture was stirred at room temperature for 1.5 hour then the solvent was removed under reduced pressure. The residue was taken up in ethyl acetate (2 L) and dichloromethane (500 mL). The organic phase was washed with 5% aqueous sodium bicarbonate solution (3×500 mL) and brine (2×500 mL), dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The residue was triturated with heptanes (2×50 mL) and the mother liquors were discarded. The solid obtained was triturated with a mixture of dichloromethane and heptanes (1:1, 40 mL) and filtered to afford methyl 3-amino-5-(trifluoromethyl)pyridine-2-carboxylate (25.25 g, 91%) as yellow solid. 1H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H), 7.28 (s, 1H), 5.98 (br. s, 2H), 4.00 (s, 3H). 19F NMR (282 MHz, CDCl3) δ−63.23 (s, 3F) ppm. ESI-MS m/z calc. 220.046, found 221.1 (M+1)+; Retention time: 1.62 minutes (LC Method E).

Intermediate 2: Preparation of 6-bromo-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic Acid

To a mixture of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-bromo-5-(trifluoromethyl)pyridine-2-carboxylate (247 g, 494.7 mmol) in THF (1.0 L) was added a solution of LiOH (47.2 g, 1.971 mol) in water (500 mL). The mixture was stirred at ambient temperature for 18 h affording a yellow slurry. The mixture was cooled with an ice-bath and slowly acidified with HCl (1000 mL of 2 M, 2 mol) keeping the reaction temperature <15° C. The mixture was diluted with heptane (1.5 L), mixed and the organic phase separated. The aqueous phase was extracted with heptane (500 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered and concentrated. The crude oil was dissolved in heptane (600 mL), seeded and stirred at ambient temperature for 18 h affording a thick slurry. The slurry was diluted with cold heptane (500 mL) and the precipitate collected using a medium frit. The filter cake was washed with cold heptane and air dried for 1 h, then in vacuo at 45° C. for 48 h to afford 6-bromo-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (158.3 g, 83%). 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.01 (s, 1H), 1.50 (s, 9H) ppm. ESI-MS m/z calc. 383.99326, found 330.9 (M+1)+; Retention time: 2.55 minutes (LC Method FF).

To a mixture of 6-bromo-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (239.2 g, 621.1 mmol) and 2-benzyloxy-2-(trifluoromethyl)hex-5-enehydrazide (hydrochloride salt) (230.1 g, 761.2 mmol) in ethyl acetate (2.2 L) at ambient temperature was added pyridine (200 mL, 2.473 mol) which afforded a precipitate. To the mixture was added 1-propanephosphonic anhydride (500 g of 50% w/w, 785.7 mmol) and the reaction mixture was stirred at ambient temperature for 12 h. The reaction was quenched with the slow addition of NaOH (149 g of 50% w/w, 1.863 mol) in water (2 L) and the mixture was stirred for 15 min. The organic phase was separated, and the aqueous phase extracted with ethyl acetate (1 L). The combined organic phases washed with brine, dried over MgSO4, filtered and concentrated in vacuo. After half of the solvent was removed, the organic phase was washed 2 times with aqueous HCl (1000 mL of 1 M, 1 mol). The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The crude product was slurried in warm heptane (2.5 L) and MTBE (0.25 L) and the mixture stirred at ambient temperature for 12 h affording a light yellow slurry. The slurry was filtered, and the resultant filter cake was washed 2 times with 1 L 10% MTBE/heptane. The off-white solid was air dried for 2 h, then in vacuo at 40° C. for 20 h giving tert-butyl N-[2-[[[2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamoyl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (379.9 g, 91%). 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.92 (s, 1H), 10.35 (s, 1H), 9.15 (s, 1H), 7.50 (d, J=7.4 Hz, 2H), 7.36 (m, 3H), 5.87 (m, 1H), 5.09 (d, J=16.9 Hz, 1H), 5.02 (d, J=10.1 Hz, 1H), 4.84 (q, J=11.4 Hz, 2H), 2.35-2.12 (m, 4H), 1.49 (s, 9H) ppm. ESI-MS m/z calc. 668.1069, found 670.9 (M+1)+; Retention time: 3.5 minutes (LC Method D).

Intermediate 5: Preparation of (2R)-2-benzyloxy-2-(trifluoromethyl)hex-5-enoic Acid

To a N2 purged jacketed reactor set to 20° C. was added isopropyl acetate (IPAC, 100 L, 0.173 M, 20 Vols), followed by previously melted 2-benzyloxy-2-(trifluoromethyl)hex-5-enoic acid (5.00 kg, 17.345 mol) and cinchonidine (2.553 kg, 8.67 mol) made into a slurry with minor amount of the reaction solvent. The reactor was set to ramp internal temperature to 80° C. over 1 hour, with solids going in solution upon heating to set temperature, then the solution was held at temperature for at least 10 minutes, then cooled to 70° C. held and seeded with chiral salt (50 g, 1.0% by wt). The mixture was stirred for 10 minutes, then ramped to 20° C. internal temperature over 4 hours, then held overnight at 20° C. The mixture was filtered, cake washed with isopropyl acetate (10.0 L, 2.0 vols) and dried under vacuum. The cake was then dried in vacuo (50° C., vacuum) to afford 4.7 kg of salt. The resulting solid salt was returned to the reactor by making a slurry with a portion of isopropyl acetate (94 L, 20 vol based on current salt wt), and pumped into reactor and stirred. The mixture was then heated to 80° C. internal, stirred hot slurry for at least 10 minutes, then ramped to 20° C. over 4-6 h, then stirred overnight at 20° C. The material was then filtered and cake washed with isopropyl acetate (9.4 L, 2.0 vol), pulled dry, cake scooped out and dried in vacuo (50° C., vacuum) to afford 3.1 kg of solid. The solid (3.1 kg) and isopropyl acetate (62 L, 20 vol based on salt solid wt) was slurried and added to a reactor, stirred under N2 purge and heated to 80° C. and held at temperature at least 10 minutes, then ramped to 20° C. over 4-6 hours, then stirred overnight. The mixture was filtered, cake washed with isopropyl acetate (6.2 L, 2 vol), pulled dry, scooped out and dried in vacuo (50° C., vac) to afford 2.25 kg of solid salt. The solid (2.25 kg) and isopropyl acetate (45 L, 20 vol based on salt solid wt) was slurried and added to a reactor, stirred under N2 purge and heated to 80° C., held at temperature at least 10 minutes, then ramped to 20° C. over 4-6 hours, then stirred overnight. The mixture was filtered, cake washed with isopropyl acetate (4.5 L, 2 vol), pulled dry, scooped out and dried in vacuo (50° C. to afford (2R)-2-benzyloxy-2-(trifluoromethyl)hex-5-enoic acid; (R)-4-quinolyl-[(2S,4S)-5-vinylquinuclidin-2-yl]methanol (1.886 kg, >98.0% ee) as off-white to tan solid. Chiral purity was determined by Agilent 1200 HPLC instrument using Phenomenex Lux i-Amylose-3 column (3 μm, 150×4.6 mm) and a dual, isocratic gradient run 30% to 70% mobile phase B over 20 minutes (Mobile phase A=H2O (0.1% CF3CO2H), mobile phase B=MeOH (0.1% CF3CO2H), flow rate=1.0 mL/min, injection volume=2 μL, and column temperature=30° C., sample concentration: 1 mg/mL in 60% acetonitrile/40% water).

Intermediate 6: Preparation of (2R)-2-benzyloxy-2-(trifluoromethyl)hex-5-enehydrazide

To a solution of (2R)-2-benzyloxy-2-(trifluoromethyl)hex-5-enoic acid (365 g, 1.266 mol) in DMF (2 L) was added HATU (612 g, 1.610 mol) and DIEA (450 mL, 2.584 mol) and the mixture was stirred at ambient temperature for 10 min. To the mixture was added tert-butyl N-aminocarbamate (200 g, 1.513 mol) (slight exotherm upon addition) and the mixture was stirred at ambient temperature for 16 h. The reaction was poured into ice water (5 L). The resultant precipitate was collected by filtration and washed with water. The solid was dissolved in ethyl acetate (2 L) and washed with brine. The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The oil was diluted with ethyl acetate (500 mL) followed by heptane (3 L) and stirred at ambient temperature for several hours affording a thick slurry. The slurry was diluted with additional heptane and filtered to collect fluffy white solid (343 g). The filtrate was concentrated and purification by silica gel chromatography (0-40% ethyl acetate/hexanes) provided tert-butyl N-[[(2R)-2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamate (464 g, 91%, combined with product from crystallization). ESI-MS m/z calc. 402.17664, found 303.0 (M+1-Boc)*; Retention time: 2.68 minutes (LC Method D).

Intermediate 8: Preparation of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-pent-4-enoyl-5-(trifluoromethyl)pyridine-2-carboxylate

Intermediate 9: Preparation of 3-(tert-butoxycarbonylamino)-6-pent-4-enoyl-5-(trifluoromethyl)pyridine-2-carboxylic Acid

Intermediate 10: Preparation of methyl 3-(tert-butoxycarbonylamino)-6-pent-4-enoyl-5-(trifluoromethyl)pyridine-2-carboxylate

Intermediate 11: Preparation of 6-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic Acid

Into a solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (5.18 g, 7.9522 mmol) in MeOH (200 mL) was added TEA (2.5410 g, 3.5 mL, 25.111 mmol). The reaction was purged with nitrogen for 5 min and then, Pd(dppf)Cl2 (604.1 mg, 0.8091 mmol) was added to the reaction mixture. The reaction was then stirred at 80° C. under 50 psi of carbon monoxide for 6 h. The reaction was cooled to rt, and then the MeOH was removed under vacuum. The residue was diluted with ethyl acetate (100 mL) and water (100 mL). Two layers were separated and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with saturated aqueous NaCl (100 mL), dried over anhydrous magnesium sulfate and concentrated under vacuum. Purification by silica gel chromatography (0 to 20% ethyl acetate/Hexanes) provided methyl 6-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (3.10 g, 62%) as a light yellow semi solid. ESI-MS m/z calc. 630.1913, found 631.5 (M+1)+; Retention time: 3.94 minutes (LC Method G).

Intermediate 12: Preparation of 6-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic Acid

Into a solution of ethyl 2-benzyloxy-2-(trifluoromethyl)pent-4-enoate (28.99 g, 95.902 mmol) in methanol (150 mL) was added a solution of NaOH (7.6714 g, 191.80 mmol) in water (50 mL). The reaction mixture was stirred at 40° C. for 3 hours. The reaction mixture was concentrated under vacuum, the residue was diluted with water (200 mL) and washed with diethyl ether (200 mL). The aqueous layer was acidified with concentrated HCl to pH 1 and extracted with diethyl ether (3×200 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to furnish 2-benzyloxy-2-(trifluoromethyl)pent-4-enoic acid (28.04 g, 99%) as a light yellow liquid. 1H NMR (250 MHz, CDCl3) δ 7.55-7.28 (m, 5H), 5.97-5.69 (m, 1H), 5.33-5.17 (m, 2H), 4.95-4.66 (m, 2H), 2.91 (d, J=7.1 Hz, 2H) ppm.

Intermediate 16: Preparation of 6-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic Acid

Intermediate 17: Preparation of 6-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic Acid

Intermediate 18: Preparation of 2-benzyloxy-6-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)hexanohydrazide

Intermediate 19: Preparation of methyl 6-[5-[1-benzyloxy-6-oxo-1-(trifluoromethyl)hexyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate

To a mixture of 6-bromo-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (11.2 g, 29.08 mmol) and 2-benzyloxy-6-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)hexanohydrazide (12.0 g, 31.88 mmol) in ethyl acetate (140 mL) at ambient temperature was added pyridine (9.5 mL, 117.5 mmol). To this solution was added T3P (24 mL of 50% w/v, 37.71 mmol) and the reaction mixture stirred at ambient temperature for 16 hours. The reaction was quenched with the slow addition of NaOH (50% in water) (7.3 mL of 50% w/v, 91.26 mmol) in water (120 mL) and the mixture stirred for 5 min in an ice bath and then at room temperature for 1 h. The organic phase was separated, and the aqueous phase extracted with ethyl acetate (100 mL). The combined organic phases washed with HCl (50 mL of 1 M), brine, dried (MgSO4), filtered and evaporated. Purification by silica gel chromatography using a gradient from 0% to 25% ethyl acetate in hexanes provided tert-butyl N-[2-[[[2-benzyloxy-6-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)hexanoyl]amino]carbamoyl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (16.4 g, 76%). ESI-MS m/z calc. 742.1437, found 645.2 (M+1)+; Retention time: 0.43 minutes (LC Method T).

To a solution of tert-butyl N-[2-[[[2-benzyloxy-6-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)hexanoyl]amino]carbamoyl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (2.298 g, 3.0908 mmol) and DIEA (1.2020 g, 1.62 mL, 9.3003 mmol) in acetonitrile (37 mL) was added tosyl chloride (707 mg, 3.7084 mmol) at 50° C. The reaction was stirred at 70° C. for 2 hours. The reaction was cooled to room temperature and then it was diluted with ethyl acetate (150 mL). The organic solution was washed with saturated ammonium chloride (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexane to furnish as a yellow foam, tert-butyl N-[2-[5-[1-benzyloxy-5-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (2.026 g, 87%). ESI-MS m/z calc. 724.1331, found 725.3 (M+1)+; Retention time: 4.33 minutes (LC Method G).

Into a solution of tert-butyl N-[2-[5-[1-benzyloxy-5-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (1.925 g, 2.5473 mmol) in methanol (50 mL) was added TEA (798.60 mg, 1.1 mL, 7.8921 mmol). The reaction was purged with argon for 2 minutes. Pd(dppf)Cl2 (186 mg, 0.2542 mmol) was added to the reaction mixture. The reaction was then stirred at 80° C. under 50 psi carbon monoxide for 6 hours. The reaction was cooled to rt, and then methanol was removed under vacuum. The residue was diluted with ethyl acetate (50 mL) and water (50 mL). Two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexane to furnish as a white foam, methyl 6-[5-[1-benzyloxy-5-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (1.678 g, 93%): ESI-MS m/z calc. 704.2281, found 705.5 (M+1)+; Retention time: 4.13 minutes (LC Method G).

Intermediate 20: Preparation of methyl 6-[5-[(1R)-1-benzyloxy-6-oxo-1-(trifluoromethyl)hexyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate

Intermediate 21: Preparation of methyl 6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate

To a solution of 2-benzyloxy-5-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)pentanoic acid (6.004 g, 16.375 mmol) in DMF (60 mL) was added HATU (8.735 g, 22.973 mmol) and triethylamine (3.6300 g, 5 mL, 35.873 mmol). The mixture was stirred for 10 minutes. Hydrazine hydrate (16.512 g, 16 mL, 329.84 mmol) was added to the reaction mixture. The reaction was stirred at room temperature for 1.5 hours. The reaction was diluted with water (200 mL) and ethyl acetate (200 mL). Two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (3×100 mL), dried over anhydrous magnesium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 10% methanol in DCM to furnish as an orange oil, 2-benzyloxy-5-(1,3-dioxolan-2-yl)-2-(trifluoromethyl)pentanehydrazide (6.075 g, 97%). ESI-MS m/z calc. 362.1453, found 363.3 (M+1)+; Retention time: 2.33 minutes (LC Method G).

A round bottom flask was charged with methyl 6-[5-[1-benzyloxy-4-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)butyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (2.308 g, 3.3421 mmol) in acetic acid (55 mL). Water (14 mL) was added to the reaction mixture. The reaction was stirred at 60° C. for 3 hours. The reaction was cooled to room temperature. Acetic acid was removed under vacuum. The residue was diluted with ethyl acetate (100 mL). The organic solution was washed with saturated sodium bicarbonate (2×20 mL) and brine (20 mL). The ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexanes to furnish as a white foam, methyl 6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (1.564 g, 72%). ESI-MS m/z calc. 646.1862, found 647.4 (M+1)+; Retention time: 3.94 minutes (LC Method G).

Intermediate 22: Preparation of methyl 5-(tert-butoxycarbonylamino)-6-[5-[2,2,2-trifluoro-1-[2-fluoro-5-(3-oxopropyl)phenyl]-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate

Triethylamine (16.6 mL) was placed in a round bottom flask and briefly chilled with ice water bath. Formic acid (11.2 mL) was added in small portions. The mixture was stirred at room temperature for 25 minutes and DMF (75 mL) was added. 3-Bromo-4-fluoro-benzaldehyde (20 g, 98.518 mmol) was added, followed by 2,2-dimethyl-1,3-dioxane-4,6-dione (14.2 g, 98.526 mmol). The mixture was heated at 100° C. for 16 h. It was then cooled to room temperature and poured into ice water (500 mL). The mixture was extracted with dichloromethane (2×300 mL). The combined organic layer was extracted with 10% aqueous NaOH (2×200 mL). The combined basic aqueous solution was acidified with concentrated HCl to pH=2. The resulted mixture was then extracted with ethyl acetate (3×150 mL). The combined organic layer washed with water (200 mL), brine (200 mL), dried over anhydrous. Sodium sulfate, filtered and concentrated to afford as white solid, 3-(3-bromo-4-fluoro-phenyl)propanoic acid (23.2 g, 86%). ESI-MS m/z calc. 245.9692, found 247.3 (M+1)+; Retention time: 3.09 minutes (LC Method G).

To a cooled (dry ice-acetone bath) solution of 3-(3-bromo-4-fluoro-phenyl)propoxy-tert-butyl-dimethyl-silane (9.4 g, 25.710 mmol) in THF (120 mL) was added n-BuLi in hexanes (11.5 mL of 2.5 M, 28.750 mmol) and stirred under N2 balloon for 25 min. Ethyl 2,2,2-trifluoroacetate (7.1460 g, 6 mL, 50.297 mmol) in THF (10 mL) was then added drop wise over 5 min. The mixture was stirred at this temperature for 15 min and with cooling bath removed. Water (˜40 mL) was added and extracted with ethyl acetate (60 mL). The organic layer was washed with brine, dried over anhydrous MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography, using 0% to 20% ethyl acetate in hexanes to afford as a white solid, 1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-ethanone (7.6 g, 73%). ESI-MS m/z calc. 364.1482, found 365.5 (M+1)+; Retention time: 4.96 minutes (LC Method G).

To a solution of 1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-ethanone (1.38 g, 3.4079 mmol) in DCM (8 mL) at room temperature was added (N-isocyanoimino)triphenylphosphorane (1.03 g, 3.4071 mmol) in DCM (8 mL) drop wise over 10 min. The brown solution was stirred at room temperature overnight and concentrated. The residue was purified by silica gel chromatography using 0% to 15% ethyl acetate in hexanes to afford as a white solid, tert-butyl N-[6-bromo-2-[5-[1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (870 mg, 47%). ESI-MS m/z calc. 772.1527, found 673.4 (M−100)+; Retention time: 5.22 minutes (LC Method G).

To a solution of tert-butyl N-[6-bromo-2-[5-[1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (3.2 g, 3.7228 mmol) in MeOH (50 mL) at room temperature was added TEA (1.0890 g, 1.5 mL, 10.762 mmol), followed by Pd(dppf)Cl2 (150 mg, 0.2050 mmol). The mixture was purged with N2 for 15 min. It was then sealed and subject to ˜100 psi CO at 80° C. temperature for 6 h. After cooling to rt, the mixture was transferred to a flask and concentrated. The residue was purified by silica gel chromatography, using 0% to 15% ethyl acetate in hexanes to afford as a white solid, methyl 5-(tert-butoxycarbonylamino)-6-[5-[1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate (1.8 g, 58%). ESI-MS m/z calc. 752.2476, found 753.6 (M+1)+; Retention time: 5.07 minutes (LC Method G).

To a solution of methyl 5-(tert-butoxycarbonylamino)-6-[5-[1-[5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenyl]-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate (1.8 g, 2.1521 mmol) in THE (10 mL) at room temperature was added tetrabutylammonium fluoride in THF (20 mL of 1 M, 20 mmol). The mixture was stirred at room temperature for 2 h. Additional tetrabutylammonium fluoride in THF (4 mL of 1 M, 4 mmol) was added and the mixture was stirred for another hour. It was then diluted with ethyl acetate (50 mL) and washed with water (3×50 mL). The organic layer was further washed with brine, dried over anhydrous MgSO4, filtered and concentrated to afford as a white solid, methyl 5-(tert-butoxycarbonylamino)-6-[5-[2,2,2-trifluoro-1-[2-fluoro-5-(3-hydroxypropyl)phenyl]-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate (1.4 g, 97%). ESI-MS m/z calc. 638.1611, found 639.6 (M+1)+; Retention time: 4.04 minutes (LC Method G).

Step 8: Preparation of methyl 5-(tert-butoxycarbonylamino)-6-[5-[2,2,2-trifluoro-1-[2-fluoro-5-(3-oxopropyl)phenyl]-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate

Intermediate 23: Preparation of methyl 6-[5-[1-benzyloxy-2,2,2-trifluoro-1-(3-oxopropoxymethyl)ethyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate

Oxalyl chloride (1.8188 g, 1.25 mL, 14.33 mmol) was added to a stirred solution of 2-(trifluoromethyl)prop-2-enoic acid (2.0 g, 14.279 mmol) and DMF (103.84 mg, 0.11 mL, 1.4206 mmol) in DCM (20 mL) at 0° C. The yellow solution was stirred at room temperature for 1 h. This provided a stock solution of 2-(trifluoromethyl)prop-2-enoyl chloride (20 mL, 0.7 M) for use in further steps.

Benzyl 2-(trifluoromethyl)prop-2-enoate (50.45 g, 214.79 mmol) was dissolved in a mixed solvents of dioxane (1000 mL) and water (200 mL). The mixture was cooled in ice water bath. With vigorous stirring, NaHCO3 (91.2 g, 1.0856 mol) was added, followed by portion-wise addition of potassium peroxymonosulfate (135.5 g) over 80 min. The mixture was stirred at the same temperature for 120 minutes at room temperature. Water (300 mL) and ethyl acetate (300 mL) were added, and layers were separated. The organic layer was washed with brine, dried over anhydrous. MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography using a gradient from 5% to 50% ethyl acetate in hexanes to afford as a colorless oil, benzyl 2-(trifluoromethyl)oxirane-2-carboxylate (57.49 g, 98%). 1H NMR (500 MHz, Chloroform-d) δ 7.62-7.28 (m, 5H), 5.33 (d, J=12.3 Hz, 1H), 5.28 (d, J=12.2 Hz, 1H), 3.30-3.18 (m, 2H) ppm.

To a solution of benzyl 2-(trifluoromethyl)oxirane-2-carboxylate (3.9 g, 15.842 mmol) in ethyl acetate (16 mL) was added 3-[tert-butyl(diphenyl)silyl]oxypropan-1-ol (11.6 g, 36.885 mmol) followed by magnesium triflate (5.8 g, 17.988 mmol). The reaction mixture was stirred at 90° C. for 16 hours. The reaction mixture was cooled to room temperature, filtered. The filtrate was concentrated under vacuum and purified by silica gel chromatography using a gradient from 0% to 15% ethyl acetate in hexanes to afford benzyl 2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-2-hydroxy-propanoate (5.633 g, 62%). ESI-MS m/z calc. 560.2206, found 561.3 (M+1)+; Retention time: 4.48 minutes (LC Method G).

Into a solution of benzyl 2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-2-hydroxy-propanoate (5.633 g, 9.5444 mmol) in anhydrous DMF (55 mL) was added NaH (572.1 mg, 60% w/w, 14.304 mmol) in mineral oil at 0° C. The reaction was stirred at the same temperature for 20 minutes, then bromoethylbenzene (2.2987 g, 1.55 mL, 13.44 mmol) and tetrabutylammonium iodide (352.5 mg, 0.9543 mmol) were added to the reaction mixture. The reaction was slowly raised to room temperature and stirred overnight. The reaction was quenched with saturated ammonium chloride (100 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with brine (2×100 mL), dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% diethyl ether in hexane to furnish as a clear liquid, benzyl 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoate (4.8 g, 77%). ESI-MS m/z calc. 650.2675, found 651.6 (M+1)+; Retention time: 4.75 minutes (LC Method G).

To a solution of benzyl 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoate (4.62 g, 6.744 mmol) in 1,4-dioxane (65 mL) was added NaOH (20.3 mL of 2 M, 40.6 mmol), and stirred at ambient temperature for overnight. The reaction mixture was acidified with aqueous HCl (1N) to ˜pH 3. The aqueous solution was extracted with ethyl acetate (3×150 mL). The combined ethyl acetate layers were washed with water (2×150 mL), brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 15% methanol in DCM to furnish as light yellow viscous oil, 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoic acid (3.63 g, 96%). ESI-MS m/z calc. 560.2206, found 561.2 (M+1)+; Retention time: 4.39 minutes (LC Method G).

Into a solution of 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoic acid (3.6 g, 5.1366 mmol) in DMF (50 mL) was added HATU (2.92 g, 7.6796 mmol) and DIEA (2.0034 g, 2.7 mL, 15.501 mmol) at ambient temperature. The reaction was stirred at room temperature for 10 minutes. hydrazine (3.2672 g, 3.2 mL, 101.96 mmol) was added to the reaction mixture. The reaction was stirred for 4 hours. Water (100 mL) and ethyl acetate (100 mL) were added to the reaction mixture. Two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×100 mL). The combined ethyl acetate layers were washed with brine (3×100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 50% to 80% diethyl ether in hexanes to furnish as a light pink oil, 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanehydrazide (2.71 g, 87%). ESI-MS m/z calc. 574.2475, found 575.4 (M+1)+; Retention time: 4.19 minutes (LC Method G).

Into a flask was charged with 2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanehydrazide (2.57 g, 4.4719 mmol) and 6-bromo-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (1.87 g, 4.8555 mmol) in ethyl acetate (50 mL). Then, pyridine (1.7604 g, 1.8 mL, 22.255 mmol) and T3P in ethyl acetate (3.5758 g, 6.69 mL of 50% w/w, 5.6191 mmol) were added to the reaction mixture. The reaction was stirred at 50° C. for 2 hours. The reaction was diluted with saturated aqueous ammonium chloride (200 mL) and ethyl acetate (150 mL). The aqueous layer was extracted with ethyl acetate (2×150 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% acetone in hexanes to furnish as a pale yellow foamy solid, tert-butyl N-[2-[[[2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoyl]amino]carbamoyl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (3.49 g, 83%). ESI-MS m/z calc. 940.2302, found 941.6 (M+1)+; Retention time: 4.94 minutes (LC Method G).

Into a solution of tert-butyl N-[2-[[[2-benzyloxy-2-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-3,3,3-trifluoro-propanoyl]amino]carbamoyl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (3.49 g, 3.3350 mmol) and DIEA (1.9663 g, 2.65 mL, 15.214 mmol) in acetonitrile (50 mL) was added TsCl (794.8 mg, 4.1690 mmol). The reaction was stirred at 70° C. for 2 hours. The solvent was removed under vacuum and the residue was directly loaded onto a column and purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexanes to furnish as a light yellow gel, tert-butyl N-[2-[5-[1-benzyloxy-1-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-2,2,2-trifluoro-ethyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (2.98 g, 95%). ESI-MS m/z calc. 922.2196, found 923.7 (M+1)+; Retention time: 5.08 minutes (LC Method G).

Into a solution of tert-butyl N-[2-[5-[1-benzyloxy-1-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-2,2,2-trifluoro-ethyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (2.93 g, 3.1716 mmol) in methanol (35 mL) was added TEA (3.2670 g, 4.5 mL, 32.286 mmol). The solution was purged with nitrogen for 5 minutes. Pd(dppf)Cl2 (465.2 mg, 0.6358 mmol) was added. The autoclave was sealed and heated at 80° C. for 5 hours under 75 psi of carbon monoxide. The volatile was removed under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexanes to furnish as a light yellow gel, methyl 6-[5-[1-benzyloxy-1-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-2,2,2-trifluoro-ethyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (2.59 g, 90%). ESI-MS m/z calc. 902.3146, found 903.8 (M+1)+; Retention time: 4.86 minutes (LC Method G).

Into a solution of methyl 6-[5-[1-benzyloxy-1-[3-[tert-butyl(diphenyl)silyl]oxypropoxymethyl]-2,2,2-trifluoro-ethyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (2.55 g, 2.8241 mmol) in anhydrous THE (70 mL) was added tetrabutylammonium fluoride in THF (7.1 mL of 1 M, 7.1 mmol) at room temperature. The reaction was stirred at room temperature for overnight. The reaction was diluted with water (150 mL) and ethyl acetate (150 mL). Two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×150 mL). The combined organic layers were washed with brine (250 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 50% ethyl acetate in hexanes to furnish as a white viscous solid, methyl 6-[5-[1-benzyloxy-2,2,2-trifluoro-1-(3-hydroxypropoxymethyl)ethyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (1.783 g, 95%). ESI-MS m/z calc. 664.1968, found 665.1 (M+1)+; Retention time: 3.91 minutes (LC Method G).

Intermediate 24: Preparation of methyl 6-chloro-3-nitro-5-(trifluoromethyl)pyridine-2-carboxylate

To an ice-cooled solution of methyl 6-hydroxy-5-(trifluoromethyl)pyridine-2-carboxylate (33.04 g, 149.41 mmol) in sulfuric acid (200 mL of 18.4 M, 3.68 mol) was added nitric acid (13 mL of 15.8 M, 205.4 mmol) dropwise. After 5 min, the ice bath was removed, and the reaction mixture was stirred at 38° C. overnight. The reaction was not completed, nitric acid (3 mL of 15.8 M, 47.4 mmol) was added dropwise at room temperature and the reaction was heated at 38° C. for 4.5 hours. The reaction was poured slowly on ice-cold water (900 mL) and the mixture was cooled at 0° C. for 15 minutes. Then the resultant solid was isolated by filtration and washed with water (600 mL). The solid was dried overnight under vacuum to give as a white solid, methyl 6-hydroxy-3-nitro-5-(trifluoromethyl)pyridine-2-carboxylate (39.49 g, 99%). 1H NMR (300 MHz, DMSO-d6) δ 8.54 (s, 1H), 3.95 (s, 3H) ppm. One exchangeable proton not observed in NMR. 19F NMR (282 MHz, DMSO-d6) δ−64.56 (s, 3F) ppm. ESI-MS m/z calc. 266.0151, found 267.1 (M+1)+; Retention time: 1.64 minutes (LC Method E).

Representative Preparation of Primary Amines

Dicyanozinc (65 mg, 0.55 mmol), Pd(dppf)Cl2 (20 mg, 0.027 mmol), and zinc dust (4 mg, 0.061 mmol) were weighed into a screwcap vial fitted with a stir bar. The vial was then capped and purged with nitrogen. DMF (2.5 mL) and 2-chloro-5-[1-(trifluoromethyl)cyclopropyl]pyridine (200 mg, 0.90 mmol) were added by syringe and the reaction was heated to 150° C. for 90 minutes. The reaction mixture was then cooled to room temperature and partitioned between 30 mL water and 30 mL ethyl acetate. The layers were separated, and the aqueous was extracted with ethyl acetate. The combined organics were washed with brine and dried over sodium sulfate, then concentrated. The resulting crude material was purified by silica gel chromatography using a gradient from 0% to 10% methanol in DCM to give 5-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carbonitrile (118 mg, 62%). ESI-MS m/z calc. 212.05614, found 213.1 (M+1)+; Retention time: 0.55 minutes (LC Method S).

5-[1-(Trifluoromethyl)cyclopropyl]pyridine-2-carbonitrile (118 mg, 0.6 mmol) was dissolved in THF (4 mL) in a nitrogen flushed round bottom flask and cooled to 0° C. in an ice bath. LAH (680 μL of 2 M, 1.36 mmol) in THF was added dropwise to the reaction mixture and stirring was continued for one hour at 0° C. after addition was complete. The reaction mixture was diluted with 5 mL diethyl ether then quenched by the sequential addition of 0.1 mL water, 0.1 mL 15% aqueous NaOH, and 0.3 mL water. The reaction mixture was then warmed to room temperature and stirred for 30 minutes. Sodium sulfate was added and the reaction mixture was filtered. The filtrate was concentrated to give as a light yellow solid, [5-[1-(trifluoromethyl)cyclopropyl]-2-pyridyl]methanamine. ESI-MS m/z calc. 216.08743, found 217.1 (M+1)+; Retention time: 0.33 minutes (LC Method T).

The residue was dissolved in methanol (3 mL) and di-tert-butyl dicarbonate (140 mg, 0.6415 mmol) and triethylamine (250 μL, 1.794 mmol) were added. The reaction was stirred at room temperature for 30 minutes then was concentrated by rotary evaporation. The residue was purified by silica gel chromatography using a gradient from 0% to 100% ethyl acetate in hexanes to give tert-butyl N-[[5-[1-(trifluoromethyl)cyclopropyl]-2-pyridyl]methyl]carbamate (65 mg, 23%). ESI-MS m/z calc. 316.13986, found 317.3 (M+1)+; Retention time: 0.53 minutes (LC Method T).

tert-Butyl N-[[5-[1-(trifluoromethyl)cyclopropyl]-2-pyridyl]methyl]carbamate (65 mg, 0.21 mmol) was combined with HCl (1 mL, 4 M, 4 mmol) as a solution in dioxane and DCM (1 mL) and stirred at room temperature for one hour. The reaction mixture was then evaporated to dryness. Dichloromethane and hexanes were added and the reaction was evaporated a second time to give [5-[1-(trifluoromethyl)cyclopropyl]-2-pyridyl]methanamine (dihydrochloride salt) (60 mg, quant.). ESI-MS m/z calc. 216.08743, found 217.1 (M+1)+; Retention time: 0.32 minutes (LC Method T).

3-Hydroxybenzonitrile (200 mg, 1.68 mmol) was combined with the [1-(trifluoromethyl)cyclopropyl]methanol (240 mg, 1.713 mmol) and triphenylphosphine (530 mg, 2.02 mmol) in 11 mL THF. The reaction mixture was cooled to 0° C. in an ice bath and diisopropyl azodicarboxylate (450 μL, 470 mg, 2.32 mmol) was added dropwise. After 15 minutes the reaction was warmed to room temperature and stirring was continued at room temperature for 16 hours. The reaction mixture was then warmed to 50° C. for an additional 2 hours. After cooling to room temperature, solvent was removed by rotary evaporation. The resulting residue was dissolved in 50 mL ethyl acetate and washed with 1 M aqueous NaOH. The organics were then washed with brine, dried over sodium sulfate, and concentrated. The crude material was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes to provide 3-[[1-(trifluoromethyl)cyclopropyl]methoxy]benzonitrile. ESI-MS m/z calc. 241.0714, found 242.0 (M+1)+; Retention time: 0.66 minutes (LC Method T).

3-(3-Chlorophenyl)cyclobutanone (500 mg, 2.768 mmol) was dissolved in DCM (25 mL) in a nitrogen-purged round bottom flask. The reaction flask was placed in a water bath and diethylaminosulfur trifluoride (1.5 mL, 11.35 mmol) was added dropwise. Stirring was continued at room temperature for 24 hours, then the reaction was placed in an ice water bath and quenched with 100 mL of saturated aqueous sodium bicarbonate (added dropwise at first). The reaction mixture was extracted 3×50 mL with ethyl acetate. The organics were combined, washed with brine, and dried over sodium sulfate then filtered and concentrated. The resulting crude material was purified by silica gel chromatography using 1% to 30% ethyl acetate in hexanes to provide as a colorless oil, 1-chloro-3-(3,3-difluorocyclobutyl)benzene (530 mg, 76%). 1H NMR (400 MHz, Chloroform-d) δ 7.28-7.24 (m, 1H), 7.24-7.19 (m, 2H), 7.11 (d, J=7.4 Hz, 1H), 3.37 (pd, J=9.2, 2.4 Hz, 1H), 3.01 (tdd, J=13.8, 8.7, 4.6 Hz, 2H), 2.66 (m, 2H) ppm.

Pd[P(tBu)3]2 (18 mg, 0.03522 mmol) was combined with zinc (8 mg, 0.1223 mmol) and dicyanozinc (42 mg, 0.3576 mmol) then purged with nitrogen. 1-Chloro-3-(3,3-difluorocyclobutyl)benzene (150 mg, 0.5922 mmol) was added as a solution in dimethylacetamide (2 mL) by syringe. The reaction was heated for 4 hours at 90° C. An additional portion of Pd[P(tBu)3]2 (18 mg, 0.03522 mmol) was added, and the reaction vial was re-purged with nitrogen, and the reaction temperature was increased to 115° C. for an additional 4 hours. The reaction was cooled and filtered through Celite and filtrate diluted with ethyl acetate. The filtrate was washed with 15 mL water, and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organics were washed with brine, dried over sodium sulfate, and concentrated. The crude material was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes to give as a colorless oil, 3-(3,3-difluorocyclobutyl)benzonitrile (50 mg, 44%). ESI-MS m/z calc. 193.07031, found 194.1 (M+1)+; Retention time: 0.59 minutes (LC Method T).

3-(3,3-Difluorocyclobutyl)benzonitrile (50 mg, 0.2588 mmol) was stirred in THF (3 mL) in a nitrogen-purged round bottom flask and cooled to 0° C. in an ice bath. LAH (200 μL of 2 M, 0.4 mmol) in THF was added dropwise via syringe. The ice bath was removed and stirring was continued at room temperature for the indicated time. The reaction mixture was then again cooled to 0° C. in an ice bath, and 0.2 mL of water was slowly added dropwise, followed by 0.2 mL of 15% aqueous NaOH, and finally 0.6 mL water. The reaction mixture was then warmed to room temperature and stirred for 3 hours. Stirring was stopped and sodium sulfate was added. The reaction mixture was filtered and concentrated, then the product was suspended in 10 mL dichloromethane and HCl (250 μL of 4 M, 1 mmol) in dioxane was added. The mixture was then concentrated by rotary evaporation, then dichloromethane and hexanes were added and the product was concentrated a second time to give as a white solid, [3-(3,3-difluorocyclobutyl)phenyl]methanamine (hydrochloride salt) (54 mg, 89%). ESI-MS m/z calc. 197.10161, found 198.1 (M+1)+; Retention time: 0.34 minutes (LC Method T).

General Methods for the Preparation of Secondary Amines

Step 1: A mixture of the primary amine (2 equivalents) and a base such as DIEA, TEA, Cs2CO3 (1 equivalent) in DMF (20 to 25 volume equivalents) was stirred at room temperature for 30 min and then 4-bromobut-1-ene or 4-iodobut-1-ene (1 eq) was added and the mixture stirred at room temperature for about 24 h. The mixture was then filtered, and the precipitate washed with ethyl acetate. The filtrate was diluted with ethyl acetate and washed with 1 M NaOH, partitioned and the organic layer extracted with ethyl acetate (3×). The combined organic extracts were washed with brine, dried (MgSO4), filtered and evaporated. Purification by silica gel chromatography or reverse-phase HPLC provided the desired secondary N-but-3-enylamine as the major product often along with a small amount of undesired tertiary amine as a side product.

Step 1: A mixture of the primary amine (1 eq) and 4-iodobut-1-ene (1 eq) in THF (20 to 25 volume equivalents) was stirred at 80° C. for about 1 to 2 h to provide the desired secondary amine along with undesired tertiary amine side product. The mixture was cooled to room temperature and TEA (3 equivalents) and di-tert-butyl dicarbonate (1.2 equivalents) were added. The mixture was stirred at room temperature for about 2 h, then diluted with ether, washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography or reverse phase HPLC to provide the desired N-boc-protected N-but-3-enylamine.

Step 2: A mixture of Boc-protected primary amine (1 eq) and HCl (4 M solution in dioxane, 20 equivalents) was stirred at room temperature for about 16 h, then the solvent evaporated to provide the desired secondary N-but-3-enylamine as hydrochloride salts.

Step 1: To a mixture of the aldehyde (1 eq), but-3-en-1-amine (1.2 equivalents) and acetic acid (0.1 equivalents) in 1,2-dichloroethane (˜30 volume equivalents) was added dry molecular sieves and the mixture was stirred at room temperature for about 1 h. A suitable reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride (1.5 equivalents) was added and the resulting mixture was stirred at room temperature overnight. After completion of the reaction, the reaction was quenched with saturated aqueous NaHCO3, filtered through Celite and extracted with dichloromethane. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. Purification by silica gel chromatography or reverse phase HPLC provided the desired secondary N-but-3-enylamine.

Step 1: A stirred suspension of a base such as potassium carbonate, cesium carbonate, cesium fluoride or cesium acetate (1.2 equivalents) and iodocopper (1 equivalent) in toluene (2 volume equivalents) in a Teflon capped vial was degassed by bubbling nitrogen through the mixture for 5 minutes and then sealed. Under nitrogen atmosphere, the halide (1 equivalent) and but-3-en-1-amine (2 equivalents) were added followed by degassed DMF (5 volume equivalents) and the resulting mixture was sealed and heated at 90° C. overnight. The mixture was cooled to room temperature and diluted with ammonium chloride (ammonium hydroxide added until a blue color persisted) then extracted with ethyl acetate. The organic fraction was washed once with brine, dried over MgSO4, filtered and concentrated to a brown syrup which was purified by silica gel chromatography or reverse phase HPLC providing the desired secondary N-but-3-enylamine.

Step 1: To a mixture of amine (hydrochloride salt) (1 equivalent) and NEt3 (3 equivalents) in THF (20 volume equivalents) was added di-tert-butyl dicarbonate (1.1 equivalents) at room temperature. The mixture was stirred at room temperature for about 1 to 2 h then diluted with ether, washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography to provide the mono-Boc protected amine.

Step 2: To a (cooled in ice bath) solution of mono-Boc protected amine (1 equivalent) in DMF (10 volume equivalents) was added a base such as [bis(trimethylsilyl)amino]sodium (2 equivalents). After stirring at 0° C. for 5 min, 3-bromo-1-propene (3 equivalents) was added dropwise. The temperature was raised to room temperature and stirred at this temperature for about 1 to 2 h. Then, the mixture was diluted with 1 M NH4Cl and ether and the aqueous layer was discarded. The organic layer was washed with water then brine, dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography or reverse phase HPLC to provide the Boc protected allyl amine.

Step 3: A mixture of the Boc protected allyl amine (1 equivalent) and HCl (10 eq, 4 M, in dioxane) was stirred at room temperature for about 2 h, then the solvent was evaporated and the residue was co-evaporated with THE to provide the hydrochloride salt of the substituted N-allyl-amine.

Step 1: To a solution of acid (1 equivalent) and but-3-en-1-amine (1.1 equivalents) in DMF (12 volume equivalents) was added DIEA (2.5 equivalents) and HATU (1.2 equivalents) were added. The resulting mixture was stirred at room temperature for 2 to 3 h. After this time, it was quenched with 1N HCl solution and extracted with ethyl acetate. The combined organic extracts were washed with H2O and saturated aqueous NaCl solution, then dried over Na2SO4, filtered, and evaporated in vacuo. Purification by silica gel chromatography using ethyl acetate and hexanes provided the desired amides.

Step 2: The amide (1 equivalent) was dissolved in diethyl ether (15 to 20 volume equivalents) and cooled to 0° C. A THE solution of LiAlH4 (1 to 2 equiv.) was added, and the resulting mixture was stirred at room temperature for about 2 h. The mixture was poured slowly into cold water and was extracted with ethyl acetate. The combined organic extracts were washed with H2O, 1N NaOH solution, H2O, and saturated aqueous NaCl solution, then dried over Na2SO4, filtered, and evaporated in vacuo. Purification by silica gel chromatography or reverse-phase preparative chromatography provided the secondary amines as free base or salt depending upon the purification methods.

Preparation of Final Compounds

Part 1: In a 250-mL sealed vial methyl 3-[bis(tert-butoxycarbonyl)amino]-6-bromo-5-(trifluoromethyl)pyridine-2-carboxylate (1.0 g, 2.003 mmol) and 2-(2-allylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (783 mg, 3.207 mmol) were combined in DMSO (10 mL). Added to the mixture were Pd(dppf)Cl2 (211 mg, 0.2884 mmol) and potassium carbonate (1.4 g, 10.13 mmol) and nitrogen was bubbled through the suspension for 1 minute. The reaction was capped and heated at 100° C. for 20 hours. The mixture was cooled to room temperature, diluted with brine and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered and concentrated. The resulting brown oil was purified by silica gel chromatography using 100% hexanes to 50% ethyl acetate in hexanes which gave as a brown oil, methyl 6-(2-allylphenyl)-3-[bis(tert-butoxycarbonyl)amino]-5-(trifluoromethyl)pyridine-2-carboxylate, ESI-MS m/z calc. 536.21344, found 537.2 (M+1)+; Retention time: 1.77 minutes (LC Method J).

Part 2: The residue was dissolved in THF (10 mL), MeOH (10 mL) and water (10 mL) followed by lithium hydroxide (340 mg, 8.102 mmol) was added. The mixture was stirred with heating at 60° C. for 3 hours. THE and methanol were removed under reduced pressure and 10 mL HCl (10%) was added (pH ˜3). The mixture was extracted with ethyl acetate (2×50 mL), combined organics dried over sodium sulfate and concentrated in vacuo to give as a tan solid, 6-(2-allylphenyl)-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (785 mg, 93%), ESI-MS m/z calc. 422.14536, found 423.2 (M+1)+; Retention time: 1.71 minutes (LC Method J).

To a solution of 6-(2-allylphenyl)-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (438 mg, 1.037 mmol) in NMP (6 mL) was added 2-benzyloxy-2-(trifluoromethyl)hex-5-enehydrazide (330 mg, 1.092 mmol), DIEA (600 μL, 3.445 mmol), followed by HATU (475 mg, 1.249 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction was diluted with ethyl acetate and washed with a saturated aqueous sodium bicarbonate solution. The organic layer was further washed with 10% citric acid solution followed by brine. The organics were separated, dried over sodium sulfate, and evaporated. The residue was purified by silica gel chromatography (80 gram column) using 100% hexanes to 60% ethyl acetate in hexanes (product elutes at 18% ethyl acetate) to afford as a white foam, tert-butyl N-[6-(2-allylphenyl)-2-[[[2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (609 mg, 83%), ESI-MS m/z calc. 706.259, found 707.2 (M+1)+; Retention time: 1.86 minutes (LC Method M).

A solution of tert-butyl N-[6-(2-allylphenyl)-2-[[[2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (605 mg, 0.8561 mmol) and DIEA (500 μL, 2.871 mmol) in acetonitrile (19 mL) was heated to 50° C., then p-toluenesulfonyl chloride (250 mg, 1.311 mmol) was added in 1 portion. The resulted mixture was heated at 70° C. for 2 hours. Reaction mixture was cooled and quenched with saturated solution of sodium bicarbonate (20 mL) and stirred for 15 minutes. The organic material was extracted with ethyl acetate (3×10 mL). The combined organics were dried over sodium sulfate and concentrated. The residue was purified by silica gel chromatography (80 gram column) using 100% hexanes to 50% ethyl acetate in hexanes to afford a tan residue which was placed under high vac pump for 18 hours which gave as a colorless oil, tert-butyl N-[6-(2-allylphenyl)-2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (574 mg, 97%). ESI-MS m/z calc. 688.2484, found 689.2 (M+1)+; Retention time: 2.14 minutes (LC Method M).

In a 500 mL round-bottom flask, a degassed solution of tert-butyl N-[6-(2-allylphenyl)-2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (574 mg, 0.8335 mmol) in 1,2-dichloroethane (145 mL) was heated to 50° C. under nitrogen atmosphere. Then, dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (98 mg, 0.1336 mmol) was added in two portions over 10 minutes. The resulting mixture was heated at 70° C. for 2 hours. Another portion of dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (98 mg, 0.1336 mmol) was added and the mixture was heated at 70° C. for 1 more hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel chromatography (80 gram column) using 100% hexanes to 40% ethyl acetate in hexanes (product elutes at 15% ethyl acetate) to afford a light pink residue. A second purification by silica gel chromatography (40 gram column) using 100% hexanes to 20% ethyl acetate in hexanes afforded a pale pink oil which was placed under high vac pump for 18 hours to produce a foam, tert-butyl N-[6-(benzyloxy)-6,19-bis(trifluoromethyl)-23-oxa-3,4,22-triazatetracyclo[16.3.1.12,5.012,17]tricosa-1(22),2,4,9,12,14,16,18,20-nonaen-21-yl]carbamate (E/Z mixture) (189 mg, 34%). ESI-MS m/z calc. 660.2171, found 661.2 (M+1)+; Retention time: 2.0 minutes (LC Method M).

tert-Butyl N-[6-hydroxy-6,19-bis(trifluoromethyl)-23-oxa-3,4,22-triazatetracyclo[16.3.1.12,5.012,17]tricosa-1(22),2,4,12,14,16,18,20-octaen-21-yl]carbamate (110 mg, 0.1921 mmol) was dissolved in dichloromethane (2 mL) and to the mixture was added TFA (1000 μL, 12.98 mmol) and stirred at room temperature for 2 hours. The mixture was evaporated, and the residue was purified by silica gel chromatography (12 gram column) using 100% hexanes to 60% ethyl acetate in hexanes (product elutes at 30% ethyl acetate). The material was further purified by reverse phase HPLC using a gradient from 1% to 99% acetonitrile in water (+5 mM HCl) over 15 minutes which gave as a white solid, 21-amino-6,19-bis(trifluoromethyl)-23-oxa-3,4,22-triazatetracyclo[16.3.1.12,5.012,17]tricosa-1(22),2,4,12,14,16,18,20-octaen-6-ol (19.7 mg, 22%). ESI-MS m/z calc. 472.1334, found 473.2 (M+1)+; Retention time: 1.48 minutes (LC Method J).

Carbonyl diimidazole (142 g, 875.7 mmol) was added to a solution of pent-4-enoic acid (71.5 mL, 700.6 mmol) in dichloromethane (2.45 L) under nitrogen, in an ice-water bath (gas evolution occurred over 34 minutes). After 1 h, added N-methoxymethanamine (hydrochloride salt) (168 g, 1.722 mol) over 3 min and then stirred for 16 h allowing the ice-water bath to eventually reach room temperature. Added a solution of citric acid (169 g, 879.6 mmol) in 1 L of ice water; temperature of the mixture dropped to 15° C. Separated the layers, washed the organic phase with HCl (700 mL of 0.25 M, 175 mmol) then a solution of potassium carbonate (24.3 g, 175.8 mmol) in 200 mL water and finally with brine. Dried the organic phase with magnesium sulfate, filtered and concentrated by rotary evaporation at 42° C. and 15 torr, then dissolved in MTBE and concentrated again giving 90.88 grams of a barely yellow liquid which was distilled (product distilled at bath temperature of 145° C., head temperature of 80° C.) providing N-methoxy-N-methyl-pent-4-enamide (81.0 g, 81%). 1H NMR (400 MHz, Chloroform-d) δ 5.94-5.79 (m, 1H), 5.13-5.03 (m, 1H), 5.03-4.95 (m, 1H), 3.69 (s, 3H), 3.19 (s, 3H), 2.60-2.47 (m, 2H), 2.44-2.34 (m, 2H) ppm.

Nitrogen was bubbled into a solution of tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-pent-4-enoyl-5-(trifluoromethyl)-3-pyridyl]carbamate (36.0 g, 53.76 mmol) in toluene (4930 mL) for 30 minutes in a 12 L flask. dichloro-[(2-isopropoxyphenyl)methylene]ruthenium; tricyclohexylphosphine (3.31 g, 5.511 mmol) was added, nitrogen was bubbled directly into the mixture while it was heated to a target temperature of 108° C., but the nitrogen bubbling was stopped once the temperature reached 50° C. After reaching 108° C. the mixture was stirred for 229 min then dichloro-[(2-isopropoxyphenyl)methylene]ruthenium; tricyclohexylphosphine (1.62 g, 2.697 mmol) was added and stirring at 108° C. was continued for 17 h 40 min. Cooled to 55° C., added 2-sulfanylpyridine-3-carboxylic acid (3.76 g, 24.23 mmol), followed by triethylamine (3.38 mL, 24.25 mmol), then heated at 55° C. and stirred for 3.5 h. Concentrated the mixture by rotary evaporation at 45° C. down to a volume of roughly 750 mL, then let sit overnight at room temperature. Filtered out the insoluble precipitate and rinsed the precipitate well with hexanes (desired product is soluble in pure hexanes). Removed all solvent by rotary evaporation at 45° C. Added 700 mL of hexanes and swirled at 45° C. to help dissolve the desired product. Cooled to room temperature, added 500 mL of saturated sodium bicarbonate/H2O 1:1 and stirred for 10 minutes then filtered and rinsed well with hexanes. Separated the layers of the filtrate, dried the organic phase with magnesium sulfate, filtered and concentrated at 48° C. giving 31.95 g of crude, darkly colored residue. The 31.95 g crude was dissolved in 100 mL MeOH and heated in a 40° C. bath (necessary to help dissolve) then 25 mL of DMSO was added to help keep the material dissolved at room temperature. The solution was loaded onto a 1.9 kg Cis reverse-phase column (column volume=1656 mL, flow rate=260 mL/min). A gradient of 65% to 93% acetonitrile/H2O was run over 96 min (15 column volumes) then 93% acetonitrile/H2O was maintained as an isocratic eluent for about 10 minutes then increased to 100% acetonitrile (after 140 min, the flow rate was increased to 450 mL/min to fully elute oligomeric side products) providing tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (17.35 g, 50%). ESI-MS m/z calc. 626.1964, found 627.1 (M+1)+; Retention time: 3.67 minutes (LC Method FF).

(This procedure was conducted in two batches, two separate pots prior to combining for purification.)

Reactor 1: tert-Butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (2.0 g, 3.160 mmol) was dissolved in acetic acid (40 mL) (in a 500 mL flask), flushed with nitrogen, treated with Pd/C (882 mg of 10% w/w as 50% water wet material, 0.414 mmol), then stirred under hydrogen at room temperature for 5 h 15 min. Evacuated the flask, backfilled with nitrogen, repeated many times then let the reaction mixture sit overnight with no stirring. Analysis showed that the reaction still progressed overnight. The mixture was put back under hydrogen atmosphere for 35 minutes then evacuated and backfilled with nitrogen multiple times. Filtered the mixture through Celite, washed the Celite with ethyl acetate and concentrated the filtrate by rotary evaporation at 45° C. to give crude material which was combined with the material obtained from reactor 2 prior to purification.

Reactor 2: tert-Butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (15.6 g, 24.65 mmol) was dissolved in acetic acid (300 mL) (in a 5 L flask), flushed with nitrogen, treated with Pd/C (6.88 g of 10% w/w as 50% water wet material, 3.24 mmol), then stirred under hydrogen at room temperature for 5.5 h. Evacuated the flask and backfilled with nitrogen. Filtered the mixture through Celite, washed the Celite with ethyl acetate and concentrated the filtrate by rotary evaporation at 45° C. to give 14.85 g of crude material which was added to the crude material from reactor 1 and purified together. The combined crude material was dissolved in 45 mL MeOH. The solution was loaded onto a 1.9 kg reverse phase C18 column (column volume=1656 mL, flow rate=260 mL/min). A gradient of 50% to 100% acetonitrile/H2O was run over 128 min (20 column volumes). The product started eluting at 81% acetonitrile, so the gradient was held at 81% acetonitrile until the product finished eluting which provided 11.72 g of the title compound with residual MTBE present. This material was dissolved in a small amount of dichloromethane, then heptane was added and concentrated again at 47° C. to remove the residual MTBE giving tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (11.17 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 9.08 (s, 1H), 7.73 (s, 1H), 3.00 (m, J=11.0, 4.7 Hz, 1H), 2.75 (m, J=11.1, 5.1 Hz, 1H), 2.27-2.13 (m, 2H), 2.12-1.97 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.43 (m, 15H) ppm. ESI-MS m/z calc. 538.1651, found 539.1 (M+1)+; Retention time: 3.28 minutes (LC Method FF).

TFA (32 mL, 415.4 mmol) was added over 3 minutes to a solution of tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (12.11 g, 22.49 mmol) in dichloromethane (75 mL) at 5° C. in a cold-water bath. The cold-water bath was removed, and the mixture was stirred at room temperature for 2 h 15 min (temperature reached 15° C. after 26 minutes). The reaction mixture was poured into a 0° C. solution of potassium bicarbonate (63.1 g, 630.3 mmol) in 500 mL water and 300 mL ethyl acetate. The layers were separated, and the organic layer was washed with a saturated sodium bicarbonate solution, dried with magnesium sulfate, filtered then concentrated. The residue was dissolved in boiling dichloromethane, then 100 mL heptane was added, resulting in a suspension. The dichloromethane was evaporated out of the suspension under vacuum then the product was collected by filtration and rinsed well with room temperature heptane. Dried the solid under vacuum with a nitrogen bleed at 40° C. over three days giving (6R)-17-amino-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-13-one (9.75 g, 96.8% ee). This material was further purified by SFC chromatography using a Phenomenex Lux-2 column (250×21.2 mm, 5 m particle size) with 16% methanol (20 mM NH3) and 84% carbon dioxide mobile phase at 70 mL/min (injection volume=650 L of 32 mg/mL solution in methanol) giving as the first enantiomer to elute, (6R)-17-amino-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-13-one (8.80 g, 90%, >98% ee). 1H NMR (400 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.77-7.37 (m, 3H), 2.99-2.84 (m, 1H), 2.77-2.62 (m, 1H), 2.28-2.10 (m, 2H), 2.10-1.94 (m, 1H), 1.85-1.70 (m, 1H), 1.70-1.40 (m, 6H) ppm. ESI-MS m/z calc. 438.11267, found 439.1 (M+1)+; Retention time: 1.76 minutes (LC Method A).

In a three necked 1 L flask, [1,3-bis-(2-tolyl)-2-imidazolidinylidene]dichloro(2-isopropoxybenzylidene)ruthenium(II) (85.6 mg, 0.1500 mmol) was dissolved in 1,2-dichloroethane (200 mL) and bubbled nitrogen gas into the solution and heated to 60° C. A solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-pent-4-enoyl-5-(trifluoromethyl)-3-pyridyl]carbamate (1.25 g, 1.91 mmol) in 1,2-dichloroethane (200 mL) was added dropwise during one hour to the catalyst solution. The mixture was heated at 75° C. for 6 hours. The reaction mixture was cooled to room temperature and solvent was evaporated. The residue was purified by silica gel chromatography using hexane and ethyl acetate (85:15) and further purified by reverse phase chromatography using a gradient from 50% acetonitrile in water to 100% acetonitrile which provided tert-butyl N-[6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (815 mg, 68%). ESI-MS m/z calc. 626.1964, found 627.0 (M+1)+; Retention time: 1.74 minutes (LC Method M).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (220 mg, 0.3511 mmol) in acetic acid (5 mL) in a Parr reactor was added Pd/C (34 mg, 10% w/w, 50% wet, 0.03195 mmol) under nitrogen gas. The Parr reactor was filled with nitrogen gas and evacuated 3 times. Finally, the reactor was evacuated and filled with hydrogen gas up to 100 psi. The reaction was stirred at room temperature for 15 hours. The reactor was evacuated and filled by nitrogen gas and the mixture was filtered through Celite pad. The solvent was evaporated, and the residue was dissolved in dichloromethane (2 mL) and pyridinium chlorochromate (75.7 mg, 0.3512 mmol) and 76 mg Celite was added and the mixture was stirred for 2 hours. The reaction mixture was filtered through a pad of Celite and washed with ethyl acetate (10 mL). The solvent was evaporated and the residue was purified by silica gel chromatography using 80:20 of hexanes:EtOAc to afford tert-butyl N-[6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (140 mg, 74%). ESI-MS m/z calc. 538.1651, found 539.0 (M+1)+; Retention time: 1.72 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 1) (40 mg, 0.06343 mmol) in acetic acid (4 mL) under nitrogen gas was added Pd/C (4.8 mg, 10% w/w, 0.004510 mmol). The flask was evacuated and filled with nitrogen gas three times and finally it was filled with hydrogen gas with two hydrogen balloons through a rubber septum. The mixture was stirred for 12 hours at room temperature. The mixture was filtered through a pad of Celite and washed with ethyl acetate. The solvent was evaporated and the residue was purified by reverse phase chromatography using a mobile gradient from 50% acetonitrile in water to 100% acetonitrile providing tert-butyl N-[6,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 1) (21 mg, 61%). ESI-MS m/z calc. 540.1807, found 541.0 (M+1)+; Retention time: 1.48 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (40 mg, 0.06343 mmol) in acetic acid (4 mL) under nitrogen gas was added Pd/C (4.8 mg, 10% w/w, 0.004510 mmol). The flask was evacuated and filled with nitrogen gas three times and finally it was capped with two balloons of hydrogen through a rubber septum creating a hydrogen gas atmosphere. The mixture was stirred for 12 h at room temperature. The mixture was purged with nitrogen then filtered through a pad of Celite eluting with ethyl acetate. The filtrate was evaporated and the resulting residue was purified by reverse phase chromatography eluting with a gradient of 50% acetonitrile in water to 100% acetonitrile giving tert-butyl N-[6,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (23 mg, 67%). ESI-MS m/z calc. 540.1807, found 541.0 (M+1)+; Retention time: 1.71 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 1) (27 mg, 0.04189 mmol) in acetic acid (4 mL) in a Parr reactor was added Pd/C (11.8 mg, 10% w/w, 50% wet, 0.005544 mmol) under nitrogen gas. The Parr reactor was set up and filled with nitrogen gas and evacuated 3 times. Finally, the reactor was evacuated and filled with hydrogen gas up to 100 psi. The reaction was stirred at room temperature for 15 h. The reactor was evacuated and filled by nitrogen gas and the mixture was filtered through Celite pad and washed with ethyl acetate (10 mL). The filtrate was evaporated and the residue was purified by reverse phase chromatography eluting with a gradient of 50% acetonitrile in water to 100% acetonitrile giving tert-butyl N-[6-hydroxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 1) (17 mg, 73%). ESI-MS m/z calc. 554.1964, found 555.0 (M+1)+; Retention time: 1.93 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (60 mg, 0.09515 mmol) in THF (4 mL) at 0° C. (ice bath) was added potassium tert-butoxide (51.3 mg of 25% w/w, 0.1143 mmol) (2 M solution in 2-methyltetrahydrofuran) under an inert atmosphere. The mixture was stirred for 5 min and then iodomethane (16.2 mg, 0.1141 mmol) in THF (1 mL) was added slowly by a syringe via a rubber septum. The mixture was stirred for additional 30 minutes at room temperature. Next, the reaction mixture was cooled down and quenched at 0° C. by adding 10 mL water and the mixture was then extracted with diethyl ether (3×20 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated then purified by silica gel chromatography eluting with 85:15 hexane/ethyl acetate which gave tert-butyl N-[6-benzyloxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (34 mg, 55%). ESI-MS m/z calc. 644.24335, found 645.0 (M+1)+; Retention time: 2.03 minutes (LC Method M).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (28 mg, 0.04344 mmol) in acetic acid (2 mL) in a high pressure Parr reactor was added Pd/C (8.5 mg, 10% w/w, 50% wet, 0.003947 mmol) under nitrogen atmosphere. The reactor evacuated and filled with nitrogen gas three times and finally filled with hydrogen gas to a pressure of 100 psi and stirred at room temperature for 16 h. The Parr reactor was then depressurized and filled with nitrogen gas. The mixture was filtered through a pad of Celite eluting with ethyl acetate. The filtrate was evaporated and the resulting residue was purified by reverse phase chromatography eluting with a gradient of 50% acetonitrile in water to 100% acetonitrile giving tert-butyl N-[6-hydroxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (20 mg, 83%). ESI-MS m/z calc. 554.1964, found 555.0 (M+1)+; Retention time: 2.04 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-hydroxy-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (diastereomer pair 2) (14 mg, 0.02525 mmol) in dichloromethane (2 mL) was added TFA (1 mL, 12.98 mmol) and the mixture was stirred for 25 minutes at room temperature. After that time, the mixture was cooled in ice bath and 2 mL of a saturated aqueous solution of sodium bicarbonate was slowly added. The product was extracted with dichloromethane (2×5 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated then purified by reverse phase chromatography eluting with a gradient of 50% acetonitrile in water to 100% acetonitrile over 7 minutes then eluting at 100% acetonitrile for 5 minutes giving a mixture of diastereomers. This material was further purified by SFC chromatography using a ChiralPak IG column (250×10 mm, 5 m particle size) with 22% methanol (20 mM NH3) and 78% carbon dioxide mobile phase at 10 mL/min (injection volume=70 μL of ˜20 mg/mL solution in methanol) which gave as a white solid and the first enantiomer to elute 17-amino-13-methoxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-6-ol (diastereomer pair 2, enantiomer 3) (2 mg, 34%). 1H NMR (500 MHz, DMSO-d6) δ 7.74 (s, 1H), 7.65 (d, J=12.7 Hz, 1H), 6.91 (s, 2H), 4.54 (d, J=7.7 Hz, 1H), 3.30 (s, 3H), 2.30-2.02 (m, 3H), 1.85-1.70 (m, 2H), 1.66-1.32 (m, 7H) ppm. ESI-MS m/z calc. 454.14395, found 455.0 (M+1)+; Retention time: 1.39 minutes (LC Method J).

In a three necked 500 mL flask, [1,3-bis-(2-tolyl)-2-imidazolidinylidene]dichloro(2-isopropoxybenzylidene)ruthenium(II) (19.2 mg, 0.03365 mmol) was dissolved in 1,2-dichloroethane (70 mL) and bubbled nitrogen gas in the solution through a long needle and the flask was heated to 60° C. A solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-(1-methylpent-4-enyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (220 mg, 0.3361 mmol) in 1,2-dichloroethane (70 mL) was added slowly dropwise during 45 min using a dropping funnel at 60° C. The reaction mixture was stirred at 75° C. for 7 hours. The reaction mixture was cooled to room temperature and solvent was evaporated. Purification by silica gel chromatography using 95:5 of hexanes and ethyl acetate provided tert-butyl N-[6-benzyloxy-13-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z mixture) (60 mg, 28%). ESI-MS m/z calc. 626.2328, found 627.0 (M+1)+; Retention time: 1.21 minutes and 1.25 minutes (LC Method M).

To a stirred solution of tert-butyl N-[6-benzyloxy-13-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (58 mg, 0.09256 mmol) in acetic acid (3 mL) was added Pd/C (50 mg, 0.02349 mmol, 50% wet, 10% w/w) in a high pressure reactor and filled with nitrogen gas and evacuated three times. The reactor was filled with hydrogen gas up to 250 psi and stirred for 20 h. The mixture was filtered through a pad of Celite by washing with ethyl acetate and solvent evaporated. Purification by silica gel chromatography using 85:15 of hexanes and ethyl acetate provided tert-butyl N-[6-hydroxy-13-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (37 mg, 74%). ESI-MS m/z calc. 538.2015, found 539.1 (M+1)+; Retention time: 1.82 minutes and 1.86 minutes (LC Method M).

To a 25 mL three neck flask with benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (5.8 mg, 0.0068 mmol) and toluene (2.35 mL) heated with a 120° C. heat bowl was added a solution of tert-butyl N-[6-[[acetyl(but-3-enyl)amino]methyl]-2-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (31 mg, 0.045 mmol) in toluene (2.35 mL) and the mixture heated at 120° C. for 1 h. Then, a solution of benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (4 mg, 0.0047 mmol) in toluene (0.5 mL) was added dropwise to the reaction mixture stirring in a 125° C. hot bowl and the mixture heated an additional 30 min at 120° C. The solvent was evaporated and the residue purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes to provide tert-butyl N-[12-acetyl-6-benzyloxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,8,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (13 mg, 44%). ESI-MS m/z calc. 655.22296, found 656.3 (M+1)+; Retention time: 0.44 minutes (LC Method T).

Part 1: A mixture of tert-butyl N-[12-acetyl-6-benzyloxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,8,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (13 mg, 0.01983 mmol), 10% palladium on carbon (6 mg, 0.005638 mmol) and acetic acid (300 μL) was stirred at room temperature under 200 psi hydrogen gas in a stainless steel vessel for 21 h then the mixture was filtered. The filtrate was evaporated to provide the intermediate tert-butyl N-[12-acetyl-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (14.7 mg, quant.). ESI-MS m/z calc. 567.19165, found 568.2 (M+1)+; Retention time: 0.7 minutes (LC Method T).

Part 2: The residue from part 1 was dissolved in TFA (0.3 mL), triisopropylsilane (14 μL, 0.06834 mmol) and water (15 μL) and stirred for 1 h at room temperature. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated to provide crude material that was purified by silica gel chromatography using a gradient from 10% to 50% ethyl acetate in hexanes which provided the racemic intended product.

Part 3: The racemic product of part 2 was separated into the single enantiomers by a normal phase SFC-MS method using an IC column (250×10 mm, 5 μm particle size) sold by Chiral Technologies (pn: 83445), eluting with an isocratic gradient of 16% mobile phase B in mobile phase A at 10 mL/min (Mobile phase A=CO2, Mobile phase B=methanol (+20 mM NH3), column temperature=40° C.).

To a 25 mL three neck flask with benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (5.8 mg, 0.006832 mmol) and toluene (2.35 mL) heated with a 120° C. heat bowl was added a solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-[[but-3-enyl(cyclobutanecarbonyl)amino]methyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (32 mg, 0.04422 mmol) in toluene (2.35 mL) and the mixture heated at 120° C. for 40 min. The solvent was evaporated and the residue purified by silica gel chromatography using a gradient from 0% to 25% ethyl acetate in hexanes to provide tert-butyl N-[6-benzyloxy-12-(cyclobutanecarbonyl)-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (14.5 mg, 46%). ESI-MS m/z calc. 695.2542, found 696.3 (M+1)+; Retention time: 0.56 minutes (LC Method T).

Part 2: The residue from part 1 was dissolved in TFA (0.3 mL), triisopropylsilane (15 μL, 0.07322 mmol) and water (15 μL) and stirred for 1 h at room temperature. The solvent was evaporated and the residue was dissolved in ethyl acetate and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated. The resulting residue was purified by silica gel chromatography using a gradient from 10% to 50% ethyl acetate in hexanes which provided the racemic intended product.

To a solution of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-bromo-5-(trifluoromethyl)pyridine-2-carboxylate (1 g, 2.003 mmol) in DMA (10 mL) in a microwave vial was added 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (684 mg, 3.287 mmol) and purged with nitrogen for 5 min. Then Pd(dppf)Cl2 (174 mg, 0.2131 mmol) was added followed by potassium carbonate (3.2 mL of 2 M, 6.4 mmol). The microwave vial was capped and heated at 100° C. in a microwave synthesizer for 30 min. The reaction mixture was cooled to room temperature and water was added. The mixture was extracted with ethyl acetate and combined organic layers washed with brine. The organics were separated, dried over sodium sulfate, filtered and evaporated. The resultant brown residue was purified by silica gel chromatography using a gradient from 100% hexanes to 100% ethyl acetate to afford as a light brown solid, methyl 3-[bis(tert-butoxycarbonyl)amino]-6-(1-methylpyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylate (762 mg, 76%). 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 7.84 (d, J=2.3 Hz, 1H), 6.70 (d, J=2.2 Hz, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 1.35 (s, 18H) ppm. ESI-MS m/z calc. 500.18826, found 501.2 (M+1)+; Retention time: 0.47 minutes (LC Method R).

To a solution of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-(1-methylpyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylate (500 mg, 0.9991 mmol) in dichloromethane (10 mL) was added TFA (250 μL, 3.245 mmol) followed by N-Iodosuccinimide (200 mg, 0.8890 mmol). After stirring the reaction mixture for 2 h at 23° C., the mixture was diluted with dichloromethane, washed with water, aqueous NaHCO3 and aqueous sodium thiosulfate. The organic layer was dried and concentrated. The resultant brown residue was purified by silica gel chromatography using a gradient from 100% hexanes to 100% ethyl acetate to afford as a brown solid, methyl 3-(tert-butoxycarbonylamino)-6-(4-iodo-1-methyl-pyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylate (423 mg, 80%). ESI-MS m/z calc. 526.0325, found 527.0 (M+1)+; Retention time: 0.56 minutes (LC Method R).

To a solution of methyl 3-(tert-butoxycarbonylamino)-6-(4-iodo-1-methyl-pyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylate (423 mg, 0.8038 mmol) in DMA (5 mL) in a microwave vial was added 2-allyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (230 μL, 1.226 mmol) and purged with nitrogen for 5 min. Then Pd(dppf)Cl2 (71 mg, 0.08694 mmol) followed by potassium carbonate (1.3 mL of 2 M, 2.600 mmol) were added. The microwave vial was capped and heated at 100° C. in a microwave synthesizer for 30 min. The reaction mixture was cooled to room temperature and filtered through Celite, then the Celite was washed with ethyl acetate. The filtrate was washed with brine, dried over sodium sulfate, filtered and concentrated. The resultant brown residue was purified by silica gel chromatography using a gradient from 100% hexanes to 100% ethyl acetate giving as light brown solid, 6-(4-allyl-1-methyl-pyrazol-3-yl)-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (296 mg, 86%). ESI-MS m/z calc. 426.1515, found 427.2 (M+1)+; Retention time: 0.53 minutes (LC Method R). The product was contaminated with inseparable side product, 3-(tert-butoxycarbonylamino)-6-(4-iodo-1-methyl-pyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylic acid; ESI-MS m/z calc. 512.01685, found 512.94 (M+1)+; Retention time: 0.51 minutes (LC Method R).

To a solution of 6-(4-allyl-1-methyl-pyrazol-3-yl)-3-(tert-butoxycarbonylamino)-5-(trifluoromethyl)pyridine-2-carboxylic acid (280 mg, 0.6567 mmol) [containing inseparable side product, 3-(tert-butoxycarbonylamino)-6-(4-iodo-1-methyl-pyrazol-3-yl)-5-(trifluoromethyl)pyridine-2-carboxylic acid] in DMF (5 mL) was added 2-benzyloxy-2-(trifluoromethyl)hex-5-enehydrazide (hydrochloride salt) (192 mg, 0.5668 mmol), DIEA (350 μL, 2.009 mmol), followed by HATU (301 mg, 0.7916 mmol). The reaction mixture was stirred at room temperature for 18 h. Water was added to the mixture and extracted with ethyl acetate. The combined organics washed with brine, and dried over MgSO4, filtered, and concentrated. The resultant brown residue was purified by silica gel chromatography using a gradient from 100% hexanes to 30% ethyl acetate in hexanes which gave as a pale yellow viscous oil, tert-butyl N-[6-(4-allyl-1-methyl-pyrazol-3-yl)-2-[[[2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (117 mg, 25%). ESI-MS m/z calc. 710.26514, found 711.26 (M+1)+; Retention time: 0.57 minutes (LC Method T).

To a degassed solution of dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (13 mg, 0.03042 mmol) in 1,2-dichloroethane (15 mL) was added a degassed solution of tert-butyl N-[6-(4-allyl-1-methyl-pyrazol-3-yl)-2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (78 mg, 0.1126 mmol) in 1,2-dichloroethane (15 mL) slowly dropwise under a stream of N2 flow bubbling through the solution over 30 min and the reaction mixture was heated at 60° C. for 5 h. The reaction mixture was cooled to room temperature and solvents removed by concentration under reduced pressure. The resultant brown residue was purified by silica gel chromatography using a gradient from 100% hexanes to 30% ethyl acetate in hexanes which gave tert-butyl N-[6-(benzyloxy)-14-methyl-6,18-bis(trifluoromethyl)-22-oxa-3,4,14,15,21-pentaazatetracyclo[15.3.1.12,5.012,16]docosa-1(21),2,4,9,12,15,17,19-octaen-20-yl]carbamate (E/Z mixture) (26 mg, 35%). ESI-MS m/z calc. 664.22327, found 665.2 (M+1)+; Retention time: 0.6 minutes (LC Method T).

To a solution of tert-butyl N-[6-(benzyloxy)-14-methyl-6,18-bis(trifluoromethyl)-22-oxa-3,4,14,15,21-pentaazatetracyclo[15.3.1.12,5.012,16]docosa-1(21),2,4,9,12,15,17,19-octaen-20-yl]carbamate (E/Z mixture) (26 mg, 0.03912 mmol) in acetic acid (5 mL) was added Pd/C (16 mg of 10% w/w, 50% wet, 0.01503 mmol) in a round bottom flask equipped with a H2 balloon using a 3-way adaptor. The mixture was subjected to vacuum and backfilled with nitrogen gas three times then subjected to vacuum again. The flask was filled with hydrogen gas then stirred the mixture for 15 hours. The mixture was then subjected to vacuum and backfilled with nitrogen gas three times then diluted with ethyl acetate and filtered over Celite. The filtrate was concentrated and dried under high vac to afford tert-butyl N-[6-hydroxy-14-methyl-6,18-bis(trifluoromethyl)-22-oxa-3,4,14,15,21-pentaazatetracyclo[15.3.1.12,5.012,16]docosa-1(21),2,4,12,15,17,19-heptaen-20-yl]carbamate (20 mg, 89%). ESI-MS m/z calc. 576.19196, found 577.1 (M+1)+; Retention time: 0.42 minutes (LC Method T).

tert-Butyl N-[6-hydroxy-14-methyl-6,18-bis(trifluoromethyl)-22-oxa-3,4,14,15,21-pentaazatetracyclo[15.3.1.12,5.012,16]docosa-1(21),2,4,12,15,17,19-heptaen-20-yl]carbamate (20 mg) was dissolved in pre-made solution of TFA (100 μL, 1.298 mmol) and dichloromethane (400 μL) and the reaction was stirred at room temperature for about 1 h. The solvents were removed, and the residue was dissolved in DMSO (1 mL) and the mixture was purified by a reverse phase HPLC using a mobile gradient from 30% to 99% acetonitrile in water (+5 mM HCl) over 15 minutes which gave as a colorless solid and as a racemic mixture, 20-amino-14-methyl-6,18-bis(trifluoromethyl)-22-oxa-3,4,14,15,21-pentaazatetracyclo[15.3.1.12,5.012,16]docosa-1(21),2,4,12,15,17,19-heptaen-6-ol (hydrochloride salt) (1 mg, 5%). ESI-MS m/z calc. 476.13956, found 477.1 (M+1)+; Retention time: 0.97 minutes (LC Method J).

To a stirred solution of methyl 3-[bis(tert-butoxycarbonyl)amino]-6-pent-4-enoyl-5-(trifluoromethyl)pyridine-2-carboxylate (500 mg, 0.9951 mmol) in THF (5 mL) was added trimethyl(trifluoromethyl)silane (1.2 g, 8.439 mmol) at 0° C. Then, tetrabutylammonium fluoride (50 μL of 1 M, 0.05 mmol, 1 M solution in THF) was added. The reaction mixture was slowly warmed to room temperature and stirred for 15 hours. After this time, 5 mL of water was added and extracted with ethyl acetate (3×10 mL). Organic phases were combined, dried over sodium sulfate, filtered and evaporated. The residue was purified by reverse phase chromatography using a mobile gradient from 40% acetonitrile in water to 100% acetonitrile over 6 minutes provided methyl 3-[bis(tert-butoxycarbonyl)amino]-5-(trifluoromethyl)-6-[1-(trifluoromethyl)-1-trimethylsilyloxy-pent-4-enyl]pyridine-2-carboxylate (265 mg, 41%). ESI-MS m/z calc. 644.2352, found 645.3 (M+1)+; Retention time: 1.98 minutes (LC Method M).

A solution of tert-butyl N-[2-[[[2-benzyloxy-2-(trifluoromethyl)hex-5-enoyl]amino]carbamoyl]-6-[1-hydroxy-1-(trifluoromethyl)pent-4-enyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (240 mg, 0.3232 mmol) and DIEA (104.5 mg, 0.8086 mmol) in acetonitrile (5 mL) was heated to 50° C., then p-TsCl (92.5 mg, 0.4852 mmol) was added. The resulted mixture was heated at 70° C. for 2 h and cooled to room temperature. The reaction mixture was quenched with saturated solution of sodium bicarbonate (10 mL) and extracted with ethyl acetate (3×20 mL). Organic layers combined and washed with brine (30 mL), dried over sodium sulfate, filtered and solvents evaporated. Purification by silica gel chromatography using 90:10 hexanes and ethyl acetate provided tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-[1-hydroxy-1-(trifluoromethyl)pent-4-enyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (145 mg, 62%). ESI-MS m/z calc. 724.2307, found 725.2 (M+1)+; Retention time: 1.93 minutes (LC Method M).

To a solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-[1-hydroxy-1-(trifluoromethyl)pent-4-enyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (280 mg, 0.3864 mmol) in toluene (100 mL) at 90° C. was added [1,3-bis-(2-tolyl)-2-imidazolidinylidene]dichloro(2-isopropoxybenzylidene)ruthenium(II) (32 mg, 0.05609 mmol) while nitrogen gas bubbled in the solution through a rubber septum. The mixture was heated at 110° C. for 3 hours. The solvent was evaporated and the residue was purified by silica gel chromatography using 90:10 hexanes and ethyl acetate) which gave two separated isomeric mixtures of the product: tert-butyl N-[6-benzyloxy-13-hydroxy-6,13,15-tris(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (isomer 1, 31 mg, 12%, ESI-MS m/z calc. 696.1994, found 697.3 (M+1)+; Retention time: 1.58 minutes (LC Method M) and isomer 2, 30 mg, 11%, ESI-MS m/z calc. 696.1994, found 697.2 (M+1)+; Retention time: 1.66 minutes (LC Method M). Because of the scale and some impurity of the compounds, both isomers were mixed and taken to the next step.

To a solution of tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (27 mg, 0.05014 mmol) in EtOH (1 mL), acetic acid (1 mL) was added O-tert-butylhydroxylamine (hydrochloride salt) (38 mg, 0.3026 mmol) followed by NaOAc (42 mg, 0.5120 mmol). The reaction mixture was heated at 80° C. for overnight. Anhydrous magnesium sulfate was added and heated the reaction at 80° C. for another 6 h, stopping the reaction at ˜60% conversion. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by reverse phase HPLC using a gradient from 50% to 99% acetonitrile in water (+5 mM HCl) over 15 minutes giving as an off-white solid and the first isomer to elute, tert-butyl N-[(6R)-13-tert-butoxyimino-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (E/Z isomer 1) (8.4 mg, 55%). ESI-MS m/z calc. 609.2386, found 610.2 (M+1)+; Retention time: 1.76 minutes (LC Method M).

tert-Butyl N-[(6R)-13-tert-butoxyimino-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (E/Z isomer 2) (6.7 mg, 0.01099 mmol) was dissolved in a (pre made solution of 1:4 TFA/dichloromethane) TFA (100 μL, 1.298 mmol) and dichloromethane (400 μL) and the reaction was stirred at room temperature for about 1 h. Solvents were removed and the residue was purified by reverse phase HPLC using a gradient from 30% to 99% acetonitrile in water (+5 mM HCl) over 15 minutes which gave as a white solid, (6R)-17-amino-13-tert-butoxyimino-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-6-ol (E/Z isomer 2) (hydrochloride salt) (2 mg, 27%). ESI-MS m/z calc. 509.18616, found 510.1 (M+1)+; Retention time: 1.93 minutes (LC Method M).

Part 1: To a solution of tert-butyl N-[(6R)-6-benzyloxy-13-hydroxyimino-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (30 mg, 0.04676 mmol) in MeOH (450 μL) was added Pd/C (20 mg of 10% w/w, 0.01879 mmol). The mixture was stirred overnight under a hydrogen atmosphere, 200 psi. The reaction mixture was filtered through Celite, washing well with ethyl acetate and then concentrated to give 25 mg of a yellow residue which was used directly in part 2.

To a stirred solution of pent-4-enoic acid (1 g, 9.988 mmol) and tert-butyl N-aminocarbamate (6.6 g, 49.94 mmol) in ethyl acetate (45 mL) was added T3P (31.8 g of 50% w/w, 49.97 mmol), followed by addition of pyridine (15.8 g, 199.7 mmol). The reaction mixture was stirred at room temperature for 16 h. Then, saturated solution of sodium bicarbonate was slowly added till no bubble formation was observed. The organic compound was extracted with ethyl acetate (3×10 mL), combined organics dried over sodium sulfate, filtered and concentrated. Purification by silica gel chromatography using 75:25 hexanes and ethyl acetate gave a pale-yellow oil. Next, the compound was dissolved in dichloromethane (15 mL) and cooled in an ice bath and TFA (10 mL, 129.8 mmol) was added slowly and let the reaction mixture warm to room temperature and stirred for an additional 1 h. After this time, saturated solution of sodium bicarbonate was added to the mixture (pH=7). The compound was extracted with ethyl acetate (4×15 mL). Organic layers were combined, dried over sodium sulfate, filtered, and concentrated under vacuum to afford pent-4-enehydrazide (418 mg, 37%). 1H NMR (400 MHz, CDCl3) δ 7.39 (bs, 1H), 5.86-5.75 (m, 1H), 5.39-4.80 (m, 2H), 3.95 (bs, 2H), 2.58-2.35 (m, 2H), 2.33-2.03 (m, 2H) ppm.

To a solution of 6-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid (205 mg, 0.3325 mmol) and pent-4-enehydrazide (45.6 mg, 0.3995 mmol) in NMP (10 mL) was added DIEA (129 mg, 0.9981 mmol), followed by addition of HATU (190 mg, 0.4997 mmol). The reaction mixture was stirred at room temperature for 35 minutes. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The organic phases combined and dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by reverse phase chromatography using a gradient from 30% to 95% acetonitrile in water (+5 mM HCl) over 10 minutes giving tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-[(pent-4-enoylamino)carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (122 mg, 51%). ESI-MS m/z calc. 712.2444, found 713.3 (M+1)+; Retention time: 1.31 minutes (LC Method M).

To a solution of tert-butyl N-[6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (850 mg, 1.316 mmol) in THF (15 mL) at 0° C. was dropwise added borane dimethyl sulfide complex (995 μL of 2 M, 1.99 mmol) and stirred for 15 min at 0° C. Allowed the reaction to warm to room temperature and stirred for 1 h. Cooled the reaction to 0° C. before quenching with aqueous NaOH (6 mL of 1 M, 6 mmol) followed by the addition of hydrogen peroxide (7 mL of 30% w/v, 61.74 mmol). This mixture was stirred for 30 min at room temperature then was extracted with ethyl acetate (2×80 mL). The organic layers were combined, washed with brine (80 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (80 gram column) using a gradient from 100% hexanes to 80% ethyl acetate in hexanes (product elutes at 30% to 40% ethyl acetate) to afford an unseparated mixture of two regioisomeric products as a white solid consisting of tert-butyl N-[6-benzyloxy-9-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (425 mg, 50%), ESI-MS m/z calc. 644.207, found 645.2 (M+1)+; Retention time: 1.73 minutes (LC Method J) and tert-butyl N-[6-benzyloxy-10-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (325 mg, 38%). ESI-MS m/z calc. 644.207, found 645.2 (M+1)+; Retention time: 1.73 minutes (LC Method J) which were taken directly to the ensuing step. Yields are estimated based on ratios of the two product peaks from HPLC.

tert-Butyl N-[6-hydroxy-9,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (130 mg, 0.2353 mmol) was dissolved in dichloromethane (3 mL) and to the mixture was added TFA (750 μL, 9.735 mmol) and stirred at room temperature. After 90 mins, the mixture was evaporated to dryness, then diluted with ether and concentrated again. The crude material was then placed under vacuum for 2 h to afford as a white solid, 17-amino-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaene-9,13-dione (105 mg, 99%). ESI-MS m/z calc. 452.09192, found 453.2 (M+1)+; Retention time: 1.41 minutes (LC Method A).

To a solution of tert-butyl N-[6-benzyloxy-10,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (70 mg, 0.1089 mmol) in acetic acid (1.89 mL) was added Pd/C (46.36 mg of 10% w/w, 0.04356 mmol). The mixture was stirred for 6 h under a hydrogen atmosphere using a Parr shaker at 150 psi. The reaction mixture was filtered through a silica plug, washing well with ethyl acetate and then concentrated to a white residue, which was purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes over 20 minutes which gave as a white solid, tert-butyl N-[6-hydroxy-10,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (20 mg, 33%). ESI-MS m/z calc. 552.14435, found 553.34 (M+1)+; Retention time: 0.32 minutes (LC Method T).

To a solution of tert-butyl N-[6-benzyloxy-10,10-difluoro-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (55 mg, 0.08276 mmol) in acetic acid (1.485 mL) was added Pd/C (35.23 mg of 10% w/w, 0.0331 mmol). The mixture was stirred for 6 h under a hydrogen atmosphere using a Parr shaker at 150 psi. Reaction mixture was filtered through a silica plug, washing well with ethyl acetate and then concentrated, which was purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes over 20 minutes which gave as a white solid, tert-butyl N-[10,10-difluoro-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (18 mg, 76%). ESI-MS m/z calc. 574.14624, found 575.35 (M+1)+; Retention time: 0.38 minutes (LC Method T).

In a pressure reactor a solution of tert-butyl N-[6-benzyloxy-9,9-difluoro-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (35 mg, 0.05267 mmol) in acetic acid (550 μL) and ethyl acetate (550 μL) was purged with nitrogen. Then, Pd/C (56.5 mg of 10% w/w, 0.05309 mmol) was added. The mixture was degassed with nitrogen, then filled with hydrogen gas and stirred at 180 psi for 4 hours. The reaction was filtered over Celite plug and washed with excess acetonitrile and ethyl acetate and concentrated. The residue was purified by silica gel chromatography (12 gram column) using a gradient from 100% hexanes to 60% ethyl acetate in hexanes (product elutes at 30% ethyl acetate) and placed under high vac pump for 2 hours which gave as a white solid, tert-butyl N-[9,9-difluoro-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (2 mg, 7%). ESI-MS m/z calc. 574.14624, found 575.2 (M+1)+; Retention time: 1.56 minutes (LC Method J) and tert-butyl N-[9,9-difluoro-6,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (26 mg, 86%). ESI-MS m/z calc. 576.16187, found 577.2 (M+1)+; Retention time: 1.28 minutes (LC Method J) contaminated with a small amount of tert-butyl N-[9,9-difluoro-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate.

To a solution of the tert-butyl N-[9,9-difluoro-6,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (30 mg, 0.05204 mmol, contaminated with a small amount of tert-butyl N-[9,9-difluoro-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate) in dichloromethane (2.5 mL) at room temperature was added DMP (65 mg, 0.1533 mmol). After 30 minutes, added additional DMP (65 mg, 0.1533 mmol) and stirred additional 2.5 h. Quenched the reaction with saturated aqueous NaHCO3 and extracted with dichloromethane (50 mL). Combined organic layers washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (24 gram column) using a gradient from 100% hexanes to 70% ethyl acetate in hexanes (product elutes at 30% ethyl acetate) which gave as a white solid, tert-butyl N-[9,9-difluoro-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (28.5 mg, 95%). ESI-MS m/z calc. 574.14624, found 575.2 (M+1)+; Retention time: 1.56 minutes (LC Method J).

To a solution of tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,9,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (3.38 g, 5.395 mmol) in THF (25 mL) at 0° C. was added dropwise borane dimethyl sulfide solution (8.10 mL of 2 M in THF, 16.2 mmol) and let the resulting mixture stir for 15 min at 0° C. Next, the reaction was allowed to warm to room temperature and further stirred for 30 min until UPLC showed starting material was consumed. Cooled the reaction to 0° C. before quenching with aqueous NaOH (10.79 mL of 2 M, 21.58 mmol) followed by the addition of hydrogen peroxide (18.35 g, 161.8 mmol). Let the resulting mixture stir for 30 min at room temperature before extracting with ethyl acetate (2×50 mL). The organic layers were combined, washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated. Purified the residue by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes to afford as a white solid and mixture of diastereomers and regioisomers, tert-butyl N-[(6R)-6-benzyloxy-9,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate mixed with tert-butyl N-[(6R)-6-benzyloxy-10,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (1.175 g, 67%). ESI-MS m/z calc. 646.2226, found 647.27 (M+1)+; Retention time: 0.31 minutes (LC Method T).

To a solution of a mixture of regioisomers/diasteromers, tert-butyl N-[(6R)-6-benzyloxy-10,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate and tert-butyl N-[(6R)-6-benzyloxy-9,13-dihydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (2.35 g, 3.635 mmol) in dichloromethane (44 mL) was added Dess-Martin periodinane (4.623 g, 10.9 mmol). The reaction was stirred for 30 min then quenched with saturated aqueous NaHCO3 and extracted with dichloromethane (2×50 mL). The organic layers were combined and washed with brine, dried over NaSO4, filtered and concentrated. The residue was purified by silica gel chromatography using a gradient from 0% to 10% ethyl acetate in hexanes to afford as the first regioisomer to elute, tert-butyl N-[(6R)-6-benzyloxy-9,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (1.1 g, 47%). ESI-MS m/z calc. 642.1913, found 643.29 (M+1)+; Retention time: 0.59 minutes (LC Method T).

To a glass sealed vial under nitrogen containing a solution of tert-butyl N-[(6R)-6-benzyloxy-10,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (350 mg, 0.5447 mmol) in 1,2-dichloroethane (1.09 mL) was added bis(2-methoxyethyl)aminosulfur trifluoride (2.41 mL of 50% w/v in THF, 5.447 mmol). Stirred the reaction for 6 h at 50° C. then quenched with saturated aqueous NaHCO3 and extracted with dichloromethane (2×25 mL). Combined the organic layers, washed with brine, dried over NaSO4, filtered and concentrated then purified via silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes to afford as a white solid, tert-butyl N-[(6R)-6-benzyloxy-13,13-difluoro-10-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (165 mg) which was contaminated with a regioisomeric product and taken directly to the next step. ESI-MS m/z calc. 664.1932, found 665.22 (M+1)+; Retention time: 0.65 minutes (LC Method T).

Part 1: To a solution of tert-butyl N-[(6R)-6-benzyloxy-13,13-difluoro-10-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (165 mg) which was contaminated with a regioisomeric product (as described in the previous step) in acetic acid (4.455 mL) was added 10% Pd/C (105.7 mg, 0.09932 mmol). The mixture was stirred for 6 h under a hydrogen atmosphere using a parr shaker at 150 psi. The reaction mixture was filtered through a silica plug, washing well with ethyl acetate and then the filtrated was concentrated to a white residue which was purified by silica chromatography using a gradient from 0% to 30% ethyl acetate in hexanes over 20 minutes to obtain a crude mixture of regioisomeric intended products and some over-reduced side-product. Carried this mixture forward to the next part.

Part 2: To the mixture obtained in part 1 (80 mg) in dichloromethane (1.107 mL) was added Dess-Martin periodinane (76 mg, 0.1792 mmol) and the resulting mixture was stirred for 15 min. Quenched the reaction with saturated aqueous NaHCO3 then extracted with dichloromethane (2×25 mL). Combined the organic layers and washed with brine, dried over NaSO4, filtered and concentrated then purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes to afford a regioisomeric mixture of products which was taken directly to the next part.

In a 20 mL sealed vial, tert-butyl N-[(6R)-6-benzyloxy-9,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (1.02 g, 1.587 mmol) was dissolved in 1,2-dichloroethane (9 mL) at room temperature. To the stirring solution was slowly added diethylaminosulfur trifluoride (6.75 mL, 51.09 mmol) allowing to stir at room temperature for 15 additional minutes. Then, the mixture was capped and heated to 85° C. for 5 h behind a blast shield. The resultant mixture was quenched slowly at 0° C. by addition of saturated aqueous NaHCO3 (20 mL). The mixture was diluted with dichloromethane, washed with water, and dried over sodium sulfate then filtered and concentrated. The crude filtrate was purified by silica gel chromatography using a gradient from 100% hexanes to 50% ethyl acetate in hexanes to afford the product containing impurities as a tan solid. The mixture was further purified by reverse-phase preparative chromatography utilizing a C18 column and a gradient from 30% to 99% acetonitrile in water (+5 mM HCl) to afford as a white solid, tert-butyl N-[(6R)-6-benzyloxy-13,13-difluoro-9-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (150 mg) which was contaminated with a regioisomeric product. ESI-MS m/z calc. 664.1932, found 665.2 (M+1)+; Retention time: 2.08 minutes (LC Method J). This material was taken directly to the ensuing step.

Part 1: In a pressure reactor, a solution of tert-butyl N-[(6R)-6-benzyloxy-13,13-difluoro-9-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (150 mg) (which was contaminated with a regioisomeric product as described in step 1) in acetic acid (2.5 mL) and ethyl acetate (2.5 mL) was purged with nitrogen. Then 10% Pd/C (235 mg, 0.2208 mmol) was added. The mixture was degassed with nitrogen, then filled with hydrogen gas and stirred at 180 psi for 4 h. The mixture was degassed with nitrogen then filled with hydrogen gas and stirred at 180 psi for 2 additional hours. The reaction was filtered, washing the celite plug with excess acetonitrile and ethyl acetate and the filtrate was concentrated. The crude residue obtained was then purified by silica gel chromatography using a gradient from 100% hexanes to 60% ethyl acetate in hexanes to afford a crude mixture of regioisomeric intended products and some over-reduced side-product (108 mg). Carried this mixture forward to the next part.

Part 2: To a solution of the mixture of products obtained in part 1 (108 mg) in dichloromethane (2.5 mL) was added Dess-Martin periodinane (150 mg, 0.3537 mmol). After 15 minutes, quenched the reaction with saturated aqueous NaHCO3 then extracted with dichloromethane (50 mL). Washed the organic dichloromethane layer with brine, dried over sodium sulfate, filtered and concentrated. The crude filtrate was then purified by silica gel chromatography using a gradient from 100% hexanes to 65% ethyl acetate in hexanes to afford a regioisomeric mixture of products as a white solid which was taken directly to the next part.

The cis and trans products obtained were combined to give tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) which was used directly in the next step.

Part 1: To a solution of tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (400 mg, 0.6384 mmol) in THF (12 mL) at 0° C. was added dropwise borane dimethyl sulfide solution (500 μL of 2 M in THF, 1 mmol) and the mixture was stirred for 15 min at 0° C. Allowed the reaction to warm to room temperature and stirred for 1 h. Added additional borane dimethyl sulfide solution (500 μL of 2 M in THF, 1 mmol) and stirred at room temperature for 30 more minutes. Cooled the reaction to 0° C. before quenching with NaOH (2.95 mL of 1 M, 2.95 mmol) followed by the addition of hydrogen peroxide (2.55 mL of 30% w/v, 22.49 mmol). Let the mixture stir for 30 min at room temperature then extracted with ethyl acetate (2×80 mL). The organic layers were combined, washed with brine (80 mL), dried over sodium sulfate, filtered and concentrated. The crude material was then purified by silica gel chromatography using a gradient from 100% hexanes to 80% ethyl acetate in hexanes to afford the intermediate product as a white solid comprised of a mixture of isomers.

In a 20-mL microwave vial, tert-butyl N-[(6R)-6-benzyloxy-8,13-dioxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (90 mg, 0.1401 mmol) was dissolved in 1,2-dichloroethane (2.25 mL) at room temperature. To the stirring solution was slowly added diethylaminosulfur trifluoride (900 μL, 6.812 mmol) allowing the mixture to stir at room temperature for 15 additional minutes. Then, the mixture was capped and heated to 80° C. for 16 hours behind a blast shield. The resultant mixture was quenched slowly at 0° C. with saturated aqueous NaHCO3 (20 mL). The mixture was diluted with dichloromethane and washed with water then dried over sodium sulfate, filtered and concentrated. The crude material was purified by reverse-phase preparative chromatography utilizing a C18 column eluting with a gradient from 30% to 99% acetonitrile in 5 mM aqueous HCl to afford as a light yellow solid, tert-butyl N-[(6R)-6-benzyloxy-8,8-difluoro-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (19 mg, 20%). ESI-MS m/z calc. 664.1932, found 665.2 (M+1)+; Retention time: 2.27 minutes (LC Method J).

A solution of [1,3-bis-(2-tolyl)-2-imidazolidinylidene]dichloro(2-isopropoxybenzylidene)ruthenium(II) (22.1 mg, 0.03874 mmol) in toluene (50 mL) was bubbled with nitrogen gas and heated to 70 to 75° C. Next, at this temperature, a solution of tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-(2,2-dimethylpent-4-enoyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (220 mg, 0.3223 mmol) in toluene (30 mL) was added dropwise over 40 min while the temperature reaching was increased to 105° C. The mixture was then stirred 90 min at this temperature and every 30 mins, the solution was bubbled with nitrogen gas for 2 min. When all starting material was converted to the desired product, the reaction mixture was cooled to 40° C. and the solvent was evaporated under reduced pressure then the residue was purified by C18 reverse-phase chromatography using a gradient from 30% acetonitrile in water to 100% water which gave tert-butyl N-[6-benzyloxy-12,12-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z mixture) (160 mg, 76%). ESI-MS m/z calc. 654.22766, found 655.1 (M+1)+; Retention time: 2.09 minutes (LC Method M).

To a solution of tert-butyl N-[6-benzyloxy-12,12-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (150 mg, 0.2291 mmol) in MeOH (2 mL) and acetic acid (2 mL) was added 10% Pd/C (49 mg, 0.02293 mmol, 50% wet) under nitrogen in a high pressure reactor. The reactor was sealed, evacuated and filled with nitrogen gas 3 times. The reactor was then evacuated and filled with hydrogen gas up to 120 psi. The mixture was stirred overnight then depressurized and filtered through a pad of celite washing with dichloromethane and MeOH. The residue was purified by C18 reverse-phase chromatography using a gradient from 30% acetonitrile in water to 100% water which gave tert-butyl N-[6-hydroxy-12,12-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (46 mg, 35%). ESI-MS m/z calc. 566.1964, found 567.2 (M+1)+; Retention time: 1.96 minutes (LC Method J). The reaction also produced a significant amount of over-reduced product (M+2).

To a nitrogen purged solution of carbonyl diimidazole (7 g, 43.17 mmol) in dichloromethane (200 mL) at 0° C. was added 3,3-dimethylhex-5-enoic acid (5 g, 35.16 mmol) under nitrogen in an ice bath. Allowed to stir while warming to room temperature. After 16 h, added N-methoxymethanamine (hydrochloride salt) (8.75 g, 89.7 mmol). Then, the stirring mixture was cooled to 0° C. and pyridine (7.5 mL, 92.73 mmol) was slowly added and on completion of addition the mixture was stirred 5 minutes then the ice-water bath was removed and the solution was stirred overnight. The dichloromethane was removed by rotary evaporation, then added 1:1 diethyl ether/dichloromethane to the residue and extracted with saturated aqueous brine (1×). Dried the organic layer over sodium sulfate, filtered and concentrated to a tan oil which was purified by silica gel chromatography using a shallow gradient from 100% dichloromethane to 10% methanol in dichloromethane which provided as a colorless oil, N-methoxy-N,3,3-trimethyl-hex-5-enamide (5.6 g, 86%). 1H NMR (400 MHz, Chloroform-d) δ 6.08-5.62 (m, 1H), 5.10-4.95 (m, 2H), 3.66 (s, 3H), 3.17 (s, 3H), 2.32 (s, 2H), 2.14 (dd, J=7.6, 1.4 Hz, 2H), 1.03 (s, 6H) ppm. ESI-MS m/z calc. 185.14159, found 186.2 (M+1)+; Retention time: 1.59 minutes (LC Method A).

A solution of tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-(3,3-dimethylhex-5-enoyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (407 mg, 0.5962 mmol) and benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (76 mg, 0.08952 mmol) in toluene (60 mL) was heated at 120° C. for 30 min. Then, benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (25 mg, 0.02945 mmol) was added and the solution was stirred at 120° C. for 50 min. Then, additional benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (25 mg, 0.02945 mmol) was added and the solution was stirred at 120° C. for 30 min. Then, additional benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (25 mg, 0.02945 mmol) was added and the solution was stirred at 120° C. for 30 min. Then, more benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (25 mg, 0.02945 mmol) was added and the solution stirred at 120° C. for 30 min. Then, additional benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (25 mg, 0.02945 mmol) was added and the solution stirred at 120° C. for 20 min. The solvent was evaporated and the residue was subjected to silica gel column chromatography using a gradient of 0% to 20% ethyl acetate in hexanes to provide partially purified material. This material was dissolved in 1 mL methanol and subjected to preparative HPLC using a Luna 75×30 mm C18 column (5 m particle size) eluting with a gradient of 70% to 99% acetonitrile in 5 mM aqueous HCl which provided tert-butyl N-[(6R)-6-benzyloxy-11,11-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,8,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (21 mg, 5%). ESI-MS m/z calc. 654.22766, found 655.3 (M+1)+; Retention time: 0.62 minutes (LC Method T).

In a 250 mL round bottom flask, a solution of tert-butyl N-[(6R)-6-benzyloxy-11,11-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,8,14,16-hexaen-17-yl]carbamate (E/Z mixture) (23 mg, 0.03514 mmol) in ethyl acetate (1 mL) was purged with nitrogen. Then, Pd/C (38 mg of 10% w/w, 0.03571 mmol) was added. The mixture was degassed with nitrogen, then filled with a balloon containing hydrogen gas and stirred at 1 atm of pressure for 4 hours. The mixture was filtered, washing the celite plug with excess ethyl acetate and then concentrated the filtrate. The crude material was purified by reverse-phase preparative chromatography utilizing a C18 column and a gradient from 30% to 99% acetonitrile in 5 mM HCl to afford as an off-white foam, tert-butyl N-[(6R)-6-hydroxy-11,11-dimethyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (12.3 mg, 62%). ESI-MS m/z calc. 566.1964, found 567.2 (M+1)+; Retention time: 2.01 minutes (LC Method J).

To tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (515 mg, 0.808 mmol) in ether (5 mL) at −78° C. was added n-BuLi (669 μL of 2.5 M, 1.672 mmol) as a solution in hexanes. After stirring a further 10 min at −78° C., a solution of 1-but-3-enyl-N-methoxy-N-methyl-cyclobutanecarboxamide (207 mg, 1.049 mmol) in ether (2.5 mL) was added dropwise via cannula needle. The mixture was stirred at −78° C. for 15 min and at 0° C. for 15 min. Then, the mixture was diluted with 1 M NH4Cl in water (2 mL) and ether then partitioned. The organic layer was separated and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography using a gradient from 5% to 20% of a solution (20% ethyl acetate in hexanes) to hexanes to provide tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-(1-but-3-enylcyclobutanecarbonyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (127 mg, 21%). ESI-MS m/z calc. 694.259, found 695.4 (M+1)+; Retention time: 0.69 minutes (LC Method T).

Part 1: A mixture of tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaene-12,1′-cyclobutane]-17-yl]carbamate (E/Z Mixture) (77 mg, 0.1155 mmol), 10% palladium on carbon (28 mg, 0.02631 mmol) and acetic acid (800 μL) was stirred at room temperature under 200 psi hydrogen gas for 13 h, then the mixture was filtered and the volatiles evaporated. The residue was dissolved in acetic acid (800 μL) was added to 10% palladium on carbon (22 mg, 0.02067 mmol) and stirred at room temperature under 200 psi hydrogen for 8 h, then the mixture was filtered and the filtrate evaporated. The residue was dissolved in ethyl acetate and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated to provide 61 mg (95% pure, 87% yield) of the ketone, tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclobutane]-17-yl]carbamate which contained as a 3% impurity by UPLC the alcohol, tert-butyl N-[(6R)-6,13-dihydroxy-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclobutane]-17-yl]carbamate.

Part 2: The 61 mg of 95% purity tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclobutane]-17-yl]carbamate was dissolved in DCM (1 mL) and Dess-Martin periodinane (9 mg, 0.02122 mmol) was added. The mixture was stirred at room temperature for 20 min then methanol (1 mL) was added and the mixture was diluted with DCM and washed with 1 M Na2S203, 1 M NaHCO3, then dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexane to provide tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclobutane]-17-yl]carbamate (54 mg, 77%). ESI-MS m/z calc. 578.1964, found 579.2 (M+1)+; Retention time: 0.44 minutes (LC Method T).

To tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-bromo-5-(trifluoromethyl)-3-pyridyl]carbamate (252 mg, 0.3954 mmol) in ether (3.8 mL) at −78° C. was added n-BuLi (330 μL of 2.5 M, 0.825 mmol) as a solution in hexanes. After stirring a further 10 min at −78° C., a solution of 1-but-3-enyl-N-methoxy-N-methyl-cyclopropanecarboxamide (145 mg, 0.7913 mmol) in ether (1.3 mL) was added dropwise via cannula needle. The mixture was stirred at −78° C. for 15 min and at 0° C. for 15 min. Then, the mixture was diluted with 1 M NH4Cl in water (2 mL) and ether and partitioned. The organic layer was separated and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated. The residue was purified silica gel chromatography using a gradient from 5% to 20% of a solution (20% ethyl acetate in hexanes) to hexanes to provide as a white solid, tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-(1-but-3-enylcyclopropanecarbonyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (100 mg, 37%). ESI-MS m/z calc. 680.24335, found 681.3 (M+1)+; Retention time: 0.62 minutes (LC Method T).

In a 100 mL 3 neck flask, a solution of tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-(1-but-3-enylcyclopropanecarbonyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (166 mg, 0.2439 mmol) and benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium (31 mg, 0.03651 mmol) in toluene (28 mL) with nitrogen gas constantly bubbling through the solution was heated at 120° C. for 30 min. Then, the solvent was evaporated. The residue was purified silica gel chromatography using a gradient from 0% to 30% of a solution (20% ethyl acetate in hexanes) to hexanes to provide tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaene-12,1′-cyclopropane]-17-yl]carbamate (E/Z Mixture) (110 mg, 69%). ESI-MS m/z calc. 652.21204, found 653.4 (M+1)+; Retention time: 0.58 minutes (LC Method T).

Part 1: A mixture of tert-butyl N-[(6R)-6-benzyloxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaene-12,1′-cyclopropane]-17-yl]carbamate (E/Z Mixture) (110 mg, 0.1686 mmol), 10% palladium on carbon (41 mg, 0.03853 mmol) and acetic acid (2.2 mL) was stirred at room temperature under 200 psi hydrogen gas for 16 h, then the mixture was filtered and the filtrate was evaporated. The residue was dissolved into ethyl acetate and washed with 1 M NaHCO3, dried (MgSO4), filtered and evaporated to provide 94 mg (90% pure, 89% yield) of the ketone, tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclopropane]-17-yl]carbamate which contained as a 1% impurity by UPLC the alcohol, tert-butyl N-[(6R)-6,13-dihydroxy-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclopropane]-17-yl]carbamate.

Part 2: The 94 mg of 90% pure ketone, tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclopropane]-17-yl]carbamate obtained in part 1 was dissolved into dichloromethane (1.5 mL) and Dess-Martin periodinane (10 mg, 0.02358 mmol) was added. The mixture was stirred at room temperature for 20 min and then more Dess-Martin periodinane (10 mg, 0.02358 mmol) was added. The mixture was stirred at room temperature for 15 min and then methanol (1 mL) was added and the mixture was diluted with dichloromethane and washed with 1 M Na2S2O3 followed by 1 M NaHCO3 then dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexane to provide tert-butyl N-[(6R)-6-hydroxy-13-oxo-6,15-bis(trifluoromethyl)spiro[19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaene-12,1′-cyclopropane]-17-yl]carbamate (71 mg, 75%). ESI-MS m/z calc. 564.1807, found 565.2 (M+1)+; Retention time: 0.8 minutes (LC Method S).

To a solution of 01-tert-butyl 03-ethyl 2-[6-methoxycarbonyl-5-nitro-3-(trifluoromethyl)-2-pyridyl]propanedioate (15.33 g, 35.133 mmol) in DCM (61 mL) was added TFA (75.48 g, 51 mL, 661.97 mmol) slowly at room temperature. The reaction mixture was cooled to 0° C. and saturated aqueous NaHCO3 (50 mL, until pH=8 to 9) was slowly added then extracted the mixture with DCM (600 mL). The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The amber residue was purified by silica gel chromatography using a gradient from 0% to 25% ethyl acetate in hexanes (hexanes modified with 0.1% acetic acid) and the fractions containing product crystallized and were filtered to provide 4.15 g of pure product. The supernatant was concentrated in vacuo to furnish an additional 3.52 g of methyl 6-(2-ethoxy-2-oxo-ethyl)-3-nitro-5-(trifluoromethyl)pyridine-2-carboxylate (7.67 g, 64%). 1H NMR (500 MHz, DMSO-d6) δ 8.95 (s, 1H), 4.24-4.20 (m, 2H), 4.13 (q, J=7.1 Hz, 2H), 3.97 (s, 3H), 1.17 (t, J=7.1 Hz, 3H) ppm. ESI-MS m/z calc. 336.0569, found 337.1 (M+1)+; Retention time: 4.92 minutes (LC Method DD).

To a solution of ethyl 2-[6-[5-[1-benzyloxy-4-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)butyl]-1,3,4-oxadiazol-2-yl]-5-nitro-3-(trifluoromethyl)-2-pyridyl]acetate (3.5 g, 5.397 mmol) in acetic acid (35 mL) was added iron powder (3.25 g, 58.197 mmol) and the mixture was stirred at 50° C. for 3 h. The reaction was allowed to cool slowly to room temperature over 1 h. The reaction was then quenched by pouring slowly into an oversized beaker containing ice cold saturated aqueous NaHCO3 (pH=8) then extracted with ethyl acetate (3×500 mL), dried over Na2SO4, filtered and concentrated in vacuo to provide ethyl 2-[5-amino-6-[5-[1-benzyloxy-4-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)butyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetate (3.12 g, 89%) as a sticky tan solid. ESI-MS m/z calc. 618.1913, found 619.4 (M+1)+; Retention time: 7.17 minutes (LC Method DD).

To a suspension of crude 2-[5-amino-6-[5-[1-benzyloxy-4-(1,3-dioxolan-2-yl)-1-(trifluoromethyl)butyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (lithium salt) (160 mg, 0.2678 mmol) in HCl (4.3 mL of 3 M, 12.9 mmol) was added acetic acid (8 mL) and stirred the mixture at room temperature for 25 min. The reaction was then diluted with ethyl acetate (50 mL) and poured into cold saturated aqueous NaHCO3 (˜25 mL, until pH=6), the organics were separated and washed with brine, dried over Na2SO4, filtered and concentrated in vacuo at 25° C. (note: the aldehyde appears unstable at higher concentrations and/or in the presence of acid) to provide 2-[5-amino-6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (148 mg, quant.). ESI-MS m/z calc. 546.1338, found 547.5 (M+1)+; Retention time: 5.63 minutes (LC Method DD). The yellow oily residue was used directly in the next step without further purification.

To a solution of DIEA (140.98 mg, 0.19 mL, 1.0908 mmol) in ethyl acetate (1.66 mL) was added T3P in ethyl acetate (175 mg, 50% w/w, 0.275 mmol) at room temperature. A separate flask containing crude 2-[5-amino-6-[5-[5-(benzylamino)-1-benzyloxy-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (166 mg, 0.1823 mmol, 70% purity) in ethyl acetate (3.3 mL) and NMP (0.83 mL) was added slowly (25 min dropwise addition) to the flask containing the T3P-DIEA at room temperature. The reaction was stirred for 1.5 h at room temperature then additional T3P (60 mg, 50% w/w, 0.0943 mmol) was added and the resulting mixture was stirred for 30 min. The reaction was diluted with ethyl acetate (50 mL), then washed with water (50 mL), 0.1 M HCl (50 mL, until pH=1), saturated aqueous NaHCO3 (20 mL, until pH=8) and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes (hexanes contained 0.5% Et3N) to provide 17-amino-11-benzyl-6-benzyloxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,11,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-12-one (52 mg, 66%). ESI-MS m/z calc. 619.2018, found 620.7 (M+1)+; Retention time: 6.9 minutes (LC Method DD).

To a solution of propan-2-amine (242.9 mg, 0.35 mL, 4.1093 mmol) in methanol (1.3 mL) and THE (2 mL) was added acetic acid (0.3 mL) and sodium cyanoborohydride (101 mg, 1.6072 mmol) at 0° C. A solution of 2-[5-amino-6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (220 mg, 0.4026 mmol) in MeOH (5.3 mL) was added dropwise at 0° C. over 5 min. The reaction was allowed to stir 30 min then quenched slowly with saturated aqueous NaHCO3 (100 mL) and diluted with MeOH (200 mL) and stirred 12 h at room temperature then concentrated in vacuo. The aqueous suspension was diluted with ethyl acetate (120 mL) and water (30 mL) and the layers were separated. The organics were washed with 0.1 M HCl (until pH=1) followed by a mixture of brine (30 mL) and water (50 mL), dried over Na2SO4, filtered and concentrated in vacuo to provide 2-[5-amino-6-[5-[1-benzyloxy-5-(isopropylamino)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (237 mg, 90%) as a pale yellow solid. ESI-MS m/z calc. 589.2124, found 590.2 (M+1)+; Retention time: 4.71 minutes. Product was used directly in the next step without further purification (LC Method DD).

To a solution of DIEA (222.6 mg, 0.3 mL, 1.7223 mmol) in ethyl acetate (2.37 mL) was added T3P in ethyl acetate (359 mg, 50% w/w, 0.5641 mmol) at room temperature. A separate flask containing crude 2-[5-amino-6-[5-[1-benzyloxy-5-(isopropylamino)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (237 mg, 0.2814 mmol, 70% purity) in ethyl acetate (4.74 mL) and NMP (1.185 mL) was added slowly (25 min dropwise addition) to the flask containing T3P-DIEA at room temperature. The reaction was stirred for 2 h at room temperature and then diluted with ethyl acetate (50 mL), washed with water (50 mL), 0.1 M HCl (50 mL, until pH=1), saturated aqueous NaHCO3 (20 mL, until pH 8) and brine. The organics were then dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes (hexanes contained 0.5% Et3N) to provide product and mixed product fractions. Mixed product fractions were combined and re-purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes (hexanes contained 0.5% Et3N) which when combined with product from the first purification gave 17-amino-6-benzyloxy-11-isopropyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,11,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-12-one (25.9 mg, 23%) as a colorless glass solid. ESI-MS m/z calc. 571.2018, found 572.4 (M+1)+; Retention time: 6.53 minutes (LC Method DD).

To a solution of 17-amino-6-benzyloxy-11-isopropyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,11,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-12-one (25.9 mg, 0.0431 mmol) in EtOH (1.5 mL) was added 10% Pd/C (14 mg, 0.0132 mmol) under nitrogen and stirred the mixture for 10 min at 0° C. The reaction was evacuated and back-filled 3 times with hydrogen gas and stirred at 30° C. at 1 atm of hydrogen gas for 17 h. The reaction was then diluted with EtOH (1 mL) and filtered through packed celite, washing with EtOH (3×1 mL) and the filtrate was concentrated in vacuo to provide 17-amino-6-hydroxy-11-isopropyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,11,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-12-one (20 mg, 92%) as a pale yellow solid and mixture of isomeric conformers. ESI-MS m/z calc. 481.1549, found 482.5 (M+1)+; Retention time: 2.21 minutes (LC Method DD).

To a solution of methyl amine in THF (2 mL of 2 M, 4 mmol) in methanol (1.3 mL) was added acetic acid (0.3 mL) and sodium cyanoborohydride (101 mg, 1.6072 mmol) at 0° C. A solution of 2-[5-amino-6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (220 mg, 0.4026 mmol) in methanol (5.3 mL) was added dropwise at 0° C. over 5 minutes. The reaction was allowed to stir 30 minutes then quenched slowly with saturated aqueous NaHCO3 (100 mL) and diluted with MeOH (200 mL) then stirred 12 h at room temperature and concentrated in vacuo. The aqueous suspension was diluted with ethyl acetate (120 mL) and water (30 mL) and the layers were separated. The organics were washed with 0.1 M HCl (until pH=1) then a mixture of brine (30 mL) and water (50 mL), dried over Na2SO4, filtered and concentrated in vacuo to provide 2-[5-amino-6-[5-[1-benzyloxy-5-(methylamino)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (226 mg, 90%) as a yellow solid. ESI-MS m/z calc. 561.1811, found 562.2 (M+1)+; Retention time: 4.5 minutes (LC Method DD). The product was used directly in the next step without further purification.

To a solution of DIEA (222.6 mg, 0.3 mL, 1.7223 mmol) in ethyl acetate (2.26 mL) was added T3P in ethyl acetate (359 mg, 50% w/w, 0.5641 mmol) at room temperature. A separate solution of crude 2-[5-amino-6-[5-[1-benzyloxy-5-(methylamino)-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)-2-pyridyl]acetic acid (226 mg, 0.2818 mmol, 70% purity) in ethyl acetate (4.5 mL) and NMP (1.13 mL) was added slowly (25 min dropwise addition) to the flask containing T3P-DIEA at room temperature. The reaction was stirred for 5 h at room temperature then diluted with ethyl acetate (50 mL), washed with water (50 mL), 0.1 M HCl (50 mL, until pH=1), saturated aqueous NaHCO3 (20 mL, until pH=8) and brine. The organic solution was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography using a gradient from 0% to 40% ethyl acetate in hexanes (hexanes contained 0.5% Et3N) to provide 17-amino-6-benzyloxy-11-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,11,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-12-one (48.4 mg, 45%). ESI-MS m/z calc. 543.1705, found 544.3 (M+1)+; Retention time: 5.87 minutes (LC Method DD).

In a 500 mL three-necked flask equipped with a nitrogen inlet and a reflux condenser, a solution of tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-6-(2-methylpent-4-enyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (468 mg, 0.6077 mmol) in 1,2-dichloroethane (250 mL) was degassed by bubbling through the solution with nitrogen gas for 18 to 19 hours. A first portion of dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (28 mg, 0.0382 mmol) was added after the temperature reached 60° C. After 40 minutes, a second portion of dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (27 mg, 0.0368 mmol) was added and the reaction was left to stir at 60° C. After a total reaction time of 4 hours the flask was cooled to room temperature, the catalyst was quenched with eight drops of DMSO and the reaction was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient from 0% to 10% ethyl acetate in heptanes to afford 426 mg of tert-butyl N-[(6R)-6-benzyloxy-12-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z mixture) as a yellow oil. This material contained a significant impurity by LCMS and was used directly in the following step without further purification.

A solution of tert-butyl N-[(6R)-6-benzyloxy-12-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,9,14,16-hexaen-17-yl]carbamate (E/Z Mixture) (426 mg) in methanol (12 mL) was purged three times (vacuum then nitrogen atmosphere). Added palladium on carbon (10% w/w, 50% wet, 207 mg, 0.0973 mmol), purged twice under hydrogen atmosphere before leaving the reaction to stir under one atmosphere of hydrogen overnight. Purged once again under nitrogen atmosphere, then the reaction mixture was filtered over a short pad of celite washing with methanol and the filtrate was concentrated. The residue was purified by silica gel chromatography using a gradient from 0% to 10% ethyl acetate in heptanes to afford tert-butyl N-[(6R)-6-hydroxy-12-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,18-triazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (288 mg, 88% over two steps) as a pale yellow oil. ESI-MS m/z calc. 538.2015, found 539.2 (M+1)+; Retention time: 4.65 minutes (LC Method BB).

To a solution of tert-butyl N-[8-fluoro-6-hydroxy-16-oxo-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.12,5.17,11]tricosa-1(21),2,4,7(22),8,10,12,17,19-nonaen-20-yl]carbamate (E/Z mixture) (52 mg, 0.08631 mmol) in ethanol (5 mL) was added Pd/C (32 mg of 10% w/w, 0.03007 mmol) in a round bottom flask equipped with a H2 balloon using a 3-way adaptor. Subjected to vacuum and backfilled with nitrogen gas three times then subjected to vacuum. Filled the flask with hydrogen gas then stirred the mixture for 15 hours. The flask was subjected to vacuum and backfilled with nitrogen gas three times then diluted with ethyl acetate and filtered over Celite. Filtrate was concentrated and the residue was dissolved in a pre-made solution of TFA (200 μL, 3.245 mmol) and dichloromethane (600 μL). Stirred the reaction for about 1 h and the solvents were evaporated. The resultant residue was dissolved in 1 mL of DMSO and purified by reverse phase HPLC using a gradient from 30% to 99% acetonitrile in water (+5 mM HCl) over 15 minutes which gave as a white solid, racemic 20-amino-8-fluoro-6-hydroxy-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.12,5.17,11]tricosa-1(21),2,4,7(22),8,10,17,19-octaen-16-one (19.7 mg, 45%). 1H NMR (400 MHz, methanol-d4) δ 8.01 (dd, J=7.7, 2.2 Hz, 1H), 7.67 (s, 1H), 7.30 (m, 1H), 7.12-6.97 (m, 1H), 2.91-2.77 (m, 4H), 1.98-1.85 (m, 2H), 1.75 (p, J=8.2 Hz, 2H) ppm. ESI-MS m/z calc. 504.10324, found 505.0 (M+1)+; Retention time: 1.21 minutes (LC Method J).

To a stirred solution of tert-butyl N-[6-(1-hydroxypent-4-enyl)-2-[5-[2,2,2-trifluoro-1-(2-fluoro-5-iodo-phenyl)-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (diastereomer pair 1) (55 mg, 0.07510 mmol) in 2-methyltetrahydrofuran (2 mL) at 0° C. (ice bath) was added t-BuOK (50 μL, 0.3832 mmol) (2 M solution in 2-methyltetrahydrofuran) under an inert atmosphere. The mixture was stirred for 5 min and then methyl iodide (10 μL, 0.1606 mmol) was added by a syringe via a rubber septum. The mixture was stirred for additional 30 minutes. The reaction was again cooled in ice bath and more reagents t-BuOK (50 μL, 0.3832 mmol), methyl iodide (10 μL, 0.1606 mmol) were added. After 40 min, the reaction was quenched at 0° C. by the addition of 10 mL water and extracted with diethyl ether (3×20 mL). Organic layers were combined, dried over Na2SO4, concentrated and purified silica gel chromatography using a gradient from 100% hexanes to 50% ethyl acetate in hexanes giving, tert-butyl N-[6-(1-methoxypent-4-enyl)-2-[5-[2,2,2-trifluoro-1-(2-fluoro-5-iodo-phenyl)-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (diastereomer pair 1) (47 mg, 84%). ESI-MS m/z calc. 746.0836, found 747.3 (M+1)+; Retention time: 1.73 minutes (LC Method M).

To a stirred solution of tert-butyl N-[6-(1-methoxypent-4-enyl)-2-[5-[2,2,2-trifluoro-1-(2-fluoro-5-iodo-phenyl)-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (diastereomer pair 1) (47 mg, 0.06297 mmol) in acetonitrile (10 mL) was added palladium (II) acetate (3 mg, 0.01336 mmol) followed by tris-o-tolylphosphane (8 mg, 0.02628 mmol) and triethylamine (60 μL, 0.4305 mmol) and the solution was bubbled with N2 for 2 min then heated at 80° C. for 16 h. Cooled the mixture to room temperature, concentrated down to about 5 mL volume and filtered through Celite and filtrate was concentrated. The resultant brown residue was purified by silica gel chromatography using a shallow gradient from 100% hexanes to 50% ethyl acetate in hexanes to afford tert-butyl N-[8-fluoro-6-hydroxy-16-methoxy-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.12,5.17,11]tricosa-1(21),2,4,7(22),8,10,12,17,19-nonaen-20-yl]carbamate (diastereomer pair 1, E/Z mixture) 24 mg, 62%). ESI-MS m/z calc. 618.1713, found 619.2 (M+1)+; Retention time: 1.41, 1.28, 1.35 and 1.44 minutes (LC Method M). Multiple isomeric products: E/Z isomers at olefin in the macrocycle along with diastereomers; the mixture was taken directly to the next step.

To a stirred solution of methyl 3-(tert-butoxycarbonylamino)-6-(1-hydroxypent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylate (580 mg, 1.434 mmol) in dichloromethane (15 mL) was added DIEA (556 mg, 4.302 mmol) at room temperature. After 15 min, methane sulfonyl chloride (231 mg, 2.017 mmol) was added dropwise through a rubber septum under inert atmosphere. The reaction mixture was stirred at room temperature and its progress was monitored by UPLC. After 2 hours, the reaction mixture was quenched by saturated solution of NaHCO3 and extracted with dichloromethane (3×30 mL). The organic phases were combined, dried over Na2SO4, concentrated and purified by silica gel chromatography using 75:25 hexanes/ethyl acetate which gave methyl 3-(tert-butoxycarbonylamino)-6-(1-methylsulfonyloxypent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylate (385 mg, 56%). ESI-MS m/z calc. 482.13345, found 483.1 (M+1)+; Retention time: 1.51 minutes (LC Method J).

To a stirred solution of methyl 3-(tert-butoxycarbonylamino)-6-(1-methylsulfonyloxypent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylate (385 mg, 0.7980 mmol) in acetonitrile (15 mL) was added tetrabutylammonium cyanide (430 mg, 1.522 mmol). The reaction mixture was heated at 70° C. for 75 minutes then cooled and quenched with a saturated aqueous solution of NaHCO3 then extracted with ethyl acetate (3×20 mL). The organic phases were combined, dried over Na2SO4, filtered and concentrated. The residue was purified by reverse phase chromatography using a gradient from 30% to 95% acetonitrile in water (+5 mM HCl) over 10 minutes which gave methyl 3-(tert-butoxycarbonylamino)-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylate (140 mg, 42%). ESI-MS m/z calc. 413.15625, found 414.1 (M+1)+; Retention time: 1.61 minutes (LC Method J).

Into a solution of methyl 3-(tert-butoxycarbonylamino)-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylate (2.648 g, 6.4056 mmol) in THF (24 mL) and water (8 mL) was added LiOH (777 mg, 32.445 mmol) at ambient temperature. The reaction mixture was stirred for 1 hour. The reaction was diluted with saturated ammonium chloride (50 mL) and the pH was adjusted to 5 with 1N HCl. The aqueous solution was extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate and concentrated under vacuum to provide 3-(tert-butoxycarbonylamino)-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylic acid (2.572 g, quant.) as a clear gel. ESI-MS m/z calc. 399.1406, found 400.2 (M+1)+; Retention time: 3.56 minutes (LC Method G).

3-(tert-Butoxycarbonylamino)-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylic acid (180 mg, 0.4507 mmol) and 1-(3-bromophenyl)-2,2,2-trifluoro-ethanone (285 mg, 1.126 mmol) were dissolved in DMF (9 mL) and heated to 45° C. Then, (isocyanoimino)triphenylphosphorane (341 mg, 1.128 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate (5 mL), added water (10 mL), and extracted with ethyl acetate (3×10 mL), washed with brine, dried over Na2SO4, filtered, and concentered under reduced pressure. The residue was purified by reverse phase chromatography using a gradient from 30% to 100% acetonitrile in water (+5 mM HCl) over 12 minutes which gave as a mixture of stereoisomers, tert-butyl N-[2-[5-[1-(3-bromophenyl)-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (159 mg, 52%). ESI-MS m/z calc. 675.0916, found 678.1 (M+1)+; Retention time: 1.55 minutes (LC Method M).

To a stirred solution of tert-butyl N-[2-[5-[1-(3-bromophenyl)-2,2,2-trifluoro-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)-3-pyridyl]carbamate (121 mg, 0.1789 mmol) in acetonitrile (7 mL) was added palladium acetate (12.1 mg, 0.05390 mmol) followed by tris-o-tolylphosphane (33.7 mg, 0.1107 mmol) and triethyl amine (56 mg, 0.5534 mmol) and the solution was bubbled with N2 for 1 min then sealed and heated in a microwave for 2 hours at 125° C. The mixture was cooled to room temperature and filtered through a pad of Celite washing with ethyl acetate. The filtrate was evaporated and the residue was purified by silica gel chromatography using 75:25 hexanes/ethyl acetate giving the desired product as a mixture of diastereomers as well as E/Z isomers. Without further purification, the residue was dissolved in MeOH (3 mL) and transferred to a high-pressure reactor then added 10% Pd/C (19 mg, 0.01785 mmol). The reactor was sealed, filled with nitrogen gas and evacuated three times. Finally, the reactor was evacuated and then filled with H2 up to 60 psi. The mixture was stirred at room temperature for 6 h. The reactor was depressurized, and the mixture was filtered through a pad of Celite washing with methanol. The filtrate was evaporated and the residue was then purified by reverse phase chromatography using a gradient from 35% to 90% acetonitrile in water (+5 mM HCl) over 10 minutes which gave as mixture of diastereomers, tert-butyl N-[16-cyano-6-hydroxy-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.12,5.17,11]tricosa-1(21),2,4,7,9,11(22),17,19-octaen-20-yl]carbamate (31 mg, 29%). ESI-MS m/z calc. 597.1811, found 598.1 (M+1)+; Retention time: 1.79 minutes and 1.84 minutes (LC Method J).

A vial was charged with 2,2,2-trifluoro-1-(3-iodophenyl)ethanone (1.078 g, 3.5931 mmol) in DCM (10 mL). (N-isocyanoimino)triphenylphosphorane (1.146 g, 3.6013 mmol) was added to the reaction mixture. The reaction was stirred at room temperature for 5 minutes before a solution of 3-(tert-butoxycarbonylamino)-6-(1-cyanopent-4-enyl)-5-(trifluoromethyl)pyridine-2-carboxylic acid (1.2 g, 3.0048 mmol) in DCM (4.7 mL) was added. The reaction was stirred at room temperature for 1 hour. The reaction was then concentrated under vacuum and purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexane to furnish tert-butyl N-[6-(1-cyanopent-4-enyl)-2-[5-[2,2,2-trifluoro-1-hydroxy-1-(3-iodophenyl)ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (1.21 g, 50%) as an off-white solid. ESI-MS m/z calc. 723.0777, found 724.1 (M+1)+; Retention time: 4.19 minutes (LC Method G).

To a solution of tert-butyl N-[6-(1-cyanopent-4-enyl)-2-[5-[2,2,2-trifluoro-1-hydroxy-1-(3-iodophenyl)ethyl]-1,3,4-oxadiazol-2-yl]-5-(trifluoromethyl)-3-pyridyl]carbamate (34 mg, 0.0470 mmol) in acetonitrile (5 mL) was added tri-o-tolylphosphine (3 mg, 0.0099 mmol) and TEA (23.958 mg, 0.033 mL, 0.2368 mmol). The reaction was purged with argon for 5 minutes. Pd(OAc)2 (1 mg, 0.0045 mmol) was added to the reaction mixture. The reaction was purged with argon for another 2 minutes. The vial was sealed and heated to 80° C. overnight. The reaction mixture was concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexanes to furnish as a complex mixture, tert-butyl N-[16-cyano-6-hydroxy-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.1{circumflex over ( )}{2,5}0.1{circumflex over ( )}{7,11}]tricosa-1(21),2,4,7,9,11(22),12,17,19-nonaen-20-yl]carbamate (mixture of isomers) (26 mg, 93%) as an orange solid. ESI-MS m/z calc. 595.1654, found 596.4 (M+1)+; Retention time: 3.84 minutes (LC Method G).

Into a solution of tert-butyl N-[16-cyano-6-hydroxy-6,18-bis(trifluoromethyl)-23-oxa-3,4,21-triazatetracyclo[15.3.1.1{circumflex over ( )}{2,5}0.1{circumflex over ( )}{7,11}]tricosa-1(21),2,4,7,9,11(22),12,17,19-nonaen-20-yl]carbamate (mixture of isomers) (623 mg, 1.0043 mmol) in methanol (50 mL) was added 10% Pd/C (200 mg, 0.1879 mmol). The reaction was hydrogenated at 1 atm of H2 for 16 hours. The catalyst was removed by filtration through a pad of Celite. The filtrate was concentrated, and the residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexane to furnish three peaks in order of elution:

Method 13: Final Product Synthesis by Ring-Closing Metathesis Route

To a solution of 6-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid, 6-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid or 6-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)pent-4-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid (1 equivalent) in DMF or THE (20 volume equivalents) was added N-substituted butenyl amine (3 equivalents), a coupling agent such as HATU (2 to 5 equivalents) or T3P (5 to 10 eq of 50% w/w solution in ethyl acetate) and then a base such as Et3N (5 to 10 equivalents) or DIEA (5 to 10 equivalents) was added. The mixture was stirred at room temperature and the reaction progress was monitored by LCMS. After the starting material was consumed, the reaction mixture was diluted with diethyl ether or ethyl acetate, washed with water, dried (MgSO4), filtered and evaporated. The residue was purified by silica gel chromatography using ethyl acetate and hexanes which gave the desired bis-olefin amide products.

To a two-neck flask with constant nitrogen bubbling through the solution was added bis-olefin (1 equivalent) in 1,2-dichloroethane (200 to 2500 volume equivalents) and the solution was heated to 50 to 60° C. Then added dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (˜0.25 to 0.4 equivalents) in 1,2-dichloroethane (˜60 volume equivalents) via syringe and the mixture was stirred at 50 to 70° C. for several hours while monitoring the reaction progress by LCMS. After most of the starting material was consumed, the reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and hexanes which gave the macrocyclization product as E/Z mixture that was taken directly to the next reaction.

To a solution of E/Z mixture of macrocycle (1 equivalent) in acetic acid (˜25 volume equivalents) was added Pd/C (˜0.5 eq of 10% w/w). The mixture was subjected to 200 psi hydrogen atmosphere in a stainless steel pressure vessel while monitoring the reaction progress for both olefin hydrogenation and de-benzylation by LCMS. After completion of the reaction, the mixture was filtered through Celite, washing well with ethyl acetate and then the filtrate was concentrated. The residue was purified by reverse phase chromatography using acetonitrile and water (+5 mM HCl) which provided the desired product.

Step 4: Removal of Boc Group

To a solution of the Boc-containing macrocycle (1 equivalent) in a solvent such as DCM or water (˜20 volume equivalents) was added TFA (˜20-200 equivalents) and triisopropylsilane (2 equivalents) and the resulting mixture was stirred at room temperature for several hours while monitoring the reaction progress by LCMS. After the starting material was consumed, the solvents were evaporated and the residue was dissolved in DCM and washed with saturated aqueous NaHCO3, brine, dried over Na2SO4, filtered, and concentrated to give the desired final product which, if needed, was further purified by C18 reverse phase HPLC using acetonitrile and water with HCl modifier.

Step 5: Chiral Separation

Racemic compounds were purified by chiral SFC using a suitable chiral column and mobile phase as indicated in Table 4 which gave as the first eluting enantiomer, enantiomer 1 and the later eluting enantiomer, enantiomer 2.

Representative Synthesis of Final Compounds by Method 13 (Ring-Closing Metathesis Route)

To a two neck flask with constant nitrogen bubbling through the solution was added tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-[but-3-enyl(methyl)carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (108 mg, 0.1613 mmol) in 1,2-dichloroethane (25 mL) and the solution was heated to 50° C. Then, via syringe, was added dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (30 mg, 0.04 mmol) in 1,2-dichloroethane (5 mL), and the mixture stirred at 70° C. for 1 h. The reaction mixture was evaporated under reduced pressure and purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes to provide as a white solid, tert-butyl N-[6-benzyloxy-12-methyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (83 mg, 80%). ESI-MS m/z calc. 641.2073, found 642.3 (M+1)+; Retention time: 0.79 minutes (LC Method R).

To a solution of tert-butyl N-[6-benzyloxy-12-methyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (80 mg, 0.1247 mmol) in acetic acid (2.16 mL) was added Pd/C (53 mg of 10% w/w, 0.05 mmol). The mixture was stirred for 16 h under 200 psi hydrogen atmosphere in a stainless steel pressure vessel. The reaction mixture was filtered through a silica plug, washing well with ethyl acetate and then the filtrate was concentrated to provide as a white solid, tert-butyl N-[6-hydroxy-12-methyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (60 mg, 87%). ESI-MS m/z calc. 553.17596, found 554.3 (M+1)+; Retention time: 0.54 minutes (LC Method R).

tert-Butyl N-[6-hydroxy-12-methyl-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (60 mg, 0.108 mmol) was dissolved in DCM (1 mL) and to the mixture was added TFA (417 μL, 5.41 mmol) and the mixture was stirred at room temperature for 2 h. The solvents were evaporated then the residue was dissolved into DCM and washed with saturated aqueous NaHCO3. The organic layer was concentrated to provide as a white solid, racemic 17-amino-6-hydroxy-12-methyl-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-13-one which was used directly in the next step.

Final Examples Prepared by Method 13 and Analogous to the

Representative Procedure Described for Example 40

Amine Structure

Method of

Amine

Example
synthesized)
Carboxylic Acid

number
Mass Spec data
Structure

Final Examples Prepared by Method 13 and Analogous to the

Representative Procedure Described for Example 40

Example

Final Product

number
Final Product structure
IUPAC Name

1final product purified by preparative HPLC eluting with a gradient from 1% to 80% acetonitrile in 5 mM aqueous HCl at 50 mL/min over 14 min through a Luna 5 μM C18 100 Å 75 × 30 mm column

2compounds were prepared by Method 13, however the diastereomeric products resulting from step 3 were separated during the reverse-phase HPLC purification and each was taken into step 4 separately

3triisopropylsilane was not used in step 4 of Method 13

4final product purified by preparative HPLC eluting with a gradient of 1% to 99% acetonitrile in 5 mM aqueous HCl

5cyclopropyl group present in starting amine opened to the n-propyl group during step 3 of Method 13

6bicyclo[1.1.1]pentane group present in starting amine opened to the trans-3-methyl cyclobutaneduring step 3 of Method 13

7purified by reverse-phase preparative chromatography using a C18 column and a gradient eluent of 1% to 50% acetonitrile in water containing 5 mM HCl

8purified by reverse-phase preparative chromatography using a C18 column and a gradient eluent of 1% to 30% acetonitrile in water containing 5 mM HCl

Characterization Data and chiral SFC Conditions (if

applicable) for Final Compounds Prepared by Method 13

SFC conditions to

Example
LCMS data and

separate enantiomers

number
method
NMR data (shifts in ppm)
from racemic final product

enantiomer to elute

enantiomer to elute

obscured by solvent.

To a solution of 6-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid (53 mg, 0.08797 mmol) and N-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]but-3-en-1-amine (25 mg, 0.08797 mmol) (prepared by Method 12) in DMF (1 mL) was added DIEA (108.3 mg, 0.838 mmol) followed by HATU (159.2 mg, 0.4187 mmol). The mixture was stirred at room temperature for 15 minutes then diluted with ether, washed with water (2×), dried (MgSO4), filtered and evaporated. The residue was purified by silica chromatography using a gradient from 0% to 25% ethyl acetate in hexanes to afford as a white solid, tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl-but-3-enyl-carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (51 mg, 71%). ESI-MS m/z calc. 811.31683, found 812.32 (M+1)+; Retention time: 0.87 minutes (LC Method T).

To a one neck flask with nitrogen gas bubbling in was added tert-butyl N-[2-[5-[(1R)-1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-6-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl-but-3-enyl-carbamoyl]-5-(trifluoromethyl)-3-pyridyl]carbamate (40 mg, 0.04927 mmol) in 1,2-dichloroethane (100 mL) and the mixture was heated to 60° C. Then, dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (11.25 mg, 0.01533 mmol) in 1,2-dichloroethane (2.5 mL) was added via syringe and the resulting mixture was heated to 60° C. and stirred for 45 min. The reaction mixture was concentrated under reduced pressure and purified by silica gel chromatography using a gradient from 0% to 30% ethyl acetate in hexanes to afford as a white solid, tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (34.1 mg, 88%). ESI-MS m/z calc. 783.2855, found 784.23 (M+1)+; Retention time: 0.87 minutes (LC Method T).

Part 1: A mixture of tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (40 mg, 0.05104 mmol) and Pd/C (5.432 mg of 10% w/w, 0.005104 mmol) in ethyl acetate (2 mL) was stirred at room temperature under a balloon of hydrogen at atmospheric pressure. After one hour, additional Pd/C (approximately 4.052 mg of 10% w/w, 0.003808 mmol) was added and the reaction mixture was stirred at the same conditions for an additional hour. The mixture was then filtered and the filtrate was evaporated to provide crude tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(3-methylcyclobutyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (20 mg, 50%) (ESI-MS m/z calc. 787.31683, found 788.41 (M+1)+; Retention time: 2.21 minutes (LC Method A)). This material was contaminated with tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (10 mg, 25%) (ESI-MS m/z calc. 785.3012, found 788.41 (M+1)+; Retention time: 2.21 minutes (LC Method A)). This crude material was used directly in the next part without further purification.

Part 2: The mixture of tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(3-methylcyclobutyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-17-yl]carbamate (20 mg, 0.0254 mmol) and tert-butyl N-[(6R)-6-benzyloxy-12-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]-13-oxo-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (10 mg, 0.01273 mmol) obtained in part 1 was stirred in TFA (1 mL) at room temperature for 16 h and then the solvent was evaporated. The residue was dissolved in methanol and filtered then purified by preparative HPLC to obtain as a white solid, (6R)-17-amino-12-[[4-(1-bicyclo[1.1.1]pentanyl)phenyl]methyl]-6-hydroxy-6,15-bis(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(18),2,4,14,16-pentaen-13-one (7 mg, 46%). ESI-MS m/z calc. 595.2018, found 596.21 (M+1)+; Retention time: 2.14 minutes (LC Method A).

To a solution of methyl 3-amino-5-bromo-pyridine-2-carboxylate (3.15 g, 13.63 mmol) in acetonitrile (272.6 mL) was added 1-bromopyrrolidine-2,5-dione (2.668 g, 14.99 mmol) and the mixture was stirred at room temperature overnight. Concentrated the reaction mixture and recrystallized the product from hot toluene to afford as a pale orange solid, methyl 3-amino-5,6-dibromo-pyridine-2-carboxylate (4.1 g, 97%). ESI-MS m/z calc. 307.8796, found 310.86 (M+1)+; Retention time: 0.49 minutes (LC Method S).

Methyl 5,6-dibromo-3-(tert-butoxycarbonylamino)pyridine-2-carboxylate was prepared from methyl 3-amino-5,6-dibromo-pyridine-2-carboxylate using a procedure analogous to step 1 of Method 11.

To a round bottom flask was added methyl 5-bromo-3-(tert-butoxycarbonylamino)-6-ethynyl-pyridine-2-carboxylate (131 mg, 0.3688 mmol), potassium peroxymonosulfate (225.6 mg, 0.3688 mmol), NaHCO3 (77.45 mg, 0.9220 mmol) and ruthenium on carbon (7.455 mg, 0.0037 mmol, 5% w/w). Added a 1:1 mixture of MeCN (1.048 mL) and water (1.048 mL) to the reaction mixture and stirred at room temperature for 3 h. Then more potassium peroxymonosulfate (225.6 mg, 0.3688 mmol) was added and the mixture stirred for another 2 hours. The mixture was filtered through Celite washing with ethyl acetate. The filtrate was diluted with citric acid (20 mL) and extracted with ethyl acetate (2×20 mL). The combined organic layers were washed with brine, dried (Na2SO4), filtered and concentrated to provide 3-bromo-5-(tert-butoxycarbonylamino)-6-methoxycarbonyl-pyridine-2-carboxylic acid (138.4 mg, quant.): ESI-MS m/z calc. 374.01135, found 376.94 (M+1)+; Retention time: 0.37 minutes (LC Method R).

To a solution of methyl 5-bromo-6-[but-3-enyl-(3,3-difluorocyclobutyl)carbamoyl]-3-(tert-butoxycarbonylamino)pyridine-2-carboxylate (96 mg, 0.19 mmol) in THF (960 μL) were added methanol (960 μL) and water (480 μL). Lithium hydroxide (84.94 mg, 3.547 mmol) was added and the mixture was heated at to 60° C. for 1 h. THF and methanol were removed under reduced pressure. 1 M HCl solution was added until acidic, then the aqueous layer was extracted with ethyl acetate (3×75 mL). The organic phases were combined, washed with brine (75 mL), dried on anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford as a white solid 5-bromo-6-[but-3-enyl-(3,3-difluorocyclobutyl)carbamoyl]-3-(tert-butoxycarbonylamino)pyridine-2-carboxylic acid (93 mg, quant.): ESI-MS m/z calc. 503.08673, found 504.12 (M+1)+; Retention time: 0.56 minutes (LC Method R).

To a nitrogen purged flask was added tert-butyl N-[2-[5-[1-benzyloxy-1-(trifluoromethyl)but-3-enyl]-1,3,4-oxadiazol-2-yl]-5-bromo-6-[but-3-enyl-(3,3-difluorocyclobutyl)carbamoyl]-3-pyridyl]carbamate (75 mg, 0.09913 mmol) in 1,2-dichloroethane (17.36 mL) and it was heated to 60° C. Then added via syringe dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][[5-[(dimethylamino)sulfonyl]-2-(1-methylethoxy-O)phenyl]methylene-C]ruthenium(II) (18.18 mg, 0.02478 mmol) in 1,2-dichloroethane (2 mL), and the solution heated to 60° C. and let stir for 45 min. The reaction mixture was concentrated and purified by silica gel chromatography using 0% to 30% ethyl acetate in hexanes to afford as a white solid, tert-butyl N-[6-benzyloxy-15-bromo-12-(3,3-difluorocyclobutyl)-13-oxo-6-(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,8,14(18),15-hexaen-17-yl]carbamate (E/Z mixture) (65 mg, 90%): ESI-MS m/z calc. 727.1429, found 728.2 (M+1)+; Retention time: 0.67 minutes (LC Method T).

TFA (800 μL) was added to tert-butyl N-[6-benzyloxy-15-bromo-12-(3,3-difluorocyclobutyl)-13-oxo-6-(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-17-yl]carbamate (42 mg, 0.05749 mmol) and stirred at room temperature overnight. After concentrating the mixture, the residue was dissolved in DCM and washed with saturated aqueous NaHCO3, then brine, dried over Na2SO4, filtered and concentrated. Purification by silica gel chromatography using 0% to 40% ethyl acetate in hexanes afford as a white solid, 17-amino-15-bromo-12-(3,3-difluorocyclobutyl)-6-hydroxy-6-(trifluoromethyl)-19-oxa-3,4,12,18-tetrazatricyclo[12.3.1.12,5]nonadeca-1(17),2,4,14(18),15-pentaen-13-one (29 mg, 93%): ESI-MS m/z calc. 539.05914, found 540.0 (M+1)+; Retention time: 0.37 minutes (LC Method R).

Method 14: Final Product Synthesis by Macrolactamization Route

The appropriate aldehyde (for example, methyl 6-[5-[1-benzyloxy-6-oxo-1-(trifluoromethyl)hexyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate, methyl 6-[5-[(1R)-1-benzyloxy-6-oxo-1-(trifluoromethyl)hexyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate, methyl 6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate, methyl 5-(tert-butoxycarbonylamino)-6-[5-[2,2,2-trifluoro-1-[2-fluoro-5-(3-oxopropyl)phenyl]-1-hydroxy-ethyl]-1,3,4-oxadiazol-2-yl]-3-(trifluoromethyl)pyridine-2-carboxylate, or methyl 6-[5-[1-benzyloxy-2,2,2-trifluoro-1-(3-oxopropoxymethyl)ethyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate) (1 eq) was combined with isopropyl amine (˜5 equivalents) in dichloromethane or MeOH-acetic acid mixture (2 to 5 volume equivalents) and stirred for about 30 minutes at room temperature. Then reducing agent, sodium triacetoxyborohydride or sodium cyanoborohydride (˜5 equivalents) was added, and the reaction mixture was stirred for an additional 30 to 60 minutes at room temperature. The reaction progress was monitored by LCMS. After completion of the reaction, the reaction was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution and brine. The organic solution was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography using ethyl acetate/hexanes or DCM/MeOH to give the desired secondary amines.

Amino ester intermediate (1 equivalent) was dissolved in dioxane/water or MeOH/THF/water (5 to 10 volume equivalents) and LiOH or NaOH (˜5 equivalents) was added. The reaction mixture was stirred at room temperature for a few hours to overnight. The reaction progress was monitored by LCMS. After completion of the reaction, diluted with ethyl acetate and saturated aqueous ammonium chloride. The aqueous pH was adjusted to ˜5 with 1N HCl. The resulting two layers were separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with 1N HCl and brine, then concentrated under vacuum to furnish the desired acid.

To a solution of amino acid (1 equivalent) in DMF (300 to 500 volume equivalents) was added DIEA (˜5 equivalents) followed by HATU (˜1.5 equivalents). The reaction mixture was stirred at room temperature for a couple of hours while monitoring the reaction progress by LCMS. After completion of the reaction, the reaction mixture was diluted with ethyl acetate and water. The two layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography using ethyl acetate in hexanes which provided the desired macrocycles.

Debenzylation of the macrocycle (1 equivalent) was performed using 10% Pd/C (0.2 to 0.5 equivalents) in ethyl acetate, ethanol or methanol (10 to 50 volume equivalents) while stirring under a hydrogen balloon (1 atmosphere of gas) for 12 to 24 hours. The catalyst was filtered off through a pad of Celite. The filtrate was concentrated under vacuum to furnish the desired alcohol.

The N-Boc protected macrocycle (1 equivalent) was dissolved in DCM or water (˜20 volume equivalents) and to the mixture was added TFA (˜20 equivalents) and the mixture was stirred at room temperature for a couple of hours while monitoring the reaction progress by LCMS. After the starting material was consumed, the solvents were evaporated and the residue was dissolved in DCM then washed with saturated aqueous NaHCO3 and brine then dried over Na2SO4, filtered and concentrated to give the desired product.

Step 6: Chiral Separation

Racemic compounds were purified by chiral SFC using a suitable chiral column and mobile phase as indicated in Table 6 giving as the first eluting enantiomer, enantiomer 1 and the later eluting enantiomer, enantiomer 2.

Representative Synthesis of Final Compounds by Method 14 (Macrolactamization Route)

Into a solution of 3,3-difluorocyclobutanamine (hydrochloride salt) (832 mg, 5.6795 mmol) in a solvent mixture of methanol (4.5 mL) and acetic acid (0.5 mL) was added sodium cyanotrihydroborate (140 mg, 2.2278 mmol) at 0° C. A solution of methyl 6-[5-[1-benzyloxy-5-oxo-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (382 mg, 0.5613 mmol) in methanol (1.8 mL) and acetic acid (0.2 mL) was added drop-wise at 0° C. The reaction was stirred at the same temperature for 10 minutes, then raised to room temperature and stirred for 1 hour. The reaction was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium bicarbonate aqueous solution (30 mL) and brine (20 mL). The organic solution was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography using 0 to 70% ethyl acetate in hexanes to furnish methyl 6-[5-[1-benzyloxy-5-[(3,3-difluorocyclobutyl)amino]-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (347 mg, 83%) as a yellow gel. ESI-MS m/z calc. 737.246, found 738.5 (M+1)+; Retention time: 3.69 minutes (LC Method G).

Into a solution of methyl 6-[5-[1-benzyloxy-5-[(3,3-difluorocyclobutyl)amino]-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylate (347 mg, 0.4657 mmol) in dioxane (3 mL) and water (3 mL) was added LiOH (69 mg, 2.8812 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction was diluted with ethyl acetate (50 mL) and saturated aqueous ammonium chloride (30 mL). The aqueous pH was adjusted to 5 with 1N HCl. Two layers were separated and the aqueous layer was extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with 1N HCl (2×20 mL) and brine (50 mL), then concentrated under vacuum to furnish 6-[5-[1-benzyloxy-5-[(3,3-difluorocyclobutyl)amino]-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid (362 mg, 91%) as a clear gel. ESI-MS m/z calc. 723.2303, found 724.5 (M+1)+; Retention time: 3.24 minutes (LC Method G).

A round bottom flask was charged with a solution of 6-[5-[1-benzyloxy-5-[(3,3-difluorocyclobutyl)amino]-1-(trifluoromethyl)pentyl]-1,3,4-oxadiazol-2-yl]-5-(tert-butoxycarbonylamino)-3-(trifluoromethyl)pyridine-2-carboxylic acid (342 mg, 0.4005 mmol) in DMF (150 mL). DIEA (259.70 mg, 0.35 mL, 2.0094 mmol) was added to the reaction mixture, followed by HATU (229 mg, 0.6023 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction was diluted with ethyl acetate (150 mL) and water (150 mL). Two layers were separated and the aqueous layer was extracted with ethyl acetate (2×150 mL). The combined organic layers were washed with brine (3×100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography using a gradient from 0% to 20% ethyl acetate in hexane to furnish tert-butyl N-[6-benzyloxy-11-(3,3-difluorocyclobutyl)-12-oxo-6,14-bis(trifluoromethyl)-18-oxa-3,4,11,17-tetrazatricyclo[11.3.1.12,5]octadeca-1(16),2,4,13(17),14-pentaen-16-yl]carbamate (93 mg, 33%) as a white foam. ESI-MS m/z calc. 705.2197, found 706.3 (M+1)+; Retention time: 4.09 minutes (LC Method G).

Into a solution of tert-butyl N-[6-benzyloxy-11-(3,3-difluorocyclobutyl)-12-oxo-6,14-bis(trifluoromethyl)-18-oxa-3,4,11,17-tetrazatricyclo[11.3.1.12,5]octadeca-1(16),2,4,13(17),14-pentaen-16-yl]carbamate (93 mg, 0.1318 mmol) in ethyl acetate (10 mL) was added 10% Pd/C (70 mg, 10% w/w, 0.0658 mmol). The reaction was stirred under 1 atmosphere of hydrogen gas for 24 hours. The catalyst was filtered off through a pad of Celite. The filtrate was concentrated under vacuum to furnish tert-butyl N-[11-(3,3-difluorocyclobutyl)-6-hydroxy-12-oxo-6,14-bis(trifluoromethyl)-18-oxa-3,4,11,17-tetrazatricyclo[11.3.1.12,5]octadeca-1(16),2,4,13(17),14-pentaen-16-yl]carbamate (82 mg, quant.) as a clear gel. ESI-MS m/z calc. 615.1728, found 616.4 (M+1)+; Retention time: 3.61 minutes (LC Method G).

Final Examples Prepared by Method 14 and Following the Procedure

Described for Representative Example 163

Example
Amine Structure Amine Method of
Carboxylic Acid
Final Product 
Final Product IUPAC

number
Preparation (if synthesized) Mass Spec data
Structure
structure
Name

found

*utilizing step 1 and the first part of step 2 of Method 1 (N-Boc protection, purification and subsequent N-Boc deprotection described in part 2 of step 2 and step 3 of Method 1 was not performed on this amine)

Characterization Data and chiral SFC Conditions (if applicable) for Final Compounds Prepared by Method 14

SFC conditions to

Example
LCMS data and

separate enantiomers from

number
method
NMR data (shifts in ppm)
racemic final product

Chiral SFC using a IG

and a gradient of 18%

minutes using a flow

rate of 70 mL/min. First

enantiomer to elute

Chiral SFC using a IG

and a gradient of 18%

minutes using a flow

rate of 70 mL/min.

Second enantiomer to

Chiral SFC using a IA

5 μm particle size) and a

gradient of 11%

minutes using a flow

rate of 10 mL/min. First

enantiomer to elute

Chiral SFC using a IA

5 μm particle size) and a

gradient of 11%

minutes using a flow

rate of 10 mL/min.

Second enantiomer to

Chiral SFC using a IA

5 μm particle size) and a

gradient of 11%

minutes using a flow

rate of 10 mL/min. First

enantiomer to elute

Chiral SFC using a IA

5 μm particle size) and a

gradient of 11%

minutes using a flow

rate of 10 mL/min.

Second enantiomer to

minutes using a flow

rate of 10 mL/min. First

enantiomer to elute

0.72 (m, 1H).
minutes using a flow

rate of 10 mL/min.

Second enantiomer to

over 14.5 minutes using

a flow rate of 40

mL/min. First

enantiomer to elute

a flow rate of 40

enantiomer to elute

(m, 1H).
a flow rate of 40

mL/min. First

enantiomer to elute

(m, 2H).
a flow rate of 40

enantiomer to elute

over 14.5 minutes using

a flow rate of 40

mL/min. First

enantiomer to elute

over 14.5 minutes using

a flow rate of 40

enantiomer to elute

over 14.5 minutes using

a flow rate of 40

mL/min. First

enantiomer to elute

mM NH3) and CO2

over 14.5 minutes using

a flow rate of 40

enantiomer to elute

Ussing chamber experiments were performed using human bronchial epithelial (HBE) cells derived from CF subjects heterozygous for F508del and a minimal function CFTR mutation (F508del/MF-HBE) and cultured as previously described (Neuberger T, Burton B, Clark H, Van Goor F; Methods Mol. Biol. 2011:741:39-54). After four days the apical media was removed, and the cells were grown at an air liquid interface for >14 days prior to use. This resulted in a monolayer of fully differentiated columnar cells that were ciliated, which are characteristic of human bronchial airway epithelia.

To isolate the CFTR-mediated short-circuit (ISC) current, F508del/MF-HBE grown on Costar® Snapwell™ cell culture inserts were mounted in an Ussing chamber and the transepithelial ISC was measured under voltage-clamp recording conditions (Vhold=0 mV) at 37° C. The basolateral solution contained (in mM) 145 NaCl, 0.83 K2HPO4, 3.3 KH2PO4, 1.2 MgCl2, 1.2 CaCl2), 10 Glucose, 10 HEPES (pH adjusted to 7.4 with NaOH) and the apical solution contained (in mM) 145 NaGluconate, 1.2 MgCl2, 1.2 CaCl2), 10 glucose, 10 HEPES (pH adjusted to 7.4 with NaOH) and 30 μM amiloride to block the epithelial sodium channel. Forskolin (20 μM) was added to the apical surface to activate CFTR, followed by apical addition of a CFTR inhibitor cocktail consisting of BPO, GlyH-101 and CFTR inhibitor 172 (each at 20 μM final assay concentration) to specifically isolate CFTR currents. The CFTR-mediated ISC (μA/cm2) for each condition was determined from the peak forskolin response to the steady-state current following inhibition.

Identification of Potentiator Compounds

The activity of the CFTR potentiator compounds on the CFTR-mediated ISC was determined in Ussing chamber studies as described above. The F508del/MF-HBE cell cultures were incubated with the potentiator compounds at a range of concentrations in combination with 10 μM (14S)-8-[3-(2-{dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione for 18-24 hours at 37° C. and in the presence of 20% human serum. The concentration of potentiator compounds and (14S)-8-[3-(2-{dispiro[2.0.2.1]heptan-7-yl}ethoxy)-1H-pyrazol-1-yl]-12,12-dimethyl-2λ6-thia-3,9,11,18,23-pentaazatetracyclo[17.3.1.111,14.05,10]tetracosa-1(22),5,7,9,19(23),20-hexaene-2,2,4-trione used during the 18-24 hours incubations was kept constant throughout the Ussing chamber measurement of the CFTR-mediated ISC to ensure compounds were present throughout the entire experiment. The efficacy and potency of the putative F508del potentiators was compared to that of the known potentiator, ivacaftor (N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide).

The following table represents CFTR modulating activity for representative compounds of the invention generated using the assay described in this example (EC50: +++ is <500 nM; ++ is 500 nM−1 μM; + is >1 μM; and ND is “not determined in this assay”).

pound

Other Embodiments

The foregoing discussion discloses and describes merely exemplary embodiments of this disclosure. One skilled in the art will readily recognize from such discussion and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of this disclosure as defined in the following claims.