TYK2 inhibitors and compositions and methods thereof

The invention provides a novel class of therapeutic agents that are safe and effective TYK2 inhibitors and pharmaceutical compositions of these compounds and methods of preparation and use thereof against various TYK2-mediated diseases and disorders.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application claims the benefit of priority to PCT International application Nos. PCT/CN2022/139649, filed Dec. 16, 2022; PCT/CN2022/106876, filed Jul. 20, 2022 and PCT/CN2021/138744, filed on Dec. 16, 2021, the entire content of each of which is incorporated herein by reference for all purposes.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to novel compounds and methods for their therapeutic use. More particularly, the invention provides a novel class of tyrosine kinase 2 inhibitors as well as pharmaceutical compositions of these compounds and methods of preparation and use thereof against various diseases and conditions.

BACKGROUND OF THE INVENTION

Janus kinase (JAK) is a family of intracellular, nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the Janus kinase-Signal Transduction Activators of Transcription (JAK-STAT) pathway. There are four members in the JAK family of enzymes in humans, i.e., JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). The family is defined by the presence of two adjacent kinase domains, JH1 and JH2, of which JH1 performs the phosphorylation involved in pathway activation whereas JH2 regulates JH1 function. (Thomas, et al., 2015British Journal of Cancer113, 365-371.)

These cytoplasmic tyrosine kinases are associated with membrane cytokine receptors such as common gamma-chain receptors and the glycoprotein 130 (gp130) transmembrane proteins. (Murray, et al. 2007Immunol.178(5):2623-2629.) About 40 cytokine receptors signal through combinations of these four JAKs and their 7 downstream substrates: the STAT family members. (Ghoreschi et al. 2009Immunol Rev.228(1):273-287.)

The selectivity against other JAK family subtypes is regarded as crucial in order to increase the intended pharmacological effects and to reduce side effects. Identifying kinase inhibitors with a high degree of TYK2 selectivity has posed a significant challenge partly due to the high sequence homology of the active site among the JAK family kinases. TYK2 specificity is critical for clinical application of TYK2 kinase inhibitors, because Tyk2 knockout mice are viable with normal blood cell counts, whereas deficiency of JAK3 results in severe combined immunodeficiency in mice, and JAK1 or JAK2 knockout mice show perinatal lethality. (Ghoreschi, et al. 2009Immunol Rev.228:273-287; Karaghiosoff, et al. 2000Immunity.13:549-560; Shimoda, et al. 2000Immunity.13:561-571.) Genetic evidence suggests that pharmacological inhibition of TYK2 should not result in acute toxicity in human patients, but careful monitoring for viral or mycobacterial infections would be warranted in patients treated for prolonged periods. (Akahane, et al. 2017Br J Haematol.177(2): 271-282.)

An urgent need exists and challenges remain across broad therapeutic areas for selective TYK2 inhibitors with improved potency and minimal side effects.

SUMMARY OF THE INVENTION

The invention provides novel, selective and potent compounds that are orally available. These therapeutic agents are safe and effective TYK2 inhibitors and exhibit fewer and/or lesser side effects than currently available drugs. The invention also provides pharmaceutical compositions of these compounds and methods of their preparation and use.

In one aspect, the invention generally relates to a compound having the structural formula (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12a, wherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3.

In another aspect, the invention generally relates to a compound having the structural formula (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or (C═O)R27;R23is

In yet another aspect, the invention generally relates to a compound having the structural formula (III):

In yet another aspect, the invention generally relates to a compound having the structural formula (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

In yet another aspect, the invention generally relates to a compound having the structural formula (V):

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a compound disclosed herein, effective to treat or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12a, wherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3,or a pharmaceutically acceptable form or an isotope derivative thereof, effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or(C═O)R27;R23is

whereineach of X4, X5, X6, X7, X8and X9is independently selected from O, C, CH, S, N and NR26;R24is H and C1-6alkyl, substituted with 0-3 R24a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R24b;R24aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3or CN, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R24bat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, C3-10cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each substituted with 0-3 R24a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R24a, C2-6alkynyl substituted with 0-3 R24a;R25is F, Cl, CN, CD3, CH2CF3, CF3, OR, NRR′, C1-C3alkyl, C3-C5cycloalkyl, substituted with 0-2 R24b;R26is H, a C1-C6alkyl, CD3, or C3-C6cycloalkyl, substituted with 0-3 R24a;R27is a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R24b;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andp is 1 or 2,effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula (III):

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

whereineach of X4, X5, X6, X7, X8, X9and X10is independently selected from C, CH, O, N and NH;R42aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3, CN, C(O)NR, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R42bis a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R42c;R42cat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl substituted with 0-3 R42a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R42a, C2-6alkynyl substituted with 0-3 R42a;R45each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, substituted with 0-3 R42a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R42c, optionally two R45s, along with the C or N atoms that they are attached to, form a 4- to 6-membered ring;R46each occurrence is independently F, Cl, CN, OR, C1-C3alkyl, C3-C5cycloalkyl, CD3, CH2CF3or CF3;R47is H, OCF3, C1-C3alkyl, C1-C3alkoxy or OCD3;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andj is 0, 1 or 2,effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (V):

In yet another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12a, wherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or (C═O)R27;R23is

whereineach of X4, X5, X6, X7, X8and X9is independently selected from O, C, CH, S, N and NR26;R24is H and C1-6alkyl, substituted with 0-3 R24a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R24b;R24aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3or CN, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R24bat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, C3-10cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each substituted with 0-3 R24a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R24a, C2-6alkynyl substituted with 0-3 R24a;R25is F, Cl, CN, CD3, CH2CF3, CF3, OR, NRR′, C1-C3alkyl, C3-C5cycloalkyl, substituted with 0-2 R24b;R26is H, a C1-C6alkyl, CD3, or C3-C6cycloalkyl, substituted with 0-3 R24a;R27is a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R24b;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andp is 1 or 2,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (III):

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

whereineach of X4, X5, X6, X7, X8, X9and X10is independently selected from C, CH, O, N and NH;R42aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3, CN, C(O)NR, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R42bis a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R42c;R42cat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl substituted with 0-3 R42a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R42a, C2-6alkynyl substituted with 0-3 R42a;R45each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, substituted with 0-3 R42a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R42c, optionally two R45s, along with the C or N atoms that they are attached to, form a 4- to 6-membered ring;R46each occurrence is independently F, Cl, CN, OR, C1-C3alkyl, C3-C5cycloalkyl, CD3, CH2CF3or CF3;R47is H, OCF3, C1-C3alkyl, C1-C3alkoxy or OCD3;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andj is 0, 1 or 2,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (V):

In yet another aspect, the invention generally relates to use of a compound disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating a disease or disorder.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 2006.

The following terms, unless indicated otherwise according to the context wherein the terms are found, are intended to have the following meanings.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 16 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

As used herein, “at least” a specific value is understood to be that value and all values greater than that value.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

Any compositions or methods disclosed herein can be combined with one or more of any of the other compositions and methods provided herein.

The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of”, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of” refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of”, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, atropisomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess of either the R- or S-configuration. For optically active compounds, it is often preferred to use one enantiomer to the substantial exclusion of the other enantiomer.

Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.

Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., —C(═O)—O— is equivalent to —O—C(═O)—.

Structures of compounds of the invention are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds that are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions (e.g., aqueous, neutral, and several known physiological conditions).

Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the present invention include, for example, hydrates.

As used herein, the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., C1-10alkyl). Whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range; e.g., “1 to 10 carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated. In some embodiments, “alkyl” can be a C1-6alkyl group. In some embodiments, alkyl groups have 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representative saturated straight chain alkyls include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, and the like. The alkyl is attached to the parent molecule by a single bond. Unless stated otherwise in the specification, an alkyl group is optionally substituted by one or more of substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, —Si(Ra)3, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, —N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tN(Ra)2(where t is 1 or 2), —P(═O)(Ra)(Ra), or —O—P(═O)(ORa)2where each Rais independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. In a non-limiting embodiment, a substituted alkyl can be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.

As used herein, the terms “aromatic” or “aryl” refer to a radical with 6 to 14 ring atoms (e.g., C6-14aromatic or C6-14aryl) that has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted aryl groups. In some embodiments, the aryl is a C6-10aryl group. For example, bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. In other embodiments, bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in“-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Whenever it appears herein, a numerical range such as “6 to 14 aryl” refers to each integer in the given range; e.g., “6 to 14 ring atoms” means that the aryl group can consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicycles, tricycles, tetracycles, and the like. In a multi-ring group, only one ring is required to be aromatic, so groups such as indanyl are encompassed by the aryl definition. Non-limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl, indanyl, and the like. Unless stated otherwise in the specification, an aryl moiety can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, —Si(Ra)3, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, —N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)N(Ra)2(where t is 1 or 2), —P(═O)(Ra)(Ra), or —O—P(═O)(ORa)2where each Rais independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein.

As used herein, the terms “cycloalkyl” and “carbocyclyl” each refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and can be saturated or partially unsaturated. Partially unsaturated cycloalkyl groups can be termed “cycloalkenyl” if the carbocycle contains at least one double bond, or “cycloalkynyl” if the carbocycle contains at least one triple bond. Cycloalkyl groups include groups having from 3 to 13 ring atoms (i.e., C3-13cycloalkyl). Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted cycloalkyl groups. Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range; e.g., “3 to 13 carbon atoms” means that the cycloalkyl group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, etc., up to and including 13 carbon atoms. The term “cycloalkyl” also includes bridged and spiro-fused cyclic structures containing no heteroatoms. The term also includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicycles, tricycles, tetracycles, and the like. In some embodiments, “cycloalkyl” can be a C3-8cycloalkyl radical. In some embodiments, “cycloalkyl” can be a C3-5cycloalkyl radical. Illustrative examples of cycloalkyl groups include, but are not limited to the following moieties: C3-6carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6) and the like. Examples of C3-7carbocyclyl groups include norbornyl (C7). Examples of C3-8carbocyclyl groups include the aforementioned C3-7carbocyclyl groups as well as cycloheptyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the like. Examples of C3-13carbocyclyl groups include the aforementioned C3-8carbocyclyl groups as well as octahydro-1H indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and the like. Unless stated otherwise in the specification, a cycloalkyl group can be optionally substituted by one or more substituents which independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, —Si(Ra)3, —ORa, —SRa, —OC(O)—Ra, —N(Ra)2, —C(O)Ra, —C(O)ORa, —OC(O)N(Ra)2, —C(O)N(Ra)2, —N(Ra)C(O)ORa, —N(Ra)C(O)Ra, —N(Ra)C(O)N(Ra)2, —N(Ra)C(NRa)N(Ra)2, —N(Ra)S(O)tN(Ra)2(where t is 1 or 2), —P(═O)(Ra)(Ra), or —O—P(═O)(ORa)2where each Rais independently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of these moieties can be optionally substituted as defined herein. The terms “cycloalkenyl” and “cycloalkynyl” mirror the above description of “cycloalkyl” wherein the prefix “alk” is replaced with “alken” or “alkyn” respectively, and the parent “alkenyl” or “alkynyl” terms are as described herein. For example, a cycloalkenyl group can have 3 to 13 ring atoms, such as 5 to 8 ring atoms. In some embodiments, a cycloalkynyl group can have 5 to 13 ring atoms.

As used herein, the term “halogen” refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). As used herein, the term “halide” or “halo”, means fluoro, chloro, bromo or iodo. The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl” and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine, such as, but not limited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl, alkynyl and alkoxy groups are as defined herein and can be optionally further substituted as defined herein.

As used herein, the term “heteroatom” refers to oxygen (O), nitrogen (N), sulfur (S), and phosphorus (Ia).

As used herein, the term “heteroalkyl” refers to an alkyl radical, which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted heteroalkyl groups. A numerical range can be given, e.g., C1-4heteroalkyl, which refers to the chain length in total, which in this example is 4 atoms long. For example, a —CH2OCH2CH3radical is referred to as a “C4” heteroalkyl, which includes the heteroatom center in the atom chain length description. Connection to the parent molecular structure can be through either a heteroatom or a carbon in the heteroalkyl chain. For example, an N-containing heteroalkyl moiety refers to a group in which at least one of the skeletal atoms is a nitrogen atom. One or more heteroatom(s) in the heteroalkyl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quaternized. For example, heteroalkyl also includes skeletal chains substituted with one or more nitrogen oxide (—O—) substituents. Exemplary heteroalkyl groups include, without limitation, ethers such as methoxyethanyl (—CH2CH2OCH3), ethoxymethanyl (—CH2OCH2CH3), (methoxymethoxy)ethanyl (—CH2CH2OCH2OCH3), (methoxymethoxy) methanyl (—CH2OCH2OCH3) and (methoxyethoxy)methanyl (—CH2OCH2CH2OCH3) and the like; amines such as (—CH2CH2NHCH3, —CH2CH2N(CH3)2, —CH2NHCH2CH3, —CH2N(CH2CH3)(CH3)) and the like.

As used herein, the term “heterocycloalkyl” refers to a cycloalkyl radical, which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted heterocycloalkyl groups. Illustrative examples of heterocycloalkyl include 2-hydroxy-aziridin−1-yl, 3-oxo-1-oxacyclobutan-2-yl, 2,2-dimethyl-tetrahydrofuran-3-yl, 3-carboxy-morpholin-4-yl, 1-cyclopropyl-4-methyl-piperazin-2-yl. 2-pyrrolinyl, 3-pyrrolinyl, dihydro-2H-pyranyl, 1,2,3,4-tetrahydropyridine, 3,4-dihydro-2H-[1,4]oxazine, etc.

As used herein, the term “heteroaryl” or, alternatively, “heteroaromatic” refers to a radical of a 5-18 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic and the like) aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-6 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous and sulfur (“5-18 membered heteroaryl”). Unless stated otherwise in the specification, the term is intended to include both substituted and unsubstituted heteroaryl groups. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. Whenever it appears herein, a numerical range such as “5 to 18” refers to each integer in the given range; e.g., “5 to 18 ring atoms” means that the heteroaryl group can consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. In some instances, a heteroaryl can have 5 to 14 ring atoms. In some embodiments, the heteroaryl has, for example, bivalent radicals derived from univalent heteroaryl radicals whose names end in “-yl” by removal of one hydrogen atom from the atom with the free valence are named by adding “-ene” to the name of the corresponding univalent radical, e.g., a pyridyl group with two points of attachment is a pyridylene.

For example, an N-containing “heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. One or more heteroatom(s) in the heteroaryl radical can be optionally oxidized. One or more nitrogen atoms, if present, can also be optionally quaternized. Heteroaryl also includes ring systems substituted with one or more nitrogen oxide (—O—) substituents, such as pyridinyl N-oxides. The heteroaryl is attached to the parent molecular structure through any atom of the ring(s).

“Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment to the parent molecular structure is either on the aryl or on the heteroaryl ring, or wherein the heteroaryl ring, as defined above, is fused with one or more cycloalkyl or heterocycyl groups wherein the point of attachment to the parent molecular structure is on the heteroaryl ring. For polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl and the like), the point of attachment to the parent molecular structure can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, phosphorous, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, phosphorous, and sulfur.

As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Suitable routes of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound.

By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies.

The compound of the invention can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).

The compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, 1995J Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations (see, e.g., Gao 1995Pharm. Res.12:857-863); or, as microspheres for oral administration (see, e.g., Eyles 1997J. Pharm. Pharmacol.49:669-674).

As used herein, the terms “disease,” “condition,” and “disorder” are used interchangeably herein and refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein.

As used herein, the term “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.

As used herein, the terms “inhibition,” “inhibit” and “inhibiting” and the like in reference to a biological target (e.g., TYK2) inhibitor interaction refers to negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

As used herein, the terms “isolated” or “purified” refer to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography.

As used herein, the term “modulate” refers to the production, either directly or indirectly, of an increase or a decrease, a stimulation, inhibition, interference, or blockage in a measured activity when compared to a suitable control. A “modulator” of a polypeptide or polynucleotide refers to a substance that affects, for example, increases, decreases, stimulates, inhibits, interferes with, or blocks a measured activity of the polypeptide or polynucleotide, when compared to a suitable control. For example, a “modulator” may bind to and/or activate or inhibit the target with measurable affinity, or directly or indirectly affect the normal regulation of a receptor activity.

As used herein, a “pharmaceutically acceptable form” of a disclosed compound includes, but is not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, isomers, prodrugs, and isotopically labeled derivatives thereof. In one embodiment, a “pharmaceutically acceptable form” includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs and isotopically labeled derivatives thereof. In some embodiments, a “pharmaceutically acceptable form” includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs and isotopically labeled derivatives thereof.

In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describes pharmaceutically acceptable salts in detail inJ. Pharmaceutical Sciences(1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchlorate acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In some embodiments, organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

The salts can be prepared in situ during the isolation and purification of the disclosed compounds, or separately, such as by reacting the free base or free acid of a parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt can be chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

In certain embodiments, the pharmaceutically acceptable form is a “solvate” (e.g., a hydrate). As used herein, the term “solvate” refers to compounds that further include a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. The solvate can be of a disclosed compound or a pharmaceutically acceptable salt thereof. Where the solvent is water, the solvate is a “hydrate.” Pharmaceutically acceptable solvates and hydrates are complexes that, for example, can include 1 to about 100, or 1 to about 10, or 1 to about 2, about 3 or about 4, solvent or water molecules. It will be understood that the term “compound” as used herein encompasses the compound and solvates of the compound, as well as mixtures thereof.

In certain embodiments, the pharmaceutically acceptable form is a prodrug. As used herein, the term “prodrug” (or “pro-drug”) refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable form of the compound. A prodrug can be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical and/or delivery properties over the parent compound. Prodrugs can increase the bioavailability of the compound when administered to a subject (e.g., by permitting enhanced absorption into the blood following oral administration) or which enhance delivery to a biological compartment of interest (e.g., the brain or lymphatic system) relative to the parent compound. Exemplary prodrugs include derivatives of a disclosed compound with enhanced aqueous solubility or active transport through the gut membrane, relative to the parent compound.

The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H.,Design of Prodrugs(1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,”A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.

Prodrug forms often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism. (See, Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, Calif., 1992.) Prodrugs commonly known in the art include well-known acid derivatives, such as, for example, esters prepared by reaction of the parent acids with a suitable alcohol, amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative, etc. Other prodrug derivatives may be combined with other features disclosed herein to enhance bioavailability. As such, those of skill in the art will appreciate that certain of the presently disclosed compounds having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds having a carbonate, carbamate, amide or alkyl ester moiety covalently bonded to any of the above substituents disclosed herein.

Exemplary advantages of a prodrug can include, but are not limited to, its physical properties, such as enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it can enhance absorption from the digestive tract, or it can enhance drug stability for long-term storage.

As used herein, the term “pharmaceutically acceptable” excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: 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; polyols, such as glycerin, sorbitol, mannitol 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; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. A subject to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example, non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), rodents (e.g., rats and/or mice), etc. In certain embodiments, the non-human animal is a mammal. The non-human animal may be a male or female at any stage of development. A non-human animal may be a transgenic animal. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

As used herein, the terms “treatment” or “treating” a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. Treating or treatment thus refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters, for example, the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. As compared with an equivalent untreated control, such reduction or degree of amelioration may be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

Treatment methods include administering to a subject a therapeutically effective amount of a compound described herein. The administering step may be a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the patient's age, the concentration of the compound, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on an unexpected discovery of novel, selective and potent compounds that are TYK2 inhibitors. The invention also provides pharmaceutical compositions of these compounds and methods of their preparation and use. The compounds are orally available and exhibit fewer and/or lesser side effects than currently available drugs.

The new class of TYK2 inhibitors disclosed herein exhibit exceptional potency profiles and are useful in treating one or more TYK2-mediated diseases and conditions, such as allergic, autoimmune, inflammatory, metabolic, neurological and proliferative diseases and conditions. Without wishing to be bound by the theory, compounds of the invention are modulators of interleukins (e.g., IL-12, IL-23) and interferons (e.g., IFN-α) by inhibiting TYK2-mediated signal transduction.

These compounds are designed to show good potency against TYK2 with good oral absorption and good in vivo stability. The invention also provides pharmaceutical compositions of these compounds and methods of preparation and use thereof. The TYK2 inhibitors disclosed herein exhibit favorable pharmacokinetic profiles and drug properties that are suitable for the target indications.

In one aspect, the invention generally relates to a compound having the structural formula (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12, is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12awherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3.

In certain embodiments of formula (I), R12is C(═O)R12′.

In certain embodiments of formula (I), R12is R12′.

In certain embodiments of formula (I), R12is an aryl.

In certain embodiments of formula (I), R12is a heteroaryl.

In certain embodiments of formula (I), each of X1and X2is CH.

In certain embodiments of formula (I), each of X4and X5is CH.

In certain embodiments of formula (I), X4is CF.

In certain embodiments of formula (I), X4is CH and X5is N.

In certain embodiments of formula (I), each of X1and X2is CH.

In certain embodiments of formula (I), each of X4and X5is CH.

In certain embodiments of formula (I), X4is CH and X5is N.

In certain embodiments of formula (I), X4is N and X5is CH, and the compound has the structural formula:

In certain embodiments of formulae (I)-(Ia), X3is O.

In certain embodiments of formulae (I)-(Ia), R12is R12′and R12′is an aryl group (e.g., an unsubstituted or substituted phenyl).

In certain embodiments of formulae (I)-(Ia), R12is R12′and R12′is a heteroaryl group (e.g., an unsubstituted or substituted pyrazolyl, pyridinyl or pyrimidyl group).

In certain embodiments of formulae (I)-(Ia), R12′is a C1-C6alkyl substituted with an amino or morpholino group.

In certain embodiments of formulae (I)-(Ia), R13is CH3.

In certain embodiments of formulae (I)-(Ia), R13is CD3.

In certain embodiments of formulae (I)-(Ia), R13is CF3.

In certain embodiments of formulae (I)-(Ia), R14is H.

In certain embodiments of formulae (I)-(Ia), k is 0 (i.e., R15is absent).

In certain embodiments of formulae (I)-(Ia), k is 1.

In certain embodiments of formulae (I)-(Ia), k is 2.

In certain embodiments of formulae (I)-(Ia), the compound has the structural formula:

In certain embodiments of formulae (I)-(Ib), j is 0 (i.e., R16is absent).

In certain embodiments of formulae (I)-(Ib), j is 1.

In certain embodiments of formulae (I)-(Ib), j is 2.

In certain embodiments of formula (Ib), j is 1 and R16is at the meta position:

In certain embodiments of formula (Ib), the compound has the structural formula:

In certain embodiments of formulae (I)-(Id), R11is CH3.

In certain embodiments of formulae (I)-(Id), R11is CD3.

In certain embodiments of formulae (I)-(Id), R15is F.

In certain embodiments of formula (I d), j is 1.

In certain embodiments of formula (Id), j is 2.

In certain embodiments of formulae (I)-(Id), each R16is independently selected from F, Cl, CN and CF3.

In certain embodiments of formulae (I)-(Id), a substituted or unsubstituted morpholino group

In another aspect, the invention generally relates to a compound having the structural formula (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or(C═O)R27;R23is

In certain embodiments of formula (II), p is 1 and R23is

In certain embodiments of formula (II), p is 2.

In certain embodiments of formula (II), Y1is CH and Y2is CH, and the compound has the structural formula:

In certain embodiments of formula (II), Y1is CH and Y2is N, and the compound has the structural formula:

In certain embodiments of formula (II), Y1is N and Y2is CH, and the compound has the structural formula:

In certain embodiments of formula (II), Y1is N and Y2is N, and the compound has the structural formula:

In certain embodiments of formulae (II)-(IId), Y1is CF.

In certain embodiments of formulae (II)-(IId), Y3is CH2.

In certain embodiments of formulae (II)-(IId), Y3is CF2

In certain embodiments of formulae (II)-(IId), R23is a group selected from:

In certain embodiments, R23is:

In certain embodiments, R23is:

In certain embodiments, R23is:

In certain embodiments, R23is:

In certain embodiments of formulae (II)-(IId), R21is F.

In certain embodiments of formulae (II)-(IId), R21is CH3.

In certain embodiments of formulae (II)-(IId), R21is CD3.

In certain embodiments of formulae (II)-(IId), R22is (C═O)R27, wherein R27is selected from C1-C6alkyl, cyclopropyl or cyclobutyl, substituted with 0-2 R24b.

In certain embodiments of formulae (II)-(IId), R22is pyridine substituted with 0-2 R24b.

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments, the compound has the structural formula:

In certain embodiments of formulae (II)-(IIl), R27is cyclopropyl.

In certain embodiments of formulae (II)-(IIl), R27is cyclobutyl.

In certain embodiments of formulae (II)-(IIl), R24is a C3-C12cycloakyl or heterocycloalkyl, optionally substituted with one or more of F, Cl, CN, OR, NRR′, CH3, CF3and OCF3.

In certain embodiments of formulae (II)-(IIl), R24is a C4-C12aryl, optionally substituted with one or more of F, Cl, CN, OR, NRR′, CH3, CF3and OCF3.

In certain embodiments of formulae (II)-(IIl), R24is a C3-C12heteroaryl, optionally substituted with one or more of F, Cl, CN, OR, NRR′, CH3, CF3and OCF3.

In certain embodiments of formulae (II)-(IIl), R25is H.

In certain embodiments of formulae (II)-(IIl), R25is F or C1.

In certain embodiments of formulae (II)-(IIl), R25is CN.

In certain embodiments of formulae (II)-(IIl), R25is OR.

In certain embodiments of formulae (II)-(IIl), i is 0 (i.e., R25is absent).

In certain embodiments of formulae (II)-(IIl), i is 1.

In certain embodiments of formulae (II)-(IIl), i is 2.

In yet another aspect, the invention generally relates to a compound having the structural formula (III):

In certain embodiments of formula (III), X1is NH, having the structural formula (III1):

In certain embodiments of formulae (III)-(III1), Ring A is a 6-membered aryl.

In certain embodiments of formulae (III)-(III1), Ring A is a 6-membered heteroaryl.

In certain embodiments of formulae (III)-(III1), the compound has the structural formula (III2):

wherein each of Z5and Z8is CH or N.

In certain embodiments of formula (III2), wherein Z8is CH and the compound has the structural formula (III3):

In certain embodiments of formula (III3), Z2and Z5are not both CH.

In certain embodiments of formulae (III) or (III3), Z7is NR. In certain embodiments, R is H and Z7is NH.

In certain embodiments of formulae (III) or (III3), Z7is CH2.

In certain embodiments of formulae (III) or (III3), Z7is CF2.

In certain embodiments of formulae (III) or (III3), each of Z3and Z4is NH.

In certain embodiments of formula (III3), the compound has the structural formula:

In certain embodiments of formula (III3), the compound has the structural formula:

In certain embodiments of formula (III3), the compound has the structural formula:

In certain embodiments of formula (III3), the compound has the structural formula:

In certain embodiments of formula (III3), the compound has the structural formula:

In certain embodiments of formulae (III3)-(III3e), R32is a 6-membered aryl or heteroaryl group comprising 0, 1 or 2 nitrogen atoms and 0 or 1 oxygen atom.

In certain embodiments of formulae (III3)-(III3e), R32is selected from:

In certain embodiments of formulae (III3)-(III3e), R32is a 3- to 6-membered cycloalkyl or heterocycloalkyl comprising 1, 2 or 3 heteroatoms wherein the heteroatoms are selected from N, O and S, substituted with 0-3 R32a.

In certain embodiments of formulae (III3)-(III3e), R32is a 3-membered cycloalkyl substituted with 0-3 R32a.

In certain embodiments of formulae (III3)-(III3e), R32is cyclopropyl substituted with 0-3 R32a.

In certain embodiments of formulae (III3)-(III3e), R32is a 5-membered heteroaryl group comprising 1, 2 or 3 nitrogen atoms and 0 or 1 oxygen atom.

In certain embodiments of formulae (III3)-(III3e), R32is selected from:

In certain embodiments of formulae (III3)-(III3e), q is 0 (i.e., R34is absent).

In certain embodiments of formulae (III3)-(III3e), q is 1.

In certain embodiments of formulae (III3)-(III3e), q is 2.

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III2), the compound has the structural formula:

In certain embodiments of formulae (III2), the compound has the structural formula:

In certain embodiments of formulae (III2), the compound has the structural formula:

In certain embodiments of formulae (III3)-(III3i), Z6is O.

In certain embodiments of formulae (III3)-(III3i), Z6is S.

In certain embodiments of formulae (III3)-(III3i), Z6is CH2.

In certain embodiments of formulae (III3)-(III3i), m=1.

In certain embodiments of formulae (III3)-(III3i), m=2.

In certain embodiments of formulae (III3)-(III3i), n=1.

In certain embodiments of formulae (III3)-(III3i), n=2.

In certain embodiments of formulae (III3)-(III3i), m=n=1.

In certain embodiments of formulae (III3)-(III3i), m=n=2.

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formulae (III3), the compound has the structural formula:

In certain embodiments of formula (III1), Z5is N and Z8is N.

In certain embodiments of formula (III1), the compound has the structural formula (III4):

In certain embodiments of formula (III4), the compound has the structural formula (III4a):

In certain embodiments of formula (III4), the compound has the structural formula (III4b):

In certain embodiments of formula (III4), the compound has the structural formula (III4c):

In certain embodiments of formula (III4), the compound has the structural formula (III4d):

In certain embodiments of formula (III4), the compound has the structural formula (III4e):

In certain embodiments of formulae (III)-(III4e) R33is OR. In certain embodiments, R is CH3and R33is OCH3. In certain embodiments, R is CD3and R33is OCD3.

In certain embodiments of formulae (III)-(III4e), R34is H.

In certain embodiments of formulae (III)-(III4e), R34is selected from F or Cl.

In certain embodiments of formulae (III)-(III4e), R34is selected from CN.

In certain embodiments of formulae (III)-(III4e), R34is selected from CH3and CF3.

In certain embodiments of formulae (III)-(III4e), R34is selected from OCF3.

In certain embodiments of formulae (III)-(III4e), R34is —(CH2)p-Q. In certain embodiments, p is 1 or 2 and Q is OH, OR or NRR′ (e.g., N(CH3)2). In certain embodiments, Q is a heterocyclic (e.g., morpholine) or heteroaryl group.

In certain embodiments of formulae (III)-(III4e), R34is —(CH2)p-Q and Q is an amino or morpholino group.

In certain embodiments of formulae (III)-(III4c), R35is CH3.

In certain embodiments of formulae (III)-(III4e), R35is CD3.

In certain embodiments of formulae (III)-(III1), Ring A is a 5-membered aryl.

In certain embodiments of formulae (III)-(III1), Ring A is a 5-membered heteroaryl.

In certain embodiments of formulae (III)-(III1), the compound has the structural formula (III5):

In certain embodiments of formula (III5), (CRR′)m, is (CH2)mand (CRR′)nis (CH2)n.

In certain embodiments of formula (III5), the compound has the structural formula:

In certain embodiments of formula (III1), the compound has the structural formula:

In certain embodiments of formulae (III5) or (III5b), R35is CH3.

In certain embodiments of formulae (III5) or (III5b), R35is CD3.

In certain embodiments of formulae (III5) or (III5b), wherein R33is OCH3.

In certain embodiments of formulae (III5) or (III5b), m is 1 and n is 2.

In yet another aspect, the invention generally relates to a compound having the structural formula (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

In certain embodiments of formula (IV), R43is selected from:

In certain embodiments of formula (IV), R43is selected from:

In certain embodiments, j is 0.

In certain embodiments, j is 1.

In certain embodiments of formula (IV), Y1is CH and Y2is CH:

In certain embodiments of formula (IV), Y1is CH and Y2is N:

In certain embodiments of formula (IV), Y1is N and Y2is CH:

In certain embodiments of formula (IV), Y1is N and Y2is N:

In certain embodiments of formula (IV), Y1is CF.

In certain embodiments of formulae (IV)-(IVd), Y3is NR.

In certain embodiments of formulae (IV)-(IVd), Y3is NH.

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formulae (IV)-(IVx), wherein R41is CH3.

In certain embodiments of formulae (IV)-(IVx), wherein R41is CD3.

In certain embodiments of formulae (IV)-(IVx), wherein R42is (C═O)R42b, wherein R42bis selected from C1-C6alkyl, cyclopropyl or cyclobutyl, substituted with 0-2 R42c.

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formula (IV), the compound has the structural formula:

In certain embodiments of formulas (IVc1)-(IVf1), R42cis H.

In certain embodiments of formulas (IVc1)-(IVf1), R42cis F.

In certain embodiments where R44is boned to N, R44is CD3, methyl or ethyl, optionally substituted with F, Cl or CN.

In certain embodiments where R44is boned to C, R44is Cl, CN, CD3, methyl or ethyl, optionally substituted with F, Cl or CN.

In yet another aspect, the invention generally relates to a compound having the structural formula (V):

In certain embodiments of formula (V), each of Z3, Z4and Z5is NH, having the structural formula (V1):

In certain embodiments of formulae (V)-(V1), wherein Ring B is a 6-membered aryl.

In certain embodiments of formulae (V)-(V1), wherein Ring B is a 6-membered heteroaryl.

In certain embodiments of formulae (V)-(V1), the compound has the structural formula (V2):

wherein each of Z7and Z8is independently CH or N.

In certain embodiments of formula (V2), the compound has the structural formula (V3):

In certain embodiments of formula (V2), the compound has the structural formula (V4):

In certain embodiments of formulae (V)-(V4), Z6is O or S.

In certain embodiments of formulae (V)-(V4), Z6is NR.

In certain embodiments of formulae (V)-(V4), each of m and n is 1.

In certain embodiments of formulae (V)-(V4), R51is CH3.

In certain embodiments of formulae (V)-(V4), R51is CD3.

In certain embodiments of formulae (V)-(V4), R57is C1-C3alkoxy.

In certain embodiments of formulae (V)-(V4), R57is OCH3.

In certain embodiments of formulae (V)-(V4), R57is OCD3.

In certain embodiments of formulae (V)-(V4), R57is OCF3.

Non-limiting examples of compounds of the invention include:

In yet another aspect, the invention generally relates to a method for preparing a compound disclosed herein, as exemplified by the synthetic schemes and experimental procedure disclosed herein.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a compound disclosed herein, effective to treat or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12a, wherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3,effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or (C═O)R27;R23iswherein

each of X4, X5, X6, X7, X8and X9is independently selected from O, C, CH, S, N and NR26;R24is H and C1-6alkyl, substituted with 0-3 R24a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R24b;R24aat each occurrence is independently H, D, halo, OIL OR, CH3, CF3, CH2CF3or CN, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R24bat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, C3-10cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each substituted with 0-3 R24a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R24a, C2-6alkynyl substituted with 0-3 R24a;R25is F, Cl, CN, CD3, CH2CF3, CF3, OR, NRR′, C1-C3alkyl, C3-C5cycloalkyl, substituted with 0-2 R24b;R26is H, a C1-C6alkyl, CD3, or C3-C6cycloalkyl, substituted with 0-3 R24a;R27is a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R24b;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andp is 1 or 2,effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (III):

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

whereineach of X4, X5, X6, X7, X8, X9and X10is independently selected from C, CH, O, N and NH;R42aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3, CN, C(O)NR, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R42bis a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R42c;R42cat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl substituted with 0-3 R42a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R42a, C2-6alkynyl substituted with 0-3 R42a;R45each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, substituted with 0-3 R42a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R42c, optionally two R45s, along with the C or N atoms that they are attached to, form a 4- to 6-membered ring;R46each occurrence is independently F, Cl, CN, OR, C1-C3alkyl, C3-C5cycloalkyl, CD3, CH2CF3or CF3;R47is H, OCF3, C1-C3alkyl, C1-C3alkoxy or OCD3;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andj is 0, 1 or 2,effective to treat, or reduce one or more diseases or disorders, in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a pharmaceutical composition comprising an amount of a compound having the structural formula of (V):

In certain embodiments, a pharmaceutical composition disclosed herein is suitable for oral administration.

In certain embodiments, a pharmaceutical composition disclosed herein is suitable for topical administration.

In certain embodiments, a pharmaceutical composition disclosed herein is suitable for GI-restricted administration.

In certain embodiments, a pharmaceutical composition disclosed herein is useful to treat or reduce one or more of inflammatory diseases, immune-mediated diseases and cancers, or a related disease or disorder. In certain embodiments, the disease or disorder is an inflammatory disease. In certain embodiments, the disease or disorder is an immune-mediated disease. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the disease or disorder is selected from: inflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), breast cancer and ovarian cancer.

In yet another aspect, the invention generally relates to a unit dosage form comprising a pharmaceutical composition disclosed herein.

In certain embodiments, the unit dosage form is a tablet.

In certain embodiments, the unit dosage form is a capsule.

In certain embodiments, the unit dosage form is a topical formulation.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound disclosed herein.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula of (I):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereineach of X1and X2is independently selected from CH and N;each of X4and X5is independently selected from CH, CF and N;X3is NR, O, CH2or CF2;R11is a H, F, C1-C3alkyl or CD3, provided that R11is not F when X3is NR or O;R12is C(═O)R12′or R12′, wherein R12′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R12a, wherein R12ais selected from the group consisting of halogen, CF3, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;R13is a C1-C3alkyl, CD3or CF3;R14is H, C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, or a 5- or 6-membered heteroaryl group comprising 1, 2 or 3 hetero atoms selected from N, O and S, or R14is OR14′, wherein R14′is C1-C6alkyl or heteroalkyl or a C3-C6cycloalkyl or heterocycloalkyl, each substituted with 0-2 R14a, wherein R14ais selected from the group consisting of halogen, R, OR, amino, CF3and CN;R15at each occurrence is independently selected from F, Cl, CN, OR, NRR′, and a C1-C3alkyl;R at each occurrence is independently H or a C1-C6alkyl; andk is 0, 1, 2 or 3,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula of (II):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R21is a H, F, C1-C3alkyl and CD3, provided that R21is not F when Y3is N or O;R22isR22′, wherein R22′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 R22a, wherein R22ais selected from the group consisting of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic;an aryl or heteroaryl group, each substituted with 0-2 R22a; or(C═O)R27;R23is

whereineach of X4, X5, X6, X7, X8and X9is independently selected from O, C, CH, S, N and NR26;R24is H and C1-6alkyl, substituted with 0-3 R24a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R24b;R24aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3or CN, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R24bat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, C3-10cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each substituted with 0-3 R24a, C1-6 haloalkyl, C2-6alkenyl substituted with 0-3 R24a, C2-6alkynyl substituted with 0-3 R24a;R25is F, Cl, CN, CD3, CH2CF3, CF3, OR, NRR′, C1-C3alkyl, C3-C5cycloalkyl, substituted with 0-2 R24b;R26is H, a C1-C6alkyl, CD3, or C3-C6cycloalkyl, substituted with 0-3 R24a;R27is a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R24b;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andp is 1 or 2,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (III):

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (IV):

or a pharmaceutically acceptable form or an isotope derivative thereof,whereinY1is CH, CF or N;Y2is CH or N;Y3is NR, O, CH2or CF2;R41is a H, F, C1-C3alkyl and CD3, provided that R41is not F when Y3is NR or O;R42isR42′, wherein R42′is a C1-C6alkyl, C3-C6cycloalkyl or heterocycloalkyl, aryl or heteroaryl, each substituted with 0-2 of halogen, CN, OR, amino, alkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;an aryl or heteroaryl group substituted with 0-2 R42a; or(C═O)R42b;R43is

whereineach of X4, X5, X6, X7, X8, X9and X10is independently selected from C, CH, O, N and NH;R42aat each occurrence is independently H, D, halo, OH, OR, CH3, CF3, CH2CF3, CN, C(O)NR, NRR′, (CH2)nNRR′ or a 4- to 6-membered heterocycle having 1-4 heteroatoms selected from N, O and S;R42bis a C1-6alkyl or C3-6cycloalkyl, aryl or heteroaryl, each substituted with 0-2 R42c;R42cat each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl substituted with 0-3 R42a, C1-6haloalkyl, C2-6alkenyl substituted with 0-3 R42a, C2-6alkynyl substituted with 0-3 R42a;R45each occurrence is independently H, halo, CN, OR, NRR′, OCF3, CF3, C1-6alkyl, substituted with 0-3 R42a, or C3-10cycloalkyl or heterocycloalkyl, C5-10aryl or heteroaryl, or a 4- to 10-membered heterocycle having 1-4 heteroatoms selected from N, O and S, each group is substituted with 0-4 R42c, optionally two R45s, along with the C or N atoms that they are attached to, form a 4- to 6-membered ring;R46each occurrence is independently F, Cl, CN, OR, C1-C3alkyl, C3-C5cycloalkyl, CD3, CH2CF3or CF3;R47is H, OCF3, C1-C3alkyl, C1-C3alkoxy or OCD3;each of R and R′ is independently H or a C1-C6alkyl, or R and R′, together with the nitrogen atom to which they are bound, form a 4- to 7-membered ring comprising 0-2 heteroatoms selected from O, NR, S and SO2;n is 0, 1, 2, 3 or 4;i is 0, 1 or 2; andj is 0, 1 or 2,wherein the disease or disorder is selected from inflammatory diseases, immune-mediated diseases, cancer, or a related disease or disorder thereof, in a mammal, including a human.

In yet another aspect, the invention generally relates to a method for treating, reducing or preventing a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the structural formula (V):

In certain embodiments, the method is used to treat an inflammatory disease. In certain embodiments, the method is used to treat an immune-mediated disease. In certain embodiments, the method is used to treat cancer. In certain embodiments, the method is used to treat a disease or disorder is selected from: inflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), breast cancer and ovarian cancer.

In certain embodiments, administration of the compound is via oral administration.

In certain embodiments, administration of the compound is via topical administration.

In certain embodiments, administration of the compound administration is via GI-restricted administration.

In yet another aspect, the invention generally relates to use of a compound disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent, in preparation of a medicament for treating a disease or disorder.

In certain embodiments, use of the compound is for treating one or more of inflammatory diseases, immune-mediated diseases and cancer. In certain embodiments, use of the compound is for treating an inflammatory disease. In certain embodiments, use of the compound is for treating an immune-mediated disease. In certain embodiments, use of the compound is for treating cancer. In certain embodiments, use of the compound is for treating a disease or disorder is selected from: inflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), breast cancer and ovarian cancer.

In certain embodiments, use of the compound is via oral administration. In certain embodiments, use of the compound is via topical administration. In certain embodiments, use of the compound is via GI restriction administration.

A list of non-limiting examples of the compounds of the invention is provided in Table #. Certain exemplary data of select compounds are provided in Table #.

As discussed herein, isotope derivative compounds having one or more hydrogen atoms (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, etc.) replaced with deuterium atoms are contemplated in the presented invention.

The term “inflammatory disease” refers to a disease or condition characterized by aberrant inflammation, e.g. an increased level of inflammation compared to a control such as a healthy person not suffering from a disease. Examples of inflammatory diseases that may be treated with a compound, pharmaceutical composition, or method described herein include autoimmune diseases, traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis. Such conditions are frequently inextricably intertwined with other diseases, disorders and conditions. A non-limiting list of inflammatory-related diseases, disorders and conditions which may, for example, be caused by inflammatory cytokines, include, arthritis, kidney failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, surgical complications (e.g., where inflammatory cytokines prevent healing), anemia, and fibromyalgia. Other diseases and disorders, which may be associated with chronic inflammation include Alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infections, inflammatory bowel disease (IBD), allergic contact dermatitis and other eczemas, systemic sclerosis, transplantation and multiple sclerosis. Some of the aforementioned diseases, disorders and conditions for which a compound of the present disclosure may be particularly efficacious (due to, for example, limitations of current therapies) are described in more detail hereafter.

In certain embodiments of the use, the disease or disorder is selected from: inflammatory bowel disease, psoriasis, vitiligo, atopic dermatitis, systemic lupus erythematosus, asthma, diabetic nephropathy, chronic myelogenous leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), breast cancer and ovarian cancer.

Isotopically-labeled compounds are also within the scope of the present disclosure. As used herein, an “isotopically-labeled compound” refers to a presently disclosed compound including pharmaceutical salts and prodrugs thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36Cl, respectively.

By isotopically-labeling the presently disclosed compounds, the compounds may be useful in drug and/or substrate tissue distribution assays. Tritiated (3H) and carbon-14 (14C) labeled compounds are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds presently disclosed, including pharmaceutical salts, esters, and prodrugs thereof, can be prepared by any means known in the art.

Further, substitution of normally abundant hydrogen (1H) with heavier isotopes such as deuterium can afford certain therapeutic advantages, e.g., resulting from improved absorption, distribution, metabolism and/or excretion (ADME) properties, creating drugs with improved efficacy, safety, and/or tolerability. Benefits may also be obtained from replacement of normally abundant12C with13C. (See, WO 2007/005643, WO 2007/005644, WO 2007/016361, and WO 2007/016431.)

Stereoisomers (e.g., cis and trans isomers) and all optical isomers of a presently disclosed compound (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers are within the scope of the present disclosure.

Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 95% (“substantially pure”), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are more than 99% pure. Solvates and polymorphs of the compounds of the invention are also contemplated herein. Solvates of the compounds of the present invention include, for example, hydrates.

Any appropriate route of administration can be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intraventricular, intracorporeal, intraperitoneal, rectal, or oral administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease or condition being treated or the nature of the therapy being used and on the nature of the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof are admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (i) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (ii) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (iii) humectants, as for example, glycerol, (iv) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (v) solution retarders, as for example, paraffin, (vi) absorption accelerators, as for example, quaternary ammonium compounds, (vii) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (viii) adsorbents, as for example, kaolin and bentonite, and (ix) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art.

The following examples are meant to be illustrative of the practice of the invention and not limiting in any way.

Representative methods of prep-HPLC: Flow rate and gradient may change.

Exemplary methods for prep-HPLC are provided below.

Representative Methods of Analytical-HPLC

Method 1: Analysis was performed on an Agilent 1200_series HPLC-6120 MS. UHPLC Long Gradient Equivalent 5% to 95% acetonitrile (containing 0.02% NH4OAc) in water run time of 6.5 minutes with a flow rate of 1.5 mL/min. A Waters Xbridge C18 column (18.5 micron, 4.6*50 mm) was used at a temperature of 40° C.

Method 2: Analysis was performed on an Agilent 1200_series HPLC-6120 MS. UHPLC Long Gradient Equivalent 5% to 95% acetonitrile (containing 0.1%_trifluoroacetic acid) in water run time of 6.5 minutes with a flow rate of 1.5 mL/min. A Waters Xbridge C18 column (18.5 micron, 4.6*50 ram) was used at a temperature of 40° C.

Method 3: Analysis was performed on an Agilent 1260_series HPLC-6120 MS. UHPLC Long Gradient Equivalent 5% to 95% acetonitrile (containing 0.02% NH4OAc) in water run time of 2.5 minutes with a flow rate of 0.5 mL/min. A diamonsil Plus C18column (18.5 micron, 4.6*30 mm) was used at a temperature of 40° C.

Method 4: Analysis was performed on an Agilent 1260_series HPLC-6125C MS. HPLC Long Gradient Equivalent 20% to 100% acetonitrile in water (containing 0.1% FA) run time of 6 minutes with a flow rate of 0.8 mL/min. Agilent ZORBAX SB-C18 column (1.8 micron, 2.1*50 mm) was used at a temperature of 30° C.

Representative Method of Prep-Chiral HPLC:

Compound 1f (50 mg, 0.23 mmol), 1h (58 mg, 0.28 mmol) and DIPEA (91 mg, 0.70 mmol) were dissolved in IPA (1 mL). The resulting reaction was stirred at 60° C. for 3 h. The reaction mixture was cooled down to r.t. and concentrated to dryness. The solid was treated with EtOAc (5 mL). The formed solid was collected by filtering and the filter cake was dried to give the title compound 1i (51.5 mg, 63% yield) as a white solid. LC-MS (Method 3) tR=1.30 min, m/z (M+H)+=347.2.

Compound 2b (1 g, 2.67 mmol) and NaOH (214 mg, 5.34 mmol) were dissolved in THF (8 mL) and H2O (4 mL). The resulting mixture was stirred at 50° C. for 3 h. The mixture was acidified with 1 N HCl to pH=2. The formed solid was filtered and the filter cake was dried to give the title compound 2c (840 mg, 87% yield) as a yellow solid. LC-MS (Method 3) tR=1.12 min, m/z (M+H)+=361.1.

To a stirred mixture of 3a (15 mg, 0.032 mmol) in THF (0.6 mL) and water (0.3 mL) was added lithium hydroxide monohydrate (3 mg, 0.064 mmol). The reaction was stirred for 12 h at r.t. The mixture was concentrated under reduced pressure to give the crude product 3b (18 mg, yield given) as a brown-yellow solid. LC-MS (Method 4) tR=3.24 min, m/z (M+H)+=452.2.

Compound 6b (1.25 g, 4.93 mmol), Fe powder (1.38 g, 24.68 mmol) and NH4Cl (1.31 g, 24.68 mmol) were dissolved in a mixture of EtOH (5 mL) and H2O (5 mL). The reaction solution was stirred at 80° C. for 2 h. The reaction mixture was cooled and filtered. The filtrate was concentrated to dryness. The residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to give the title compound 6c (450 mg, 41% yield) as a red oil. LC-MS (Method 3) tR=1.18 min, m/z (M+H)+=224.1.

Compound 9a (5 g, 17.09 mmol) and C2H5I (10 mL) were dissolved in EtOH (10 mL) in a sealed tube. The resulting mixture was stirred at 80° C. for 18 h. After cooling to r.t., the reaction mixture was cooled and concentrated to dryness. The residue was used to the next step without purification. LC-MS (Method 3) tR=1.48 min, m/z M+=321.1.

A mixture of 15c (71 mg, 0.33 mmol) and 11c (67 mg, 0.33 mmol) in EtOH (2 mL) and conc. HCl (0.2 mL) was stirred overnight at 80° C. After cooling to r.t., the formed solid was filtered and the filter cake was dried to afford 15d (60 mg, 53% yield) as a yellow solid.

To a solution of 17a (1 g, 4.35 mmol) in DMF (10 mL) was added NaH (150 mg, 6.52 mmol, 60% in mineral oil) at 0° C. The resulting mixture was stirred at r.t. for 0.5 h. Then CH3CH2I (813 mg, 5.22 mmol) was added to the mixture. After stirring at r.t. overnight, the reaction mixture was poured into water (30 mL) and extracted with EtOAc (50 mL*2). The combined organic layer was dried over Na2SO4, filtered and evaporated under vacuum. The crude product was purified by silica gel flash flash chromatography (PE/EtOAc=4/1) to afford 17b (810 mg, 72%) as a black oil. LCMS (Method 3) tR=1.43 min, m/z (M+H)+=260.1.

Compound 23a (100 mg, 0.28 mmol) was dissolved in a solution of methanamine (5 mL, 2 M in THF). The reaction mixture was stirred at r.t. for 2 h. The reaction was washed with water (5 mL) and extracted with EtOAc (10 mL*3). The combined organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to afford the title compound 23b (80 mg, 80% yield). LC-MS (Method 3) tR=1.25 min, m/z (M+H)+=361.1.

Compound 33a (2.0 g, 16.24 mmol), 2,4-dimethoxybenzaldehyde (8.10 g, 48.72 mmol) and AcOH (975 mg, 16.24 mmol) were dissolved in MeOH (30 mL). The above reaction was stirred at r.t. for 10 min. Then NaBH3CN (5.10 g, 81.20 mmol) was added to the mixture. The mixture was stirred at r.t. for 4 h. The mixture was diluted with H2O (30 mL) and extracted with EtOAc (50 mL*2). The combined organic layer was concentrated to dryness. The residue was purified by flash chromatography on silica gel (PE/EtOAc=30/1) to give the title compound 33b (1.1 g, 16% yield) as a yellow oil. LC-MS (Method 3) tR=1.66 min, m/z (M+H)+=424.3.

To a mixture of 33b (311 mg, 0.73 mmol) in DMF (2 mL) was added NaH (35 mg, 0.88 mmol, 60% in mineral oil) at 0° C. The mixture was stirred at r.t. for 30 min. Then 22c (200 mg, 0.61 mmol) was added at 0° C. The mixture was stirred at r.t. for 3 h. The mixture was quenched with H2O (5 mL) and extracted with EtOAc (15 mL*2). The combined organic layer was washed with brine (15 mL), dried over Na2SO4and filtered. The filtrate was concentrated. The residue was purified by flash chromatography on silica gel (EtOAc) to give the title compound 33c (150 mg, 37% yield) as a yellow solid. LC-MS (Method 3) tR=1.78 min, m/z (M+H)+=670.4.

To a mixture of 33f (20 mg, 0.04 mmol) in EtOH (6 mL) was added 1 drop of conc. HCl. The mixture was stirred at 60° C. for 2 h. The mixture was concentrated to give the crude title compound 33g (20 mg, 99% yield) as a yellow solid. LC-MS (Method 3) tR=1.56 min, m/z (M−H)−=486.5.

Compound 35b (50 mg, 0.08 mmol) was dissolved in a solution consisting of TFA (0.5 mL) and DCM (0.5 mL). The resulting reaction was stirred at r.t. for 1 h. The reaction mixture was purified by Prep-HPLC (Method C) to give the title compound 35c (45 mg, 99% yield) as a yellow solid. LC-MS (Method 3) tR=1.31 min, m/z (M+H)+=510.5.

Compound 38b (100 mg, 0.38 mmol) was dissolved in a mixture of TFA and DCM (2 mL, v/v=1/3). The above solution was stirred at r.t. for 2 h. The reaction mixture was concentrated to dryness to give 38c (105 mg, yield given) as a brown oil. LC-MS (Method 3) tR=0.29 min, m/z (M+H)+=165.1.

A solution of LiBH4(4.1 mL, 8.2 mmol, 2 M in THF) in THF (3.0 mL) was treated with trimethylchlorosilane (1.78 g, 16.4 mmol) dropwise at r.t. under nitrogen atmosphere, followed by the addition of 39a (480 mg, 2.05 mmol) dropwise in THF (6 mL). The resulting mixture was stirred overnight at r.t. The mixture was quenched with MeOH and basified by 4 N NaOH to pH=8 to 9 and extracted with EA. The organic phases were combined, dried over Na2SO4, concentrated under reduced pressure to afford compound 39b (280 mg, 68% yield) as a light-yellow oil. LC-MS (Method 4) tR=1.25 min, m/z (M+H)+=206.1.

A solution of 39b (210 mg, 1.01 mmol) in DCM (9 mL) was treated with triphosgene (120 mg, 0.4 mmol) in DCM (0.5 mL) dropwise at 0° C., followed by the addition of saturated sodium bicarbonate (2.5 mL) solution dropwise at 0° C. The resulting mixture was stirred for 1 h at 0° C., dried over Na2SO4and concentrated to afford the crude compound 39c (250 mg, yield given). The crude was used for next step without purification.

To a stirred solution of 39f (20 mg, 0.067 mmol) in DCM (0.5 mL) was added TFA (148.00 mg, 1.30 mmol, 0.1 mL) dropwise at 0° C. The mixture was stirred for 2 h at r.t. The mixture was dilute with DCM and concentrated under reduced pressure to get the crude compound 39g (25 mg, yield given). The crude was used for next step without purification. LC-MS (Method 4) tR=1.83 min, m/z (M+H)+=197.1.

To a stirred mixture of 39i (12 mg, 0.029 mmol) in THF (0.9 mL) and water (0.3 mL) was added lithium hydroxide monohydrate (8 mg, 0.21 mmol). The reaction was stirred for 12 h at r.t. The mixture was concentrated under reduced pressure to give the crude compound 39j (19 mg, yield given) as a brown-yellow solid. LC-MS (Method 4) tR=2.89 min, m/z (M+H)+=401.1.

To a solution of 42a(1.1 g, 5.23 mmol) in DCM (20 mL) was added methan-d3-amine hydrochloride (406 mg, 5.75 mmol) and TEA (2.64 g, 26.14 mmol) at 0° C. Then the mixture was stirred at r.t. for 1 h. The mixture was diluted with H2O (20 mL) and extracted with DCM (20 mL). The organic layer was separated and washed with brine (20 mL), dried over Na2SO4and filtered. The filtrate was concentrated to dryness to give the title compound 42b (800 mg, 74% yield) as an off-white solid. LC-MS (Method 4) tR=2.18 min, m/z (M+H)+=208.0.

To a solution of 42e (300 mg, 1.07 mmol) and K2CO3(296 mg, 2.14 mmol) in DMF (3 mL) was added iodomethane (228 mg, 1.61 mmol). The mixture was stirred at 25° C. for 2 h, then poured into water (20 mL) and extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under vacuum to give the title product 42f (300 mg, 95% yield) as a yellow solid. LC-MS (Method 4) tR=4.00 min, m/z (M+H)+=294.9.

To a solution of 42g (270 mg, 0.72 mmol) in DMF (2.5 mL) was added K2CO3(300 mg, 2.17 mmol) and CuI (14 mg, 0.072 mmol). The mixture was stirred at 100° C. for 16 h. Water (40 mL) was added to the above mixture. The solution was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 42h (150 mg, 81% yield) as a pale-yellow solid. LC-MS (Method 4) tR=3.99 min, m/z (M+H)+=255.0.

To a solution of 42i (31 mg, 0.1 mmol) in dioxane (0.5 mL) was added a solution of HCl (g) in dioxane (4 M, 0.5 mL). The mixture was stirred at r.t. for 30 min. The mixture was concentrated to dryness. The residue was diluted with H2O (10 mL) and adjusted to pH>7 with aq Na2CO3, then extracted with EtOAc (10 mL*3). The organic layers were washed with aq. Na2CO3(15 mL) and brine (15 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 42j (20 mg, 98% yield) as a yellow solid. LC-MS (Method 4) tR=1.73 min, m/z (M+H)+=192.3.

To a solution of 42j (20 mg, 0.10 mmol) and 42b (26 mg, 0.13 mmol) in THF (1 mL) was added NaHMDS (0.35 mL, 0.7 mmol, 2 M in THF) at 0° C., then the mixture was stirred at r.t. for 30 min. The mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4and filtered. The filtrate was concentrated to dryness to give the title compound 42k (30 mg, 79% yield) as a yellow solid. LC-MS (Method 4) tR=3.52 min, m/z (M+H)+=363.2.

A solution of 45b (2.1 g, 5.91 mmol) in TFA (10 mL) was stirred at 80° C. for 16 h. Then the mixture was concentrated and diluted with EA (10 mL), filtered and wash with EA (5 mL*2), then the solid was dried to get the compound 45c (1.8 g, 87% yield, TFA salt) as an off-white solid. LC-MS (Method 4) tR=1.28 min, m/z (M+H)+=236.2.

To a stirred solution of 45d (1 g, 5.24 mmol) in DCM (30 mL) were added TEA (2.65 g, 26.18 mmol, 3.65 mL) and 2,2-dimethoxyethanamine (826 mg, 7.85 mmol) at room temperature. The reaction mixture was cooled to 0° C. and T3P (4.7 mL, 7.85 mmol, 50% in ethyl acetate) was added and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (60 mL) and extracted with dichloromethane (30 mL*3). The combined organic layer was dried over Na2SO4, filtered and concentrated and purified by flash chromatography (PE/EtOAc=10/1 to 1/1) to get the compound 45e (500 mg, 34% yield) as a white solid. LC-MS (Method 4) tR=2.28 min, m/z (M−H)−=276.0.

To a solution of methyl 45j (100 mg, 0.22 mmol) in the solvent (2.5 mL, MeOH/THF/H2O=2/2/1) was added LiOH·H2O (28 mg, 0.67 mmol), then the mixture was stirred at r.t. for 16 h. The mixture was diluted with H2O (2 mL) and acidified to pH=2 with aq HCl (1 N), then concentrated to get the compound 45k (100 mg, yield given) as a white solid. LC-MS (Method 4) tR=2.40 min, m/z (M+H)+=437.1.

To a solution of 46a (200 mg, 1.27 mmol) and 2-aminoethanol (93.33 mg, 1.53 mmol) in toluene was added SOCl2(454 mg, 3.82 mmol) and DMF (9 mg, 0.13 mmol) then the mixture was stirred at 50° C. for 2 h. Then stirred at 70° C. overnight. The mixture was concentrated and dissolved in DMF (10 mL) and TEA (644 mg, 6.37 mmol) was added into the mixture, then the mixture was stirred at 70° C. for 2 h. The mixture was diluted with H2O (50 mL), extracted with EtOAc (20 mL*3), washed with brine, dried over Na2SO4, concentrated to get the crude product 46b (180 mg, 78% yield) as a yellow solid. LC-MS (Method 4) tR=1.10 min, m/z (M+H)+=183.0.

To a solution of 46c (70 mg, 0.26 mmol) in THF (2 mL) was added Pd/C (7 mg, 10% wt), then the mixture was stirred at r.t. under H2for 2 h. The mixture was filtered and the filtrate was concentrated to get the compound 46d (52 mg, 84% yield) as a colorless solid. LC-MS (Method 4) tR=1.58 min, m/z (M+H)+=235.1.

To a solution of 47b (400 mg, 1.72 mmol) in MeOH (5 mL) was added HCl (2 N in H2O, 1 mL). Then the mixture was stirred at 60° C. for 1 h. The mixture was concentrated to get the compound 47c (350 mg, 95% yield) as a white solid. LC-MS (Method 4) tR=0.76 min, m/z (M+H)+=214.0.

To a stirred solution of 48b (150 mg, 0.69 mmol) in methanol (6 mL) was added 12 (265 mg, 2.07 mmol) at 0° C. The mixture was stirred for 48 h at 55° C. The mixture was quenched by addition of saturated sodium thiosulfate solution dropwise at r.t. The mixture was diluted with H2O (30 mL), extracted with EtOAc (20 mL*3). The organic layer was concentrated and purified by Prep-HPLC (Method E) to obtain 48c (186 mg, 78% yield) as a light yellow solid. LC-MS (Method 4) tR=2.94 min, m/z (M+H)+=343.9.

A solution of 49a (300 mg, 0.86 mmol) and methylamine (5 mL, 2.0 M in THF) was stirred at 50° C. overnight in a sealed tube. The mixture was cooled to r.t. and concentrated to afford the title compound 49b (210 mg, 70% yield) as a yellow solid. LC-MS (Method 3) tR=1.26 min, m/z (M+H)+=347.0.

A solution of methyl 56a (200 mg, 0.52 mmol) in TFA (4 mL) was stirred at 40° C. for 2 h. The mixture was concentrated and then diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, extracted with EtOAc (15 mL*3), washed with brine (20 mL), dried over Na2SO4, concentrated and purified by flash chromatography (DCM/MeOH=100/1 to 25/1) to get the compound 56b (55 mg, 40% yield) as a yellow solid. LC-MS (Method 4) tR=2.13 min, m/z (M+H)+=263.1.

Compound 56d (2.2 g, 10.33 mmol) was dissolved in DCM (20 mL) and the mixture was cooled to 5° C., triethylamine trihydrofluoride (4.99 g, 30.98 mmol) and NBS (2.21 g, 12.39 mmol) were added to the mixture in one portion and the mixture was stirred at r.t. overnight. The mixture was washed with 10% aq. solution of NaHCO3(200 mL*2) and brine (100 mL), dried over Na2SO4, and evaporated in vacuo at 45° C. to get the compound 56e (2.5 g, 78% yield) as a colorless oil.

To a solution of 56h (10 mg, 0.023 mmol) in the solvent (MeOH/THF/H2O=2/2/1, 0.5 mL) was added LiOH·H2O (3 mg, 0.070 mmol), then the mixture was stirred at r.t. for 4 h. The mixture was acidified to pH=4 with 1 N HCl, then the mixture was concentrated to get the crude compound 56i (9 mg, 93% yield) as a yellow solid. LC-MS (Method 4) tR=3.44 min, m/z (M+H)+=413.1.

To a solution of 4-bromopyridin-3-ol (3.0 g, 17.24 mmol) in H2O (30 mL) was added Na2CO3(3.91 g, 36.21 mmol) and 12 (4.38 g, 17.24 mmol), then the mixture was stirred at r.t. for 16 h. The mixture was adjusted to pH=4 with aq HCl (2 N), the mixture was filtered and the solid was dried to get the compound 57a (5.1 g, 99% yield) as a white solid. LC-MS (Method 4) tR=2.67 min, m/z (M+H)+=299.9.

To a solution of 57b (4 g, 12.74 mmol) in THF (40 mL) was addediPrMgCl (7.7 mL, 15.4 mmol, 2 M in THF) at 0° C., and the mixture was stirred at 0° C. for 1 h. A solution of N-methoxy-N-methyl-propanamide (1.81 g, 15.4 mmol) in THF (5 mL) was added into the mixture at 0° C. and stirred at 0° C. for 1 h and stirred at r.t. for 4 h. The mixture was diluted with H2O (200 mL), extracted with EtOAc (60 mL*3), washed with brine (60 mL), dried over Na2SO4, concentrated and purified by flash chromatography (PE/EA=100/1 to 2/1) to get the compound 57c (2.7 g, 87% yield) as a colorless oil. LC-MS (Method 4) tR=3.80 min, m/z (M+H)+=244.0.

A mixture of 58a (7.00 g, 37.12 mmol) and N2H4·H2O (5.57 g, 111.37 mmol) in isobutanol (5 mL) was stirred at 80° C. for 2 h. A yellow suspension was formed. The reaction mixture was concentrated and triturated with MeCN (30 mL) to give 58b as a pale brown solid (6.8 g, yield given), which was used for the next step directly without further purification. LC-MS (Method 4) tR=0.61 min, m/z (M+H)+=185.1.

To a mixture of 58c (1.20 g, 5.35 mmol) in DCM (15 mL) and MeOH (5 mL), was added PhI(AcO)2(1.72 g, 5.35 mmol) at 20° C. The resulting mixture was stirred at 20° C. for 12 h. A yellow solution was formed. The reaction mixture was quenched with water (50 mL) and extracted with DCM (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 10-60%) to give 58d as a yellow solid (410 mg, 34% yield). LC-MS (Method 4) tR=2.95 min, m/z (M+H)+=223.1.

To a solution of 58d (130 mg, 0.585 mmol) in ethanol (10 mL) was added aq. Na2S2O4(1 M, 5 mL). The resulting mixture was stirred at 80° C. under N2atmosphere for 10 min. A white suspension was formed. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (MeOH in DCM is 0-10%) to give 58e (40 mg, 36% yield) as a brown solid. LC-MS (Method 4) tR=0.72 min, m/z (M+H)+=193.1.

A mixture of 58f (80 mg, 0.195 mmol) and LiOH·H2O (25 mg, 0.585 mmol) in co-solvent of methanol (4 mL) and water (1 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was concentrated and dried in vacuo to give 58g (80 mg, yield given) as a yellow solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=2.05 min, m/z (M+H)+=397.2.

To a solution of 63a (46 mg, 0.1 mmol) in MeOH/H2O (1 mL/0.5 mL) was added LiOH·H2O (8.5 mg, 0.2 mmol) and the mixture was stirred at r.t. for 16 h. The mixture was diluted with 0.5 N HCl aq. (2 mL), extracted with EA (3 mL*3), washed with brine, dried over Na2SO4, concentrated to get the compound 63b (50 mg, yield given) as an off-white solid.

To a mixture of 66f (3.9 g, 13.34 mmol) in THF (40 mL) was added LiAlH4(557 mg, 14.68 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 0.5 h under N2. The reaction mixture was quenched with ice-water (4 mL) followed by 15% ag. NaOH (4 mL), water (12 mL). To the mixture was added Na2SO4(40 g) and EtOAc (150 mL) and the mixture was stirred at r.t. for 16 h. The mixture was filtered and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (EtOAc) to afford the title compound 66g (1.7 g, 48% yield) as a yellow solid. LC-MS (Method 3) tR=1.26 min, m/z (M+H)+=265.2.

To a mixture of 66g (2.09 g, 7.91 mmol) and TEA (2.40 g, 23.73 mmol) in DCM (20 mL) was added methanesulfonyl chloride (1.36 g, 11.86 mmol) dropwise at 0° C. The reaction was stirred at 0° C. for 1 h under N2and diluted with ice-water (10 mL). The mixture was extracted with DCM (30 mL*2) and the combined organic layer was dried over Na2SO4and filtered. The filtrate was concentrated to afford the title compound 66h (2.71 g, yield given) as a yellow oil. LC-MS (Method 3) tR=1.38 min, m/z (M+H)+=343.2.

To a solution of 66h (500 mg, 1.46 mmol) and 66b (316 mg, 1.53 mmol) in THF (5 mL) was added NaH (84 mg, 2.19 mmol, 60% purity in mineral oil) at 0° C. After stirring at r.t. for 30 min, the mixture was quenched with brine (8 mL) and extracted with EtOAc (10 mL*3). The combined organic layer was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=1/1) to afford the title compound 66i (400 mg, 61% yield) as a yellow oil. LC-MS (Method 3) tR=1.58 min, m/z (M+H)+=452.2.

A mixture of 69b (1.62 g, 5.92 mmol) and CaCO3(2.96 g, 29.60 mmol) in H2O/1,4-dioxane (10 mL/10 mL) was stirred at 115° C. for 36 h. The reaction mixture was cooled, diluted with water (15 mL) and extracted with EtOAc (20 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under the reduced pressure to afford the title compound 69c (900 mg, 72% yield) as a yellow solid. LC-MS (Method 3) tR=1.46 min, m/z (M+H)+=212.0.

A mixture of 6-bromo-3-methylpicolinic acid (1.0 g, 4.63 mmol) in THF (10 mL) was added BH3·THF (9.2 mL, 9.26 mmol, 1.0 M in THF) at 0° C. The reaction mixture was stirred at 55° C. for 24 h. The reaction mixture was diluted with MeOH (10 mL) and 6 M HCl (10 mL) and stirred at 60° C. for 1 h. The reaction mixture was cooled to room temperature and extracted with EtOAc (30 mL*2). The combined organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=4/1) to afford the title compound 70b (280 mg, 30% yield) as a colorless oil. LC-MS (Method 3) tR=1.12 min, m/z (M+H)+=202.1.

To a mixture of 2-bromo-6-methylpyridin-4-amine (5.0 g, 26.73 mmol) in H2O (40 mL) and conc. HCl (25 mL) was added NaNO2(3.69 g, 53.47 mmol) at 0° C. over 10 min. After stirring for 10 min at this temperature, to the reaction mixture was added hexafluorophosphoric acid solution (8.58 g, 58.81 mmol, 65% purity) dropwise. The reaction mixture was stirred at 0° C. for 0.5 h and the formed solid was collected by filtering. The filter cake was washed with ice- water (30 mL), diethyl ether (30 mL) and dried in the air for 24 h. The solid was slowly heated to 100° C. and a dark-red oily material was formed after 10 min. After cooling to r.t., the oil was basified with 1M aq. NaOH to pH=10 and extracted with DCM (50 mL*2). The combined organic layer was dried over Na2SO4, filtered and concentrated to afford the title compound 71b (1.24 g, 24% yield) as a black oil.1H NMR (400 MHz, CDCl3) δ 7.10-7.07 (m, 1H), 6.87 (dd, J=9.2 Hz, 2.0 Hz, 1H), 2.54 (s, 3H).

A mixture of 71b (1.1 g, 5.79 mmol), NBS (1.03 g, 5.79 mmol) and BPO (70 mg, 0.29 mmol) in CCl4(10 mL) was stirred at illumination for 2 h. The reaction mixture was diluted with water (10 mL) and extracted with DCM (20 mL*2). The combined organic phase was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=10/1) to afford the title compound 71c (710 mg, 46% yield) as a colorless oil. LC-MS (Method 3) tR=1.48 min, m/z (M+H)+=267.9.

To a solution of 22d (90 mg, 0.32 mmol) and 66a (66 mg, 0.32 mmol) in MeOH (2 mL) was added NaBH3CN (102 mg, 1.62 mmol). The reaction mixture was stirred at r.t. for 5 min. Then AcOH (2 mg, 0.03 mmol) was added to the solution. After stirring at r.t. for 2 h, the reaction mixture was quenched with water (5 mL) and extracted with EtOAc (20 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=1/1) to afford the title compound 72a (80 mg, 53% yield) as a yellow oil. LC-MS (Method 3) tR=1.51 min, m/z (M+H)+=465.1.

To a solution of 73d (6.65 g, 17.03 mmol) in MeOH (65 mL) was added NaBH4(1.29 g, 34.06 mmol) portionwise at 0° C. The reaction mixture was stirred at r.t. for 1 h. The reaction mixture was quenched with water (80 mL) and extracted with EtOAc (70 mL*3). The combined organic layer was concentrated to afford the title compound 73e (5.4 g, 87% yield) as a white solid. LC-MS (Method 1) tR=1.55 min, m/z (M+H)+=363.3.

To a solution of methyl 3-amino-4-methoxy-5-nitrobenzoate (1.3 g, 5.75 mmol) in THF (15 mL) was added N-(tosyloxy)acetimidamide (1.44 g, 6.32 mmol) followed by trimethoxymethane (915 mg, 8.62 mmol). After the reaction mixture was stirred at 60° C. for 2 h, the reaction mixture was cooled and concentrated. The residue was dissolved in DCM (50 mL), washed with sat. NaHCO3(50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=1/1) to afford the title compound 75b (1.68 g, yield given) as a yellow solid. LC-MS (Method 3) tR=1.53 min, m/z (M+H)+=293.0.

To a solution of 75b (1 g, 3.42 mmol) in THF (40 mL) was added diisobutylaluminum hydride (20 mL, 20.53 mmol, 1.0 M in hexane) at −70° C. The reaction mixture was stirred at −70° C. for 30 min and then stirred at r.t. for 4 h. The reaction mixture was quenched with sat. NH4Cl (50 mL) and the temperature was maintained below 25° C. The separated aqueous phase was extracted with DCM (100 mL*2). The combined organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (EtOAc) to afford the title compound 75c (450 mg, 50% yield) as a colorless oil. LC-MS (Method 3) tR=1.31 min, m/z (M+H)+=265.0.

To a mixture of methan-d3-amine hydrochloride (1.40 g, 19.86 mmol) in DCM (200 mL) was added 1g (3.5 g, 16.55 mmol) slowly followed by TEA (1.68 g, 16.55 mmol, 2.31 mL) at −78° C. After stirring for 1 h at this temperature, the reaction was quenched with water (30 mL). The organic layer was separated and concentrated. The residue was purified by flash chromatography using silica gel (PE/EtOAc=5/1) to afford the title compound 76a (1.38 g, 35% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.85 (s, 1H).

A mixture of 81a (41 mg, 0.06 mmol) in TFA (0.5 mL) and DCM (1.5 mL) was stirred at 35° C. for 0.5 h. The solvent was removed by pumping through N2and the residue was purified via reverse flash (C-18) (5% to 95% acetonitrile in water containing 0.1% HCl) to afford the title compound 81b (36 mg, 98% yield) as a yellow solid. LC-MS (Method 3) tR=1.35 min, m/z (M+H)+=531.3.

To a solution of 82c (8.2 g, 22.56 mmol) in DCM (10 mL) was added TFA (30 mL) at 0° C. The mixture was stirred at rt for 5 h. After the reaction was completed, saturated NaHCO3solution (100 mL) was added. The mixture was stirred at r.t. for 20 min. DCM (100 mL) was added to the mixture. The organic layer was separated, washed with brine (100 mL), dried over Na2SO4and filtered. The filtrate was concentrated to afford the title compound 82d (5.2 g, 99% yield) as a white solid. LC-MS (Method 3) tR=1.31 min, m/z (M+H)+=234.0.

To a solution of 66a (500 mg, 2.45 mmol), (2,4-dimethylphenyl)methanamine (410 mg, 2.45 mmol) in MeOH (5 mL) was added acetic acid (14.71 mg, 0.245 mmol). After stirring at 0° C. for 15 min, NaBH3CN (616 mg, 9.80 mmol) was added to the mixture. The mixture was stirred for 2 h at r.t. Then the reaction was quenched with water (5 mL) and extracted with EtOAc (8 mL*3). The combined organic layer was concentrated to afford the title compound 87a (375 mg, 43% yield) as a yellow oil. LC-MS (Method 3) tR=1.64 min, m/z (M+H)+=355.1.

To a mixture of 90b (140 mg, 0.24 mmol) and 67a (50 mg, 0.24 mmol) in anhydrous THF (14 mL) was added LiHMDS (0.48 mL, 0.48 mmol, 1.0 M in THF) at −40° C. The mixture was stirred at −40° C. for 1 h. The mixture was diluted with H2O (5 mL), extracted with EtOAc (15 mL*2). The separated organic layer was dried over MgSO4, filtered and concentrated to afford the crude title compound 90c (180 mg, 99% yield) as a yellow solid which was used directly in next step without further purification. LC-MS (Method 3) tR=1.67 min, m/z (M+H)+=752.3.

To a mixture of 92c (420 mg, 1.60 mmol) and 92b (407 mg, 1.60 mmol) in THF (20 mL) was added 1.5 M NaOH (1.60 mL, 2.4 mmol) at 0° C. The reaction mixture was stirred at r.t. for 4 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (30 mL*2). The combined organic layer was concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=1/1) to afford the title compound 92d (444 mg, 56% yield) as a yellow solid. LC-MS (Method 3) tR=1.70 min, m/z (M+H)+=499.2.

The mixture of 92d (300 mg, 0.60 mmol), NaBH4(23 mg, 0.60 mmol) and Pd(OAc)2(7 mg, 0.03 mmol) were combined in a round-bottom flask. MeOH (6 mL) was slowly added into the flask through a syringe under H2atmosphere. The reaction mixture was stirred at r.t. for 18 h. The reaction mixture was concentrated and the residue was purified by flash chromatography on silica gel (DCM/MeOH=20/1) to afford the title compound 92e (80 mg, 28% yield) as a yellow solid. LC-MS (Method 3) tR=1.38 min, m/z (M+H)+=473.2.

To a mixture of 9e (150 mg, 0.66 mmol), 11c (203 mg, 0.99 mmol) in EtOH (3 mL) was added conc. HCl (64 mg, 0.66 mmol). The reaction mixture was stirred at 80° C. for 12 h. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel (DCM/MeOH=50/1) to give the title compound 98a (50 mg, 21% yield) as a yellow solid. LC-MS (Method 3) tR=1.65 min, m/z (M+H)+=360.3.

To a solution of 106b (10.8 g, 72.89 mmol) in DMF (80 mL) was added KOH (8.18 g, 145.79 mmol) at 0° C. for 5 min. Then 12 (18.44 g, 72.89 mmol) was added to the mixture. The reaction mixture was stirred at 0° C. for 30 minutes. The reaction mixture was used directly in next step without working up. LC-MS (Method 3) tR=1.33 min, m/z (M+H)+=275.1.

A mixture of 106e (6.0 g, 21.64 mmol), LiCl (1.19 g, 28.13 mmol) and TsOH·H2O (5.34 g, 28.13 mmol) in DMSO (50 mL) was stirred at 60° C. for 1 h. The reaction mixture was diluted with water (150 mL) and extracted with EtOAc (50 mL*3). The combined organic phase were dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=2/1) to give the title compound 106f (4.1 g, 72% yield) as a white solid. LC-MS (Method 3) tR=1.28 min, m/z (M+H)+=264.2.

To a solution of 107c (500 mg, 2.08 mmol) in anhydrous DMF (5 mL) was added NaH (74.9 mg, 1.87 mmol, 60% purity in mineral oil) at 0° C. After stirring at 0° C. for 30 min, to the mixture was added iodomethane (265.9 mg, 1.87 mmol) at 0° C. The reaction was stirred at r.t. for 5 h. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (5 mL*2). The organic layer was dried over Na2SO4, filtered, and the filtrate was concentrated. The title compound was purified by Prep-HPLC (Method A) to afford 107d (130 mg, 25% yield) as a yellow oil. LC-MS (Method 3) tR=1.27 min, m/z (M+H)+=255.0.

To a solution of 108a (400 mg, 1.32 mmol) in DCM (4 mL) was added TFA (1 mL). The mixture was stirred at r.t. for 1 h. The mixture was concentrated to give the title compound 108b (400 mg, 96% yield) as a brown solid. LC-MS (Method 3) tR=0.29 min, m/z (M+H)+=204.3.

To a solution of 106f (400 mg, 1.06 mmol) and Cs2CO3(1.04 g, 3.19 mmol) in DMF (4 mL) was added 2,2,2-trifluoroethyl methanesulfonate (227 mg, 1.28 mmol) at r.t. The mixture was stirred at 80° C. for 16 h. The reaction mixture was diluted with H2O (5 mL) and extracted with EtOAc (8 mL*3). The combined organic phase was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to afford the title compound 110a (135 mg, 37% yield) as a yellow solid. LC-MS (Method 3) tR=1.51 min, m/z (M+H)+=346.3.

To a stirred solution of 123c (500 mg, 2.37 mmol) in DMF (5 mL) was added NaH (181 mg, 4.74 mmol, 60% in mineral oil). After stirring for 30 min at r.t., to it was added iodoethane (554 mg, 3.55 mmol). The reaction mixture was stirred at r.t. for 3 h. The reaction mixture was diluted with H2O (30 mL) and extracted with EtOAc (50 mL*3). The organic phase was evaporated under reduced pressure to afford 125a (500 mg, 88% yield) as a brown solid. LC-MS (Method 3) tR=1.10 min, m/z (M+H)+=240.0.

To a solution of 126a (1.1 g, 2.85 mmol) in DMF (15 mL) was added K2CO3(982 mg, 7.11 mmol) and CuI (54 mg, 0.28 mmol). The mixture was stirred at 100° C. for 16 h. Water (50 mL) was added to above mixture. The solution was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 126b (700 mg, 2.60 mmol, 91% yield) as a yellow oil. LC-MS (Method 4) tR=4.69 min, m/z (M+H)+=269.0.

To a solution of 126c (600 mg, 1.96 mmol) in dioxane (6 mL) was added a solution of HCl (g) in dioxane (4 M, 6 mL). The mixture was stirred at r.t. for 30 min. The mixture was concentrated to dryness. The residue was diluted with H2O (30 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (30 mL*3). The organic layers were washed with aq Na2CO3(30 mL) and brine (30 mL), dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 126d (350 mg, 87% yield) as a yellow solid. LC-MS (Method 4) tR=2.80 min, m/z (M+H)+=206.1.

To a solution of 127d (600 mg, 1.40 mmol) in DMF (10 mL) was added K2CO3(580 mg, 4.20 mmol) and CuI (27 mg, 0.14 mmol). The mixture was stirred at 100° C. for 16 h. Water (30 mL) was added to above mixture. The solution was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 127e (250 mg, 57% yield) as a yellow oil. LC-MS (Method 4) tR=4.72 min, m/z (M+H)+=311.2.

To a solution of 127f (200 mg, 0.58 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (30 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (30 mL*3). The organic layers were washed with aq Na2CO3(30 mL) and brine (30 mL), dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA from 1/1 to 1/10) to give the title compound 127g (130 mg, 91% yield) as a yellow solid. LC-MS (Method 4) tR=2.40 min, m/z (M+H)+=248.2.

To a solution of 129a (3.5 g, 7.53 mmol) in DMF (50 mL) was added K2CO3(2.6 g, 18.83 mmol) and CuI (143 mg, 0.75 mmol). The mixture was stirred at 100° C. for 16 lh. Water (200 mL) was added to above mixture. The solution was extracted with EtOAc (60 mL*3). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 129b (2.4 g, 91% yield) as a yellow oil. LC-MS (Method 4) tR=4.81 min, m/z (M+H)+=347.1.

129b (2.4 g, 6.91 mmol) was dissolved in TFA (20 mL), then the mixture was stirred at 90° C. for 4 h. Then the mixture was concentrated, and diluted with H2O (50 mL), adjusted pH to 7 with aq NaHCO3, then extracted with EtOAc (50 mL*3), washed with brine (50 mL), dried over Na2SO4, concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to get the compound 129c (1.4 g, 89% yield) as a yellow solid.

To a solution of 129e (42 mg, 0.12 mmol) in dioxane (0.5 mL) was added a solution of HCl (g) in dioxane (4 M, 0.5 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL), dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to give the title compound 129f (20 mg, 67% yield) as a yellow solid. LC-MS (Method 4) tR=3.06 min, m/z (M+H)+=246.1.

To a solution of 130b (200 mg, 0.62 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to give the title compound 130c (90 mg, 65% yield) as a yellow solid. LC-MS (Method 4) tR=2.13 min, m/z (M+H)+=222.1.

To a solution of 131b (100 mg, 0.31 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to give the title compound 131c (50 mg, 72% yield) as a yellow solid. LC-MS (Method 4) tR=2.66 min, m/z (M+H)+=228.1.

To a solution of 132b (400 mg, 1.07 mmol) in DMF (15 mL) was added K2CO3(515 mg, 3.73 mmol) and CuI (20 mg, 0.11 mmol). The mixture was stirred at 100° C. for 16 h. Water (50 mL) was added to above mixture. The solution was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 132c (250 mg, 91% yield) as a yellow oil. LC-MS (Method 4) tR=4.49 min, m/z (M+H)+=258.1.

To a solution of 132d (110 mg, 0.37 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 30 min. The mixture was concentrated to dryness. The residue was diluted with H2O (30 mL), adjusted pH to 7-9 with aq Na2CO3and extracted with EtOAc (30 mL*3). The organic layers were washed with aq Na2CO3(30 mL) and brine (30 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 132e (40 mg, 55% yield) as a yellow solid. LC-MS (Method 4) tR=2.34 min, m/z (M+H)+=195.3.

To a solution of 133a (1.19 g, 3.06 mmol) in DMF (15 mL) was added K2CO3(1.56 mg, 7.65 mmol) and CuI (59 mg, 0.31 mmol). The mixture was stirred at 100° C. for 16 h. Water (50 mL) was added to above mixture. The solution was extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 133b (700 mg, 2.60 mmol, 85% yield) as a yellow oil. LC-MS (Method 4) tR=4.69 min, m/z (M+H)+=272.0.

To a solution of 133c (600 mg, 1.96 mmol) in dioxane (6 mL) was added a solution of HCl (g) in dioxane (4 M, 6 mL). The mixture was stirred at r.t. for 30 min. The mixture was concentrated to dryness. The residue was diluted with H2O (30 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (30 mL*3). The organic layers were washed with aq Na2CO3(30 mL) and brine (30 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 133d (350 mg, 87% yield) as a yellow solid. LC-MS (Method 4) tR=2.80 min, m/z (M+H)+=209.1.

To a solution of 134a (1.7 g, 3.94 mmol) in DMF (20 mL) was added K2CO3(1.36 g, 18.83 mmol) and CuI (75 mg, 0.39 mmol). The mixture was stirred at 100° C. for 16 h. Water (200 mL) was added to above mixture. The solution was extracted with EtOAc (60 mL*3). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under vacuum to give crude compound 134b (1.0 g, 72% yield) as a yellow oil. LC-MS (Method 4) tR=4.84 min, m/z (M+H)+=350.1.

134b (1.0 g, 2.86 mmol) was dissolved in TFA (10 mL), then the mixture was stirred at 90° C. for 4 h. Then the mixture was concentrated and diluted with H2O (50 mL), adjusted pH to 7 with aq NaHCO3, then extracted with EtOAc (50 mL*3), washed with brine (50 mL), dried over Na2SO4, concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to get the compound 134c (500 mg, 76% yield) as a yellow solid.

To a solution of 134e (200 mg, 0.67 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to give the title compound 134f (120 mg, 90% yield) as a yellow solid. LC-MS (Method 4) tR=2.32 min, m/z (M+H)+=200.3.

To a solution of 129c (200 mg, 0.88 mmol) in DMF (5 mL) was added NaH (135 mg, 3.52 mmol, 60% purity in mineral oil) at 0° C. After stirring at 25° C. for 30 min, to the reaction mixture was added a solution of CD3I (255 mg, 1.76 mmol) in DMF (0.2 mL). The reaction was stirred at 25° C. for 16 h and quenched with water (15 mL). The resultant mixture was extracted with EtOAc (30 mL*3) and the combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=10/1) to afford the title compound 140a (148 mg, 69% yield) as a yellow solid.1H NMR (300 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.26 (d, J=8.7 Hz, 1H), 4.04 (s, 3H).

To a solution of 141a (2.0 g, 7.10 mmol) in MeOH/THF/H2O (20 mL, v/v/v=2/2/1) was added LiOH·H2O (1.49 g, 35.50 mmol). After stirring at r.t. for 12 h, the reaction mixture was concentrated to dryness and acidified with 1 N HCl to pH=2. The formed solid was collected by filtering and filter cake was dried to give the crude compound 141b (1.8 g, 95% yield) as a yellow solid. LC-MS (Method 3) tR=0.88 min, m/z (M+H)+=268.2.

A mixture of 141b (400 mg, 1.50 mmol), trideuteriomethanamine hydrochloride (529 mg, 7.50 mmol) and DIPEA (1.16 g, 9.00 mmol) in T3P (2 mL, 50% wt in DMF) was stirred at 50° C. for 16 h. After cooling to r.t., the reaction mixture was poured into water (10 mL) and the formed solid was collected by filtering. The filter cake was slurried with MeOH (5 mL) for 30 min. The solid was filtered and dried to afford the title compound 141c (380 mg, 90% yield) as a yellow solid. MS (Method 3) tR=1.10 min, m/z (M+H)+=284.1.

To a mixture of 142a (700 mg, 2.59 mmol) and K2CO3(537.68 mg, 3.89 mmol) in DMF (10 mL) was added CH3I (405 mg, 2.85 mmol) and at 20° C. The resulting mixture was stirred at 20° C. for 1 h. A yellow solution was formed. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 0-10%) to give 142b (710 mg, 96% yield) as a white solid. LC-MS (Method 4) tR=3.82 min, ink (M+H)+=283.9.

To a mixture of 142b (710 mg, 2.50 mmol) in THF (10 mL), was added LDA (2 M, 1.50 mL) at −78° C. The resulting mixture was stirred at −78° C. for 15 min. Then the DMF (192 mg, 2.63 mmol, 203 μL) was added into the above mixture at −78° C. The reaction mixture was further stirred at −78° C. for 1 h. A yellow solution was formed. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 0-20%) to give 142c (564 mg, 72% yield) as a yellow solid. LC-MS (Method 4) tR=3.52 min, m/z (M+H)+=311.9.

A mixture of 142c (364 mg, 1.17 mmol) and tert-butyl N-aminocarbamate (170 mg, 1.28 mmol) in ethanol (10 mL) was stirred at 80° C. for 2 h. A yellow suspension was formed. The reaction mixture was concentrated to give a yellow solid, which was further purified by flash chromatography (EA in PE is 10-30%) to give 142d (497 mg, 99% yield) as a light-yellow solid. LC-MS (Method 4) tR=4.66 min, m/z (M+H)+=425.9.

A solution of 142d (200 mg, 0.469 mmol), K2CO3(130 mg, 0.934 mmol) and CuI (45 mg, 0.235 mmol) in DMF (2 mL) was degassed and purged with nitrogen for 3 times. The resulting mixture was stirred at 100° C. under N2atmosphere for 12 h. A black suspension was formed. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was dried in vacuo to give 142e (162 mg, crude) as a yellow solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=4.52 min, m/z (M+H)+=345.0.

To a mixture of 142e (162 mg, 0.469 mmol) in DCM (3 mL), was added TFA (1 mL). The resulting mixture was stirred at 20° C. for 1 h. A brown suspension was formed. The reaction mixture was quenched with aq. NaHCO3(40 mL) and extracted with DCM (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 20-50%) to give 142f (67 mg, 58% yield for 2 steps) as a yellow solid. LC-MS (Method 4) tR=3.75 min, m/z (M+H)+=245.0.

To a mixture of 142h (38 mg, 0.121 mmol) in DCM (3 mL), was added TFA (1 mL). The resulting mixture was stirred at 20° C. for 1 h. A brown suspension was formed. The reaction mixture was quenched with aq. NaHCO3(40 mL) and extracted with DCM (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 10-50%) to give 142i (15 mg, 58% yield) as a yellow solid. LC-MS (Method 4) tR=3.07 min, m/z (M+H)+=215.1.

A mixture of methyl 142j (30 mg, 0.069 mmol) and LiOH·H2O (9 mg, 0.208 mmol) in co-solvent of MeOH (3 mL) and water (1 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was diluted with water (5 mL), adjusted to pH=6 with 1 M aq. HCl, concentrated and dried in vacuo to give 142k (29 mg, yield given) as a yellow solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=3.10 min, m/z (M+H)+=419.2.

To a stirred solution of 42f (1.0 g, 3.42 mmol) in EtOH (20 mL) was added cyclopropylhydrazine hydrochloride (500 mg, 4.26 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated and slurried with EtOH (10 mL) to give 143a (1.0 g, 84% yield) as a white solid. LCMS (Method 4) tR=4.58 min, m/z (M+H)+=347.0.

To a solution of 144a (100 mg, 0.41 mmol) in MeOH (1 mL) and H2O (1 mL) was added LiOH·H2O (34 mg, 0.82 mmol), then the mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated and used for next step directly without further purification. LC-MS (Method 4) tR=1.44 min, m/z (M+H)+=242.1.

To a stirred solution of 132a (1 g, 3.38 mmol) in EtOH (20 mL) was added cyclopropylhydrazine hydrochloride (500 mg, 4.26 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated and slurried with EtOH (2 mL) to give the compound 145a (1 g, 77% yield) as a white solid. tR=4.58 min, m/z (M+H)+=350.0.

To a solution of 142c (108 mg, 0.35 mmol) in EtOH (10 mL) was added ethylhydrazine hydrochloride (43 mg, 0.45 mmol). The resulting mixture was stirred at r.t. for 1 h. Then the solvent was removed under vacuum, and the residue was purified by flash chromatography on silica gel (PE/EA=10/1) to afford compound 147a (104 mg, 77% yield) as a white solid. LC-MS (Method 4) tR=4.67 min, m/z (M+H)+=352.9.

To a solution of 147a (240 mg, 0.61 mmol) in DMF (5 mL) was added CuI (13 mg, 0.07 mmol) and K2CO3(260 mg, 1.88 mmol). The resulting mixture was stirred at 100° C. under nitrogen for 16 h. Then the mixture was allowed to cooled down to r.t. and quenched with water (10 mL), extracted with EtOAc (10 mL*3). The organic layers were combined, dried over anhydrous Na2SO4and concentrated. The residue was purified by chromatography on silica gel (PE/EA=8/1) to give the compound 147b (95 mg, 57% yield) as a light-yellow oil. LC-MS (Method 4) tR=4.70 min, m/z (M+H)+273.0.

To a solution of 147b (95 mg, 0.35 mmol) and tert-butyl carbamate (54 mg, 0.46 mmol) in anhydrous 1,4-dioxane (5 mL) was added Pd2(dba)3(16 mg, 0.02 mmol), XantPhos (24 mg, 0.04 mmol) and Cs2CO3(230 mg, 0.70 mmol). The resulting mixture was stirred at 100° C. under nitrogen for 16 h. Then the mixture was allowed to cooled down to r.t. The solid was removed by filtration, and the filtrate was concentrated. The residue was purified by chromatography on silica gel (PE/EA=6/1) to provide the compound 147c (83 mg, 77% yield) as a light-yellow oil. LC-MS (Method 4) tR=4.71 min, m/z (M+H)+=310.2.

To a stirred solution of compound 42h (0.5 g, 1.96 mmol) and 1-dodecanethiol (845 mg, 4.17 mmol) in DMF (5 mL) was added t-BuOLi (343 mg, 4.28 mmol). The resulting mixture was stirred at 100° C. for 2.5 h. Then the mixture was cooled to room temperature and quenched with water (20 mL). The pH was adjusted to 5 with 2 N HCl aqueous solution and extracted with EtOAc (13 mL*3). The organic layers were combined, dried over anhydrous Na2SO4and concentrated. The residue was purified by chromatography on silica gel (PE/EA=10/1) to give the compound 148a (0.45 g, 95% yield) as a light-yellow solid. LC-MS (Method 4) tR=3.62 min, m/z (M+H)+=241.0.

To a solution of 148a (0.45 g, 1.87 mmol) in DMF (5 mL) was added NaH (247 mg, 6.17 mmol, 60% in mineral oil) at 0° C. After stirred at r.t. for 0.5 h, dibromodifluoromethane (3.92 g, 18.6 mmol) was added, and the resulting reaction mixture was stirred at r.t. for 16 h. Then it was quenched with water (20 mL) and extracted with EtOAc (15 mL*3). The organic layers were combined, dried over anhydrous Na2SO4and concentrated. The residue was purified by chromatography on silica gel (PE/EA=15/1) to give the compound 148b (0.60 g, 87% yield) as a yellow solid. LC-MS (Method 4) tR=5.11 min, m/z (M+H)+=369.0.

To a solution of 148b (0.6 g, 1.62 mmol) in DCE (10 mL) was added AgBF4(1.1 g, 5.64 mmol) at 0° C. Then the mixture was stirred at 65° C. for 4 h under nitrogen atmosphere. Then it was quenched with water (10 mL) and extracted with DCM (20 mL*3). The organic layers were combined, dried over anhydrous Na2SO4and concentrated. The residue was purified by chromatography on silica gel (PE/EA=9/1) to give the compound 148c (428 mg, 77% yield) as a dark-yellow solid. LC-MS (Method 4) tR=5.15 min, m/z (M+H)+=309.0.

To a solution of 149a (2.0 g, 8.58 mmol) in anhydrous THF (25 mL) was added EtMgBr (2 M in THF, 6.5 mL, 13 mmol) dropwise at 0° C. under nitrogen atmosphere. Then the mixture was allowed to warm up to r.t. and stirred for 1 h, cautiously quenched with sat. NH4Cl (20 mL). After the separation of the layers, the aqueous layer was extracted with DCM (20 mL*3). The organic layers were combined, dried over anhydrous Na2SO4and concentrated. The residue was purified by silica gel chromatography (PE/EA=5/1) to give 149b (1.11 g, 49% yield) as a colorless liquid. LC-MS (Method 4) tR=3.86 min, m/z (M+H−18)+=245.0.

To a solution of 149b (1.11 g, 4.22 mmol) in DCM (15 mL) was added Dess-Martin (2.3 g, 5.42 mmol), and the mixture was stirred at r.t. for 3 h. The resulting reaction mixture was concentrated under vacuum. The residue was purified by chromatography on silica gel (PE/EA=5/1) to give 149c (880 mg, 80% yield) as a colorless liquid.

A solution of 149c (880 mg, 3.37 mmol) and hydrazine hydrate (6 mL, 80% in H2O) in EtOH (15 mL) was stirred at 90° C. for 3 h. Then the reaction solution was cooled down to r.t. and concentrated under vacuum. The residue was purified by chromatography on silica gel (PE/EA=7/1) to afford 149d (215 mg, 25% yield) as a light yellow solid. LC-MS (Method 4) tR=3.94 min, m/z (M+H)+=255.0.

To a stirred mixture of 149d (215 mg, 0.84 mmol) and K2CO3(242 mg, 1.75 mmol) in DMF (6 mL) was added MeI (136 mg, 0.95 mmol). After stirred at 70° C. for 2 h, the mixture was quenched with water (15 mL), extracted with EA (12 mL*3) and The combined organic layer was dried over anhydrous Na2SO4and concentrated. The residue was purified by chromatography on silica gel (PE/EA=7/1) to give 149e (144 mg, 64% yield) as a yellow oil. LC-MS (Method 4) tR=4.48 min, m/z (M+H)+=269.0.

To a solution of 149 (22 mg, 0.05 mmol) in THF (0.8 mL) and H2O (0.2 mL) was added LiOH·H2O (52 mg, 1.24 mmol), and the mixture was stirred at 65° C. for 16 h. Then it was cooled down to r.t. and adjusted pH to 5 with 2 N HCl aqueous solution. The acidified solution was extracted with DCM/MeOH=6:1 (3 mL*7) and The combined organic layer was dried over anhydrous Na2SO4. Then it was concentrated to afford the crude product 150a (15 mg, 71% yield) as a light yellow solid. LC-MS (Method 4) tR=2.64 min, m/z (M+H)+=410.3.

To a mixture of 147d (280 mg, 1.14 mmol), 42b (237 mg, 1.14 mmol) in THF (5 mL) was added LiHMDS (9.12 mL, 1 M in THF) at −60° C. After stirring for 20 min at 20° C., the reaction was quenched with ice water (5 mL) and the organic layer was removed under vacuo. The formed solid was collected by filtering and dried to afford the title compound 151a (340 mg, 81% yield) as a brown solid. LC-MS (Method 3) tR=1.71 min, m/z (M+H)+=381.1.

To a solution of 155a (2.30 g, 18.27 mmol) and (3-methoxy-2-trimethylsilyl-phenyl) trifluoromethanesulfonate (5 g, 15.23 mmol) in THF (20 mL) was added TBAF (6.37 g, 24.36 mmol) at 0° C. After stirring at r.t. for 16 h, the mixture was poured into sat. NaHCO3(20 mL) and extracted with EtOAc (20 mL*2). The separated organic layer was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=3/1) to afford the title compound 155b (1.5 g, 52% yield) as a white solid. LC-MS (Method 3) tR=1.17 min, m/z (M+H)+=189.1.

A mixture of 164c (550 mg, 2.20 mmol), DIPEA (569 mg, 4.40 mmol) and ethanamine (1.32 mL, 2.64 mmol, 2.0 M in THF) in ethanol (10 mL) was stirred at 20° C. for 12 h. A white suspension was formed. The reaction mixture was diluted with water (40 mL) and extracted with EtOAc (40 mL*3). The combined organic layer was washed with water (40 mL*3) and brine (40 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (EtOAc in PE is 0-10%) to give 164d (600 mg, 99% yield) as an orange oil. LCMS (Method 4) tR=4.82 min, m/z (M+H)+=275.0.

To a solution of 164d (600 mg, 2.18 mmol) in ethanol (10 mL), was added aq. Na2S2O 4 (8.55 mL, 8.55 mmol, 1 M). The resulting mixture was stirred at 80° C. under N2atmosphere for 10 min. A white suspension was formed. The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (MeOH in DCM is 0-10%) to give 164e (248 mg, 35% yield) as a brown solid. LCMS (Method 4) tR=2.23 min, m/z (M+H)+=247.0.

A mixture of 164i (110 mg, 0.268 mmol) and LiOH·H2O (34 mg, 0.804 mmol) in co-solvent of MeOH (6 mL) and water (2 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The mixture was adjusted pH=2 with 2N HCl, The reaction mixture was concentrated and dried in vacuo to give 164j (106 mg, crude) as a yellow solid, which was used for the next step directly without further purification. LCMS (Method 4) tR=2.51 min, m/z (M+H)+=397.1.

A mixture of 165b (1.45 g, 6.47 mmol) and O-(2,4-dinitrophenyl) hydroxylamine (1.42 g, 7.11 mmol) in MeCN (50 mL) was stirred at 50° C. for 16 h. A yellow solution was formed. The reaction was concentrated to give 165c (2.73 g, crude) as a yellow solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=1.66 min, m/z M+=240.1.

A mixture of 165f (40 mg, 0.098 mmol) and LiOH·H2O (9 mg, 0.208 mmol) in co-solvent of THF (3 mL) and water (1 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was adjusted pH=2 with 1 N HCl and concentrated and dried in vacuo to 165g (40 mg, yield given) as a yellow solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=2.35 min, m/z (M+H)+=393.2.

To a solution of 166b (130 mg, 0.45 mmol) in dioxane (2 mL) was added a solution of HCl (g) in dioxane (4 M, 2 mL). The mixture was stirred at r.t. for 16 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 166c (80 mg, 93% yield) as a brown solid. LC-MS (Method 4) tR=2.75 min, m/z (M+H)+=192.1.

To a solution of 167f (40 mg, 0.14 mmol) in dioxane (1 mL) was added HCl/dioxane (4 M, 1 mL). The mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (10 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (10 mL*3). The organic layers were washed with aq Na2CO3(10 mL) and brine (10 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 167g (25 mg, 80% yield) as a yellow solid. LC-MS (Method 4) tR=1.07 min, m/z (M+H)+=192.1.

To a solution of 168a (1.6 g, 6.48 mmol, CAS 89677-51-0) in DMF (8 mL) was added NaH (993 mg, 25.91 mmol, 60% purity in mineral oil) at 0° C. After stirring at 25° C. for 30 min, to it was added a solution of Boc2O (4.24 g, 19.43 mmol) in DMF (0.5 mL). The reaction mixture was stirred at 25° C. for 16 h and quenched with water (30 mL). The resultant mixture was extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (30 mL*3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was dissolved in MeOH (20 mL) and to it was added K2CO3(1.73 g, 12.52 mmol). The reaction mixture was stirred at 50° C. for 16 h. After cooling to r.t., iodoethane (3.03 g, 19.44 mmol) was added to the reaction. The reaction was stirred at stirred at 70° C. for 16 h. After cooling to r.t., the reaction mixture was concentrated and the residue was diluted with water (30 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel (PE/EtOAc=10/1) to afford the title compound 168b (780 mg, 32% yield) as a yellow oil.1H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J=8.8 Hz, 1H), 7.76 (d, J=8.8 Hz, 1H), 3.76 (s, 3H), 3.64-3.61 (m, 1H), 3.46-3.60 (m, 1H), 1.23 (s, 9H), 1.07 (t, J=7.2 Hz, 3H).

To a solution of 169a (150 mg, 0.448 mmol) in DCM (5 mL) was cooled to 0° C. and added TFA (1 mL) dropwise and then stirred at 25° C. for 2 h. The mixture was concentrated and the residue was diluted with DCM (10 mL), added sat. NaHCO3solution to adjust pH to 8 and extracted with DCM (10 mL*3). The combined organic layer was washed by brine (10 mL*2), dried over sodium sulphate and evaporated in vacuo to give 169b (70.0 mg, 66% yield) as a yellow solid. LC-MS (Method 5) tR=1.40 min, m/z (M+H)+=236.2.

A mixture of 160c (198 mg, 0.87 mmol), K2CO3(240 mg, 1.74 mmol) and CD3I (189 mg, 1.30 mmol) in DMF (2 mL) was stirred at 50° C. overnight. After cooling to r.t., the mixture was diluted with H2O (8 mL), extracted with DCM (10 mL*2) and washed with brine (5 mL). The combined organic layer was dried over Na2SO4, filtered and the filtrate was concentrated. The residue was slurried with EtOAc (5 mL) and filtered. The filter cake was dried to afford the title compound 170a (199 mg, 94% yield) as a green solid. LC-MS (Method 3) tR=1.12 min, m/z (M+H)+=245.0.

A mixture of 17a (1.5 g, 6.52 mmol) and NCS (1.13 g, 8.48 mmol) in ACN (10 mL) was stirred at 90° C. for 5 h. The reaction mixture was cooled, poured into ice-water (20 mL) and extracted with EtOAc (50 mL*2). The combined organic phase was dried over Na2SO4, filtered and concentrated to give the title compound 171a (2.0 g, yield given) as a brown solid. LC-MS (Method 3) tR=1.34 min, m/z (M+H)+=264.0.

A mixture of 171a (500 mg, 1.89 mmol) in DMF (5 mL) was added NaH (145 mg, 6.05 mmol, 60% in mineral oil) at 0° C. After stirring at r.t. for 30 min, to it was added MeI (537 mg, 3.78 mmol). The reaction was stirred at r.t. for 1 h and poured into ice-water. The mixture was extracted with EtOAc (50 mL*2) and the combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to afford 171b (310 mg, 59% yield) as a brown solid.1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.87 (s, 1H), 3.51 (s, 3H).

To a solution of 173b (4 g, 10.38 mmol) in DCM (30 mL) was added TFA (10 mL) at 0° C. The reaction mixture was stirred at 30° C. for 2 h. The solvent was removed by pumping through N2. The residue was diluted in water (20 mL) and basified with saturated Na2CO3to adjust pH to above 7. The mixture was extracted with EtOAc (80 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated to afford the title compound 173c (1.4 g, 57% yield) as an off-white solid. LC-MS (Method 3) tR=1.29 min, m/z (M+H)+=236.0.

A mixture of ethylhydrazine hydrochloride (1.71 g, 17.72 mmol) and KOH (994 mg, 17.72 mmol) in EtOH (20 mL) was stirred at r.t. for 0.5 h. The white solid was filtered off and the filtrate was added to the solution of 178a (3.5 g, 14.76 mmol) in 10 mL of EtOH. The resultant mixture was stirred at 100° C. for 16 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was purified by chromatography on silica gel (DCM/MeOH=20/1) to afford 178b (3.15 g, 82% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.16 (s, 1H), 4.15 (q, J=7.2 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H).

To a solution of 185b (250 mg, 0.52 mmol) and LiOH·H2O (109 mg, 2.60 mmol) in THF/MeOH/H2O (5 mL, v/v/v=3/1/1 mL) was stirred at 60° C. for 12 h. The organic solvent was removed under reduced pressure and the aqueous layer was acidified with 1 N HCl to pH=2. The formed solid was collected by filtering and was dried to afford compound 185c (230 mg, 95% yield) as a yellow solid. LC-MS (Method 3) tR=1.23 min, m/z (M+H)+=468.0.

To a solution of 187a (5 g, 23.92 mmol) and hexamethylphosphoramide (857 mg, 4.78 mmol) in THF (100 mL) was added LDA dropwise (26.3 mL, 52.62 mmol, 2 M in THF) at −60° C. After stirring for 1 h at −60° C., to the reaction mixture was added N-methoxy-N-methyl-acetamide (4.19 g, 40.66 mmol) at the same temperature. Then the reaction mixture was stirred at r.t. for 1 h. The reaction mixture was quenched with 1 N HCl to adjust to pH=1 and extracted with EtOAc (50 mL*2). The combined organic layer was washed with brine (30 mL) and dried over Na2SO4and filtered. The filtrate was concentrated to afford 187b (3.5 g, 58% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 3.62 (s, 3H).

A mixture of 189b (600 mg, 1.45 mmol) in HCl/EtOAc (12 mL, 2 M) was stirred for 2 h at r.t. The formed solid was filtered and the filter cake was dried to afford the title compound 189c (260 mg, 63% yield) as a yellow solid. LC-MS (Method 3) tR=1.52 min, m/z (M+H)+=249.9.

A solution of 190d (3 g, 8.56 mmol) in THF (30 mL) was added n-BuLi (7 mL, 17.5 mmol, 2.5 M in THF) dropwise at −30° C. After stirring for 30 min at this temperature, to it was added anhydrous DMF (1.25 g, 17.12 mmol). The reaction mixture was stirred for 30 mins at −30° C. Then aq. HCl (6.5 mL, 2 M) was added to the reaction mixture slowly and the temperature was kept below −5° C. The reaction mixture was stirred for 2 h at r.t. and poured into water (50 mL). The solution was extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated to afford compound 190e (2.85 g, 92% yield) as a black oil. LC-MS (Method 3) tR=0.75 min, m/z (M+H)+=360.8.

To a solution of 190f (200 mg, 0.87 mmol) in DCM (2 mL) was added conc. sulfuric acid (109 mg, 1.09 mmol) followed by nitric acid (21 mg, 0.22 mmol) at 0° C. The reaction mixture was stirred for 1 h at r.t. The reaction mixture was poured into water (5 mL) and the formed solid was collected by filtering and dried to afford compound 190g (200 mg, 84% yield). LC-MS (Method 3) tR=0.63 min, m/z (M+H)+=275.8.

Compound 190 (17.2 mg) was separated by Prep-Chiral HPLC to afford the title compound 190A (8.1 mg) as a white solid and 190B (8.2 mg) as a white solid.

To a mixture of 194a (2 g, 7.16 mmol) and K2CO3(1.98 g, 14.31 mmol) in DMF (20 mL) was added CH3I (1.52 g, 10.73 mmol). After stirring at r.t. for 16 h, the mixture was diluted with water (10 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4and filtered. The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to afford the title compound 194b (408 mg, 19% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 7.95 (s, 1H), 3.82 (s, 3H).

To a solution of 15c (270 mg, 1.50 mmol) and 67a (313 mg, 1.50 mmol) in THF (2 mL) was added LiHMDS (6.0 mL, 6.00 mmol, 1 M in THF) at −60° C. and stirred at 0° C. for 15 min. The reaction was quenched with ice-water (3 mL) and the organic solvent was removed under vacuo. The formed solid was filtered and dried to afford the title compound 196a (300 mg, 57% yield) as a yellow solid. LC-MS (Method 3) tR=1.12 min, m/z (M+H)+=353.2.

To a solution of 17d (400 mg, 2.06 mmol) and 67a (344 mg, 1.65 mmol) in THF (4 mL) was added LiHMDS (8.2 mL, 8.2 mmol, 1 M in THF) at −60° C. and stirred at r.t. for 30 min. The reaction was quenched with ice-water (0.5 mL) and the organic solvent was removed under vacuo. The formed solid was filtered and dried to afford the title compound 197a (500 mg, 66% yield) as a yellow solid. LC-MS (Method 3) tR=1.21 min, m/z (M+H)+=367.3.

To a solution of 198a (1 g, 6.00 mmol) in DMF (10 mL) was added KOH (674 mg, 12.0 mmol) at 0° C. for 5 min. Then 12 (1.52 g, 6.0 mmol) was added to the reaction and the reaction mixture was stirred at 0° C. for 30 min. To the reaction mixture was added iodomethane (1.28 g, 9.03 mmol). After stirring at 0° C. for 30 min, the reaction mixture was quenched with water (20 mL) and the formed solid was collected by filtering. The filter cake was dried to afford the title compound 198b (1.55 g, 84% yield) as a white solid.1H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1H), 7.68 (s, 1H), 4.05 (s, 3H), 2.63 (s, 3H).

To a mixture of 190g (1.0 g, 3.63 mmol) in DMF (15 mL) was added NaH (291 mg, 7.27 mmol, 60% in mineral oil) at 0° C. After stirring for 30 min at 25° C., to the reaction was added 1-bromo-2-methoxyethane (758 mg, 5.45 mmol). The reaction was stirred at 50° C. for 16 h. After cooling to r.t., the reaction mixture was poured into water (30 mL). The formed solid was collected by filtering and the filter cake was dried to afford the title compound 201a (300 mg, 25% yield) as a brown solid. LC-MS (Method 3) tR=1.25 min, m/z (M+H)+=334.3.

Compound 201 (11 mg) was separated by Prep-Chiral HPLC to afford the title compound 201A (4.7 mg, 43% yield) as a yellow solid and 201B (4.1 mg, 37% yield) as a yellow solid.

To a solution of 202b (80 mg, 0.26 mmol) in DCM (1 mL) was added TFA (0.5 mL), then the mixture was stirred at r.t. for 2 h. The mixture was concentrated to dryness. The residue was diluted with H2O (10 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (10 mL*3). The organic layers were washed with aq Na2CO3(10 mL) and brine (10 mL) and separated. The solution was dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography (PE/EA=1/1 to 1/10) to give the title compound 202c (30 mg, 56% yield) as a yellow solid. LC-MS (Method 4) tR=1.30 min, m/z (M+H)+=203.1.

A solution of 204b (5 g, 18.31 mmol) in conc. H2SO4(30 mL) was stirred at r.t. for 12 h. The mixture was diluted with H2O (40 mL) and the formed solid was collected by filtering. The filter cake was dried to afford the title compound 204c (3.19 g, 68% yield) as a brown solid. LC-MS (Method 3) tR=1.01 min, m/z (M+H)+=256.2.

To a solution of 1a (1.0 g, 6.55 mmol) and KOH (1.10 g, 19.66 mmol) in DMF (10 mL) was added 12 (1.66 g, 6.55 mmol) at 0° C. After stirring for 0.5 h at this temperature, to the reaction was added 2,2,2-trifluoroethyl methanesulfonate (1.73 g, 9.70 mmol). The resultant mixture was stirred at r.t. for 2 h and diluted with water (30 mL). The mixture was extracted with EtOAc (30 mL*3). The combined organic phase was washed with brine (30 mL) and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to afford 209a (2.2 g, 94% yield) as a yellow solid. LC-MS (Method 3) tR=1.33 min, m/z (M+H)+=361.0.

A mixture of 209b (1.33 g, 2.57 mmol) and Na2CO3(816 mg, 7.70 mmol) in EtOH (10 mL) and H2O (10 mL) was stirred at 70° C. for 5 h. The reaction mixture was cooled and concentrated. The residue was diluted with water (30 mL) and extracted with EtOAc (30 mL*3). The combined organic phase was washed with brine (30 mL) and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=1/2) to afford 209c (600 mg, 63% yield) as a white solid. LC-MS (Method 3) tR=1.14 min, m/z (M+H)+=371.2.

To a solution of 210a (5 g, 21.3 mmol) in CCl4(80 mL) was added N-bromosuccinimide (11.4 g, 64 mmol) and benzoyl peroxide (1.54 g, 6.4 mmol) and the mixture was stirred at 85° C. for 15 h. The mixture was cooled to room temperature and filtered, and the filtrate was concentrated. The residue was dissolved in ethyl acetate (100 mL), washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated and purified by flash chromatography on silica gel (PE/EtOAc=100/1 to 1/1) to give 210b (4 g, 60% yield) as a yellow solid. LC-MS (Method 4) tR=4.21 min, m/z (M+H)+=312.0.

To a solution of 211b (4.5 g, 18.59 mmol) in DMF (30 mL) was added 1-iodopyrrolidine-2,5-dione (6.27 g, 27.89 mmol) at r.t. The mixture was stirred at 25° C. for 12 h. H2O (30 mL) was added and the mixture was extracted by EA (50 mL). The combined organic layer was washed by brine, dried over Na2SO4, filtered and concentrated. The crude was purified by flash chromatography on silica gel (PE/EA=10/1 to 1/1) to afford 211c (3.6 g, 61% yield) as a brown solid. LC-MS (Method 4) tR=4.18 min, m/z (M+H)+=320.0.

To a solution of 211c (1.5 g, 4.69 mmol) in dioxane (12 mL) was added NaOH (2 M, 23.5 mL) at r.t. The mixture was stirred at 100° C. for 16 h. The reaction mixture was concentrated. The aqueous residue was diluted with water (30 mL) and acidified to pH 4-6 using 1.5 N hydrochloric acid solution. The precipitated solid was filtered, washed with hexane and concentrated to afford 211d (1.3 g, 92% yield) as a white solid. LC-MS (Method 4) tR=2.58 min, m/z (M+H)+=302.0.

To a solution of 211f (80 mg, 0.18 mmol) in THF (6 mL) and water (2 mL) was added LiOH·H2O (7.7 mg, 0.18 mmol) at r.t. The mixture was stirred at r.t. for 16 h. The resulting solution was diluted with water (10 mL) and acidified to pH 4-6 using 2 N hydrochloric acid solution. The solution was extracted by EA (30 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated to afford 211g (70 mg, 90% yield) as a light-yellow solid. LC-MS (Method 4) tR=2.56 min, m/z (M+H)+=423.3.

To a solution of 194a (2 g, 7.16 mmol) in DMF (30 mL) was added sodium hydride (343.5 mg, 8.59 mmol, 60% purity in mineral oil) at 0° C. under nitrogen protection. 10 min later, iodoethane (1.34 g, 8.59 mmol, 0.69 mL) was added. The mixture was stirred from 0° C. to r.t. for 2 h. The resulting solution was added into H2O (40 mL) and extracted by EA (40 mL*3). The combined organic layer was washed by brine, dried over Na2SO4and filtered. The filtrate was concentrated to get the crude product. The crude was purified by flash chromatography on silica gel (PE/EA=10/1 to 1/1) to afford 212a (1.61 g, 73% yield) as a light-yellow solid. LC-MS (Method 4) tR=4.19 min, m/z (M+H)+=308.0.

To a solution of 212b (850 mg, 2.94 mmol), K2CO3(1.15 g, 3.53 mmol) in DMF (12 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (819 mg, 3.53 mmol, 0.51 mL) at r.t. The mixture was stirred at 100° C. for 3 h. The reaction mixture was cooled to room temperature. The resulting solution was added into H2O (30 mL) and extracted by EA (30 mL*3). The combined organic layer was washed with brine (30 mL*3), dried over Na2SO4and filtered. The filtrate was concentrated and the crude was purified by flash chromatography on silica gel (PE/EA=10/1 to 1/1) to afford 212c (900 mg, 82% yield) as a white solid. LC-MS (Method 4) tR=3.01 min, m/z (M+H)+=372.3.

To a solution of 194a (2 g, 7.16 mmol), Cs2CO3(2.80 g, 8.59 mmol) in DMF (4 mL) and ACN (6 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (1.99 g, 8.59 mmol, 1.24 mL) at r.t. The mixture was stirred at r.t. for 4 h under nitrogen protection. The solution was then added into H2O (30 mL) and extracted by EA (50 mL). The combined organic layer was washed by brine, dried over Na2SO4and filtered. The filtrate was concentrated to get the crude product. The crude was purified by flash chromatography (PE/EA=10/1 to 2/1) to afford 214a (1.58 g, 61% yield) as a yellow solid. LC-MS (Method 4) tR=4.34 min, m/z (M+H)+=362.0.

To a solution of pent-1-yn-3-ol (200 mg, 2.38 mmol) in acetone (2 mL) was added Jones reagent (4.76 mmol, 3 M, 1.6 mL) at an ice-bath, then the mixture was stirred at r.t. for 4 h. The mixture was quenched with i-PrOH (0.5 mL) at an ice-bath, and the mixture was stirred at r.t. for 30 min. Then the mixture was diluted with H2O (15 mL), extracted with EtOAc (5 mL*3). The combined organic layer was washed with aq Na2CO3(10 mL), and brine (10 mL), dried over Na2SO4to get the crude compound 215a (15 mL, 2.38 mmol, 0.16 M in EtOAc) as a solution.

To a solution of 215c (110 mg, 0.36 mmol) in dioxane (1.5 mL) was added a solution of HCl (g) in dioxane (4 M, 1.5 mL). The mixture was stirred at r.t. for 16 h. The mixture was concentrated to dryness. The residue was diluted with H2O (20 mL), adjusted pH to 7-9 with aq Na2CO3, and extracted with EtOAc (20 mL*3). The organic layers were washed with aq Na2CO3(20 mL) and brine (20 mL). The solution was dried over Na2SO4and filtered. The filtrate was concentrated to give the title compound 215d (51 mg, 69% yield) as a brown solid. LC-MS (Method 4) tR=2.27 min, m/z (M+H)+=206.1.

A mixture of 217b (1.4 g, 3.89 mmol) and HCl (g) in EtOH (20 mL, 1 M) was stirred for 3 h at r.t. The formed solid was filtered and the filter cake was dried to afford 217c (977 mg, 85% yield) as a white solid. LC-MS (Method 3) tR=0.88 min, m/z (M+H)+=261.1.

A mixture of 218a (400 mg, 1.43 mmol), Cs2CO3(932 mg, 2.87 mmol) and C2H5I (447 mg, 2.87 mmol) in DMF (5 mL) was stirred at 40° C. for 2 h. After cooling to r.t., the mixture was diluted with water (20 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was concentrated and the residue was purified by flash chromatography on silica gel (PE/EtOAc=3/1) to afford 218b (380 mg, 86% yield) as a yellow solid. LC-MS (Method 3) tR=1.26 min, m/z (M+H)+=307.9.

To a solution of 220a (200 mg, 0.62 mmol) in DCM (3 mL) was added TFA (1 mL) at 0° C. After stirring for 2 h, the reaction mixture was concentrated to dryness and basified with sat. Na2CO3to adjust pH to above 8. The mixture was then extracted with EtOAc (5 mL*3). The combined organic layer was washed with brine (5 mL), dried over Na2SO4and filtered. The filtrate was concentrated to afford 220b (120 mg, 87% yield) as a brown oil. LC-MS (Method 3) tR=0.88 min, m/z (M+H)+=222.3.

A mixture of 221a (180 mg, 0.49 mmol), C2H5I (1 mL) and EtOH (1 mL) in a sealed tube was stirred at 70° C. for 4 h. After cooling to r.t., the reaction mixture was concentrated to afford 221b (194 mg, yield given) as a yellow solid. The crude product was used in the next step without purification. LC-MS (Method 3) tR=1.34 min, m/z M+=391.1.

To a solution of 9e (170 mg, 0.75 mmol) and 67a (156 mg, 0.75 mmol) in THF (2 mL) was added LiHMDS (3 mL, 3.0 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h. The reaction was quenched with H2O (2 mL) and extracted with ethyl acetate (10 mL*3). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography on silica gel (DCM/MeOH=20/1) to afford compound 223a (90 mg, 33% yield) as a yellow solid. LC-MS (Method 3) tR=1.07 min, m/z (M+H)+=364.3.

To a mixture of 225a (400 mg, 1.24 mmol) in DCM (3 mL) was added TFA (1 mL) at 20° C. The resulting mixture was stirred at 20° C. for 1 h. A yellow solution was formed. The reaction mixture was concentrated to give methyl 5-amino-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylate hydrochloride (280 mg, crude) as a yellow solid.

To a mixture of methyl 5-amino-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylate hydrochloride (280 mg, crude) in MeCN (5 mL) was added tert-butyl nitrite (196 mg, 1.90 mmol, 0.23 mL) at 0° C. After stirring for 10 min, CuCl (250 mg, 2.54 mmol) was added into the above mixture, and the resulting mixture was stirred at 80° C. for 12 h. A yellow solution was formed. The reaction mixture was concentrated and purified by column chromatograph (EA in PE is 10-30%) to give 225a (173 mg, 57% yield) as a yellow solid. LC-MS (Method 4) tR=2.31 min, m/z (M+H)+=241.2.

A mixture of 225c (50 mg, 0.12 mmol) and LiOH·H2O (15 mg, 0.36 mmol) in co-solvent of THF (3 mL) and water (1 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was concentrated and dried in vacuo to give 225d (48 mg, yield given) as a yellow solid, which was used for the next step directly without further purification. LCMS (Method 4) tR=0.86 min, m/z (M+H)+=398.2.

A mixture of 226b (168 mg, 0.395 mmol) and LiOH·H2O (50 mg, 1.18 mmol) in co-solvent of THF (6 mL) and water (2 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was concentrated and dried in vacuo to give 226c (162 mg, 99% yield) as a yellow solid, which was used for the next step directly without further purification. LCMS (Method 4) tR=1.73 min, m/z (M+H)+=412.3.

To a mixture of 227a (1.00 g, 4.06 mmol) in THF (30 mL), was added DIBAL-H (1.5 M, 6.2 mL) at −5° C. The resulting mixture was stirred at 0° C. for 1 h. The reaction mixture was warmed to 20° C. and stirred for 12 h. A yellow solution was formed. The reaction mixture was quenched with aq. Na2CO3(50 mL) and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated to give 227b (886 mg, yield given) as a white solid, which was used for the next step directly without further purification. LC-MS (Method 4) tR=2.99 min, m/z (M+H)+=218.0.

To a mixture of 227b (886 mg, 4.06 mmol) and isoindoline-1,3-dione (658 mg, 4.47 mmol) in THF (15 mL) was added PPh3(1.28 g, 4.88 mmol) and DIAD (986 mg, 4.88 mmol) at 0° C. The resulting mixture was stirred at 20° C. for 12 h. A yellow solution was formed. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (EA in PE is 10-30%) to give 227c (2.50 g, yield given) as a yellow solid. LCMS (Method 4) tR=3.46 min, m/z (M+H)+=347.0.

A mixture of 227c (2.50 g, 4.06 mmol) and N2H4·H2O (1.35 g, 21.60 mmol, 80% purity) in ethanol (40 mL) was stirred at 80° C. for 2 h. A white suspension was formed. The reaction mixture was diluted with EtOAc (50 mL) and filtered through a pad of celite. The filtrate was concentrated and purified by flash chromatography (MeOH in DCM is 0-10%) to give 227d (1.56 g, yield given) as a yellow oil. LCMS (Method 4) tR=1.01 min, m/z (M+H)+=217.0.

To a mixture of 227d (1.89 g, 8.71 mmol) in DCM (30 mL) was added TEA (1.32 g, 13.06 mmol, 1.82 mL) and propionyl chloride (1.05 g, 11.32 mmol, 0.99 mL) at 0° C. The resulting mixture was stirred at 20° C. for 2 h. A yellow solution was formed. The reaction mixture was quenched with water (50 mL) and extracted with DCM (50 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatograph (MeOH in DCM is 0-10%) to give 227e (1.79 g, 6.55 mmol, 75% yield) as a yellow oil. LCMS (Method 4) tR=2.94 min, m/z (M+H)+=273.1.

To a mixture of 227f (90 mg, 0.21 mmol) in dioxane (10 mL) was added PPA (300 mg) at 20° C. The resulting mixture was stirred at 100° C. for 2 h. A black suspension was formed. The reaction mixture was concentrated in vacuo and diluted with water (50 mL), basified with 2 M aq. NaOH to pH=11 and extracted with EtOAc (50 mL*2). The combined organic layer was washed with water (50 mL*2), brine (50 mL*2), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (MeOH in DCM is 0-10%) to give 227g (50 mg, 58% yield) as a green solid. LCMS (Method 4) tR=2.23 min, m/z (M+H)+=410.2.

A mixture of 227g (50 mg, 0.122 mmol) and LiOH·H2O (15 mg, 0.366 mmol) in co-solvent of THF (3 mL) and water (1 mL) was stirred at 40° C. for 12 h. A yellow solution was formed. The reaction mixture was concentrated and dried in vacuo to give 227h (48 mg, 99% yield) as a yellow solid, which was used for the next step directly without further purification. LCMS (Method 4) tR=0.85 min, m/z (M+H)+=396.2.

To a solution of 232b (7 mg, 0.016 mmol) in THF (0.4 mL) and H2O (0.1 mL) was added LiOH·H2O (14 mg, 0.036 mmol), and the mixture was stirred at 65° C. for 16 h. The reaction mixture was cooled down to r.t. and adjusted to pH=5 with 2 N HCl aqueous solution. The acidified solution was extracted with DCM/MeOH=6/1 (3 mL*7) and the combined organic layer was dried over anhydrous Na2SO4. Then it was concentrated to afford the crude compound 232c (5 mg, 76% yield) as a light yellow solid. LC-MS (Method 4) tR=2.64 min, m/z (M+H)+=410.3.

To a solution of 233a (5 g, 25.23 mmol) in THF (50 mL) was added n-BuLi (12 mL, 30 mmol, 2.5 M) at 0° C. The reaction mixture was stirred at 0° C. for 1 h under N2atmosphere, then propionic anhydride (13.27 g, 102 mmol) was added to the reaction mixture at −78° C., the reaction mixture was stirred at −78° C. for 1 h. After quenching with water, the mixture was extracted with ethyl acetate. The collected organic layer was dried over sodium sulphate, filtered and evaporated. The crude product was purified by flash chromatography on silica gel (PE/EtOAc=10/1 to 1/3) to afford 233b (3 g, 47% yield) as a yellow oil. LC-MS (Method 4) tR=3.46 min, m/z (M+H)+=255.2.

To a solution of 233e (210 mg, 1.29 mmol) in CCl4(5 mL) was added Br2(102.8 mg, 0.64 mmol) at −30° C. The reaction mixture was stirred at −30° C. for 1 h. The reaction mixture was concentrated and purified by flash chromatography on silica gel (PE/EtOAc=10/1 to 1/5) to give 233f (105 mg, 34% yield) as a yellow solid. LC-MS (Method 4) tR=2.64 min, m/z (M+H)+=242.0.

To a solution of 237a (300 mg, 2.33 mmol) in 12 mL DCM was added DMAP (427.6 mg, 3.50 mmol). Cyclopropanecarboxylic acid (200.9 mg, 2.33 mmol) and EDCI (671 mg, 3.50 mmol) was added after a while. After stirring at room temperature for 12 h, the resulting solution was added into H2O (40 mL) and extracted by EA (40 mL). The organic layer was washed by brine (20 mL), dried over Na2SO4and filtered. The filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (PE/EA=10/1 to 1/1) to afford 237b (330 mg, 72% yield) as a white solid. LC-MS (Method 4) tR=2.26 min, m/z (M+H)+=197.1.

To a solution of 244a (5 g, 21.64 mmol) in THF (50 mL) at −40° C. under N2atmosphere was added vinylmagnesium bromide (64.93 mL, 64.93 mmol). The resulting mixture was stirred at −20° C. for 30 min. The reaction was then quenched with aq. NH4Cl (150 mL) and extracted with EtOAc (200 mL*3). The combined organic layer was washed by brine (200 mL*2), dried over sodium sulphate and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (PE/EA=30/1 to 8/1) to give 244b (1.5 g, 26% yield) as a white solid. LC-MS (Method 5) tR=2.84 min, m/z (M+H)+=225.9.

To a solution of 244b (1.5 g, 6.64 mmol) in DMF (15 mL) was added 60% NaH (292 mg, 7.30 mmol) at 0° C. and stirred for 0.5 h under N2atmosphere. Then C2H5I (1.55 g, 9.96 mmol) was added. The reaction mixture was stirred at 25° C. for 5 h. The reaction was quenched with aq. NH4Cl (30 mL) and extracted with EtOAc (10 mL*3). The combined organic layer was washed by brine (30 mL*2), dried over sodium sulphate and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (PE/EA=30/1 to 5/1) to give 244c (600 mg, 33% yield) as a yellow solid. LC-MS (Method 5) tR=3.19 min, m/z (M+H)+=253.9.

To a solution of 244d (150 mg, 0.367 mmol) in THF/MeOH/H2O=3/2/1 (2 mL) was added LiOH·H2O (30.9 mg, 0.735 mmol) and stirred at 40° C. for 16 h under N2atmosphere. The mixture was diluted with water (10 mL) and extracted with EtOAc (2 mL*3). The combined organic layer was washed by brine (2 mL*2), dried over sodium sulphate and evaporated in vacuo. The residue was purified by Prep-TLC (DCM/MeOH=10/1) to give 244e (100 mg, 59% yield) as a yellow solid. LC-MS (Method 5), tR=1.55 min, m/z (M+H)+=395.0.

To a solution of 1a (5.00 g, 32.8 mmol) in DMF (100 mL) was added cyclopropylboronic acid (8.46 g, 98.4 mmol), Cu(OAc)2(17.8 g, 98.4 mmol) and TEA (16.5 g, 164 mmol). The reaction mixture was stirred at 120° C. under O2atmosphere for 5 h. The mixture was cooled to room temperature and diluted with water (250 mL), extracted with EtOAc (200 mL*3). The combined organic layer was washed by brine (200 mL*2), dried over sodium sulphate and evaporated in vacuo. The residue was purified by flash chromatography on silica gel (PE/EA=50/1 to 10/1) to give 245a (5.10 g, 81% yield) as a yellow solid. LC-MS (Method 5), tR=1.28 min, m/z (M+H)+=193.1.

To a stirred solution of 247a (10.0 g, 52.47 mmol) and TEA (21 mL, 151.08 mmol) in DCM (100 mL) was added propionyl chloride (5.7 mL, 65.24 mmol) dropwise at 0° C. After stirring at 0° C. for 5 min, the reaction solution was allowed to stirred at r.t. for 3 h. Then the reaction was quenched with 2 N HCl (30 mL) and neutralized with sat. NaHCO3. The organic layer was separated and washed with brine (10 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated and the residue was recrystallized from EA to give 247b (9.27 g, 84% yield) as a white solid. LC-MS (Method 4) tR=2.14 min, m/z (M+H)+=211.1.

A solution of 247b (9.27 g, 44.10 mmol) in DMF-DMA (50 mL) and DMF (100 mL) was stirred at 90° C. for 16 h. After cooling down to r.t., the reaction solution was quenched with H2O (150 mL) and extracted with DCM (80 mL*3). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under vacuum to give a residue which was purified by flash chromatography on silica gel (PE/EA=5/1) to afford the title compound 247c (7.13 g, 72% yield) as a white solid. LC-MS (Method 4) tR=2.97 min, m/z (M+H)+=225.2.

To a sealed tube was added 247c (7.13 g, 31.79 mmol) and NH3·H2O (100 mL, 28-30% w.t. in H2O). The reaction mixture was stirred for 7 h under 115° C. After cooling down to r.t., the reaction solution was concentrated and the residue was purified by C-18 column (5-25% MeCN/H2O (0.5% NH4HCO3), 30 min) to give the title compound 247d (632 mg, 11% yield) as a white solid. LC-MS (Method 4) tR=0.71 min, m/z (M+H)+=178.1.

To a solution of 247e (330 mg, 1.72 mmol) in conc. H2SO4(4 mL) was added HNO3(0.5 mL) at 0° C. The resulting mixture was stirred at 0° C. for 1 h and quenched with H2O (15 mL) at 0° C. After neutralized with sat. NaHCO3at 0° C., the mixture solution was extracted with DCM (10 mL*3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated to give the title compound 247f (298 mg, 73% yield) as a light yellow solid. LC-MS (Method 4) tR=0.96 min, m/z (M+H)+=237.1.

To a solution of 247f (298 mg, 1.26 mmol) in EtOH (4 mL) and H2O (1 mL) was added Fe powder (340 mg, 6.10 mmol) and NH4Cl (675 mg, 12.62 mmol). The resulting mixture was stirred at 80° C. for 1 h. After cooling down to r.t., the reaction mixture was filtered. The filtrate was diluted with H2O (8 mL) and extracted with DCM (10 mL*3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated to afford the title compound 247g (203 mg, 78% yield) as a brown solid. LC-MS (Method 4) tR=0.69 min, m/z (M+H)+=207.2.

A mixture of 248c (1.6 g, 8.32 mmol) and FeCl3(135 mg, 0.83 mmol) in DMF (16 mL) was stirred at 60° C. for 4 h. After cooling to r.t, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (20 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=10/1) to afford 248d (600 mg, 38% yield) as a yellow solid. LC-MS (Method 3) tR=1.14 min, m/z M+H)+=189.2.

To a solution of 248d (200 mg, 0.85 mmol) in DMF (2 mL) was added NBS (121 mg, 0.68 mmol) at 0° C. After stirring at 0° C. for 1 h, the mixture was diluted with water (4 mL). The formed solid was collected by filtering and was dried to afford 248e (200 mg, 88% yield) as a yellow solid. LC-MS (Method 3) tR=1.19 min, m/z (M+H)+=267.1.

A mixture of 248e (200 mg, 0.75 mmol) and KNO3(151 mg, 1.50 mmol) in TFA (2 mL) was stirred at 70° C. for 16 h. After cooling to r.t., the solvent was removed by pumping through N2. The residue was diluted with water (2 mL) and extracted with EtOAc (5 mL*3). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated to to afford 248f (130 mg, 56% yield) as a yellow solid. LC-MS (Method 3) tR=1.22 min, m/z (M+H)+=312.2.

A mixture of 248f (400 mg, 1.28 mmol) and Pd/C (40 mg, 10% wt wet in 50% water) in MeOH (4 mL) was stirred at r.t. overnight under H2(50 Psi) atmosphere. The mixture was filtered and the filter cake was washed with MeOH (4 mL). The combined filtrate was concentrated and the residue was purified by Prep-HPLC (Method A) to afford 248g (60 mg, 23% yield) as a white solid. LC-MS (Method 3) tR=0.85 min, m/z (M+H)+=204.0.

To a solution of 249b (200 mg, 0.63 mmol) in DCM (10 mL) was added TFA (720.9 mg, 6.32 mmol, 0.48 mL) at r.t. The mixture was stirred at 25° C. for 2 h and concentrated to dryness. The residue was diluted with H2O (10 mL), adjusted pH to 7-9 with 1 M Na2CO3solution and extracted with EA (20 mL*3). The organic layer was washed with brine (10 mL), dried over Na2SO4and filtered. The filtrate was concentrated and purified by flash chromatography on silica gel (PE/EA=10/1 to 1/2) to afford 249c (80 mg, 58% yield) as an off-white solid. LC-MS (Method 4) tR=1.81 min, m/z (M+H)+=217.1.

A mixture of 220a (1.79 g, 5.57 mmol) in ACN (20 mL) was added potassium trimethylsilanolate (2.86 g, 22.27 mmol) and stirred at 50° C. for 3 h. The mixture was concentrated and the residue was dissolved with water (10 mL). The aqueous layer was acidified with 2 M HCl to pH=2. The aqueous layer was extracted with EtOAc (30 mL*3), washed with brine (10 mL). The combined organic layer was concentrated. The residue was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford compound 250a (1.32 g, 77% yield) as a yellow solid. LC-MS (Method 3) tR=1.16 min, m/z (M+H)+=308.2.

A mixture of 250a (1 g, 3.25 mmol) in DMF (10 mL) was added NaHCO3(820 mg, 9.76 mmol) and NIS (952 mg, 4.23 mmol). The mixture was stirred at r.t. for 2 h and diluted with water (10 mL). The aqueous layer was extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (20 mL) and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=5/1) to afford compound 250b (722 mg, 57% yield) as a blue solid. LC-MS (Method 3) tR=1.32 min, m/z (M+H)+=390.2.

A mixture of 235b (90 mg, 0.25 mmol) in ACN (5 mL) was cooled to 0° C. and to it was added selectfluor (88 mg, 0.25 mmol). After stirring for 3 h at r.t., the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (PE/EtOAc=3/1) to afford compound 253a (30 mg, 32% yield) as a purple solid. LC-MS (Method 3) tR=1.34 min, m/z (M+H)+=382.4.

A mixture of 129c (1.1 g, 4.84 mmol), Cs2CO3(3.16 g, 9.69 mmol) and methyl bromoacetate (1.11 g, 7.27 mmol) in DMF (10 mL) was stirred at 40° C. for 6 h. After cooling to r.t., the mixture was filtered and the filtrate was diluted with water (20 mL). The aqueous layer was extracted with EtOAc (30 mL*3) and the combined organic layer was washed with brine (30 mL*3), concentrated and purified by flash chromatography on silica gel (PE/EtOAc=2/1) to afford compound 259a (568 mg, 39% yield) as a yellow solid. LC-MS (Method 3) tR=1.31 min, m/z (M+H)+=299.0.

To a solution of 260a (1.4 g, 4.92 mmol) in THF (20 mL) was added NaH (394 mg, 9.84 mmol, 60% purity in mineral oil) at 0° C., then the mixture was stirred at 0° C. for 1 h. Iodoethane (768 mg, 4.92 mmol) was added into the mixture and the mixture was stirred at r.t. for 16 h. The mixture was diluted with H2O (40 mL) at an ice-bath, extracted with EtOAc (30 mL*3), washed with brine (30 mL), dried over Na2SO4, concentrated and purified by flash chromatography (PE/EA=100/1 to 1/1) to get compound 260b (1.0 g, 65% yield) as a yellow oil. LC-MS (Method 4) tR=5.40 min, m/z (M+H)+=313.0.

To a solution of 260c (700 mg, 2.18 mmol) in DCM (10 mL) was added TFA (3 mL), and the mixture was stirred at r.t. for 16 h. The mixture was concentrated and diluted with H2O (30 mL), adjusted to pH>7 with aq Na2CO3and extracted with EtOAc (30 mL*3). The combined organic layer was washed with brine (50 mL*2), dried over Na2SO4, concentrated and purified by flash chromatography (DCM/MeOH=100/1 to 10/1) to get compound 260d (300 mg, 68% yield) as a yellow solid. LC-MS (Method 4) tR=1.22 min, m/z (M+H)+=202.1.

To a solution of 260f (120 mg, 0.38 mmol) in DCM (1 mL) was added TFA (2 mL), and the mixture was stirred at r.t. for 24 h. The mixture was concentrated and diluted with H2O (20 mL), adjusted to pH>7 with aq Na2CO3and extracted with EtOAc (20 mL*3). The combined organic layer was washed with brine (30 mL*2), dried over Na2SO4, concentrated and purified by flash chromography (DCM/MeOH=100/1 to 5/1) to get compound 260g (60 mg, 81% yield) as a yellow solid. LC-MS (Method 4) tR=0.38 min, m/z (M+H)+=193.1.

To a solution of n-BuLi (5.5 mL, 13.93 mmol, 2.5 M in THF) in THF (25 mL) was added 2,2,6,6-tetramethylpiperidine (2.3 g, 16.72 mmol) at −78° C. The reaction mixture was stirred at −78° C. for 15 min. Then to the reaction mixture was added a solution of 261a (2.0 g, 13.93 mmol) in THF (25 mL). The reaction mixture was stirred at −78° C. for 4 h. A solution of hexachloroethane (6.6 g, 27.86 mmol) in THF (50 mL) was added dropwise to the reaction and the temperature was kept below −60° C. After stirring for another 2 h at −78° C., the reaction mixture was quenched with sat. NH4Cl (20 mL), diluted with water (40 mL) and extracted with EtOAc (30 mL*3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EtOAc=10/1) to afford compound 261b (700 mg, 28% yield) as a yellow oil.1H NMR (400 MHz, CDCl3) δ 8.06 (d, J=5.2 Hz, 1H), 7.29 (d, J=5.6 Hz, 1H), 3.95 (s, 3H).

To a solution of 262a (150 mg, 0.78 mmol) and 42b (178 mg, 0.86 mmol) in THF (3 mL) was added LiHMDS (3.1 mL, 3.1 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was dried to afford compound 262b (90 mg, 32% yield) as a red solid. LC-MS (Method 3) tR=1.25 min, m/z (M+H)+=364.2.

To a solution of 265c (14 g, 83 mmol) in conc·H2SO4(250 mL) at 0° C. was added NBS (29.65 g, 160 mmol) slowly at 0° C. Then the reaction mixture was allowed to warm to 60° C. and stirred for 16 h. The mixture was adjusted to pH=7 with saturated Na2CO3(2000 mL). Then the resulting mixture was extracted with EtOAc (600 mL*3). The combined organic layer was washed with brine (100 mL*2), dried over anhydrous Na2SO4and concentrated to give the crude product. The residue was purified by flash chromatography on silica gel (PE/EtOAc=20/1 to 10/1) to give 265d (8.3 g, 38% yield) as a yellow oil. LC-MS (Method 5) tR=2.3 min, m/z (M+H)+=247.0.1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 4.00 (s, 1H), 2.45 (s, 3H).

A solution of 265f (1 g, 4.4 mmol) and Cs2CO3(2.8 g, 8.8 mmol) in DMF (20 mL) was stirred and cooled to 0° C. Iodoethane (1.37 g, 8.8 mmol) was added dropwise and the reaction was stirred at 25° C. for 1 h. The resulting mixture was poured into water (20 mL) and extracted with EtOAc (10 mL*3). The combined organic layer was washed with brine (20 mL*2), dried over anhydrous Na2SO4and concentrated to give the crude product. The residue was purified by flash chromatography on silica gel (PE/EtOAc=50/1 to 10/1) to give 265g (110 mg, 9% yield) as a yellow oil and 6-bromo-2-ethyl-7-methoxy-211-pyrazolo[4,3-b]pyridine (1 g, 86% yield) as a yellow solid. LC-MS (Method 5) tR=1.69 min, m/z (M+H)+=256.0.

To a solution of 173c (50 mg, 0.21 mmol), 265h (108 mg, 0.42 mmol) and Cs2CO3(208 mg, 0.64 mmol) in dioxane (1 mL) was added BrettPhos Pd G3 (19 mg, 0.021 mmol) and BrettPhos (23 mg, 0.042 mmol). Then the reaction mixture was heated to 100° C. and stirred for 16 h under N2. TLC (PE/EA=1/1) showed the reaction mixture was completed. The mixture was cooled to room temperature and poured into water (10 mL) and extracted with EtOAc (5 mL*3). The combined organic layer was washed with brine (5 mL*2), dried over anhydrous Na2SO4and evaporated in vacuo to give the crude product. The residue was purified by Prep-TLC (PE/EtOAc=1/1) to give 265h (34 mg, 37% yield) as a yellow solid. LC-MS (Method 5) tR=2.04 min, m/z (M+H)+=411.1.

To a solution of 265h (34 mg, 0.083 mmol) in MeOH (5 mL), THF (5 mL) and H2O (2 mL) was added LiOH·H2O (10 mg, 0.25 mmol) at 0° C. Then the reaction mixture was allowed to 25° C. and stirred for 1 h. The mixture was added into H2O (10 mL) and adjusted to pH=7 with 1 N HCl (10 mL). Then the resulting mixture was extracted with EtOAc (10 mL*3). The combined organic layer was washed with brine (10 mL*2), dried over anhydrous Na2SO4and concentrated to give crude compound 265i (30 g, 88% yield) as a yellow solid. LC-MS (Method 5) tR=1.53 min, m/z (M+H)+=397.2.

To a solution of 266b (45 mg, 0.18 mmol) and 42b (46 mg, 0.22 mmol) in THF (0.5 mL) was added LiHMDS (0.7 mL, 0.7 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was dried to afford compound 266c (12 mg, 16% yield) as a white solid. LC-MS (Method 3) tR=1.63 min, m/z (M+H)+=418.2.

To a solution of 267b (57 mg, 0.28 mmol) and 42b (64 mg, 0.31 mmol) in THF (1 mL) was added LiHMDS (1.1 mL, 1.1 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was dried to afford compound 267c (30 mg, 29% yield) as a brown solid. LC-MS (Method 3) tR=1.37 min, m/z (M+H)+=376.2.

To a solution of 230 (60 mg, 0.15 mmol) in THF (1 mL) was added HCl (1 mL, 2 M in H2O) at 25° C. The reaction mixture was stirred at 60° C. for 5 h and was adjusted to pH>7 with aq Na2CO3solution (5 mL). Then the mixture was extracted with EA (10 mL*3), washed with brine (10 mL), dried over Na2SO4, concentrated and purified by flash chromatography (DCM/MeOH=100/1 to 8/1) to get compound 271a (48 mg, 89% yield) as a white solid. LC-MS (Method 4) tR=1.74 min, m/z (M+H)+=344.2.

To a solution of 272a (300 mg, 1.84 mmol) in DCM (12 mL) was added DMAP (337.3 mg, 2.76 mmol) at r.t. Cyclopropanecarboxylic acid (158.4 mg, 1.84 mmol) and EDCI (423.4 mg, 2.21 mmol) was added after a while. The mixture was stirred at 25° C. for 3 h. The resulting solution was added H2O (20 mL) and extracted by EA (30 mL). The combined organic layer was washed by brine (15 mL*3), dried over Na2SO4and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography (PE/EA=10/1 to 2/1) to afford 272b (160 mg, 37% yield) as a white solid. LC-MS (Method 4) tR=2.83 min, m/z (M+H)+=231.0.

To a stirred solution of 129c (100.0 mg, 0.44 mmol) and 2-chloro-N,N-dimethylethan-1-amine hydrochloride (72 mg, 0.5 mmol) in DMF (4 mL) was added Cs2CO3(275 mg, 0.85 mmol). The resulting reaction mixture was stirred at 85° C. for 1.5 h. Then it was allowed to cool down to r.t., and was quenched with water (10 mL), extracted with DCM (15 mL*3). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4and filtered. The filtrate was concentrated under vacuum to give a residue which was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford compound 274a (86 mg, 65% yield) as a yellow oil. LC-MS (Method 4) tR=1.42 min, m/z (M+H)+=298.1.

To a solution of 274a (75 mg, 0.25 mmol) and 129c (52 mg, 0.16 mmol) in anhydrous 1,4-dioxane (3.5 mL) was added BrettPhos Pd G3 (22 mg, 0.024 mmol) and Cs2CO3(182 mg, 0.56 mmol). The resulting mixture was refluxed at 105° C. under nitrogen atmosphere for 16 h. Then the mixture was allowed to cool down to r.t. The solvent was removed, and the residue was purified by flash chromatography on silica gel (DCM/MeOH=15/1) to get a crude product, which was further purified by Prep-HPLC (Method E) to afford compound 274b (42.5 mg, 37% yield) as a white solid. LC-MS (Method 4) tR=1.60 min, m/z (M+H)+=453.3.

To a solution of 274b (42.5 mg, 0.094 mmol) in THF (1 mL) and H2O (0.2 mL) was added LiOH·H2O (58 mg, 1.38 mmol), and the mixture was stirred at 65° C. for 6 h. Then it was cooled down to r.t. and adjusted to pH=5 with 2 N aq. HCl. The acidified solution was concentrated to afford compound 274c (40 mg, 97% yield) as a light yellow solid. LC-MS (Method 4) tR=0.56 min, m/z (M+H)+=439.3.

To a solution of 275b (80 mg, 0.31 mmol) and 42b (77 mg, 0.37 mmol) in THF (0.8 mL) was added LiHMDS (1.2 mL, 1.2 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was dried to afford compound 275c (50 mg, 38% yield) as a red solid. LC-MS (Method 3) tR=1.31 min, m/z (M+H)+=432.1.

To a solution of 276d (90 mg, 0.37 mmol) and 42b (93 mg, 0.45 mmol) in THF (0.8 mL) was added LiHMDS (1.5 mL, 1.5 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (1 mL). The organic solvent was evaporated under reduce pressure. The formed solid was collected by filtering and was dried to afford 276e (50 mg, 38% yield) as a red solid. LC-MS (Method 3) tR=1.60 min, m/z (M+H)+=414.2.

To a solution of 277b (35 mg, 0.19 mmol) and 42b (48 mg, 0.23 mmol) in THF (0.4 mL) was added LiHMDS (0.8 mL, 0.8 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was purified by flash chromatography on silical gel (DCM/MtOH=20/1) to afford compound 277c (6 mg, 9% yield) as a red solid. LC-MS (Method 3) tR=1.18 min, m/z (M+H)+=353.1.

A solution of 278b (25 mg, 0.09 mmol) in toluene (1 mL) and MeOH (0.1 mL) was added into TMSCHN2(1 mL, 2 M in hexane). The mixture was stirred at r.t. for 12 h. The mixture was diluted with water (3 mL) and the aqueous layer was extracted with EtOAc (5 mL*3). The combined organic layer was concentrated under reduced pressure and purified by flash chromatography on silica gel (PE/EtOAc=3/1) to afford 278c (22 mg, 84% yield) as a white solid. LC-MS (Method 3) tR=1.29 min, m/z (M+H)+=294.2.

To a solution of 279e (15 g, 30.1 mmol) in EtOH (150 ml) was added DMF-DMA (17.93 g, 150.5 mmol). The reaction mixture was stirred at 80° C. for 16 h. The mixture was concentrated in vacuum. The residue was purified by flash chromatography on silica gel (PE/EA=30/1 to 3/1) to give 279f (5.5 g, 32% yield) as a yellow solid. LC-MS (Method 4) tR=1.75 min, m/z (M+H)+=567.3.

To a solution of 279j (9 mg, 1.24 mmol) and 42b in THF (2 mL) was added LiHMDS (0.40 mmol, 0.40 mL, 1 M in THF) at −40° C. The resulting mixture was stirred at 0° C. for 1 h. A yellow solution was formed. The reaction mixture was quenched with water (30 mL) and extracted with EtOAc (30 mL*3). The combined organic layer was washed with water (30 mL) and brine (30 mL), dried over anhydrous Na2SO4and concentrated to give the crude product. The crude product was purified by Prep-TLC (PE/EA=1/1) to give 279k (15 mg, 85% yield) as a yellow solid. LC-MS (Method 3) tR=1.21 min, m/z (M+H)+=350.2.

A mixture of 280b (12.00 g, 38.74 mmol) and MeSNa (4.07 g, 58.11 mmol) in DMF (50 mL) was stirred at 80° C. for 5 h. After cooling to r.t., the reaction mixture was diluted with water (80 mL) and extracted with EtOAc (100 mL*3). The combined organic layer was concentrated and the residue was purified by flash chromatography on silica gel (PE/EA=5/1) to afford 280c (4.3 g, 35% yield) as a yellow oil. LC-MS (Method 3) tR=0.28 min, m/z (M+H)+=322.3.

To a solution of 280d (1.5 g, 4.45 mmol) and but-3-yn-2-one (363 mg, 5.33 mmol) in THF (15 mL) was added MnO2(580 mg, 6.67 mmol) and K2CO3(922 mg, 6.67 mmol) at 0° C. After stirring at 35° C. for 4 h, the reaction mixture was filtered. The filtrate was diluted with water (25 mL) and extracted with EtOAc (50 mL*2). The combined organic layer was concentrated and the residue was purified by flash chromatography on silica gel (PE/EA=2/1) to afford 280e (330 mg, 18% yield) as a yellow solid. LC-MS (Method 3) tR=1.24 min, m/z (M+H)+=403.3.

A mixture of 280e (320 mg, 0.79 mmol) and Pd/C (45 mg, 10% wt wetted in 50% water) in Et3SiH (3 mL) and THF (3 mL) was stirred at 80° C. for 6 h. After cooling to r.t., the reaction mixture was diluted with water (5 mL) and extracted with EtOAc (8 mL*2). The combined organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (DCM/EtOAc=3/1) to afford 280f (97 mg, 34% yield) as a yellow solid. LC-MS (Method 3) tR=1.16 min, m/z (M+H)+=357.3.

A mixture of 280g (42 mg, 0.12 mmol) in TFA/DCM (0.4 mL/1.2 mL) was stirred at 8° C. for 30 min. The solvent was removed. The residue was diluted with DCM (1 mL), adjusted to pH>7 with NH3/1,4-dioxane (2 mL, 2 M). Then the mixture was concentrated and the residue was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford 280h (14 mg, 59% yield) as a yellow oil. LC-MS (Method 3) tR=1.09 min, m/z (M+H)+=193.0.

To a solution of 280h (14 mg, 0.073 mmol) and 42b (18 mg, 0.087 mmol) in THF (0.2 mL) was added LiHMDS (0.3 mL, 0.3 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was purified by flash chromatography on silical gel (DCM/MeOH=20/1) to afford 280i (6 mg, 9% yield) as a brown solid. LC-MS (Method 3) tR=1.22 min, m/z (M+H)+=364.2.

To a solution of tert-butyl methyl malonate (13.08 g, 75.08 mmol) in THF (200 mL) was added NaH (6.01 g, 150.16 mmol, 60% in mineral oil) at 0° C. The mixture was stirred at 0° C. for 30 min. Then a solution of 279a (11.2 g, 62.57 mmol) in THF (20 mL) was added to the mixture at 0° C. The mixture was stirred at 80° C. for 3 h and poured into ice-water (150 mL). The mixture was acidified with 2 N HCl to pH=2 and extracted with EtOAc (300 mL*2). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered and concentrated to afford 281a (19.8 g, yield given) as a yellow oil. LC-MS (Method 3) tR=1.26 min, m/z (M+H−56)+=261.2.

To a mixture of 281e (4.26 g, 11.44 mmol) in DCM (15 mL) was added TFA (15 mL) dropwise at r.t. The reaction mixture was stirred at r.t. for 1 h and concentrated under vacuum at 30° C. The residue was dissolved in DCM (80 mL) and the solution was basified with 1 M NaOH to pH=12. The mixture was extracted with DCM (80 mL). The organic layer was concentrated and the residue was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford 281f (1.94 g, 76% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.27 (s, 1H), 6.68 (brs, 2H), 3.77 (s, 3H), 3.71 (s, 3H).

A mixture of 281h (240 mg, 0.59 mmol) in diphenyl ether (3 mL) was stirred at 170° C. for 4 h. After cooling to r.t., the mixture was diluted with PE (5 mL) and filtered. The filtrate was concentrated and the residue was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford the title compound 281i (40 mg, 42% yield) as a brown solid. LC-MS (Method 3) tR=0.27 min, m/z (M+H)+=165.1.

To a solution of 281j (50 mg, 0.25 mmol) and 42b (105 mg, 0.50 mmol) in THF (0.4 mL) was added LiHMDS (1.0 mL, 1.0 mmol, 1 M in THF) at −40° C. The reaction was stirred at −40° C. to r.t. for 1 h and quenched with H2O (2 mL). The organic solvent was evaporated under reduced pressure. The formed solid was collected by filtering and was dried to afford 281k (20 mg, 22% yield) as a brown solid. LC-MS (Method 3) tR=1.21 min, m/z (M+H)+=370.2.

To a solution of tert-butyl 2-cyanoacetate (9.46 g, 67.04 mmol) in THF (200 mL) was added NaH (5.36 g, 134.08 mmol, 60% in mineral oil) at 0° C. After stirring at 0° C. for 30 min, to it was added a solution of 279a (10 g, 55.86 mmol) in THF (20 mL). The mixture was stirred at 80° C. for 2 h. After cooling to r.t., the reaction mixture was poured into ice-water (150 mL) and acidified with 2 N HCl to pH=2. The mixture was extracted with EtOAc (300 mL*2). The combined organic layer was washed with brine (100 mL), dried over Na2SO4, filtered. The filtrate was concentrated under reduced pressure to afford 282a (15 g, 95% yield) as a yellow solid. LC-MS (Method 3) tR=1.13 min, m/z (M+H−56)+=228.2.

To a solution of 282e (400 mg, 1.18 mmol) in DCM (2 mL) was added TFA (2 mL) dropwise at r.t. The reaction mixture was stirred at r.t. for 1 h and concentrated under vacuum at 30° C. The residue was dissolved in H2O (80 mL) and the solution was adjusted to pH=12 with 1 M NaOH solution. The mixture was extracted with DCM (80 mL*3). The combined organic layer was concentrated and the residue was purified by flash chromatography on silica gel (DCM/MeOH=10/1) to afford 282f (100 mg, 44% yield) as a yellow solid. LC-MS (Method 2) tR=0.60 min, m/z (M+H)+=190.2.

A mixture of 282g (58 mg, 0.14 mmol) and LiOH·H2O (18 mg, 0.43 mmol) in THF (3 mL), MeOH (0.6 mL) and water (0.6 mL) was stirred at 40° C. for 3 h. The reaction mixture was diluted with water (5 mL). The aqueous layer was acidified with 1 N HCl to pH=4 and was extracted with EtOAc (15 mL*3). The combined organic layer was washed with brine (15 mL), dried over Na2SO4, filtered, concentrated and purified by flash chromatography on silica gel (DCM/MeOH=50/1 to 10/1) to get compound 282h (20 mg, 35% yield) as a white solid.

Summary of representative compounds are shown in Table 1.

Table 1. Exemplary Compounds

Binding Activity Test for JH2 Domain of JAK1 and TYK2

The compounds are prepared in DMSO for 200× top dose, then serial dilute it with 27-fold for 3 more points. Add 8 μL/well in echo source plate, echo will add 75 nL/well with 3-fold serial dilution for 11 points in assay plate. 75 nL DMSO for high control and low control.

Prepare 3× working solutions (1.5 nM) of JAK1-JH2 domain or TYK2-JH2 domain enzyme in assay buffer, add 5 μL of enzyme working solutions per well to the assay plate including high control. For low control, add 5 μL/well assay buffer. And then spin down at 1000 rpm and centrifuge for 30 seconds. After enzyme system prepared, add 5 μL of Tb-antibody solution into each wells of assay plate. Spin down at 1000 rpm and centrifuge for 30 sec. After Tb-antibody added, also add 5 μL of tracer into the assay plate. Spin down at 1000 rpm and centrifuge for 30 sec. Incubate for 60 mins at 25° C. firstly and then continue to incubate the plate at 4° C. overnight. Read by envision in FRET mode. The Luminescence value was recorded by a multi-label reader Envision (PerkinElmer). Inhibition Rate was calculated relative to vehicle (DMSO) treated control wells using following formula:

%⁢Inhibition=100-RLUcompound-RLU⁢lowcontrolRLU⁢high⁢control-RLU⁢low⁢control×1⁢0⁢0⁢%
whereinRLU compound=the relative light unit of cells treated with test compoundsRLU_low control=the relative light unit of medium with DMSO onlyRLU_high control=the relative light unit of cells treated with DMSO only

The dose-response (percent inhibition) curve was plotted and IC50 values (the concentration that causes 50% growth inhibition) were determined by GraphPad software. The IC50 of tested compounds are shown in Table 2.

TABLE 2IC50 on JH2 domain activity (nM)TYK2-JH2JAK1-JH2JAK1/TYK2Example(nM)(nM)selectivity170.202——190.098——231.332——270.48——41(BMS986165)0.1330.6314.7440.2869.88834.6530.293——575.215——680.4——974.381>298.507—1000.887——1050.733——1100.92——1163.347——1251.442——1290.41620.7749.91310.3313.27740.21351.46——1390.52412.37923.61441.291——148>298.5——14962.806——1501.872——1510.4613.12828.51520.17110.70562.61530.45515.11833.21541.191——1570.35725.19570.61580.948——1650.27818.62567.01660.15413.59888.31682.15——1690.318.76328.31730.817——1904.033——190A0.622——1923.365——1931.414——1940.756——2030.17318.462106.72051.305——2060.62310.96317.62070.407——2080.24834.737140.12090.15811.1970.82110.71——2120.601——2130.25——2140.557——2150.532——2160.525——2171.313——2180.343——2192.862——2250.86815.21517.52261.45656.51738.82277.528——2280.2886.48422.52291.68105.03562.52303.00958.70219.52310.979——2321.576——234A0.52124.3546.7234B5.784244.24842.22350.2626.74425.72360.96943.77345.22390.1885.41228.82400.2605.07119.52410.53415.34428.72421.17845.32938.52433.446129.98537.72440.1254.57636.62450.1574.34427.72460.15611.84875.92490.78225.43632.5249A0.71517.97925.15249B0.73828.96639.252500.2337.03530.192510.3045.57018.32251A1.03497.23094.03251B0.2664.60117.302521.683211.529125.692530.78319.04724.33254A0.86844.10150.81254B11.646251.57721.602550.3234.42013.72560.26510.81840.82570.1353.17823.52580.83414.89517.92596.29379.89212.72600.1249.78178.92610.95645.3647.42620.22014.75667.12630.29025.35187.42641.77273.73141.426569.234——2660.28744.2301542670.1819.25351.127125.572——272137.846——2730.1662.67816.127434.415——2750.44927.02360.22760.253110.323436.12771.10761.38355.52780.3859.62625.02790.330——2800.180——2810.250——2820.270——

Conclusion: Most the exemplified compounds with different scaffolds have better JH2 TYK2 over JH2 JAK1 selectivity than BMS986165.

Biochemical Assay

Testing for JAK1, JAK2 and TYK2 Kinase Activities

JAK activity was determined in the reaction buffer 50 mM HEPES, 0.01% Brij35, 10 mM MgCl2, 2 mM DTT by a microfluidic assay. The phosphorylation of a FAM labeled peptide substrate was monitored in the Caliper EZ Reader II (Perkin Elmer). The assay condition for each batch of enzyme (Carna Biosciences) was optimized to obtain 10% conversion rate of peptide substrate.

The test compounds were dissolved in DMSO to a stock concentration of 10 mM. Three-fold serially diluted compounds with top concentration of 5 μM were pre-incubated with JAK1, JAK2 or TYK2 for 10 min at ambient temperature. The final DMSO concentration of assay mixture was 1%. FAM labeled peptide substrate (final concentration 3 μM) and ATP (Km concentration or 1 mM) were sequentially added to initiate the kinase reaction at 28° C. for 90 min (JAK), 15 min (JAK2) and 30 min (TYK2), respectively. The reaction was stopped by adding 50 mM EDTA.

The well in the test plate without enzyme was defined as 100% inhibition. And the well without compound but with equivalent DMSO was defined as no inhibition. The percent inhibition was calculated by the following formula:

The dose-response (percent inhibition) curve was plotted and IC50 values were determined by GraphPad software. The IC50 values of tested compounds were list in Table 3.

Conclusion: Most the exemplified compounds with different scaffolds have better selectivity in JAK1, JAK2 and TYK2 kinases inhibition than BMS986165.

Dimerization domain of Tel protein fused with JAK kinase domain was permanently transduced into Ba/F3 cells, whose proliferation is dependent on JAK activity in the absence of IL-3 induction. These engineered Ba/F3 cells (Ba/F3-FL-TYK2-P760L, Ba/F3-TEL-TYK2 and Ba/F3-TEL-JAK2) were used to monitor JAK inhibitory activities of the compounds in the cellular. Ba/F3 cells were cultured in RPMI-1640 (Corning) containing 10% fetal bovine serum. Cells were seeded at 2000/well of white flat bottom 96-well plates. The well containing medium only was used as background control. After 24h growth, cells were treated with compounds. The test compounds were dissolved in DMSO to a stock concentration of 20 mM. 3-fold serially diluted compounds for 9 concentrations with top concentration of 20 μM was added into the each well. The final DMSO concentration was 0.1%. The cells continued to grow at 37° C. in 5% CO2for 72 h after compound treatment. The viability was measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent (Promega). The Luminescence value was recorded by a multi-label reader Envision (PerkinElmer). Inhibition Rate was calculated relative to vehicle (DMSO) treated control wells using following formula:

%⁢Inhibition=100-RLU_compound-RLU_blankRLU_control-RLU_blank×1⁢0⁢0⁢%
whereinRLU compound=the relative light unit of cells treated with test compoundsRLU blank=the relative light unit of medium with DMSO onlyRLU_control=the relative light unit of cells treated with DMSO only

The dose-response (percent inhibition) curve was plotted and IC50 values (the concentration that causes 50% growth inhibition) were determined by GraphPad software. The IC50 of tested compounds are shown in Table 4.

The inhibitory potential of test articles in human PBMC and human whole blood assay was evaluated by the method of flow cytometry.

IFN-alpha will activate JAK1 and TYK2 kinase in T cells by binding to IFN receptors, and then lead to phosphorylation of STAT1 and STAT2. The phosphorylated STAT1 enter nuclear and promote transcription and expression of IFN-gamma. To evaluate the efficacy of TYK2i in T cells, freshly prepared human PBMC or freshly collected human whole blood will be stimulated with certain unit of human IFN-alpha (Universal Type I IFN (1MU), R&D, 11200-2) for 20 mins in incubator. After stimulation, fix cells with Phosflow™ Fix Buffer I (BD, 557870), and then collect fixed cells by centrifugation (500 g, 8 mM). Wash cells once with pre-cooled PBS, and then permeabilize cells with cold Perm Buffer III buffer (BD, 558050) for 45 mins on ice. Collect cells by centrifugation (600 g, 8 min) and washed twice with cold PBS. Stain cells with anti-human CD3(FITC Mouse Anti-Human CD3, BD, 555332) and anti-pSTAT1_Y701(Alexa Fluor 647 Mouse Anti-Statl (pY701), BD, 612597) antibodies. Detect pSTAT1 by flow cytometry (CytoFlex S) and analyze data with FlowJo and GraphPad Prism 8 software. IC50 values of test articles were determined using 4-parameter logistic equation.

Exemplary results are summarized in Table 5.

Conclusion: Most the exemplified compounds have decent pSTAT1 inhibition in both hWB and hPBMC.

Microsomal Metabolic Stability Assay

Microsomes were pre-incubated with test compound or control compounds for 5 min at 37° C. in 100 mM potassium phosphate buffer, pH 7.4, 1.0 mM EDTA. The reaction was initiated by addition of 15 μL of the NADPH regenerating system to 30 μL of each incubation mixture per time point. The final incubation condition was composed of 0.5 mg/mL microsomal protein, 1 μM test article/positive control, 2 mM NADPH. The 0-minute samples were prepared by addition of a 30 μL aliquot of each incubation mixture to 135 μL quench reagent to precipitate proteins. And then a 15 μL aliquot of the NADPH regenerating system was added. At 5, 15, 30 and 45 minutes, the reaction will be stopped by the addition of cold acetonitrile solution containing internal standard. The samples taken at all time points were centrifuged at 5000×g for 15 minutes. 50 μL of supernatant are taken into 96-well assay plates pre-added with 50 μL ultrapure water, and then analyzed by LC/MS/MS.

Concentrations of test articles, control compounds in the samples were determined by using LC/MS/MS method. Plotting of the chromatograms and peak area integrations are carried out by Analyst (AB Sciex).

In the determination of the in vitro elimination constant (ke) of the control compounds, the analyte/internal standard peak area ratios will be converted to percentage remaining (% Remaining) with the following equation:

The CLintof microsomes was calculated using the formula: CLint (mic)=0.693/T1/2/mg microsome protein per mL. The slope was measured by the natural logarithm of the percentage of the residual compound and time, T1/2 was calculated according to the following formula.

Exemplary results are summarized in Table 6.

The vials of cryopreserved hepatocytes were removed from the liquid nitrogen container and immediately immersed in a 37° C. water bath for approximately 2 min. The melted ice pellets were transferred into tubes containing 50 mL of pre-warmed thawing medium and mixed well by gently inverting the tubes, and then centrifuged at 500 rpm for 3 minutes at room temperature. The supernatants were discarded, and cell pellets were re-suspended by adding appropriate volumes of pre-warmed incubation medium. The cells viability of each species was determined using Trypan Blue exclusion. Add 50 μL of pre-warmed 2× dosing solution to the wells designated for different time points. Then 50 μL of cells suspension (2×106cells/mL) were added into appropriate wells at corresponding time points of T15, T30, T60, T120. Start timing and put the plate in an incubator at 37° C. For T0 sample, add 300 μL of ACN containing internal standard to the wells, mix gently, then add 50 μL of pre-warmed hepatocytes solution (2×106cells/mL). The concentration of test compound was 1 μM and 1.0×106cells/mL in the final incubation. At each corresponding time point, test and control compounds reaction samples were stopped by adding 300 μL acetonitrile containing internal standard. All sample plates were thoroughly mixed and centrifuged at 5000×g for 15 minutes. The supernatants were diluted at ratio of 1:1 with ultra-pure water for test and positive controls. Then submitted for LC-MS/MS analysis.

The analyte/internal standard peak area ratios were converted to percentage remaining with the following equation:

The in vitro elimination constant, k of test compounds and control compounds were calculated from a log linear plot of % Remaining versus time, then the half-life (T1/2), the estimation of the in vitro hepatic intrinsic clearance (CLint) values were calculated from substrate disappearance rate in hepatocytes incubation:T1/2=0.693/kCLint (hep)=k/million cells per mL

Exemplary results are summarized in Table 7.

TABLE 7HumanHumanHumanHumanhepatocytehepatocytehepatocytehepatocytestabilityClint(liver)stabilityClint(liver)ExampleT1/2 (min)(mL/min/kg)ExampleT1/2 (min)(mL/min/kg)41>216.8<17.8151193.699.14489.443.1152>240067>240<7.35157127.1113.87105202.938.69165>240<7.3513293.0718.94166156.6811.2513498.1617.96169206.148.60137122.5914.38197>240<7.35139>240<7.3526295.2218.52
Plasma Protein Binding by Equilibrium Dialysis

Aliquots of test compound stock solution and warfarin stock solution were spiked into plasma to give the matrix solutions with theoretical concentrations of test compound at 0.2 and 2 μM or warfarin at 2 μM. Immediately transfer 50 μL of the spiked plasma solution suspension to a 96-well plate to act as T=0 control sample. Assemble the dialysis set up following the manufacturer's instructions. Load cells with 150 μL of plasma sample and dialyzed against equal volume of dialysis buffer (PBS). The assay is performed in duplicate. Cover the unit with gas permeable lid and incubate for 5 hours at 37° C. at 100 rpm in a constant temperature shaking box. At the end of incubation, remove lid and pipette 50 μL of post-dialysis samples from both buffer and plasma solution chambers into separated 96-well plate for analysis, respectively. Add 50 μL of plasma solution to the buffer samples, and an equal volume of PBS to the collected plasma solution samples. Shake the plate at 1000 rpm for 2 minutes and add 600 μL of 50% ACN/MeOH containing an appropriate internal standard (IS) to precipitate protein and release compound. Vortex at 1000 rpm for 10 minutes. Centrifuge for 10 minutes at 4000 rpm. Then transfer 75 μL of the supernatant to new 96-well plates for analysis. Add 150 μL of distilled water to each sample and mix for analysis by LC-MS/MS.

Exemplary results are summarized in Table 8

Aliquots of 8 μL of reference and test compound stock solutions (10 mM/5 mM) are added into 792 μL of 100 mM pH 7.4 phosphate buffer. The final DMSO concentration is 1%. Sample tubes are shaken for 2 hours at 1000 rpm at room temperature. Samples are centrifuged at 12000 rpm for 10 min to precipitate un-dissolved particles. And transfer the supernatants to a new tube or plate. Add 5 μL of samples (no diluted, 10 times diluted and 100 times diluted) and standard curve samples to 95 μL of ACN containing IS for LC-MS/MS analysis.

Exemplary results are summarized in Table 9

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.