HETEROCYCLIC COMPOUNDS AS IMMUNOMODULATORS

Disclosed are compounds of Formula (I), methods of using the compounds as immunomodulators, and pharmaceutical compositions comprising such compounds. The compounds are useful in treating, preventing or ameliorating diseases or disorders such as cancer or infections.

FIELD OF THE INVENTION

The present application is concerned with pharmaceutically active compounds. The disclosure provides compounds as well as their compositions and methods of use. The compounds modulate PD-1/PD-L1 protein/protein interaction and are useful in the treatment of various diseases including infectious diseases and cancer.

BACKGROUND OF THE INVENTION

The immune system plays an important role in controlling and eradicating diseases such as cancer. However, cancer cells often develop strategies to evade or to suppress the immune system in order to favor their growth. One such mechanism is altering the expression of co-stimulatory and co-inhibitory molecules expressed on immune cells (Postow et al, J. Clinical Oncology 2015, 1-9). Blocking the signaling of an inhibitory immune checkpoint, such as PD-1, has proven to be a promising and effective treatment modality.

Programmed cell death-1 (PD-1), also known as CD279, is a cell surface receptor expressed on activated T cells, natural killer T cells, B cells, and macrophages (Greenwald et al, Annu. Rev. Immunol 2005, 23:515-548; Okazaki and Honjo, Trends Immunol 2006, (4): 195-201). It functions as an intrinsic negative feedback system to prevent the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance. In addition, PD-1 is also known to play a critical role in the suppression of antigen-specific T cell response in diseases like cancer and viral infection (Sharpe et al,Nat Immunol2007 8, 239-245; Postow et al, J. Clinical Oncol 2015, 1-9).

The structure of PD-1 consists of an extracellular immunoglobulin variable-like domain followed by a transmembrane region and an intracellular domain (Parry et al, Mol Cell Biol 2005, 9543-9553). The intracellular domain contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates T cell receptor-mediated signals. PD-1 has two ligands, PD-L1 and PD-L2 (Parry et al, Mol Cell Biol 2005, 9543-9553; Latchman et al, Nat Immunol 2001, 2, 261-268), and they differ in their expression patterns. PD-L1 protein is upregulated on macrophages and dendritic cells in response to lipopolysaccharide and GM-CSF treatment, and on T cells and B cells upon T cell receptor and B cell receptor signaling. PD-L1 is also highly expressed on almost all tumor cells, and the expression is further increased after IFN-γ treatment (Iwai et al, PNAS 2002, 99(19):12293-7; Blank et al, Cancer Res 2004, 64(3):1140-5). In fact, tumor PD-L1 expression status has been shown to be prognostic in multiple tumor types (Wang et al, Eur J Surg Oncol 2015; Huang et al, Oncol Rep 2015; Sabatier et al, Oncotarget 2015, 6(7): 5449-5464). PD-L2 expression, in contrast, is more restricted and is expressed mainly by dendritic cells (Nakae et al, J Immunol 2006, 177:566-73). Ligation of PD-1 with its ligands PD-L1 and PD-L2 on T cells delivers a signal that inhibits IL-2 and IFN-γ production, as well as cell proliferation induced upon T cell receptor activation (Carter et al, Eur J Immunol 2002, 32(3):634-43; Freeman et al, J Exp Med 2000, 192(7): 1027-34). The mechanism involves recruitment of SHP-2 or SHP-1 phosphatases to inhibit T cell receptor signaling such as Syk and Lck phosphorylation (Sharpe et al, Nat Immunol 2007, 8, 239-245). Activation of the PD-1 signaling axis also attenuates PKC-θ activation loop phosphorylation, which is necessary for the activation of NF-κB and API pathways, and for cytokine production such as IL-2, IFN-γ and TNF (Sharpe et al, Nat Immunol 2007, 8, 239-245; Carter et al, Eur J Immunol 2002, 32(3):634-43; Freeman et al, J Exp Med 2000, 192(7):1027-34).

Several lines of evidence from preclinical animal studies indicate that PD-1 and its ligands negatively regulate immune responses. PD-1-deficient mice have been shown to develop lupus-like glomerulonephritis and dilated cardiomyopathy (Nishimura et al, Immunity 1999, 11:141-151; Nishimura et al, Science 2001, 291:319-322). Using an LCMV model of chronic infection, it has been shown that PD-1/PD-L1 interaction inhibits activation, expansion and acquisition of effector functions of virus-specific CD8 T cells (Barber et al, Nature 2006, 439, 682-7). Together, these data support the development of a therapeutic approach to block the PD-1-mediated inhibitory signaling cascade in order to augment or “rescue” T cell response. Accordingly, there is a need for new compounds that block PD-1/PD-L1 protein/protein interaction.

SUMMARY

The present disclosure provides, inter alia, a compound of Formula (I):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein constituent variables are defined herein.

The present disclosure further provides a pharmaceutical composition comprising a compound of the disclosure, or a pharmaceutically acceptable salt or a stereoisomer thereof, and at least one pharmaceutically acceptable carrier or excipient.

The present disclosure further provides methods of modulating or inhibiting PD-1/PD-L1 protein/protein interaction, which comprises administering to an individual a compound of the disclosure, or a pharmaceutically acceptable salt or a stereoisomer thereof.

The present disclosure further provides methods of treating a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt or a stereoisomer thereof.

DETAILED DESCRIPTION

The present disclosure provides a compound of Formula (I):

X2is N or C;

X4is N or CR4;

X5is N or CR5;

X6is N or CR6;

Y is C or N;

at least one of X1, X2, X3and Y is a heteroatom selected from N, O and S;

Cy is C6-10aryl, C3-10cycloalkyl, 5- to 14-membered heteroaryl, or 4- to 10-membered heterocycloalkyl, each of which is optionally substituted with 1 to 5 independently selected R7substituents;

or two adjacent R7substituents on the Cy ring, taken together with the atoms to which they are attached, form a fused phenyl ring, a fused 5-, 6- or 7-membered heterocycloalkyl ring, a fused 5- or 6-membered heteroaryl ring or a fused C3-6cycloalkyl ring, wherein the fused 5-, 6- or 7-membered heterocycloalkyl ring and fused 5- or 6-membered heteroaryl ring each have 1-4 heteroatoms as ring members selected from N, O and S and wherein the fused phenyl ring, fused 5-, 6- or 7-membered heterocycloalkyl ring, fused 5- or 6-membered heteroaryl ring and fused C3-6cycloalkyl ring are each optionally substituted with 1, 2 or 3 independently selected Rbsubstituents;

or two R13substituents attached to the same carbon atom, taken together with the carbon atom to which they are attached, form a C3-6cycloalkyl ring or 4-, 5-, 6- or 7-membered heterocycloalkyl ring, wherein the C3-6cycloalkyl ring and 4-, 5-, 6- or 7-membered heterocycloalkyl ring are each optionally substituted with 1, 2 or 3 independently selected Rbsubstituents;

or two Rhgroups attached to the same carbon atom of the 4- to 10-membered heterocycloalkyl taken together with the carbon atom to which they are attached form a C3-6cycloalkyl or 4- to 6-membered heterocycloalkyl having 1-2 heteroatoms as ring members selected from O, N or S;

is a single bond or a double bond to maintain ring A being aromatic;

the subscript n is an integer of 1, 2, 3, 4, 5 or 6; and

when R9is OH, Cy is other than 6-carbamimidoyl-1H-benzo[d]imidazol-2-yl.

The compounds, or pharmaceutically acceptable salts or stereoisomers thereof, as described herein are useful as inhibitors of the PD-1/PD-L1 protein/protein interaction. For example, compounds or pharmaceutically acceptable salts or stereoisomers thereof as described herein can disrupt the PD-1/PD-L1 protein/protein interaction in the PD-1 pathway.

In some embodiments of compounds of Formula (I), when R9is OH, Cy is other than 1H-benzo[d]imidazol-2-yl optionally substituted with a R7substituent.

In some embodiments of compounds of Formula (I), two adjacent R7substituents on the Cy ring, taken together with the atoms to which they are attached, form a fused phenyl ring, a fused 5-, 6- or 7-membered heterocycloalkyl ring, a fused 5- or 6-membered heteroaryl ring or a fused C3-6cycloalkyl ring, wherein the fused 5-, 6- or 7-membered heterocycloalkyl ring and fused 5- or 6-membered heteroaryl ring each have 1-4 heteroatoms as ring members selected from N, O and S and wherein the fused phenyl ring, fused 5-, 6- or 7-membered heterocycloalkyl ring, fused 5- or 6-membered heteroaryl ring and fused C3-6cycloalkyl ring are each optionally substituted with 1, 2 or 3 independently selected Rqsubstituents.

In some embodiments of compounds of Formula (I), Cy is C6-10aryl, optionally substituted with 1 to 5 independently selected R7substituents. In certain embodiments, Cy is phenyl or naphthyl, each of which is optionally substituted with 1 to 4 independently selected R7substituents. In certain embodiments, Cy is phenyl optionally substituted with 1 to 5 independently selected R7substituents. In certain embodiments, Cy is unsubstituted phenyl. In certain embodiments, Cy is 2,3-dihydro-1,4-benzodioxin-6-yl, optionally substituted with 1 to 5 independently selected R7substituents.

In some embodiments of compounds of Formula (I), Cy is C3-10cycloalkyl, optionally substituted with 1 to 5 independently selected R7substituents. In certain embodiments, Cy is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl or cyclooctyl, each of which is optionally substituted with 1 to 5 independently selected R7substituents.

In some embodiments of compounds of Formula (I), Cy is phenyl, 5- or 6-membered heteroaryl, C3-6cycloalkyl or 5- or 6-membered heterocycloalkyl, each of which is optionally substituted with 1 to 5 independently selected R7substituents. In certain instances, Cy is phenyl, 2-thiophenyl, 3-thiophenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, C3-6cycloalkyl or 3,6-dihydro-2H-pyran-4-yl, each of which is optionally substituted with 1 to 5 R7substituents.

In some embodiments of compounds of Formula (I), X4is CR4, X5is N and X6is N. In certain instances, R4is H.

In some embodiments of compounds of Formula (I), X4is CR4, X5is N and X6is CR6. In certain instances, R4and R6are each H.

In some embodiments of compounds of Formula (I), X4is N, X5is N and X6is CR6. In certain instances, R6is H.

In some embodiments, the present disclosure provides compounds having Formula (II):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the variables of Formula (II) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In one embodiment of compounds of Formula (II), R9is halo, CN or C1-4alkyl optionally substituted with 1 or 2 Rqgroups. In another embodiment, R9is Cl, CH3or CN.

In some embodiments, the present disclosure provides compounds having Formula (IIa):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the variables of Formula (IIa) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In one embodiment, Cy is phenyl optionally substituted with 1 to 5 R7groups. In one embodiment, R9is halo, CN or C1-4alkyl optionally substituted with 1 or 2 Rqgroups. In another embodiment, R9is Cl, CH3or CN.

In some embodiments, the present disclosure provides compounds having Formula (III):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the variables of Formula (III) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In one embodiment, Cy is phenyl optionally substituted with 1 to 5 R7groups. In one embodiment, R5and R6are H. In one embodiment, R9is halo, CN or C1-4alkyl optionally substituted with 1 or 2 Rqgroups. In another embodiment, R9is Cl, CH3or CN.

In some embodiments, the present disclosure provides compounds having Formula (IV):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the variables of Formula (IV) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In one embodiment, Cy is phenyl optionally substituted with 1 to 5 R7groups. In one embodiment, R4and R5are H. In one embodiment, R9is halo, CN or C1-4alkyl optionally substituted with 1 or 2 Rqgroups. In another embodiment, R9is Cl, CH3or CN.

In some embodiments, the present disclosure provides compounds having Formula (V):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the variables of Formula (V) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In one embodiment, R9is halo, CN or C1-4alkyl optionally substituted with 1 or 2 Rqgroups. In another embodiment, R9is Cl, CH3or CN.

In some embodiments, the present disclosure provides compounds having Formula (VI):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the subscript m is an integer of 1, 2, 3 or 4 and the variables of Formula (VI) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. The moiety

in Formula (VI) is

In certain embodiments, the present disclosure provides compounds having Formula (VIa):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the subscript m is an integer of 1, 2, 3 or 4 and the variables of Formula (VIa) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein.

In certain embodiments, the present disclosure provides compounds having Formula (VIb):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the subscript m is an integer of 1, 2, 3 or 4 and the variables of Formula (VIb) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein.

In certain embodiments, the present disclosure provides compounds having Formula (VIc):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the subscript m is an integer of 1, 2, 3 or 4 and the variables of Formula (VIc) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein.

In some embodiments, the present disclosure provides compounds having Formula (VII):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein m is an integer of 1, 2 or 3 and the variables of Formula (VII) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. The moiety

in Formula (VII) is

In some embodiments, the present disclosure provides compounds having Formula (VIIa):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein m is an integer of 1, 2 or 3 and the variables of Formula (VIIa) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein.

In some embodiments, the present disclosure provides compounds having Formula (VIIb):

or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein m is an integer of 1, 2 or 3 and the variables of Formula (VIIb) are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein.

In some embodiments of compounds of any of the Formulas as disclosed herein or a pharmaceutically acceptable salt or a stereoisomer thereof, the moiety:

is selected from:

wherein the substituents R1, R3, R13, R14and the subscript n are as defined in Formula (I) or any embodiment of compounds of Formula (I) as described herein. In certain embodiments, at each occurrence, R1and R3are each H. In other embodiments, R13is H or C1-6alkyl. In one embodiment, the subscript n is 2.

In some embodiments of compounds of any of the Formula as disclosed herein or a pharmaceutically acceptable salt or a stereoisomer thereof, the moiety:

is selected from:

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is S and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is S, X2is C, X3is N, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is CR3, and Y is N. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is NR3, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is S, X6is N, and Y is C. In some instances, X4and X5are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is N, X3is CR3, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is CR1, X2is N, X3is N, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is CR1, X2is C, X3is N, and Y is N. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is NR1, X2is C, X3is N, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is O, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is O, X2is C, X3is N, and Y is C. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is N, and Y is N. In some instances, X4, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, X1is N, X2is C, X3is S, X4is N, and Y is C. In some instances, X5and X6are each CH.

In some embodiments of compounds of any of the Formulas as disclosed herein, R13is H or C1-6alkyl.

In some embodiments of compounds of any of the Formulas as disclosed herein, two R13substituents attached to the same carbon atom, taken together with the carbon atom to which they are attached, form a C3-6cycloalkyl ring or 4-, 5-, 6- or 7-membered heterocycloalkyl ring, wherein the C3-6cycloalkyl ring and 4-, 5-, 6- or 7-membered heterocycloalkyl ring are each optionally substituted with 1, 2 or 3 independently selected Rqsubstituents. Exemplary spiro C3-6cycloalkyl ring formed by two R13substituents include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In some embodiments of compounds of any of the Formulas as disclosed herein, R14is 2-hydroxyethyl, 2-hydroxypropyl, (R)-2-hydroxypropyl, (S)-2-hydroxypropyl, tetrahydro-2H-pyran-4-yl, 4-carboxycyclohexyl, 3-carboxypropyl, 2-carboxycyclopropylmethyl, 1H-pyrazol-4-ylmethyl or 4-cyanomethylcyclohexyl.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (while the embodiments are intended to be combined as if written in multiply dependent form). Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. Thus, it is contemplated as features described as embodiments of the compounds of Formula (I) can be combined in any suitable combination.

At various places in the present specification, certain features of the compounds are disclosed in groups or in ranges. It is specifically intended that such a disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-6alkyl” is specifically intended to individually disclose (without limitation) methyl, ethyl, C3alkyl, C4alkyl, C5alkyl and C6alkyl.

At various places in the present specification, variables defining divalent linking groups may be described. It is specifically intended that each linking substituent include both the forward and backward forms of the linking substituent. For example, —NR(CR′R″)n-includes both —NR(CR′R″)n- and —(CR′R″)nNR— and is intended to disclose each of the forms individually. Where the structure requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” or “aryl” then it is understood that the “alkyl” or “aryl” represents a linking alkylene group or arylene group, respectively.

The term “substituted” means that an atom or group of atoms formally replaces hydrogen as a “substituent” attached to another group. The term “substituted”, unless otherwise indicated, refers to any level of substitution, e.g., mono-, di-, tri-, tetra- or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. It is to be understood that substitution at a given atom is limited by valency. It is to be understood that substitution at a given atom results in a chemically stable molecule. The phrase “optionally substituted” means unsubstituted or substituted. The term “substituted” means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent, e.g., oxo, can replace two hydrogen atoms.

The term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include C1-4, C1-6and the like.

The term “alkyl” employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chained or branched. The term “Cn-malkyl”, refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C—H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and the like.

The term “alkenyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more double carbon-carbon bonds. An alkenyl group formally corresponds to an alkene with one C—H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. The term “Cn-malkenyl” refers to an alkenyl group having n to m carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl and the like.

The term “alkynyl” employed alone or in combination with other terms, refers to a straight-chain or branched hydrocarbon group corresponding to an alkyl group having one or more triple carbon-carbon bonds. An alkynyl group formally corresponds to an alkyne with one C—H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. The term “Cn-malkynyl” refers to an alkynyl group having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

The term “alkylene”, employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C—H bond replaced by points of attachment of the alkylene group to the remainder of the compound. The term “Cn-malkylene” refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl and the like.

The term “alkoxy”, employed alone or in combination with other terms, refers to a group of formula —O-alkyl, wherein the alkyl group is as defined above. The term “Cn-malkoxy” refers to an alkoxy group, the alkyl group of which has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

The term “amino” refers to a group of formula —NH2.

The term “carbonyl”, employed alone or in combination with other terms, refers to a —C(═O)— group, which also may be written as C(O).

The term “cyano” or “nitrile” refers to a group of formula —C≡N, which also may be written as —CN.

The terms “halo” or “halogen”, used alone or in combination with other terms, refers to fluoro, chloro, bromo and iodo. In some embodiments, “halo” refers to a halogen atom selected from F, Cl, or Br. In some embodiments, halo groups are F.

The term “haloalkyl” as used herein refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by a halogen atom. The term “Cn-mhaloalkyl” refers to a C-m alkyl group having n to m carbon atoms and from at least one up to {2(n to m)+1}halogen atoms, which may either be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the haloalkyl group has 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF3, C2F5, CHF2, CCl3, CHCl2, C2Cl5and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

The term “haloalkoxy”, employed alone or in combination with other terms, refers to a group of formula —O-haloalkyl, wherein the haloalkyl group is as defined above. The term “Cn-mhaloalkoxy” refers to a haloalkoxy group, the haloalkyl group of which has n to m carbons. Example haloalkoxy groups include trifluoromethoxy and the like. In some embodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

The term “oxo” refers to an oxygen atom as a divalent substituent, forming a carbonyl group when attached to carbon, or attached to a heteroatom forming a sulfoxide or sulfone group, or an N-oxide group. In some embodiments, heterocyclic groups may be optionally substituted by 1 or 2 oxo (═O) substituents.

The term “sulfido” refers to a sulfur atom as a divalent substituent, forming a thiocarbonyl group (C═S) when attached to carbon.

The term “aromatic” refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e., having (4n+2) delocalized 71 (pi) electrons where n is an integer).

The term “aryl,” employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2 fused rings). The term “Cn-maryl” refers to an aryl group having from n to m ring carbon atoms.

Aryl groups include, e.g., phenyl, naphthyl, indanyl, indenyl and the like. In some embodiments, aryl groups have from 6 to about 10 carbon atoms. In some embodiments aryl groups have 6 carbon atoms. In some embodiments aryl groups have 10 carbon atoms. In some embodiments, the aryl group is phenyl. In some embodiments, the aryl group is naphthyl.

The term “heteroaryl” or “heteroaromatic,” employed alone or in combination with other terms, refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-14 ring atoms including carbon atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-10 ring atoms including carbon atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six-membered heteroaryl ring. In other embodiments, the heteroaryl is an eight-membered, nine-membered or ten-membered fused bicyclic heteroaryl ring. Example heteroaryl groups include, but are not limited to, pyridintl (pyridyl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, thiazolyl, imidazolyl, furanyl, thiophenyl, quinolinyl, isoquinolinyl, naphthyridinyl (including 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3- and 2,6-naphthyridine), indolyl, benzothiophenyl, benzofuranyl, benzisoxazolyl, imidazo[1,2-b]thiazolyl, purinyl, and the like.

A six-membered heteroaryl ring is a heteroaryl group having six ring atoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independently selected from N, O and S. Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

The term “cycloalkyl,” employed alone or in combination with other terms, refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic or polycyclic), including cyclized alkyl and alkenyl groups. The term “Cn-mcycloalkyl” refers to a cycloalkyl that has n to m ring member carbon atoms. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4, 5, 6 or 7 ring-forming carbons (C3-7). In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to 5 ring members, or 3 to 4 ring members. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic.

In some embodiments, the cycloalkyl group is a C3-6monocyclic cycloalkyl group. Ring-forming carbon atoms of a cycloalkyl group can be optionally oxidized to form an oxo or sulfido group. Cycloalkyl groups also include cycloalkylidenes. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, e.g., benzo or thienyl derivatives of cyclopentane, cyclohexane and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbomyl, norpinyl, norcarnyl, bicyclo[1.1.1]pentanyl, bicyclo[2.1.1]hexanyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term “heterocycloalkyl,” employed alone or in combination with other terms, refers to a non-aromatic ring or ring system, which may optionally contain one or more alkenylene groups as part of the ring structure, which has at least one heteroatom ring member independently selected from nitrogen, sulfur oxygen and phosphorus, and which has 4-10 ring members, 4-7 ring members, or 4-6 ring members. Included within the term “heterocycloalkyl” are monocyclic 4-, 5-, 6- and 7-membered heterocycloalkyl groups.

Heterocycloalkyl groups can include mono- or bicyclic (e.g., having two fused or bridged rings) ring systems. In some embodiments, the heterocycloalkyl group is a monocyclic group having 1, 2 or 3 heteroatoms independently selected from nitrogen, sulfur and oxygen. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally oxidized to form an oxo or sulfido group or other oxidized linkage (e.g., C(O), S(O), C(S) or S(O)2, N-oxide etc.) or a nitrogen atom can be quaternized. The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the heterocycloalkyl ring, e.g., benzo or thienyl derivatives of piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Examples of heterocycloalkyl groups include azetidinyl, azepanyl, dihydrobenzofuranyl, dihydrofuranyl, dihydropyranyl, morpholino, 3-oxa-9-azaspiro[5.5]undecanyl, 1-oxa-8-azaspiro[4.5]decanyl, piperidinyl, piperazinyl, oxopiperazinyl, pyranyl, pyrrolidinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, tropanyl, and thiomorpholino.

At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas an azetidin-3-yl ring is attached at the 3-position.

In some embodiments, the compounds of the invention have the (R)-configuration. In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral centers, each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated.

The term, “compound,” as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the structures depicted. The term is also meant to refer to compounds of the inventions, regardless of how they are prepared, e.g., synthetically, through biological process (e.g., metabolism or enzyme conversion), or a combination thereof.

All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated. When in the solid state, the compounds described herein and salts thereof may occur in various forms and may, e.g., take the form of solvates, including hydrates. The compounds may be in any solid state form, such as a polymorph or solvate, so unless clearly indicated otherwise, reference in the specification to compounds and salts thereof should be understood as encompassing any solid state form of the compound.

The expressions, “ambient temperature” and “room temperature,” as used herein, are understood in the art, and refer generally to a temperature, e.g., a reaction temperature, that is about the temperature of the room in which the reaction is carried out, e.g., a temperature from about 20° C. to about 30° C.

Compounds of the invention, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, such as those in the Schemes below.

Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups is described, e.g., in Kocienski,Protecting Groups, (Thieme, 2007); Robertson,Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al.,March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure,6thEd. (Wiley, 2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,”J Chem. Educ.,1997, 74(11), 1297; and Wuts et al.,Protective Groups in Organic Synthesis,4th Ed., (Wiley, 2006).

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

The Schemes below provide general guidance in connection with preparing the compounds of the invention. One skilled in the art would understand that the preparations shown in the Schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention.

Compounds of Formula (I) can be prepared, e.g., using a process as illustrated in Schemes 1-7.

Compounds of formula 1-7 can be synthesized as shown in Scheme 1. A selective coupling of the iodide 1-1 with compounds of formula 1-2 [M is B(OR)2, Sn(Alkyl)3, or Zn-Hal] under suitable Suzuki coupling conditions, Stille coupling conditions, or Negishi coupling conditions can give derivatives of formula 1-3. The resulting chloride 1-3 can be converted to its boronic esters or stannanes of formula 1-4 in the presence of a suitable palladium catalyst. Another palladium catalyzed coupling of the resulting compounds of formula 1-4 with a commercially available bromide or iodide building block 1-5 (e.g. Hal is Br or I) under suitable Suzuki or Stille coupling conditions can give compounds of formula 1-6. After removal of Boc on the piperidine under acidic condition (trifluoroacetic acid or hydrochloric acid), the substitution of R14can be introduced to the resulting secondary amine by a reductive amination with the corresponding aldehydes or ketones or an alkylation with the corresponding alkyl halides to provide the desired compounds of formula 1-7.

Similarly, compounds of formula 2-4, with C—N bonding between the five- and six-membered aromatic rings, can be synthesized as shown in Scheme 2. Compounds of formula 2-1 (e.g., Hal is Cl or Br) can be prepared using similar conditions as described in Scheme 1. The C—N bond can be formed under suitable Buchwald-Hartwig amination conditions with a commercial amine moiety of formula 2-2 to give compounds of formula 2-3. After removal of Boc on the piperidine under acidic condition, the substitution of R14can be introduced to the resulting secondary amine by a reductive amination with the corresponding aldehydes or ketones to provide the desired compounds of formula 2-4.

Alternatively, compounds of formula 3-7 can be synthesized as shown in Scheme 3. Selective conversion of the L group in compound 3-1 (L is Br, I or OTf) to boronic ester can be achieved in the presence of a suitable palladium catalyst and bis(pinacolato)diboron to give boronic ester of formula 3-2. Selective Suzuki coupling of heteroaryl bromide 3-3 with boronic ester 3-2 can give biaryl chloride 3-4. Installation of Cy ring can be achieved using similar conditions as described in Scheme 1 by coupling biaryl chloride 3-4 with compound 3-5 to give compounds of formula 3-6. Removal of Boc protecting group followed by reductive amination with the corresponding aldehydes or ketones can provide the desired compounds of formula 3-7.

Thioazole compounds of formula 4-7, with substitutions on the piperidine ring, can be synthesized as shown in Scheme 4. The Boc protected oxo-piperidine of formula 4-1 can be brominated at the ketone a position either by treatment with bromine, or by a sequence of TMS enol ether formation and NBS bromination. The resulting bromide 4-2 can be converted to the aminothioazole 4-3 via reacting with thiourea in alcoholic solvents at elevated temperature. The amine group in 4-3 can be converted to halide under Sandermeyer conditions (e.g., in the presence oftBuONO and CuBr2) to give bromothiazole 4-4. Compound of formula 4-5 [M′ is B(OR)2or SnBu3] can be prepared using similar conditions as described in Scheme 1. Coupling of bromothiazole 4-4 with compound 4-5 can be achieved under suitable Suzuki coupling conditions or Stille coupling conditions to give compounds of formula 4-6. After removal of Boc on the coupling product 4-6 under acidic condition (trifluoroacetic acid or hydrochloric acid), the substitution of R14can be introduced to the resulting secondary amine by a reductive amination with the corresponding aldehydes or ketones to provide the desired compounds of formula 4-7.

Alternatively, oxazole derivatives of formula 5-7 can be synthesized according to the synthetic route as outlined in Scheme 5. Condensation of carboxylic acid 5-1 with amino, hydroxyl-disubstituted pyridine 5-2 in the presence of a condensation reagent (such as cyanuric chloride) can produce compounds of formula 5-3. Alkylation of the pyridine in 5-3 with benzyl bromide can give the quaternary salt 5-4 and subsequent reduction of 5-4 with NaBH4can lead to compound 5-5. Removal of the benzyl group using Pd/C under hydrogenation conditions can give compound 5-6. The R14group can be introduced under standard alkylation conditions or reductive amination conditions to give the final product 5-7.

Compounds of formula 6-5 can also be synthesized using conditions as shown in Scheme 6. Cyclization of α-bromo ketone derivatives of formula 6-1 with amino pyrazine 6-2 can give the heteroaryl compounds 6-3. Selective reduction of the pyrazine ring in compound 6-3 can be achieved by treating with LiBH4or using a similar reaction sequence as described in Scheme 5 to give compound 6-4. Similarly, the R14group can be introduced under alkylation conditions or reductive amination conditions to give the desired product 6-5.

Alternatively, compounds of formula 7-5 can be synthesized as shown in Scheme 7. Coupling of compound 7-1 [M′ is B(OR)2or SnBu3] with heteroaryl halide 7-2 (Hal is Cl, Br or I) can be achieved under suitable Suzuki coupling conditions or Stille coupling conditions to give compounds of formula 7-3. Selective reduction of the heteroaryl ring in 7-3 using similar conditions as described in Scheme 5 or Scheme 6 can give compound 7-4. Installation of R14group can be achieved similarly under alkylation conditions or reductive amination conditions to give compound 7-5.

III. Uses of the Compounds

Compounds of the present disclosure can inhibit the activity of PD-1/PD-L1 protein/protein interaction and, thus, are useful in treating diseases and disorders associated with activity of PD-1 and the diseases and disorders associated with PD-L1 including its interaction with other proteins such as PD-1 and B7-1 (CD80). In certain embodiments, the compounds of the present disclosure, or pharmaceutically acceptable salts or stereoisomers thereof, are useful for therapeutic administration to enhance, stimulate and/or increase immunity in cancer or chronic infection, including enhancement of response to vaccination. In some embodiments, the present disclosure provides a method for inhibiting or blocking the PD-1/PD-L1 protein/protein interaction. The method includes administering to an individual or a patient a compound of Formula (I) or any of the formulas as described herein or of a compound as recited in any of the claims and described herein, or a pharmaceutically acceptable salt or a stereoisomer thereof. The compounds of the present disclosure can be used alone, in combination with other agents or therapies or as an adjuvant or neoadjuvant for the treatment of diseases or disorders, including cancer or infection diseases. For the uses described herein, any of the compounds of the disclosure, including any of the embodiments thereof, may be used.

The compounds of the present disclosure inhibit the PD-1/PD-L1 protein/protein interaction, resulting in a PD-1 pathway blockade. The blockade of PD-1 can enhance the immune response to cancerous cells and infectious diseases in mammals, including humans. In some embodiments, the present disclosure provides treatment of an individual or a patient in vivo using a compound of Formula (I) or a salt or stereoisomer thereof such that growth of cancerous tumors is inhibited. A compound of Formula (I) or of any of the formulas as described herein, or a compound as recited in any of the claims and described herein, or a salt or stereoisomer thereof, can be used to inhibit the growth of cancerous tumors. Alternatively, a compound of Formula (I) or of any of the formulas as described herein, or a compound as recited in any of the claims and described herein, or a salt or stereoisomer thereof, can be used in conjunction with other agents or standard cancer treatments, as described below. In one embodiment, the present disclosure provides a method for inhibiting growth of tumor cells in vitro. The method includes contacting the tumor cells in vitro with a compound of Formula (I) or of any of the formulas as described herein, or of a compound as recited in any of the claims and described herein, or of a salt or stereoisomer thereof. In another embodiment, the present disclosure provides a method for inhibiting growth of tumor cells in an individual or a patient. The method includes administering to the individual or patient in need thereof a therapeutically effective amount of a compound of Formula (I) or of any of the formulas as described herein, or of a compound as recited in any of the claims and described herein, or a salt or a stereoisomer thereof.

In some embodiments, provided herein is a method for treating cancer. The method includes administering to a patient in need thereof, a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a salt thereof. Examples of cancers include those whose growth may be inhibited using compounds of the disclosure and cancers typically responsive to immunotherapy.

In some embodiments, the present disclosure provides a method of enhancing, stimulating and/or increasing the immune response in a patient. The method includes administering to the patient in need thereof a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a salt thereof.

Examples of cancers that are treatable using the compounds of the present disclosure include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. The compounds of the present disclosure are also useful for the treatment of metastatic cancers, especially metastatic cancers that express PD-L1.

In some embodiments, cancers treatable with compounds of the present disclosure include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer). Additionally, the disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the disclosure.

In some embodiments, cancers that are treatable using the compounds of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, hepatic cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancers of the head and neck, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL, mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma or multiple myeloma) and combinations of said cancers.

PD-1 pathway blockade with compounds of the present disclosure can also be used for treating infections such as viral, bacteria fungus and parasite infections. The present disclosure provides a method for treating infections such as viral infections. The method includes administering to a patient in need thereof, a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, a salt thereof. Examples of viruses causing infections treatable by methods of the present disclosure include, but are not limit to, human immunodeficiency virus, human papillomavirus, influenza, hepatitis A, B, C or D viruses, adenovirus, poxvirus, herpes simplex viruses, human cytomegalovirus, severe acute respiratory syndrome virus, ebola virus, and measles virus. In some embodiments, viruses causing infections treatable by methods of the present disclosure include, but are not limit to, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, comovirus, respiratory syncytial virus, mumpsvirus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

The present disclosure provides a method for treating bacterial infections. The method includes administering to a patient in need thereof, a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a salt thereof. Non-limiting examples of pathogenic bacteria causing infections treatable by methods of the disclosure includechlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci,klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme's disease bacteria.

The present disclosure provides a method for treating fungus infections. The method includes administering to a patient in need thereof, a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a salt thereof. Non-limiting examples of pathogenic fungi causing infections treatable by methods of the disclosure includeCandida(albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus(fumigatus, niger, etc.), GenusMucorales(mucor, absidia, rhizophus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitisandHistoplasma capsulatum.

The present disclosure provides a method for treating parasite infections. The method includes administering to a patient in need thereof, a therapeutically effective amount of a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a salt thereof. Non-limiting examples of pathogenic parasites causing infections treatable by methods of the disclosure includeEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp.,Giardia lambia, Cryptosporidiumsp.,Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, andNippostrongylus brasiliensis.

The phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

In some embodiments, the compounds of the invention are useful in preventing or reducing the risk of developing any of the diseases referred to herein; e.g., preventing or reducing the risk of developing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease.

Combination Therapies

Cancer cell growth and survival can be impacted by multiple signaling pathways. Thus, it is useful to combine different enzyme/protein/receptor inhibitors, exhibiting different preferences in the targets which they modulate the activities of, to treat such conditions. Targeting more than one signaling pathway (or more than one biological molecule involved in a given signaling pathway) may reduce the likelihood of drug-resistance arising in a cell population, and/or reduce the toxicity of treatment.

The compounds of the present disclosure can be used in combination with one or more other enzyme/protein/receptor inhibitors for the treatment of diseases, such as cancer or infections. Examples of cancers include solid tumors and liquid tumors, such as blood cancers. Examples of infections include viral infections, bacterial infections, fungus infections or parasite infections. For example, the compounds of the present disclosure can be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, CSFIR, KIT, FLK-II, KDR/FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR/Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK and B-Raf. In some embodiments, the compounds of the present disclosure can be combined with one or more of the following inhibitors for the treatment of cancer or infections. Non-limiting examples of inhibitors that can be combined with the compounds of the present disclosure for treatment of cancer and infections include an FGFR inhibitor (FGFR1, FGFR2, FGFR3 or FGFR4, e.g., INCB54828, INCB62079 and INCB63904), a JAK inhibitor (JAK1 and/or JAK2, e.g., ruxolitinib, baricitinib or INCB39110), an IDO inhibitor (e.g., epacadostat and NLG919), an LSD1 inhibitor (e.g., INCB59872 and INCB60003), a TDO inhibitor, a PI3K-delta inhibitor, a PI3K-gamma inhibitor such as PI3K-gamma selective inhibitor (e.g., INCB50797), a Pim inhibitor, a CSF1R inhibitor, a TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer), an angiogenesis inhibitor, an interleukin receptor inhibitor, bromo and extra terminal family members inhibitors (for example, bromodomain inhibitors or BET inhibitors such as INCB54329 and INCB57643) and an adenosine receptor antagonist or combinations thereof.

Compounds of the present disclosure can be used in combination with one or more immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, PD-1, PD-L1 and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, and VISTA. In some embodiments, the compounds provided herein can be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors and TGFR beta inhibitors.

In some embodiments, the inhibitor of an immune checkpoint molecule is anti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, or AMP-224. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the anti PD-1 antibody is SHR-1210.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG3, e.g., an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016 or LAG525.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of GITR, e.g., an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518 or MK-4166.

In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of OX40, e.g., an anti-OX40 antibody or OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383.

Compounds of the present disclosure can be used in combination with one or more agents for the treatment of diseases such as cancer. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of an alkylating agent include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

Other anti-cancer agent(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies to costimulatory molecules such as CTLA-4 (e.g., ipilimumab), 4-1BB, antibodies to PD-1 and PD-L1, or antibodies to cytokines (IL-10, TGF-β, etc.). Examples of antibodies to PD-1 and/or PD-L1 that can be combined with compounds of the present disclosure for the treatment of cancer or infections such as viral, bacteria, fungus and parasite infections include, but are not limited to, nivolumab, pembrolizumab, MPDL3280A, MEDI-4736 and SHR-1210.

The compounds of the present disclosure can further be used in combination with one or more anti-inflammatory agents, steroids, immunosuppressants or therapeutic antibodies.

The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with another immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with a vaccination protocol for the treatment of cancer. In some embodiments, the tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include the proteins from viruses implicated in human cancers such as Human Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). In some embodiments, the compounds of the present disclosure can be used in combination with tumor specific antigen such as heat shock proteins isolated from tumor tissue itself. In some embodiments, the compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be combined with dendritic cells immunization to activate potent anti-tumor responses.

The compounds of the present disclosure can be used in combination with bispecific macrocyclic peptides that target Fe alpha or Fe gamma receptor-expressing effectors cells to tumor cells. The compounds of the present disclosure can also be combined with macrocyclic peptides that activate host immune responsiveness.

The compounds of the present disclosure can be used in combination with bone marrow transplant for the treatment of a variety of tumors of hematopoietic origin.

The compounds of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or salts thereof can be used in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self antigens. Examples of pathogens for which this therapeutic approach may be particularly useful, include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to, HIV, Hepatitis (A, B, & C), Influenza, Herpes,Giardia, Malaria,Leishmania, Staphylococcus aureus, Pseudomonas Aeruginosa.

When more than one pharmaceutical agent is administered to a patient, they can be administered simultaneously, separately, sequentially, or in combination (e.g., for more than two agents).

IV. Formulation, Dosage Forms and Administration

When employed as pharmaceuticals, the compounds of the present disclosure can be administered in the form of pharmaceutical compositions. Thus the present disclosure provides a composition comprising a compound of Formula (I) or any of the formulas as described herein, a compound as recited in any of the claims and described herein, or a pharmaceutically acceptable salt thereof, or any of the embodiments thereof, and at least one pharmaceutically acceptable carrier or excipient. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is indicated and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, e.g., by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can be prepared by processes known in the art see, e.g., WO 2002/000196.

In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% silicon dioxide w/w.

In some embodiments, the composition is a sustained release composition comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and hydroxypropyl methylcellulose. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel KOOLV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).

In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to produce the composition.

The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration. For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.

Topical formulations can contain one or more conventional carriers. In some embodiments, ointments can contain water and one or more hydrophobic carriers selected from, e.g., liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g., glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, e.g., glycerol, hydroxyethyl cellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2 or at least about 5 wt % of the compound of the invention. The topical formulations can be suitably packaged in tubes of, e.g., 100 g which are optionally associated with instructions for the treatment of the select indication, e.g., psoriasis or other skin condition.

V. Labeled Compounds and Assay Methods

The compounds of the present disclosure can further be useful in investigations of biological processes in normal and abnormal tissues. Thus, another aspect of the present invention relates to labeled compounds of the invention (radio-labeled, fluorescent-labeled, etc.) that would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for localizing and quantitating PD-1 or PD-L1 protein in tissue samples, including human, and for identifying PD-L1 ligands by inhibition binding of a labeled compound.

Accordingly, the present invention includes PD-1/PD-L1 binding assays that contain such labeled compounds.

The present invention further includes isotopically-labeled compounds of the disclosure. An “isotopically” or “radio-labeled” compound is a compound of the invention where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to deuterium,3H (also written as T for tritium),11C,13C,14C,13N,15N,15O,17O,18O,18F,35S,36Cl,82Br,75Br,76Br,77Br,123I,124I,125I and131I. The radionuclide that is incorporated in the instant radio-labeled compounds will depend on the specific application of that radio-labeled compound. For example, for in vitro PD-L1 protein labeling and competition assays, compounds that incorporate3H,14C,82Br,125I,131I,35S or will generally be most useful. For radio-imaging applications11C,18F,125I,123I,124I,131I,75Br,76Br or77Br will generally be most useful.

It is to be understood that a “radio-labeled” or “labeled compound” is a compound that has incorporated at least one radionuclide. In some embodiments the radionuclide is selected from the group consisting of3H,14C,125I,35S and82Br. In some embodiments, the compound incorporates 1, 2, 3, 4, 5, 6, 7 or 8 deuterium atoms. Synthetic methods for incorporating radio-isotopes into organic compounds are known in the art.

Specifically, a labeled compound of the invention can be used in a screening assay to identify and/or evaluate compounds. For example, a newly synthesized or identified compound (i.e., test compound) which is labeled can be evaluated for its ability to bind a PD-L1 protein by monitoring its concentration variation when contacting with the PD-L1 protein, through tracking of the labeling. For example, a test compound (labeled) can be evaluated for its ability to reduce binding of another compound which is known to bind to a PD-L1 protein (i.e., standard compound). Accordingly, the ability of a test compound to compete with the standard compound for binding to the PD-L1 protein directly correlates to its binding affinity.

Conversely, in some other screening assays, the standard compound is labeled and test compounds are unlabeled. Accordingly, the concentration of the labeled standard compound is monitored in order to evaluate the competition between the standard compound and the test compound, and the relative binding affinity of the test compound is thus ascertained.

The present disclosure also includes pharmaceutical kits useful, e.g., in the treatment or prevention of diseases or disorders associated with the activity of PD-L1 including its interaction with other proteins such as PD-1 and B7-1 (CD80), such as cancer or infections, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I), or any of the embodiments thereof. Such kits can further include one or more of various conventional pharmaceutical kit components, such as, e.g., containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

EXAMPLES

Experimental procedures for compounds of the invention are provided below. Open Access Preparative LCMS Purification of some of the compounds prepared was performed on Waters mass directed fractionation systems. The basic equipment setup, protocols and control software for the operation of these systems have been described in detail in literature. See, e.g., Blom, “Two-Pump At Column Dilution Configuration for Preparative LC-MS”, K. Blom,J. Combi. Chem.,2002, 4, 295-301; Blom et al., “Optimizing Preparative LC-MS Configurations and Methods for Parallel Synthesis Purification”,J. Combi. Chem.,2003, 5, 670-83; and Blom et al., “Preparative LC-MS Purification: Improved Compound Specific Method Optimization”,J. Combi. Chem.,2004, 6, 874-883.

A mixture of 3-chloro-2-methylbiphenyl (1.44 mL, 8.08 mmol) (Aldrich, cat#361623), 4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl] (6.15 g, 24.2 mmol), palladium acetate (72.5 mg, 0.323 mmol), K3PO4(5.14 g, 24.2 mmol) and 2-(dicyclohexylphosphino)-2′,6′-dimethoxy-1,1′-biphenyl (332 mg, 0.808 mmol) in 1,4-dioxane (30 mL) was degassed and stirred at r.t. for 48 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 5% ethyl acetate in methylene chloride to give the desired product (1.60 g, 68%). LCMS calculated for C19H24BO2(M+H)+: m/z=295.2; found 295.1.

To a solution of tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[4,5-c]pyridine-5(4H)-carboxylate (9.9 mg, 31 μmol) (Astatech, cat#27671), 4,4,5,5-tetramethyl-2-(2-methylbiphenyl-3-yl)-1,3,2-dioxaborolane (10 mg, 34 μmol) and sodium carbonate (8.2 mg, 77.2 μmol) in tert-butyl alcohol (0.3 mL) and water (0.1 mL) was added dichloro[1,1′-bis(dicyclohexylphosphino)ferrocene]palladium(II) (Pd-127: 4.7 mg, 6.2 μmol). The mixture was purged with N2, then heated at 110° C. for 2 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C24H27N2O2S (M+H)+: m/z=407.2; found 407.2.

The crude product from Step 2 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H19N2S (M+H)+: m/z=307.2; found 307.2.

This compound was prepared using similar procedures as described for Example 1, Step 2 with tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (Astatech, cat#AB1021) replacing tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[4,5-c]pyridine-5 (4H)-carboxylate. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C24H27N2O2S (M+H)+: m/z=407.2; found 407.2.

The crude product from Step 1 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H19N2S (M+H)+: m/z=307.2; found 307.2.

Formaldehyde (37 wt. % in water, 16 μL, 0.2 mmol) was added to a solution of 2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro[1,3]thiazolo[5,4-c]pyridine (Example 2: 15 mg, 0.049 mmol) and N,N-diisopropylethylamine (20 μL, 0.1 mmol) in methylene chloride (1.0 mL), then the reaction mixture was allowed to stir at r.t. for 5 min before sodium triacetoxyborohydride (30 mg, 0.1 mmol) was added to the reaction mixture. The resulting mixture was stirred for another 2 h then concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H21N2S (M+H)+: m/z=321.2; found 321.2.

This compound was prepared using similar procedures as described for Example 3 with tetrahydro-4H-pyran-4-one (Aldrich, Cat#198242) replacing formaldehyde. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C24H27N2OS (M+H)+: m/z=391.2; found 391.2.

This compound was prepared using similar procedures as described for Example 3 with 4-oxocyclohexanecarboxylic acid (Aldrich, Cat#751294) replacing formaldehyde. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C26H29N2O2S (M+H)+: m/z=433.2; found 433.2.

This compound was prepared using similar procedures as described for Example 3 with 4-oxobutanoic acid (Aldrich, Cat#14075) replacing formaldehyde. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C23H25N2O2S (M+H)+: m/z=393.2; found 393.2.

This compound was prepared using similar procedures as described for Example 3 with trans-ethyl 2-formylcyclopropanecarboxylate (Aldrich, Cat#157279) replacing formaldehyde. The resulting mixture was concentrated to dryness and used in the next step without further purification. LC-MS calculated for C26H29N2O2S (M+H)+: m/z=433.2; found 433.2.

The crude product in Step 1 was treated with 1 N aq. NaOH (0.5 mL) in methanol (1.0 mL) at 50° C. and stirred for 15 h. The reaction mixture was cooled to room temperature then purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C24H25N2O2S (M+H)+: m/z=405.2; found 405.2.

This compound was prepared using similar procedures as described for Example 3 with 1H-pyrazole-4-carbaldehyde (Ark Pharm, Cat#AK-25836) replacing formaldehyde. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C23H23N4S (M+H)+: m/z=387.2; found 387.2.

This compound was prepared using similar procedures as described for Example 3 with (4-oxocyclohexyl)acetonitrile (ArkPharm, Cat#AK-46872) replacing formaldehyde. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C27H30N3S (M+H)+: m/z=428.2; found 428.2.

This compound was prepared using similar procedures as described for Example 1, Step 2 with tert-butyl 2-iodo-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxylate (Aurum Pharmatech, cat#10451833) replacing tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[4,5-c]pyridine-5(4H)-carboxylate. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C24H28N3O2(M+H)+: m/z=390.2; found 390.2.

The crude product from Step 1 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N3(M+H)+: m/z=290.2; found 290.2.

To a solution of 2-bromo-6-iodobenzonitrile (207 mg, 0.674 mmol) (Astatech, cat#CL8155), 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid (127 mg, 0.707 mmol) (Aldrich, cat#635995) and sodium carbonate (178 mg, 1.68 mmol) in tert-butyl alcohol (3 mL) and water (1 mL) was added Pd-127 (51 mg, 67 μmol). The reaction mixture was purged with N2, and then heated at 90° C. for 2 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 10 to 20% ethyl acetate in hexanes to give the desired product (130 mg, 61%). LCMS calculated for C15H11BrNO2(M+H)+: m/z=316.2; found 316.2.

A mixture of 4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl] (106 mg, 0.418 mmol), 2-bromo-6-(2,3-dihydro-1,4-benzodioxin-6-yl)benzonitrile (120 mg, 0.380 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane (1:1) (20 mg, 20 μmol) and potassium acetate (112 mg, 1.14 mmol) in 1,4-dioxane (3 mL) was purges with nitrogen and heated at 90° C. for 16 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 50% ethyl acetate in hexanes to give the desired product (70 mg, 51%). LCMS calculated for C21H23BNO4(M+H)+: m/z=364.2; found 364.2.

This compound was prepared using similar procedures as described for Example 2, Step 1 with 2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (Step 2) replacing 4,4,5,5-tetramethyl-2-(2-methylbiphenyl-3-yl)-1,3,2-dioxaborolane. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C26H26N3O4S (M+H)+: m/z=476.2; found 476.2.

The crude product from Step 3 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H18N3O2S (M+H)+: m/z=376.2; found 376.2.

To a solution of 2-chloro-4-iodo-3-methylpyridine (303 mg, 1.20 mmol) (Aldrich, cat#724092), phenylboronic acid (160 mg, 1.32 mmol) (Aldrich, cat#78181) and sodium carbonate (317 mg, 2.99 mmol) in tert-butyl alcohol (10 mL) and water (6 mL) was added Pd-127 (181 mg, 0.239 mmol). The resulting mixture was purged with N2, and then heated at 80° C. for 2 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 10 to 20% ethyl acetate in hexanes to give the desired product (225 mg, 92%). LCMS calculated for C12H11C1N (M+H)+: m/z=204.2; found 204.2.

A solution of 2-chloro-3-methyl-4-phenylpyridine (40.0 mg, 0.196 mmol) in 1,4-dioxane (2.0 mL) was bubbled with N2, then hexabutyldistannane (129 μL, 0.255 mmol), lithium chloride (51.6 mg, 1.22 mmol), dichloro[bis(triphenylphosphoranyl)]palladium (14 mg, 20 μmol) and tetrakis(triphenylphosphine)palladium(0) (23 mg, 20 μmol) were added in sequence. The resulting mixture was heated at 90° C. for 90 min before a solution of tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (94.0 mg, 0.294 mmol) in 1,4-dioxane (1.5 mL) was pumped in over 1.5 h at 95° C. The resulted mixture was stirred at the same temperature for another 12 h, then cooled to room temperature, diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C23H26N3O2S (M+H)+: m/z=408.2; found 408.2.

This compound was prepared using similar procedures as described for Example 14, Step 1-3 with 3-methoxyphenylboronic acid (Aldrich, cat#441686) replacing phenylboronic acid in Step 1. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N30S (M+H)+: m/z=338.2; found 338.2.

This compound was prepared using similar procedures as described for Example 14, Step 1-3 with 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid (Combi-blocks, cat#BB-8311) replacing phenylboronic acid in Step 1. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H20N3O2S (M+H)+: m/z=366.2; found 366.2.

This compound was prepared using similar procedures as described for Example 13, Step 1 with 1-bromo-3-iodo-2-methylbenzene (Oakwood, cat#037475) replacing 2-bromo-6-iodobenzonitrile, and phenylboronic acid replacing 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 5% ethyl acetate in hexanes to give the desired product.

The crude product from Step 2 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N3(M+H)+: m/z=290.2; found 290.2.

This compound was prepared using similar procedures as described for Example 17, Step 2 with tert-butyl 1,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine-6-carboxylate (Ark Pharm, cat#AK-39955) replacing tert-butyl 1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridine-5-carboxylate. The resulting mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C24H28N3O2(M+H)+: m/z=390.2; found 390.2.

The crude product from Step 1 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N3(M+H)+: m/z=290.2; found 290.2.

To a solution of tert-butyl 3,3-dimethyl-4-oxopiperidine-1-carboxylate (107 mg, 469 μmol) (Combi-blocks, cat#QA-1430) in chloroform (2.0 mL) was added bromine (24.2 μL, 469 μmol) in chloroform (0.5 mL) at 0° C. After stirred at the same temperature for 15 min, it was allowed to warm up to r.t. and stirred for another 30 min. The resulted mixture was concentrated to dryness and used in the next step without further purification. LC-MS calculated for C12H21BrNO3(M+H)+: m/z=306.2; found 306.2.

To a solution of above crude product in ethanol (0.5 mL) was added thiourea (53.5 mg, 703 μmol). The resulted mixture was heated at 80° C. for 3 h then concentrated to dryness and used in the next step without further purification. LC-MS calculated for C13H22N3O2S (M+H)+: m/z=284.2; found 284.2.

To a solution of the crude product from Step 2 in acetonitrile (1.0 mL) was added tert-butyl nitrite (94.8 μL, 797 μmol) and copper(II) bromide (157 mg, 703 μmol). After the reaction mixture was stirred for 3 h, it was diluted with methylene chloride and washed over water. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C13H20BrN2O2S (M+H)+: m/z=347.2; found 347.2.

This compound was prepared using similar procedures as described for Example 1, Step 2 with tert-butyl 2-bromo-7,7-dimethyl-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (Step 3) replacing tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[4,5-c]pyridine-5(4H)-carboxylate. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C26H31N2O2S (M+H)+: m/z=435.2; found 435.2.

The crude product from Step 4 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H23N2S (M+H)+: m/z=335.2; found 335.2.

To a solution of 1-(3-bromo-2-methylphenyl)ethanone (500 mg, 2.35 mmol) (Astatech, cat#CL9266), phenylboronic acid (300 mg, 2.46 mmol) and sodium carbonate (622 mg, 5.87 mmol) in tert-butyl alcohol (10 mL) and water (4 mL) was added Pd-127 (178 mg, 235 μmol). The resulted mixture was heated at 105° C. for 2 h, and then was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 50% ethyl acetate in hexanes to give the desired product (400 mg, 80%). LC-MS calculated for C15H15O (M+H)+: m/z=211.2; found 211.2.

To a solution of 1-(2-methylbiphenyl-3-yl)ethanone (1.25 g, 5.94 mmol) in ethyl acetate (30 mL) was added copper(II) bromide (5.3 g, 24 mmol) then stirred at 80° C. for 2 hours, then it was filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 50% ethyl acetate in hexanes to give the desired product (1.50 g, 87%). LC-MS calculated for C15H14BrO (M+H)+: m/z=289.2; found 289.2.

A solution of 2-bromo-1-(2-methylbiphenyl-3-yl)ethanone (20 mg, 69 μmol), aminopyrazine (9.87 mg, 104 μmol) in acetonitrile (0.4 mL) was heated at 100° C. for 2 h, then it was concentrated to dryness under reduced pressure. The residue was used in the next step without further purification. LC-MS calculated for C19H16N3(M+H)+: m/z=286.2; found 286.2.

To the solution of the crude product from Step 3 in methanol (2.0 mL) was added Pd/C (10 mg) and the resulting mixture was stirred at r.t. for 4 h under an atmosphere of H2. The resulting mixture was filtered and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N3(M+H)+: m/z=290.2; found 290.2.

To a solution of (2-methylbiphenyl-3-yl)methanol (TCI, Cat#:H0777: 4.12 g, 20.8 mmol) in methylene chloride (60 mL) was slowly added Dess-Martin periodinane (9.25 g, 21.8 mmol). The resulting mixture was stirred at r.t. for 30 min, and then washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 30% ethyl acetate in hexanes to give the desired product (3.30 g, 80%). LC-MS calculated for C14H13O (M+H)+: m/z=197.2; found 197.2.

To a solution of pyridine-3,4-diamine (15 mg, 0.14 mmol) and 2-methylbiphenyl-3-carbaldehyde (30 mg, 0.15 mmol) in methanol (0.69 mL) was added catalytic amount of zinc triflate (5 mg), then heated at 70° C. for 36 h. The resulting mixture was filtered and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H16N3(M+H)+: m/z=286.2; found 286.2.

To a solution of 2-(2-methylbiphenyl-3-yl)-1H-imidazo[4,5-c]pyridine (10 mg, TFA salt) in DMF (0.3 mL) was added benzylbromide (10 μL) and DIPEA (10 μL). The resulting mixture was heated at 100° C. for 2 h, then concentrated to dryness. The crude mixture was dissolved in methanol (2.0 mL) and NaBH4(10 mg) was added at r.t. The resulting mixture was stirred for 30 min and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C26H26N3(M+H)+: m/z=380.2; found 380.2.

To a solution of 5-benzyl-2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine (5 mg, TFA salt) in methanol (2.0 mL) was added Pd/C (10 mg) and stirred at r.t. for 4 h under under an atmosphere of H2. The resulting mixture was filtered and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N3(M+H)+: m/z=290.2; found 290.2.

To a mixture of 2-methylbiphenyl-3-carboxylic acid (100 mg, 471 μmol) (Combi-Blocks, cat#YA-8643) and triethylamine (65.7 μL, 471 μmol) in methylene chloride (2.0 mL) was added cyanuric chloride (28.9 mg, 157 μmol). The resulting mixture was heated at 60° C. for 20 min then 4-aminopyridin-3-ol (51.9 mg, 471 μmol) was added. The resulting mixture was heated at the same temperature for 18 h then cooled to room temperature and concentrated. The residue was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H15N20(M+H)+: m/z=287.2; found 287.2.

This compound was prepared using similar procedures as described for Example 21, Step 3 with 2-(2-methylbiphenyl-3-yl)[1,3]oxazolo[5,4-c]pyridine (Step 1) replacing 2-(2-methylbiphenyl-3-yl)-1H-imidazo[4,5-c]pyridine. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C26H25N20(M+H)+: m/z=381.2; found 381.3.

This compound was prepared using similar procedures as described for Example 21, Step 4 with 5-benzyl-2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro[1,3]oxazolo[5,4-c]pyridine (Step 2) replacing 5-benzyl-2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H19N20(M+H)+: m/z=291.2; found 291.2.

This compound was prepared using similar procedures as described for Example 22 with 3-aminopyridin-4-ol replacing 4-aminopyridin-3-ol in Step 1. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H19N20(M+H)+: m/z=291.2; found 291.2.

This compound was prepared using similar procedures as described for Example 3 with 2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro[1,3]oxazolo[4,5-c]pyridine (Example 23) replacing 2-(2-methylbiphenyl-3-yl)-4,5,6,7-tetrahydro[1,3]thiazolo[5,4-c]pyridine. The resulting mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H21N2O (M+H)+: m/z=305.2; found 305.2.

To a solution of aminopyrazine (200 mg, 2.10 mmol) in 1,4-dioxane (10 mL) was added ethoxycarbonyl isothiocyanate (273 μL, 2.42 mmol). The reaction mixture was stirred at r.t. for 15 h. The resulted mixture was concentrated to dryness and used in the next step without further purification. LC-MS calculated for C8H11N4O2S (M+H)+: m/z=227.2; found 227.2.

To a solution of the crude product from Step 1 in methanol (7.0 mL) and ethanol (7.0 mL) was added hydroxyaminehydrochoride (438 mg, 6.31 mmol) and N,N-diisopropylethylamine (733 μL, 4.20 mmol). The resulting mixture was heated at 75° C. for 7 h. After cooled to room temperature, the precipitated product (yellow solid) was filtered and washed with small amount of methanol. LC-MS calculated for C8H6N5(M+H)+: m/z=136.2; found 136.2.

This compound was prepared using similar procedures as described for Example 19, Step 3 with [1,2,4]triazolo[1,5-a]pyrazin-2-amine (Step 2) replacing tert-butyl 2-amino-7,7-dimethyl-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate. After stirred for 3 h, the reaction mixture was diluted with methylene chloride and washed over water. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C5H4BrN4(M+H)+: m/z=199.2; found 199.2.

This compound was prepared using similar procedures as described for Example 1, Step 2 with 2-bromo[1,2,4]triazolo[1,5-a]pyrazine (Step 3) replacing tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[4,5-c]pyridine-5 (4H)-carboxylate. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C18H15N4(M+H)+: m/z=287.2; found 287.2.

The crude product from Step 4 was dissolved in methanol (1.0 mL) then treated with LiBH4(10 mg) at 50° C. for 30 min. The resulting mixture was quenched with TFA before concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C18H19N4(M+H)+: m/z=291.2; found 291.2.

A mixture of 2-chloro-4-iodo-3-methylpyridine (250 mg, 986 μmol) (AstaTech, cat#22441) and boric acid, trimethyl ester (224 μL, 1.97 mmol) in tetrahydrofuran (5.0 mL) was added 2.5 M n-butyllithium in hexanes (789 μL, 1.97 mmol) dropwise at −78° C. The reaction mixture was allowed to warm up to r.t. after 90 min and stirred for another 30 min. The resulting mixture was concentrated and acetonitrile (5 mL) was added. The resulting suspension was filtered through celite then concentrated to give the desired product. LCMS calculated for C6H8BClNO2(M+H)+: m/z=172.2; found 172.2.

To a solution of (2-chloro-3-methylpyridin-4-yl)boronic acid (Example 26, Step 1: 170 mg, 1.0 mmol), tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (AstaTech, cat#AB1021: 320 mg, 1.0 mmol) and sodium carbonate (314 mg, 2.96 mmol) in tert-butyl alcohol (10 mL) and water (5 mL) was added Pd-127 (75 mg, 0.10 mmol). The resulting mixture was purged with N2, then heated at 105° C. for 2 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C17H21C1N3O2S (M+H)+: m/z=366.1; found 366.2.

To a solution of 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid (Combi-blocks, Cat#BB-8311: 36 mg, 0.20 mmol), tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (Example 26, Step 2: 32 mg, 0.10 mmol) and sodium carbonate (31 mg, 0.30 mmol) in tert-butyl alcohol (1.0 mL) and water (0.6 mL) was added Pd-127 (15 mg, 0.020 mmol). The resulting mixture was purged with N2, then heated at 105° C. for 1.5 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C25H28N3O4S (M+H)+: m/z=466.1; found 466.2.

The crude product from Step 3 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min then concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H20N3O2S (M+H)+: m/z=366.2; found 366.2.

To a solution of (3-chloro-2-methylphenyl)boronic acid (Combi-blocks, cat#BB-2035: 64 mg, 0.38 mmol), tert-butyl 2-bromo-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (AstaTech, cat#AB1021: 100 mg, 0.31 mmol) and sodium carbonate (100 mg, 0.94 mmol) in tert-butyl alcohol (3.2 mL) and water (2 mL) was added Pd-127 (47 mg, 0.063 mmol). The resulting mixture was purged with N2, then heated at 105° C. for 2 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 40% ethyl acetate in hexanes to give the desired product (114 mg, 83%). LC-MS calculated for C18H22C1N2O2S (M+H)+: m/z=365.1; found 365.2.

A mixture of tert-butyl 2-(3-chloro-2-methylphenyl)-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (Example 26, Step 1: 95 mg, 0.26 mmol), 4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl] (200 mg, 0.78 mmol), palladium acetate (2.5 mg, 0.014 mmol), K3PO4(170 mg, 0.78 mmol) and 2-(dicyclohexylphosphino)-2′,6′-dimethoxy-1,1′-biphenyl (11 mg, 0.026 mmol) in 1,4-dioxane (1 mL) was degassed and stirred at r.t. for 3 d. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography on a silica gel column eluting with 0 to 5% ethyl acetate in methylene chloride to give the desired product (108 mg, 90%). LC-MS calculated for C24H34BN2O4S (M+H)+: m/z=457.2; found 457.2.

To a solution of tert-butyl 2-[2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-6,7-dihydro[1,3]thiazolo[5,4-c]pyridine-5(4H)-carboxylate (Example 26, Step 2: 15 mg, 0.033 mmol), thiophene, 3-bromo- (6.2 μL, 0.066 mmol) and sodium carbonate (8.7 mg, 0.082 mmol) in tert-butyl alcohol (0.3 mL) and water (0.2 mL) was added Pd-127 (5.0 mg, 0.0066 mmol). The resulting mixture was purged with N2, then heated at 105° C. for 1.5 h. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C22H25N2O2S2(M+H)+: m/z=413.1; found 413.2.

The crude product from Step 3 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min then concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C17H17N2S2(M+H)+: m/z=313.1; found 313.2.

This compound was prepared using similar procedures as described for Example 27, Step 1-4 with 1-bromo-3-methoxybenzene replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H21N2OS (M+H)+: m/z=337.2; found 337.2.

This compound was prepared using similar procedures as described for Example 27, Step 1-4 with 4-bromo-3,6-dihydro-2H-pyran (Combi-blocks, cat#OT-0686) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C18H21N2OS (M+H)+: m/z=313.2; found 313.2.

This compound was prepared using similar procedures as described for Example 27, Step 1-4 with 4-bromo-2-methoxypyridine (Ark Pharm, cat#AK-47404) replacing 3-bromothiophene in Step 3. The reaction mixture purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H20N30S (M+H)+: m/z=338.2; found 338.2.

This compound was prepared using similar procedures as described for Example 27, Step 1-4 with 2-bromo-5-fluoropyridine (Aldrich, cat#595675) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C18H17FN3S (M+H)+: m/z=326.2; found 326.2.

This compound was prepared using similar procedures as described for Example 27 with 1-bromocyclohexene (Combi-blocks, cat#OT-0350) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C19H23N2S (M+H)+: m/z=311.2; found 311.2.

This compound was prepared using similar procedures as described for Example 27 with 1-bromo-3-ethoxybenzene (Aldrich, cat#453250) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H23N2OS (M+H)+: m/z=351.2; found 351.2.

This compound was prepared using similar procedures as described for Example 27 with 3,5-dimethoxybromobenzene (Aldrich, cat#569313) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H23N2O2S (M+H)+: m/z=367.2; found 367.2.

This compound was prepared using similar procedures as described for Example 16 with 2-chloro-4-iodonicotinonitrile (Aurum Pharmatech, cat#A-6061) replacing 2-chloro-4-iodo-3-methylpyridine. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H17N4O2S (M+H)+: m/z=377.2; found 377.2.

To a solution of 3-bromophenol (100 mg, 0.58 mmol) and 2-bromoethanol (36 mg, 0.29 mmol) in methanol (1 mL) was added potassium carbonate (80 mg, 0.58 mmol). The reaction mixture was heated at 55° C. for 4 h, and then diluted with methylene chloride, washed with water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification.

This compound was prepared using similar procedures as described for Example 27, Steps 1-4 with 2-(3-bromophenoxy)ethanol (Step 1) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H23N2O2S (M+H)+: m/z=367.2; found 367.2.

To a stirred slurry of 2,6-difluoro-3,5-dimethoxyaniline (500 mg, 2.64 mmol) in 6.0 M hydrogen chloride in water (4 mL, 24 mmol) was added a solution of sodium nitrite (191 mg, 2.78 mmol) in water (1 mL) dropwise over 15 min at 0° C. After stirring the resulting mixture at 0° C. for another 15 min, a solution of potassium iodide (1.8 g, 10. mmol) in water (2 mL) was slowly added to the resulting orange-red slurry at 0° C. with vigorus stirring. After completion of the addition, the reaction mixture was allowed to warm up to r.t. for 1 hour. The solid was collected by filtration, washed with water and dried under vacuum. 570 mg solid was collected and used directly in the next step.

This compound was prepared using similar procedures as described for Example 27, Step 1-4 with 2,4-difluoro-3-iodo-1,5-dimethoxybenzene (Step 1) replacing 3-bromothiophene in Step 3. The reaction mixture was purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C21H21F2N2O2S (M+H)+: m/z=403.2; found 403.2.

This compound was prepared using similar procedures as described for Example 27, Step 3 with 3-bromobenzoic acid nitrile (Aldrich, cat#B58202) replacing 3-bromothiophene. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C25H28N3O3S (M+H)+: m/z=450.2; found 450.2.

The crude product from Step 1 was dissolved in methylene chloride (0.6 mL) then treated with TFA (0.3 mL). The resulting mixture was stirred at room temperature for 30 min then concentrated and purified by prep-HPLC (pH=2, acetonitrile/water+TFA) to give the desired product as the TFA salt. LC-MS calculated for C20H20N3OS (M+H)+: m/z=350.2; found 350.2.

This compound was prepared using similar procedures as described for Example 27, Step 3 with (3-bromophenyl)acetonitrile (Aldrich, cat#260088) replacing 3-bromothiophene. The reaction mixture was diluted with methylene chloride, washed with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was used in the next step without further purification. LC-MS calculated for C26H30N3O3S (M+H)+: m/z=464.2; found 464.2.

To a solution of 4-bromo-1H-indazole (Aldrich, cat#776610: 100. mg, 0.508 mmol) in acetone (2.5 mL) was added potassium hydroxide (85.4 mg, 1.52 mmol). The resulting mixture was stirred at room temperature for 10 min then methyl iodide (63.2 μL, 1.02 mmol) was added. The mixture was stirred at room temperature overnight then concentrated to give a mixture of 4-bromo-2-methyl-2H-indazole and 4-bromo-1-methyl-1H-indazole, which was used in the next step without further purification. LC-MS calculated for CsHsBrN2(M+H)+: m/z=211.0; found 211.1.

The assays were conducted in a standard black 384-well polystyrene plate with a final volume of 20 μL. Inhibitors were first serially diluted in DMSO and then added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1%. The assays were carried out at 25° C. in the PBS buffer (pH 7.4) with 0.05% Tween-20 and 0.1% BSA. Recombinant human PD-L1 protein (19-238) with a His-tag at the C-terminus was purchased from AcroBiosystems (PD1-H5229). Recombinant human PD-1 protein (25-167) with Fc tag at the C-terminus was also purchased from AcroBiosystems (PD1-H5257). PD-L1 and PD-1 proteins were diluted in the assay buffer and 10 μL was added to the plate well. Plates were centrifuged and proteins were preincubated with inhibitors for 40 minutes. The incubation was followed by the addition of 10 μL of HTRF detection buffer supplemented with Europium cryptate-labeled anti-human IgG (PerkinElmer-AD0212) specific for Fc and anti-His antibody conjugated to SureLight®-Allophycocyanin (APC, PerkinElmer-AD0059H). After centrifugation, the plate was incubated at 25° C. for 60 min. before reading on a PHERAstar FS plate reader (665 nm/620 nm ratio). Final concentrations in the assay were—3 nM PD1, 10 nM PD-L1, 1 nM europium anti-human IgG and 20 nM anti-His-Allophycocyanin. IC50determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.

Compounds of the present disclosure, as exemplified in Examples 1-40, showed IC50values in the following ranges: +=IC50≤100 nM; ++=100 nM<IC50≤500 nM; +++=500 nM<IC50≤10000 nM

Data obtained for the Example compounds using the PD-1/PD-L1 homogenous time-resolved fluorescence (HTRF) binding assay described in Example A is provided in Table 1.