LACTONE AND LACTAM CONTAINING COMPOUNDS USEFUL AS IMMUNOMODULATORS

Abstract

The present disclosure generally relates to lactone and lactam containing compounds useful as immunomodulators. Provided herein are compounds, compositions comprising such compounds. and methods of their use. The disclosure further pertains to pharmaceutical compositions comprising at least one compound according to the disclosure that are useful for the treatment of various diseases, including cancer and infectious diseases.

Claims

1. A compound of Formula (I): ##STR00119## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is independently (O).sub.m(CH.sub.2).sub.nR.sup.1a or (CH.sub.2).sub.n(O).sub.mR.sup.1b; R.sup.1a is independently a 5- to 6-membered heterocycle with one to two heteroatoms selected from O, N, S, and NR.sup.a; wherein said heterocycle is substituted with 0 to 3 R.sup.b; R.sup.1b is phenyl or a 5- to 6-membered heteroaryl with one to four heteroatoms selected from O, N, S, and NR.sup.a; wherein said phenyl and heteroaryl are substituted with 0 to 3 R.sup.1c. R.sup.1c is independently halogen, CN, OH, SH, NH.sub.2, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkoxy, C.sub.1-C.sub.4 alkyl substituted with 0 to 1 OH, or C.sub.3-C.sub.6 cycloalkyl, (O).sub.m(CH.sub.2).sub.nR.sup.1d, or (CH.sub.2).sub.nNR.sup.7R.sup.2a; R.sup.1d is phenyl or a 5- to 6-membered heteroaryl with one to four heteroatoms selected from O, N, S, and NR.sup.a; wherein said phenyl and heteroaryl are substituted with 0 to 3 R.sup.d; Z is a bond or a C.sub.1-C.sub.2 alkylene; R.sup.2 is independently a 4- to 8-membered lactone or lactam substituted with 0 to 4 R.sup.c; R.sup.3, R.sup.4 and R.sup.5 are, at each occurrence, independently halogen, CN, OH, SH, NH.sub.2, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkoxy, or C.sub.3-C.sub.6 cycloalkyl; R.sup.6 is independently hydrogen, C.sub.1-C.sub.4 alkyl, or (CH.sub.2).sub.nR.sup.6a; R.sup.6a is independently phenyl or a 5- to 6-membered heteroaryl with one to four heteroatoms selected from O, N, S, and NR.sup.a; wherein said phenyl and heteroaryl are substituted with 0 to 3 R.sup.6b; R.sup.6b is independently halogen, CN, OH, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4 alkoxy, or C.sub.1-C.sub.4 haloalkoxy; R.sup.7 is independently hydrogen, C.sub.1-C.sub.4 alkyl, C(O)C.sub.1-C.sub.4 alkyl, (CH.sub.2).sub.nC.sub.3-C.sub.6 cycloalkyl, or (CH.sub.2).sub.n-phenyl; alternatively, R.sup.6 and R.sup.7 can be joined to form W; and W is a 1 to 4 membered linker with elements independently selected from carbon, oxygen, and nitrogen; wherein said linker is substituted with 0 to 2 R.sup.e; R.sup.a is independently halogen, C.sub.1-C.sub.4 alkyl, (CH.sub.2).sub.nC.sub.3-C.sub.6 cycloalkyl, or (CH.sub.2).sub.n-phenyl; R.sup.b and R.sup.c are, at each occurrence, independently oxo, halogen, CN, OH, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4 alkoxy; R.sup.d is, at each occurrence, independently halogen, CN, OH, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4 alkoxy; R.sup.e is independently oxo, ?CH.sub.2, halogen, CN, OH, C.sub.1-C.sub.4 alkyl, or C.sub.1-C.sub.4 alkoxy; m is, at each occurrence, independently 0 or 1; n is, at each occurrence, independently 0, 1, or 2; r, s, and t are each independently 0, 1, or 2.

2. The compound of claim 1, wherein: R.sup.2 is independently a 5- to 6-membered lactone or lactam substituted with 0 to 4 R.sup.a; R.sup.a is independently oxo, halogen, OH, or C.sub.1-C.sub.3 alkyl; W is a 2 to 3 membered linker with elements independently selected from carbon, oxygen, and nitrogen; wherein said linker is substituted with 0 to 2 R.sup.e; and R.sup.e is independently oxo, CH.sub.2, OH, or C.sub.1-C.sub.4 alkyl.

3. The compound of claim 2, wherein the compound is of Formula (II): ##STR00120## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is independently OCH.sub.2R.sup.1a or CH.sub.2OR.sup.1b; R.sup.1a is N(C.sub.1-C.sub.3 alkyl)-piperidinyl; R.sup.1b is phenyl substituted with 1 to 3 R.sup.1c; R.sup.1c is independently halogen, CH.sub.2OH, OCH.sub.2R.sup.1d, or CH.sub.2NR.sup.7R.sup.2a; R.sup.1d is cyano-substituted pyridyl; R.sup.2 and R.sup.2a are, at each occurrence, independently ##STR00121## R.sup.3 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; R.sup.4 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; R.sup.5 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; R.sup.6 is independently hydrogen, C.sub.1-C.sub.3 alkyl, or CH.sub.2-(cyano-substituted pyridyl); and R.sup.7 is independently hydrogen, C.sub.1-C.sub.3 alkyl, C(O)C.sub.1-C.sub.3 alkyl or CH.sub.2-cyclopropyl.

4. The compound of claim 3, wherein: R.sup.1 is OCH.sub.2R.sup.1a; R.sup.1a is N(C.sub.1-C.sub.3 alkyl)-piperidinyl; R.sup.6 is CH.sub.2-(cyano-substituted pyridyl); and R.sup.7 is hydrogen.

5. The compound of claim 3, wherein: R.sup.1 is CH.sub.2OR.sup.1b; R.sup.1b is phenyl substituted with 1 to 3 R.sup.1c; R.sup.1c is independently halogen, OCH.sub.2R.sup.1d, or CH.sub.2NR.sup.7R.sup.2a; R.sup.1d is cyano-substituted pyridyl; and R.sup.7 is hydrogen.

6. The compound of claim 2, wherein the compound is of Formula (III): ##STR00122## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is independently OCH.sub.2R.sup.1a or CH.sub.2OR.sup.1b; R.sup.1a is N(C.sub.1-C.sub.3 alkyl)-piperidinyl; R.sup.1b is phenyl substituted with 1 to 3 R.sup.1c; R.sup.1c is independently halogen, OCH.sub.2R.sup.1d, or CH.sub.2NR.sup.7R.sup.2a; R.sup.1d is cyano-substituted pyridyl; R.sup.2 and R.sup.2a are, at each occurrence, independently ##STR00123## R.sup.3 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; R.sup.4 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; R.sup.5 is independently hydrogen, C.sub.1-C.sub.3 alkyl or halogen; and W is independently CH.sub.2C(O), CH.sub.2C(?CH.sub.2)CH.sub.2, or CH.sub.2C(OH)CH.sub.2.

7. A compound of claim 1, wherein the compound is selected from the exemplified Examples, Compound 1001 to 1054, or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. (canceled)

10. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to claim 1.

11-12. (canceled)

Description

EXAMPLES

[0177] The invention is further defined in the following Examples. It should be understood that the Examples are given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain the essential characteristics of the invention, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the invention to various uses and conditions. As a result, the invention is not limited by the illustrative examples set forth hereinbelow, but rather is defined by the claims appended hereto.

[0178] The compounds may be made by methods known in the art including those described below and including variations within the skill of the art. Some reagents and intermediates are known in the art. Other reagents and intermediates can be made by methods known in the art using readily available materials. The variables (e.g. numbered R substituents) used to describe the synthesis of the compounds are intended only to illustrate how to make the compounds and are not to be confused with variables used in the claims or in other sections of the specification. The following methods are for illustrative purposes and are not intended to limit the scope of the invention.

[0179] Abbreviations used in the schemes generally follow conventions used in the art. Chemical abbreviations used in the specification and examples are defined as follows: THF for tetrahydrofuran; DMF for N,N-dimethylformamide; MeOH for methanol; EtOH for ethanol; n-PrOH for 1-propyl alcohol or propan-1-ol; i-PrOH for 2-propyl alcohol or propan-2-ol; Ar for aryl; TFA for trifluoroacetic acid; DMSO for dimethylsulfoxide; EtOAc for ethyl acetate; Et.sub.2O for diethyl ether; DMAP for 4-dimethylaminopyridine; DCE for 1,2-dichloroethane; ACN for acetonitrile; DME for 1,2-dimethoxyethane; h for hours; rt for room temperature or retention time (context will dictate); min for minutes; HOBt for 1-hydroxybenzotriazole hydrate; HCTU for 1-[bis(dimethylamino)methylen]-5-chlorobenzotriazolium 3-oxide hexafluorophosphate or N,N,N,N-tetramethyl-O)-(6-chloro-1H-benzotriazol-1-yl)uronium hexafluorophosphate; HATU for 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate or N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; DIEA and iPrNEt.sub.2 for diisopropylethylamine; Et.sub.3N for triethyl amine.

[0180] Abbreviations are defined as follows: 1? for once, 2? for twice, 3? for thrice, ? C. for degrees Celsius, eq for equivalent or equivalents, g for gram or grams, mg for milligram or milligrams, L for liter or liters, mL for milliliter or milliliters, ?L for microliter or microliters, N for normal, M for molar, mmol for millimole or millimoles, min for minute or minutes, h for hour or hours, rt for room temperature, RT for retention time, atm for atmosphere, psi for pounds per square inch, conc. for concentrate, sat or sat'd for saturated, MW for molecular weight, mp for melting point, ee for enantiomeric excess, MS or Mass Spec for mass spectrometry, ESI for electrospray ionization mass spectroscopy, HR for high resolution, HRMS for high resolution mass spectrometry, LC for liquid chromatography, LCMS for liquid chromatography mass spectrometry, HPLC for high pressure liquid chromatography, RP HPLC for reverse phase HPLC, TLC or tlc for thin layer chromatography, NMR for nuclear magnetic resonance spectroscopy, .sup.1H for proton, ? for delta, s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad, Hz for hertz, and ?, ?, R, S, E, and Z are stereochemical designations familiar to one skilled in the art.

##STR00006##

[0181] General Procedure for the preparation of compounds of the present invention, from Intermediates 1 to 5 and amino amino lactones or lactams:

[0182] A mixture of any one of Intermediates 1 to 5 (1 eq.) and an agent (1-20 eq.) in THF or dioxane or DME or MeOH or EtOH or their co-solvents, with or without AcOH (1-20 eq.), was stirred at room temperature for 0.5 to 48 hours, before NaCNBH.sub.3 (1-20 eq.) was added. The reaction was stirred at room temperature to 100? C. for 0.5 to 48 hours, before being quenched with methanol or water. After all the solvents were 10 removed under vacuum, the residue was purified by the preparative HPLC to give a compound of the present invention.

TABLE-US-00001 Compound 1001 [00007] Starting Material Intermediate 1 Agent Used [00008] MS MS (M + H).sup.+ Calcd. 715.2 MS (M + H).sup.+ Observ. 715.3 Retention Time 1.89 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00002 Compound 1002 [00009] Starting Material Intermediate 1 Agent Used [00010] MS MS (M + H).sup.+ Calcd. 715.2 MS (M + H).sup.+ Observ. 715.3 Retention Time 1.83 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00003 Compound 1003 [00011] Starting Material Intermediate 1 Agent Used [00012] MS MS (M + H).sup.+ Calcd. 715.2 MS (M + H).sup.+ Observ. 715.3 Retention Time 1.89 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00004 Compound 1004 [00013] Starting Material Intermediate 1 Agent Used [00014] MS MS (M + H).sup.+ Calcd. 715.2 MS (M + H).sup.+ Observ. 715.3 Retention Time 1.88 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00005 Compound 1005 [00015] Starting Material Intermediate 1 Agent Used [00016] MS MS (M + H).sup.+ Calcd. 729.3 MS (M + H).sup.+ Observ. 729.3 Retention Time 2.05 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00006 Compound 1006 [00017] Starting Material Intermediate 1 Agent Used [00018] MS MS (M + H).sup.+ Calcd. 729.3 MS (M + H).sup.+ Observ. 729.1 Retention Time 2.04 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00007 Compound 1007 [00019] Starting Material Intermediate 1 Agent Used [00020] MS MS (M + H).sup.+ Calcd. 743.3 MS (M + H).sup.+ Observ. 743.4 Retention Time 2.12 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00008 Compound 1008 [00021] Starting Material Intermediate 1 Agent Used [00022] MS MS (M + H).sup.+ Calcd. 743.3 MS (M + H).sup.+ Observ. 743.3 Retention Time 2.17 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00009 Compound 1009 [00023] Starting Material Intermediate 1 Agent Used [00024] MS MS (M + H).sup.+ Calcd. 757.3 MS (M + H).sup.+ Observ. 757.3 Retention Time 2.17 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00010 Compound 1010 [00025] Starting Material Intermediate 1 Agent Used [00026] MS MS (M + H).sup.+ Calcd. 714.3 MS (M + H).sup.+ Observ. 714.2 Retention Time 1.87 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00011 Compound 1011 [00027] Starting Material Intermediate 1 Agent Used [00028] MS MS (M + H).sup.+ Calcd. 714.3 MS (M + H).sup.+ Observ. 714.3 Retention Time 1.87 min LC Condition Solvent A 5:95 acetonitrile:water with 10 mM ammonium acetate Solvent B 95:5 acetonitrile:water with 10 mM ammonium acetate Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00012 Compound 1012 [00029] Starting Material Intermediate 1 Agent Used [00030] MS MS (M + H).sup.+ Calcd. 728.3 MS (M + H).sup.+ Observ. 728.3 Retention Time 1.93 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00013 Compound 1013 [00031] Starting Material Intermediate 1 Agent Used [00032] MS MS (M + H).sup.+ Calcd. 728.3 MS (M + H).sup.+ Observ. 728.3 Retention Time 1.96 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00014 Compound 1014 [00033] Starting Material Intermediate 1 Agent Used [00034] MS MS (M + H).sup.+ Calcd. 728.3 MS (M + H).sup.+ Observ. 728.2 Retention Time 1.81 min LC Condition Solvent A 5:95 acetonitrile:water with 10 mM ammonium acetate Solvent B 95:5 acetonitrile:water with 10 mM ammonium acetate Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00015 Compound 1015 [00035] Starting Material Intermediate 1 Agent Used [00036] MS MS (M + H).sup.+ Calcd. 742.2 MS (M + H).sup.+ Observ. 742.3 Retention Time 1.84 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 30x2, 3u

TABLE-US-00016 Compound 1016 [00037] Starting Material Intermediate 2 Agent Used [00038] MS MS (M + H).sup.+ Calcd. 981.3 MS (M + H).sup.+ Observ. 981.5 Retention Time 3.06 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 40 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00017 Compound 1017 [00039] Starting Material Intermediate 2 Agent Used [00040] MS MS (M + H).sup.+ Calcd. 884.3 MS (M + H).sup.+ Observ. 884.1 Retention Time 2.66 min LC Condition Solvent A 5:95 acetonitrile:water with 10 mM ammonium acetate Solvent B 95:5 acetonitrile:water with 10 mM ammonium acetate Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00018 Compound 1018 [00041] Starting Material Intermediate 2 Agent Used [00042] MS MS (M + H).sup.+ Calcd. 953.3 MS (M + H).sup.+ Observ. 953.1 Retention Time 2.02 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00019 Compound 1019 [00043] Starting Material Intermediate 2 Agent Used [00044] MS MS (M + H).sup.+ Calcd. 953.3 MS (M + H).sup.+ Observ. 953.1 Retention Time 1.99 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00020 Compound 1020 [00045] Starting Material Intermediate 2 Agent Used [00046] MS MS (M + H).sup.+ Calcd. 953.3 MS (M + H).sup.+ Observ. 953.4 Retention Time 3.37 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 20 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00021 Compound 1021 [00047] Starting Material Intermediate 2 Agent Used [00048] MS MS (M + H).sup.+ Calcd. 1009.3 MS (M + H).sup.+ Observ. 1009.5 Retention Time 3.68 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 20 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00022 Compound 1022 [00049] Starting Material Intermediate 2 Agent Used [00050] MS MS (M + H).sup.+ Calcd. 1009.3 MS (M + H).sup.+ Observ. 1009.5 Retention Time 3.25 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 20 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00023 Compound 1023 [00051] Starting Material Intermediate 2 Agent Used [00052] MS MS (M + H).sup.+ Calcd. 1009.3 MS (M + H).sup.+ Observ. 1009.2 Retention Time 2.71 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00024 Compound 1024 [00053] Starting Material Intermediate 2 Agent Used [00054] MS MS (M + H).sup.+ Calcd. 981.3 MS (M + H).sup.+ Observ. 981.2 Retention Time 3.71 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 20 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00025 Compound 1025 [00055] Starting Material Intermediate 2 Agent Used [00056] MS MS (M + H).sup.+ Calcd. 1937.4 MS (M + H).sup.+ Observ. 1937.2 Retention Time 4.11 min LC Condition Solvent A 5% ACN:95% Water:10 mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium Actetate Start % B 20 Final % B 100 Gradient Time 4 min Flow Rate 0.8 mL/min Wavelength 220 Temperature 40? C. Column Phenomenex LUNA C18, 50x2, 3u

TABLE-US-00026 Compound 1026 [00057] Starting Material Intermediate 3 Agent Used [00058] MS MS (M + H).sup.+ Calcd. 993.2 MS (M + H).sup.+ Observ. 993.1 Retention Time 1.67 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm x 50 mm, 1.7 ?m particles

TABLE-US-00027 Compound 1027 [00059] Starting Material Intermediate 1 Agent Used [00060] MS MS (M + H).sup.+ Calcd. 714.3 MS (M + H).sup.+ Observ. 714.2 Retention Time 1.37 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00028 Compound 1028 [00061] Starting Material Intermediate 4 Agent Used [00062] MS MS (M + H).sup.+ Calcd. 599.2 MS (M + H).sup.+ Observ. 599.2 Retention Time 1.34 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00029 Compound 1029 [00063] Starting Material Intermediate 4 Agent Used [00064] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.2 Retention Time 1.40 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00030 Compound 1030 [00065] Starting Material Intermediate 4 Agent Used [00066] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.2 Retention Time 1.40 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00031 Compound 1031 [00067] Starting Material Intermediate 4 Agent Used [00068] MS MS (M + H).sup.+ Calcd. 599.2 MS (M + H).sup.+ Observ. 599.2 Retention Time 1.41 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00032 Compound 1032 [00069] Starting Material Intermediate 5 Agent Used [00070] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.1 Retention Time 1.41 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00033 Compound 1033 [00071] Starting Material Intermediate 5 Agent Used [00072] MS MS (M + H).sup.+ Calcd. 627.2 MS (M + H).sup.+ Observ. 627.2 Retention Time 1.45 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00034 Compound 1034 [00073] Starting Material Intermediate 5 Agent Used [00074] MS MS (M + H).sup.+ Calcd. 641.2 MS (M + H).sup.+ Observ. 641.2 Retention Time 1.49 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00035 Compound 1035 [00075] Starting Material Intermediate 5 Agent Used [00076] MS MS (M + H).sup.+ Calcd. 626.2 MS (M + H).sup.+ Observ. 626.2 Retention Time 1.39 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00036 Compound 1036 [00077] Starting Material Intermediate 5 Agent Used [00078] MS MS (M + H).sup.+ Calcd. 627.2 MS (M + H).sup.+ Observ. 627.2 Retention Time 1.42 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 um particles

TABLE-US-00037 Compound 1037 [00079] Starting Material Intermediate 5 Agent Used [00080] MS MS (M + H).sup.+ Calcd. 641.2 MS (M + H).sup.+ Observ. 641.2 Retention Time 1.49 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00038 Compound 1038 [00081] Starting Material Intermediate 5 Agent Used [00082] MS MS (M + H).sup.+ Calcd. 612.2 MS (M + H).sup.+ Observ. 612.2 Retention Time 1.37 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00039 Compound 1039 [00083] Starting Material Intermediate 5 Agent Used [00084] MS MS (M + H).sup.+ Calcd. 655.3 MS (M + H).sup.+ Observ. 655.2 Retention Time 1.51 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00040 Compound 1040 [00085] Starting Material Intermediate 5 Agent Used [00086] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.1 Retention Time 1.45 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00041 Compound 1041 [00087] Starting Material Intermediate 5 Agent Used [00088] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.2 Retention Time 1.41 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00042 Compound 1042 [00089] Starting Material Intermediate 5 Agent Used [00090] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.2 Retention Time 1.40 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min/ Wavelength 220 Termperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00043 Compound 1043 [00091] Starting Material Intermediate 5 Agent Used [00092] MS MS (M + H).sup.+ Calcd. 613.2 MS (M + H).sup.+ Observ. 613.1 Retention Time 1.40 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00044 Compound 1044 [00093] Starting Material Intermediate 5 Agent Used [00094] MS MS (M + H).sup.+ Calcd. 612.2 MS (M + H).sup.+ Observ. 612.2 Retention Time 1.39 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00045 Compound 1045 [00095] Starting Material Intermediate 5 Agent Used [00096] MS MS (M + H).sup.+ Calcd. 626.2 MS (M + H).sup.+ Observ. 626.2 Retention Time 1.45 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00046 Compound 1046 [00097] Starting Material Intermediate 5 Agent Used [00098] MS MS (M + H).sup.+ Calcd. 640.2 MS (M + H).sup.+ Observ. 640.2 Retention Time 1.39 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00047 Intermediate 6 [00099] Starting Material Intermediate 4 Agent Used [00100] MS MS (M + H).sup.+ Calcd. 599.2 MS (M + H).sup.+ Observ. 599.2 Retention Time 1.09 min LC Condition Solvent A 10% Acetonitrile/90% Water/0.1% TFA Solvent B 10% Water/90% Acetonitrile/0.1% TFA Start % B 0 Final % B 100 Gradient Time 1.5 min Flow Rate 1 mL/min Wavelength 220 Temperature 40? C. Column Acquity BEH 21. ? 50 mm 1.7 um

TABLE-US-00048 Intermediate 7 [00101] Starting Material Intermediate 2 Agent Used [00102] MS MS (M + H).sup.+ Calcd. 953.3 MS (M + H).sup.+ Observ. 953.3 Retention Time 2.54 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Acquity BEH C18 2.1 ? 50 mm; 1.7 um
General Procedure for the preparation of compounds of the present invention from Intermediate 6: A mixture of Intermediate 6 (1 eq.), an agent (1 eq.), iPr.sub.2NEt (1-10 eq.) and Cs.sub.2CO.sub.3 or K.sub.2CO.sub.3 (1-20 eq.) in THF or dioxane or DME or their co-solvents was stirred at room temperature to 100? C. for 0.5 to 48 hours, before being quenched with methanol or water. After all the solvents were removed under vacuum, the residue was purified by the preparative HPLC to give a compound of the present invention.
General Procedure for the preparation of compounds of the present invention from Intermediate 7: A mixture of Intermediate 7 (1 eq.) and an agent (1-20 eq.) in THF or dioxane or DME or their co-solvents, with or without iPr.sub.2NEt or Et.sub.3N (1-20 eq.) was stirred at room temperature to 100? C. for 0.5 to 48 hours, before being quenched with methanol or water. After all the solvents were removed under vacuum, the residue was purified by the preparative HPLC to give a compound of the present invention.

TABLE-US-00049 Compound 1047 [00103] Starting Material Intermediate 6 Agent Used [00104] MS MS (M + H).sup.+ Calcd. 639.2 MS (M + H).sup.+ Observ. 639.2 Retention Time 1.75 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00050 Compound 1048 [00105] Starting Material Intermediate 6 Agent Used [00106] MS MS (M + H).sup.+ Calcd. 651.2 MS (M + H).sup.+ Observ. 651.1 Retention Time 1.51 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00051 Compound 1049 [00107] Starting Material Intermediate 6 Agent Used [00108] MS MS (M + H).sup.+ Calcd. 655.2 MS (M + H).sup.+ Observ. 655.1 Retention Time 1.45 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles

TABLE-US-00052 Compound 1050 [00109] Starting Material Intermediate 6 Agent Used [00110] MS MS (M ? H).sup.+ Calcd. 1035.3 MS (M +?? H).sup.+ Observ. 1035.1 Retention Time 2.10 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Waters XBridge C18, 2.1 mm ? 50 mm, 1.7 ?m particles
General Procedure for the preparation of compounds of the present invention, from Reference Compounds 1 to 5 and amino amino lactones or lactams, in the presence of aldehyde or ketone: A mixture of any one of Reference Compounds 1 to 5 (1 eq.), an agent (1-20 eq.) in DCM or THF or dioxane or DME or MeOH or EtOH or their co-solvents, with or without iP2NEt, was stirred at room temperature for 0.5 to 48 hours, before AcOH (1-20 eq.) was added. The mixture was stirred at room temperature for 0.5 to 48 hours, then NaCNBH.sub.3 (1-20 eq.) was added. The reaction was stirred at room 10 temperature to 100? C. for 0.5 to 48 hours, before an aldehyde or ketone (1-20 eq.) was added. After be stirred at room temperature to 100? C. for 0.5 to 48 hours, the reaction was quenched with methanol or water. After all the solvents were removed under vacuum, the residue was purified by the preparative HPLC to give a compound of the present invention.

TABLE-US-00053 Compound 1051 [00111] Starting Material Intermediate 2 Agent Used [00112] Aldehyde Used formaldehyde MS MS (M + H).sup.+ Calcd. 1009.3 MS (M + H).sup.+ Observ. 1009.3 Retention Time 2.72 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Acquity BEH C18 2.1 ? 50 mm; 1.7 um

TABLE-US-00054 Compound 1052 [00113] Starting Material Intermediate 2 Agent Used [00114] Aldehyde Used cyclopropanecarbaldehyde MS MS (M + H).sup.+ Calcd. 1089.4 MS (M + H).sup.+ Observ. 1089.3 Retention Time 2.94 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Acquity BEH C18 2.1 ? 50 mm; 1.7 um

TABLE-US-00055 Compound 1053 [00115] Starting Material Intermediate 2 Agent Used [00116] Aldehyde Used formaldehyde MS MS (M + H).sup.+ Calcd. 1009.3 MS (M + H).sup.+ Observ. 1009.3 Retention Time 2.56 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Acquity BEH C18 2.1 ? 50 mm; 1.7 um

TABLE-US-00056 Compound 1054 [00117] Starting Material Intermediate 2 Agent Used [00118] Aldehyde Used acetaldehyde MS MS (M + H).sup.+ Calcd. 1037.4 MS (M + H).sup.+ Observ. 1037.3 Retention Time 2.68 min LC Condition Solvent A 5:95 acetonitrile:water with 0.1% trifluoroacetic acid Solvent B 95:5 acetonitrile:water with 0.1% trifluoroacetic acid Start % B 0 Final % B 100 Gradient Time 3 min Flow Rate 1 mL/min Wavelength 220 Temperature 50? C. Column Acquity BEH C18 2.1 ? 50 mm; 1.7 um

Biological Assays

[0183] The ability of the compounds of the invention to bind to PD-L1 was investigated using a PD-1/PD-L1 Homogenous Time-Resolved Fluorescence (HTRF) binding assay.

[0184] The interaction of PD-1 and PD-L1 can be assessed using soluble, purified preparations of the extracellular domains of the two proteins. The PD-1 and PD-L1 protein extracellular domains were expressed as fusion proteins with detection tags, for PD-1, the tag was the Fc portion of Immunoglobulin (PD-1-Ig) and for PD-L1 it was the 6 histidine motif (PD-L1-His). All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (with) bovine serum albumin and 0.05% (v/v) Tween-20. For the h/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 ?l of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 ?l of assay buffer and further incubation for 15 m. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 ?l was dispensed on top of the binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and the resulting signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between the human proteins PD-1-Ig/PD-L2-His (20 & 5 nM, respectively) and CD80-His/PD-L1-Ig (100 & 10 nM, respectively).

[0185] Recombinant Proteins: Human PD-1 (25-167) with a C-terminal human Fc domain of immunoglobulin G (Ig) epitope tag [hPD-1 (25-167)-3S-IG] and human PD-L1 (18-239) with a C-terminal His epitope tag [hPD-L1(18-239)-TVMV-His] were expressed in HEK293T cells and purified sequentially by ProteinA affinity chromatography and size exclusion chromatography. Human PD-L2-His and CD80-His was obtained through commercial sources.

Methods

[0186] Homogenous Time-Resolved Fluorescence (HTRF) Assays of Binding of Soluble PD-1 to Soluble PD-L1. Soluble PD-1 and soluble PD-L1 refers to proteins with carboxyl-end truncations that remove the transmembrane-spanning regions and are fused to heterologous sequences, specifically the Fc portion of the human immunoglobuling G sequence (Ig) or the hexahistidine epitope tag (His). All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 ?l of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 ?l of assay buffer and further incubation for 15 m. PD-L1 fusion proteins from either human, cynomologous macaques, mouse, or other species were used. HTRF detection was achieved using europium crypate-labeled anti-Ig monoclonal antibody (1 nM final) and allophycocyanin (APC) labeled anti-His monoclonal antibody (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 ?l was dispensed on top of binding reaction. The reaction was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 and 5 nM, respectively), CD80-His/PD-L1-Ig (100 and 10 nM, respectively) and CD80-His/CTLA4-Ig (10 and 5 nM, respectively).

[0187] Binding/competition studies between biotinylated Compound No. 71 and human PD-L1-His were performed as follows. The compounds of the present invention were pre-incubated with PD-L1-His (10 nM final) for 60 minutes in 4 ?l of assay buffer followed by addition of biotinylated Compound No. 71 (0.5 nM final) in 1 ?l of assay buffer. Binding was allowed to equilibrate for 30 minutes followed by addition of europium crypated labeled Streptavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 ?l of HTRF buffer. The reaction was allowed to equilibrate for 30m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.

[0188] Recombinant Proteins. Carboxyl-truncated human PD-1 (amino acids 25-167) with a C-terminal human Ig epitope tag [hPD-1 (25-167)-3S-IG] and human PD-L1 (amino acids 18-239) with a C-terminal His epitope tag [hPD-L1(19-239)-tobacco vein mottling virus protease cleavage site (TVMV)-His] were expressed in HEK293T cells and purified sequentially by recombinant Protein A affinity chromatography and size exclusion chromatography. Human PD-L2-His (Sino Biologicals), CD80-His (Sino Biologicals), CTLA4-Ig (RnD Systems) were all obtained through commercial sources.

[0189] The table below lists the IC.sub.50 values for representative examples of this disclosure measured in the PD-1/PD-L1 Homogenous Time-Resolved Fluorescence (HTRF) binding assay.

TABLE-US-00057 Compound Number HTRF IC.sub.50 (?M) 1001 0.0027 1002 0.0035 1003 0.0020 1004 0.0031 1005 0.0017 1006 0.0026 1007 0.0035 1008 0.0044 1009 0.0030 1010 0.0023 1011 0.0015 1012 0.0029 1013 0.0040 1014 0.0032 1015 0.0024 1016 0.1103 1017 0.1117 1018 0.0150 1019 0.0183 1020 0.0073 1021 0.1209 1022 0.1454 1023 >10 1024 0.0214 1025 >10 1026 0.0104 1027 0.0025 1028 0.0445 1029 0.1317 1030 0.0801 1031 0.0393 1032 0.0078 1033 0.0115 1034 0.0074 1035 0.0021 1036 0.0073 1037 0.1362 1038 0.0048 1039 0.0196 1040 0.0067 1041 0.0091 1042 0.0056 1043 0.0058 1044 0.0056 1045 0.0036 1046 0.0031 1047 0.3877 1048 3.8320 1049 0.0115 1050 0.0473 1051 0.3833 1052 2.5000 1053 1.4696 1054 2.5151

[0190] The compounds of the present invention tested possess activity as inhibitors of the PD-1/PD-L1 interaction, and therefore, may be used in the treatment of diseases or deficiencies associated with the PD-1/PD-L1 interaction. Via inhibition of the PD-1/PD-L1 interaction, the compounds of the present disclosure may be employed to treat infectious diseases such as HIV, septic shock, Hepatitis A, B, C, or D and cancer.

[0191] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0192] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.