BIOMARKER ASSAYS FOR DETECTING OR MEASURING INHIBITION OF TOR KINASE ACTIVITY

WIPO Patent Application WO/2012/016113

A1

Provided herein are methods for detecting and/or measuring the inhibition of TOR kinase activity in a subject and uses associated therewith.

WONG, Lilly (871 Viva Court, Solana Beach, CA, 92075, US)
XU, Shuichan (12426 Dormouse Road, San Diego, CA, 92129, US)
DING, Jian-Hua (8024 Camino Jonata, San Diego, CA, 92122, US)

US2011/045842

February 02, 2012

July 29, 2011

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SIGNAL PHARMACEUTICALS, LLC (4550 Towne Centre Court, San Diego, CA, 92121, US)
WONG, Lilly (871 Viva Court, Solana Beach, CA, 92075, US)
XU, Shuichan (12426 Dormouse Road, San Diego, CA, 92129, US)
DING, Jian-Hua (8024 Camino Jonata, San Diego, CA, 92122, US)

G01N33/573

INSOGNA, Anthony, M. et al. (Jones Day, 222 East 41st StreetNew York, NY, 10017-6702, US)

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CLAIMS What is claimed is: 1. A method for detecting or measuring the inhibition of TOR kinase activity in a subject, comprising the use of flow cytometry to measure the amount of phosphorylated 4EBP 1 in a biological sample from said subject prior to and after the administration of a TOR kinase inhibitor. 2. A method for determining a dose-response relationship for the administration of a TOR kinase inhibitor to a subject, wherein said subject is administered varying doses of said TOR kinase inhibitor and the amount of TOR kinase activity inhibition in said subject resulting from each dose of said TOR kinase inhibitor is determined by the use of flow cytometry to measure the amount of phosphorylated 4EBP1 in a biological sample from said subject prior to and after each administration of said TOR kinase inhibitor. 3. A method for determining whether a subject is sensitive to a TOR kinase inhibitor, comprising administering said subject said TOR kinase inhibitor and determining whether or not TOR kinase is inhibited in said subjecting by the use of flow cytometry to measure the amount of phosphorylated 4EBP 1 in a biological sample from said subject prior to and after the administration of said TOR kinase inhibitor. 4. A method for determining the effective amount of a TOR kinase inhibitor for the treatment or management of a disease in a patient, comprising administering said patient varying doses of said TOR kinase inhibitor and determining the amount of TOR kinase activity inhibition in said patient resulting from each dose of said TOR kinase inhibitor by the use of flow cytometry to measure the amount of phosphorylated 4EBP1 in a biological sample from said subject prior to and after each administration of said TOR kinase inhibitor. 5. A method for treating or managing a disease associated with TOR kinase in a patient having a disease associated with TOR kinase, comprising administering to said patient an effective amount of a TOR kinase inhibitor, wherein the effective amount of said TOR inhibitor is determined using the method of claim 4. 6. A method for detecting or measuring the inhibition of TOR kinase activity in a biological sample from a subject, comprising the use of flow cytometry to measure the amount of phosphorylated 4EBP1 in said biological sample prior to and after contacting said biological sample with a TOR kinase inhibitor ex vivo. 7. A kit comprising one or more containers filled with reagents for detecting p4EBPl using flow cytometry and instructions for detecting p4EBPl using flow cytometry. 8. The method of claim 1 , wherein the TOR kinase inhibitors has the formula (I): (I) or a pharmaceutically acceptable salt, clathrate, solvate, stereoisomer, tautomer, or prodrug thereof, wherein: R1 is substituted or unsubstiiuted C(.g alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl; R2 is H, substituted or unsubstituted C|.g alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl. substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkylalkyl; and R3 and R4 are each independently H, substituted or unsubstituted Ci- alky I, substituted or unsubstituted aryl, substituted or unsubstituted cycloaikyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylaikyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkylalkyl, or R3 and R4, together with the atoms to which they are attached, form a substituted or unsubstituted cycloaikyl or substituted or unsubstituted heterocyclyl; or R2 and one of R3 and R4, together with the atoms to which they are attached, form a substituted or unsubstituted heterocyclyl. 9. The method of claim 8, wherein the TOR kinase inhibitor is: 6-(l H-pyrrolo[2,3-b]pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(4-methyl-6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one: 6-(5-fluoro-2-methyl-4-( lH-l ,2,4-triazol-3-yl)phenyl)-4-((/ram-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)-4-((cw-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-4-((/ra«.y-4-methoxycyclohexyl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-4-((/ram'-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-( l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-( (c 1v-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-( l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((/ram-4-hydroxycyclohexyi)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-( l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(m-4-hydroxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(l H-l,2,4-triazol-3-yl)pyridin-3-yl)-4-((c,w-4-hydroxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(5-fluoro-2-methyl-4-( I H- 1 ,2,4-triazol-3-y l)pheny l)-4-(/ram-4- methoxycyclohexyI)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 6-(6-(l H-l ,2,4-tria2ol-3-yl)pyridin-3-yl)-4-(/r m-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-4-(/ram-4-hydroxycyclohexyl )-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)-4-((c/j-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(c7'5'-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-isopropyl-3.4-dihydropyrazino[2,3- b]pyrazin-2f 1 H)-one; 6-(5-fluoro-2-methyl-4-(lH-l ,2,4-triazol-3-yl)phenyl)-4-(cw-4-hydroxycyclohexyl)- 3,4-dihydropyrazino[2,3-b pyrazin-2(l H)-one; 6-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)-4-(cw-4-methoxycyclohexyl)- 3,4-dihydropyrazino[2,3-blpyrazin-2( 1 H)-one; 6-(5-fluoro-2-meihyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(tetrahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(l H- l,2,4-triazol-3-yl)pyridin-3-yI)-4-ethyl-3,4-dihydropyrazino[2,3-b]pyrazin- 2( lH)-one; 6-(5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-4-(iram-4- hydroxycyclohexyl)-3.4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-f5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yr)phenyl)-4-(tetrahydro-2H-pyran-4-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-( 5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazoI-3-y l)phenyI)-4-isopropyI-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one: 4-ethyl-6-(5-fluoro-2-methyl-4-(l H- l ,2,4-triazol-3-yl)phenyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(3-fluoro-2-methyl-4-( lH-l,2,4-triazol-3-yi)phenyl)-4-(tetrahydro-2H-pyran-4-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(3-fluoro-2-methyl-4-( l H-l ,2,4 riazol-3-yl)phenyl)-4-( /5-4-rnethoxycyclohexyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(3-fluoro-2-methyl-4-( 1H- 1 ,2,4-triazol-3-yl)phenyl)-4-(/rara-4- methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4- (2-methoxyethyl)-6-(4-methyl-6-( 1 H- 1 ,2,4-triazol-3 -yl)pyridin-3 -yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-(3-(lH-l ,2,4-triazol-5-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino [2,3 -b]pyrazin-2( 1 H)-one; 5- (8-(2-methoxyethyl)-6-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]pyrazin-2-yl)-4- methylpicolinamide; 3-(6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)benzamide; 3- (6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)benzonitrile; 5- (8-(/ram'-4-meihoxycyclohexy])-6-oxo-5,6,7.8-ietrahydropyrazino[2,3-b]pyrazin- 2-yl)-4-methylpicolinamide; 6- (lH-imidazo[4,5-b]pyridin-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-( l H-indazol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 4- (( l R,3S)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l ,2.4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4-(( Ί S,3R)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4-((lR,3R)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-(( lS,3S)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4-ethyl-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 6-(lH-pyrrolo[2,3-b]pyridin-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 6-(lH-indol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 6-(l H-indol-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 4-((( 1 R,3 S)-3 -methoxycyclopentyl )methyl)-6-(2-methyl-6-(4H- 1 ,2,4-triazol-3- yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 4-(((lS,3R)-3-methoxycyclopentyl)methyl)-6-(2-methyl-6-(4H-l,2,4-triazol-3- yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 6-(3-fluoro-2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 6-(3-fluoro-2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 3J-dimeihyl-6-(4-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-i(tetrahydro-2H- pyran-4-yl)methyl)-3,4-dihydropyrazino[2.3-b]pyrazin-2(lH)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((lR.3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((lS,3R)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-bjpyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((l S,3S)-3- methoxycyclopentyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((l R,3R)-3- methoxycyclopentyl)methyl)-3,4-dihydropyrazino[2J-b]pyrazin-2( lH)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(( l S.3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((lR,3R)-3-methoxycyc[opentyD-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((l R,3S)-3- methoxycyclopentyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((l S,3R)-3- methoxycyclopentyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-(3-fluoro-4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(3-iluoro-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3-y l)phenyl)- 1 '-((tetrahydro-2H-pyran-4- yl)methyl)- 1 'H-spiro[cyclopentane- 1 ,2'-pyrazino[2,3-b]pyrazin]-3'(4'H)-one; 7'-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-l'-((tetrahydro-2H-pyran-4- yl)methyl)-rH-spiro[cyclobutane-l,2'-pyrazino[2,3-b]pyrazin]-3'(4'H)-one; 4-(cyclopropy lmethyl)-6-( 6-( 2-hydroxypropan-2-y l)pyridin-3 -y l)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 7'-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-l 'H-spiro[cyclopentane-l,2'- pyrazino[2,3-b]pyrazin]-3'(4'H)-one; 7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3-yl )phenyl)- 1 'H-spirojcyclobutane- 1 ,2'- pyrazino[2,3-b]pyrazin]-3'(4'H)-one; 7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3~yl)phenyl)- 1 'H-spiro[cyclopropane- 1 ,2'- pyrazino[2,3-b]pyrazin]-3'(4'H)-one; (R)-6-(4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-((tetrahydroiliran-2-yI)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; (S)-6-(4-(4H-l ,2,4 riazol-3-yl)phenyl)-4-((tetrahydrofuran-2-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-( 1 H-indazol-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl )ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one: 4-(6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6.7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)benzamide; 4-(2-methoxyethyl)-3,3-dimethyl-6-(2-methyl-4 4H-1.2,4-triazol-3-yI)phenyI)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-ethyl-3,3-dimethyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(2-methyl-4-(4H-1.2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(l H)-one; 3,3-dimethyl-6-(2-methyl-6-(4H-l ,2.4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H- pyran-4-yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; (R)-6-(6-( l -hydroxyethyl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 3,3-dimethyl-6-(2-methyl-4-(4H- l ,2,4-triazol-3-yr)phenyl)-4-((tetrahydro-2H-pyran- 4-yl)methyl)-3.4-dihydropyrazino[2,3 -b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)-4-methylpyridin-3-yl)-4-(/ram-4-methoxycyclohexyl)- 3,4-dihydropyrazino[2.3-b]pyrazin-2(l H)-one: 6-(6-(2-hydroxypropan-2-yl)-4-methylpyridin-3-yl)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 3,3-dimethyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3- bjpyrazin-2( 1 H)-one; 3,3-dimethyl-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(2-(tetrahydro- 2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)-2-methylpyridin-3-yI)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3.4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 6-(6-(2-hydroxypropan-2-yl)-2-methylpyridin-3-yl)-4-(/r W5-4-methoxycyclohexyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; (S)-6-(6-( l -hydroxyethyl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 3,3-dimethyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H- pyran-4-yl)ethyl)-3.4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,3-dimethyl-4-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4-dihydropyrazino[2,3-bJpyrazin-2( 1 H)-one; 6-(4-(2-hydroxypropan-2-yl)phenyl)-4-(ir ns-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(4-(2-hydroxypropan-2-yl)phenyl)-4-((irans-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-(c7 4-methoxycyclohexyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4-(/r m'-4-methoxycyclohexyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 6-(4-(2-hydroxypropan-2-yl)phenyl)-4-((tetrahydro-2H-pyran-4-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4- (2-methoxyethyl)-6-(2-methyl-6-(4H- l,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 9-(6-(4H- l ,2,4-triazol-3-yl)-3-pyridyl)-6,l l ,4a-trihydromoipholino[4,3- e]pyrazino[2,3-b]pyrazin-5-one; 6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 5- (8-(cw-4-methoxycyclohexyl)-6-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]pyrazin-2- yl)-6-methylpicolinonitrile; 6- (6-(4H- l ,2,4-triazoI-3-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 9-(4-(4H- l ,2,4-triazol-3-yl)-2-methylphenyl)-3-(2-methoxyacetyl)-6, l l ,4a- trihydropiperazino[ 1 ,2-e]pyrazino[2,3-b]pyrazin-5-one; 9-(4-(4H- 1.2,4-triazol-3-yl)-2-methylphenyl)-6, l l ,4a-trihydropiperazino[l ,2- e]pyrazino[2,3-b]pyrazin-5-one; 9-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)-3-f2-methoxyethyl)-6, l 1 ,4a- trihydropiperazino[ l ,2-e]pyrazino[2,3-b]pyrazin-5-one; 4-(cyclopentylmethyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl )pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 9-(6-(4H- 1 ,2,4-triazol-3-yl)-2-methyl-3-pyridyl)-6, 1 1 ,4a-trihydromorpholino[43- e]pyrazino 2,3-bJpyrazin-5-one; 4-(/ram-4-hydroxycyclohexyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-(c ts-4-hydroxycyclohexyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydrofuran-3-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( I H)-one; 4-(cyclopentylmethyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-neopentyl-3.4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-isobutyl-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 3-methyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(piperidin-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-3-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 8-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)(3aS,2R)-2-methoxy-5, 10,3a- trihydropyrazino[2,3-b]pyrrolidino[ 1 ,2-e]pyrazin-4-one; 8-(4-(4H-K2,4-triazol-3-yl)-2-methylphenyl)(2R,3aR)-2-methoxy-5, 10,3a- trihydropyrazino[2,3-b]pyiTolidino[1.2-e]pyrazin-4-one; 8-(4-(4H-l ,2,4-triazol-3-yl)-2-methyIphenylX2S,3aR)-2-methoxy-5J0,3a- trihydropyrazino[2,3-b]pyrrolidino[l ,2-e]pyrazin-4-one; 8-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)(2S,3aS)-2-methoxy-5.10,3a- trihydropyrazino[2,3-b]pyrrolidino[l ,2-e]pyrazin-4-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(3-methoxypropyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; (S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydro uran-2-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; (R)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydro uran-2-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4- y )ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 9.(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)-3-methyl-6,l l ,4a- trihydropiperazino[l ,2-e]pyrazino[2,3-b]pyrazin-5-one; 9-(4-(4H-l ,2,4-triazol-3-yl)phenyl)-6, l l ,4a-trihydromo holino[4,3-e]pyrazino[2,3- b]pyrazin-5-one; 9-(4-(4H- 1 ,2,4-triazol-3-yl)-2-methylphenyl)-6, 1 1 ,4a-trihydropiperidino[ 1 ,2- e]pyrazino[2,3-b]pyrazin-5-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(/ra«.s-4-methoxycyc!ohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(c s-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-morpholinoethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-phenethyl-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-(cyclohexylmethyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yI)-4-((/ra«5-4-methoxycyclohexyI)methyi)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((c/s-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; (R)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydrofuran-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; (S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydrofiiran-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yI)-4-phenyl-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; (S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3-methyl-4-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 9-[6-( l -hydroxy-isopropyl)-3-pyridyl]-6,l l ,4a-trihydromorpholino[4,3- e]pyrazino[2,3-b]pyrazin-5-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4-yl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3 -b]pyrazin-2( 1 H)-one; 6-(2-amino-7-methyl-l H-benzo[d]imidazol-5-yl)-4-(3-(trifluoromethyl)benzy dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 9-(4-(4H-l,2,4-triazol-3-yl)-2-methylphenyl)-6,l 1 ,4a-trihydromorpholino[4,3- e]pyrazino[2,3-b]pyrazin-5-one; 6-(4-methyl-2-(methylamino)- l H-benzo[d]imidazol-6-yl)-4-(2-(tetrahydro-2H- pyran-4-y l)ethyl)-3 ,4-dihydropyrazino [2,3 -b] pyrazin-2( 1 H)-one ; 8-(4-(4H-l ,2,4-triazo]-3-yl)-2-methy]phenyl)-5, 10,3a-trihydropyrazino[2,3- b]pyrrolidino[l ,2-ejpyrazin-4-one; 6-(4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-ethyl-3,4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one; 6-(4- 4H-l ,2,4-triazol-3-yl)phenyI)-4-((tetrahydro-2H-pyran-4-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 6-(4-(4H-l ,2.4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 6-(4-(4H-K2,4-triazol-3-yl)phenyl)-4-(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(2-methyl-4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyI)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 6-(4-methyl-lH-benzo[d]imidazol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[23-b]pyrazin-2(l H)-one; 6-(4-(2-hydroxypropan-2-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(4-( 1 H- 1 ,2,4-triazol-5-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; or a pharmaceutically acceptable salt thereof. 10. The method of claim 1 , wherein the TOR kinase inhibitors has the formula (II): (II) or a pharmaceutically acceptable salt, clathrate, solvate, stereoisomer, tautomer, or prodrug thereof, wherein: R1 is substituted or unsubstituted Ci. alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cvcloalkyl, substituted or unsubstituted heterocvclyl, or substituted or unsubstituted heterocyclylalkyl; R2 is H. substituted or unsubstituted C|.g alkyl. substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl. or substituted or unsubstituted cycloalkylalkyl; and R3 is H. or a substituted or unsubstituted C i.g alkyl. 1 1 . The method of claim 10, wherein the TOR kinase inhibitor is: 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -((/ram-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(6-(l H-l ,2,4-tria2ol-3-yl)pyridin-3-yl)-l-( /5'-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(l H-pyrrolo[2,3-b]pyridin-3-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino [2,3 -b] pyrazin-2( 1 H)-one ; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -((c«-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l-ethyl-7-(l H-pyrrolo[3,2-b]pyridin-5-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)- one; 7-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-l-((c5-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-( 1 H-benzo[d]imidazol-4-yl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(lH-pyrrolo[2J-b]pyridin-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(l H-l ,2^-triazol-3-yr)pyridin-3-yl)-l-((/ra75-4-methoxycyclohexyl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3 -yl)- 1 -((/ra/7i--4-hydroxycyclohexyl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-( I H- 1 ,2,4-triazol-3-yl)pyridin-3 -yl )- 1 -( ,v-4-hydroxycyclohexy l)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(5-fluoro-2-methyl-4-( 1 H- 1.2,4-triazol-3-yi)phenyl)- l-(cz 4-hydroxycyclohexyl)- 3.4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-l-(tetrahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b pyrazin-2( 1 H)-one; 7-(6-( l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-l -ethyl-3,4-dihydropyrazino[2,3-b]pyrazin- 2( l H)-one; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -({ is-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 7-(5-fluoro-2-methyl-4-( 1 H-l ,2,4-triazol-3-yl)phenyl)- 1 -(tetrahydro-2H-pyran-4-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 7-(l H-indol-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b ]pyrazin-2( 1 H)-one ; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -{{trans A- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 7-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)- l -(( 75-4-hydroxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-l-(/rara-4-hydroxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3 -yl)- 1 -(irarcs-4-methoxycyclohexyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(lH-l ,2,4-triazol-3-yl)pyridin-3-yl)-l-isopropyl-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -(/rara-4- methoxycyclohexyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(5-fluoro-2-methy]-4-( l H-l ,2,4-triazol-3-yl)pbeny])-l -(/ram-4- hydroxycyclohexyl )-3 ,4-dihydropyrazino[2,3 -b]pyrazin-2( 1 H)-one; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -(2-methoxyetliyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazoI-3-yl)phenyl)- 1 -isopropyl-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l -ethyl-7-(5-tluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(2-hydroxypyridin-4-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2 ,3-b]pyrazin-2( 1 H)-one; l -isopropyl-7-(4-methyl-6-( lH-l ,2,4-triazol-3-yI)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 5-(8-isopropyl-7-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]pyrazin-2-yl)-4- methylpicolinamide; 7-( l H-indazol-4-yr)-l -(2-(tetrahydro-2H-pyTan-4-yl)ethyl)-3.4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(2-aminopyrimidin-5-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(2-aminopyridin-4-yl)- l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2( 1 H)-one; 7-(6-(methylamino)pyridin-3-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-hydroxypyridin-3-yl)- l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2(l H)-one; 7-(4-(lH-pyrazol-3-yl)phenyl)-l-(2-methoxyethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(pyridin-3-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-( l H-indazol-4-yl)- l-(2-methoxyethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)- one; 7-( 1 H-indazol-6-yl)- 1 -(2-methoxyethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)- one; 7-(pyrimidin-5-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(6-methoxypyridin-3-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l-(2-methoxyethyl)-7-(l H-pyrrolo[2,3-b]pyridin-5-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; l -ethyl-7-( l H-pyrrolo[2,3-b]pyridin-5-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)- one; 1 -ethyl-7-( 1 H-indazol-4-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(pyridin-4-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(6-aminopyridin-3-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 1 - methyI-7-(2-methyl-6-(4H-1.2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihy dropy r azino [2 , 3 -b] pyr azin-2( 1 H)-one ; 2- (2-hydroxypropan-2-yl)-5-(8-( mn.s--4-methoxycyclohexyr)-7-oxo-5,6,7,8- tetrahydropyrazino[2,3-b]pyrazin-2-yl)pyridine 1 -oxide; 4- methyl-5-(7-oxo-8-((tetrahydro-2H-pyran-4-yI)methyl)-5,6,7,8- tetrahydropyrazino[2,3-b]pyrazin-2-yl)picolinamide; 5- (8-(( /5-4-methoxycyclohexyl)methyl)-7-oxo-5,6,7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)-4-methylpicolinamide; 7-( l H-pyrazol-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; l-(/ram-4-methoxycyclohexyl)-7-(4-methyl-6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 3- ((7-(2-methyl-6-(4H-l ,2,4-triazoi-3-yl)pyridin-3-yl)-2-oxo-3,4- dihydropyrazino[2,3-b]pyrazin-l(2H)-yl)methyl)benzonitrile; l -((/rara-4-methoxycyclohexyl)methyl)-7-(4-methy]-6-(lH-l ,2,4-triazol-3- yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 3-(7-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)benzamide; 5-(8-((ira«s-4-methoxycyclohexyl)methyl)-7-oxo-5,6,7,8-tetrahydropyrazino[2,3- b]pyrazin-2-yl)-4-methylpicolinamide; 3-((7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-2-oxo-3,4-dihydropyrazino[2,3- b]pyrazin- l(2H)-yl)methyl)benzonitrile; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -((l R,3R)-3-methoxycyclopentyi)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- l -((l S.3R)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- l -((l S,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- 1 -((1 R,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(lH-indazol-6-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(2-morpholinoethyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l-( ra¾v-4-hydroxycyclohexyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)- 3 ,4-dihydropyrazino [2,3 -b]pyrazin-2( 1 H)-one; l-(cw-4-hydroxycyclohexyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(2-morpholinoethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 1 -isopropyl-7-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3 -yl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(lH-imidazo[4,5-b]pyridin-6-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 1 -((cM-4-methoxycyclohexyl)methyl)-7-(2-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin- 3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l -(/ran*-4-hydroxycyclohexyl)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; l -(c/i,-4-hydroxycyclohexyl)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yi)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 4-(7-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetrahydropyrazino[2,3- bjpyrazin-2-yl)benzamide; 7-(l H-indazol-5-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(l H-pyrrolo[2,3-b]pyridin-5-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(2-methyl-6-(4H- l ,2,4-triazoI-3-yl)pyridin-3-yl)- l-(tetrahydro-2H-pyran-4-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l -((lS R)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l -(( l R,3R)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 1 -(( 1 R,3 S)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l -((l S,3S)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(lH-indol-5-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; l -ethyl-7-(2-methyl-6-(4H-l ,2.4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(lH-indol-6-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(4-(2-hydroxypropan-2-yl)phenyl)-l-(/ra/75-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(tetrahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l-((rr m'-4-methoxycyclohexyl)methyl)-7-(2-methyl-6-(lH-l ,2,4-triazol-3- yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- i-(( 'i-4-rnethoxycyciohexyl)methyJ)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; l-(2-methoxyethyl)-7-(4-methyl-2-(methylamino)-l H-benzo[d]imidazol-6-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(7-methyl-2-oxo-2,3-dihydro-l H-benzo[d]imidazol-5-yl)-l -((tetrahydro-2H- pyran-4-yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 7-(2-methyl-4-(4H-l,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2( l H)-one; l-(2-methoxyethyl)-7-(4-methyl-6-( l H- l,2,4-triazol-3-y[)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l -benzyl-7-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(3-fluoro-4-(4H-l ,2,4-triazol-3-yl)phenyl)-l-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(3-fluoro-4-(4H-l ,2,4-triazol-3-yl)phenyl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2.3-b]pyrazin-2(l H)-one; 7-(3-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; l-(iraw-4-methoxycyclohexyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yI)pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(i,ram--4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one; 7-(5-fluoro-2-methyl-4-(4H- 1.2,4-triazol-3-yl)phenyl)- 1 -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(3-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one; l -(2-methoxyethy])-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3.4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -((/mm-4-methoxycyclohexyl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l -(cyclopentylmethyl)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yi)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(4-(2-hydroxypropan-2-yl)phenyl)- l -(2-methoxyethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one; (S)-7-(6-(l -hydroxyethyl)pyridin-3-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; (R)-7-(6-( 1 -hydroxyethyl)pyridin-3-yl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(2-methyl-6-(4H- 1.2,4-triazol-3-yl)pyridin-3-yl)- 1 -((tetrahydro-2H-pyran-4- yl)methyl)-3.4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(4-(2-hydroxypropan-2-yl)phenyl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(4-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-ylJ- l -(3-methoxypropyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(4-methyl-6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)- l -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -((tetrahydro-2H-pyran-4-yl)methyI)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(4-methyl-2-(methylamino)-l H-benzo[d]imidazol-6-yl)-l -((tetrahydro-2H-pyran- 4-yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one: 7-(2-amino-4-methyl- 1 H-benzo[d]imidazol-6-yl)- 1 -((tetrahydro-2H-pyran-4- yl)methyl)-3.4-dihydropyrazino[2,3-b pyrazin-2(lH)-one; 7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)- l-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; (R)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3-methyl- l -(2-(tetrahydro-2H-pyran- 4-y l)ethyl)-3 ,4-dihydropyrazino [2,3 -b]pyrazin-2( I H)-one; (S)-7-(6-(2-hydroxypropai 2-yl)pyridin-3-yl)-3-methyl- l-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 7-( 6-(2-hydroxypropan-2-yl)pyridin-3 -yl)-3 ,3 -dimethyl- 1 -( 2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4-dihydropyrazino[2,3-bjpyrazin-2( 1 H)-one; 7-(2-amino-4-methyl- 1 H-benzo[d]imidazol-6-yl)- 1 -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-( 2-hydroxypropan-2-yl)pyridin-3-yl)- l -(2-(tetral ydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one; 7-(2-methyl-4-( 1 H-l ,2,4-triazol-3-yl)phenyl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-ohe; 7-(4-( 1 H- 1.2,4-triazol-5-yl)phenyl)- 1 -( 2-(tetrahydro-2H-pyran-4-y l)ethy l)-3 ,4- dihydropyrazino[2,3-bJpyrazin-2( 1 H)-one; 1 -( 1 -hydroxypropan-2-yl)-7-(2-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; l -(2-hydroxyethyl)-7-(2-methyl-6-( l H- l ,2,4-triazo!-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( l H)-one; or a pharmaceutically acceptable salt thereof.

BIOMARKER ASSAYS FOR DETECTING OR MEASURING INHIBITION OF

TOR KINASE ACTIVITY

[0001] This application claims the benefit of U.S. Provisional Application No.

61/369,455, filed July 30, 2010, the entire contents of which are incorporated herein by reference.

1. FIELD

[0002] Provided herein are methods for detecting and/or measuring the inhibition of

TOR kinase activity in a subject and uses associated therewith.

2. BACKGROUND

J0003] The connection between abnormal protein phosphorylation and the cause or consequence of diseases has been known for over 20 years. Accordingly, protein kinases have become a very important group of drug targets. See Cohen, Nature Reviews Drug Discovery, 1 :309-315 (2002). Various protein kinase inhibitors have been used clinically in the treatment of a wide variety of diseases, such as cancer and chronic inflammatory diseases, including diabetes and stroke. See Cohen, Eur. J. Biochem., 268:5001-5010 (2001 ), Protein Kinase Inhibitors for the Treatment of Disease: The Promise and the Problems, Handbook of Experimental Pharmacology, Springer Berlin Heidelberg, 167 (2005).

[0004] The protein kinases are a large and diverse family of enzymes that catalyze protein phosphorylation and play a critical role in cellular signaling. Protein kinases may exert positive or negative regulatory effects, depending upon their target protein. Protein kinases are involved in specific signaling pathways which regulate cell functions such as, but not limited to, metabolism, cell cycle progression, cell adhesion, vascular function, apoptosis, and angiogenesis. Malfunctions of cellular signaling have been associated with many diseases, the most characterized of which include cancer and diabetes. The regulation of signal transduction by cytokines and the association of signal molecules with protooncogenes and tumor suppressor genes have been well documented. Similarly, the connection between diabetes and related conditions, and deregulated levels of protein kinases, has been demonstrated. See e.g., Sridhar et al. Pharmaceutical Research,

17(1 1 ): 1345- 1353 (2000). Viral infections and the conditions related thereto have also been associated with the regulation of protein kinases. Park et al. Cell 101 (7): 777-787 (2000).

[0005] Protein kinases can be divided into broad groups based upon the identity of the amino acid(s) that they target (serine/threonine, tyrosine, lysine, and histidine). For example, tyrosine kinases include receptor tyrosine kinases (RTKs), such as growth factors and non-receptor tyrosine kinases, such as the src kinase family. There are also dual- specific protein kinases that target both tyrosine and serine/threonine, such as cyclin dependent kinases (CDKs) and mitogen-activated protein kinases (MAPKs).

[0006] Because protein kinases regulate nearly every cellular process, including metabolism, cell proliferation, cell differentiation, and cell survival, they are attractive targets for therapeutic intervention for various disease states. For example, cell-cycle control and angiogenesis, in which protein kinases play a pivotal role are cellular processes associated with numerous disease conditions such as but not limited to cancer, inflammatory diseases, abnormal angiogenesis and diseases related thereto, atherosclerosis, macular degeneration, diabetes, obesity, and pain.

[0007] Protein kinases have become attractive targets for the treatment of cancers.

Fabbro et al.. Pharmacology & Therapeutics 93:79-98 (2002). It has been proposed that the involvement of protein kinases in the development of human malignancies may occur by: (1 ) genomic rearrangements (e.g., BCR-ABL in chronic myelogenous leukemia), (2) mutations leading to constitutively active kinase activity, such as acute myelogenous leukemia and gastrointestinal tumors, (3) deregulation of kinase activity by activation of oncogenes or loss of tumor suppressor functions, such as in cancers with oncogenic RAS, (4) deregulation of kinase activity by over-expression, as in the case of EGFR and (5) ectopic expression of growth factors that can contribute to the development and

maintenance of the neoplastic phenotype. Fabbro et al.. Pharmacology & Therapeutics 93:79-98 (2002). [0008] The elucidation of the intricacy of protein kinase pathways and the complexity of the relationship and interaction among and between the various protein kinases and kinase pathways highlights the importance of developing pharmaceutical agents capable of acting as protein kinase modulators, regulators or inhibitors that have beneficial activity on multiple kinases or multiple kinase pathways. Accordingly, there remains a need for new kinase modulators.

[0009] The protein named mTOR (mammalian target of rapamycin), which is also called FRAP, RAFTI or RAPTl), is a 2549-amino acid Ser/Thr protein kinase, that has been shown to be one of the most critical proteins in the mTOR/PDK/Akt pathway that regulates cell growth and proliferation. Georgakis and Younes Expert Rev. Anticancer Ther.

6(1): 131- 140 (2006). mTOR exists within two complexes, mTORC 1 and mTORC2. While mTORCl is sensitive to rapamycin analogs (such as temsirolimus or everolimus), mTORC2 is largely rapamycin-insensitive. Notably, rapamycin is not a TOR kinase inhibitor. Several mTOR inhibitors have been or are being evaluated in clinical trials for the treatment of cancer. Temsirolimus was approved for use in renal cell carcinoma in 2007 and everolimus was approved in 2009 for renal cell carcinoma patients that have progressed on vascular endothelial growth factor receptor inhibitors. In addition, sirolimus was approved in 1999 for the prophylaxis of renal transplant rejection. The interesting but limited clinical success of these mTORC l inhibitory compounds demonstrates the usefulness of mTOR inhibitors in the treatment of cancer and transplant rejection, and the increased potential for compounds with both mTORC 1 and mTORC2 inhibitory activity.

[0010] Due to the potential pharmaceutical applications for inhibitors of TOR kinase activity, there is a need for methods for detecting and/or measuring the inhibition of TOR kinase activity in vivo.

[0011 ] Citation or identification of any reference in Section 2 of this application is not to be construed as an admission that the reference is prior art to the present application.

3. SUMMARY

[0012] Provided herein are methods for detecting or measuring the inhibition of

TOR kinase activity in a subject, comprising measuring the amount of phosphorylated 4EBP1 (also referred to herein as ''p4EBP l in a biological sample from said subject, for example a peripheral blood sample, prior to and after the administration of a TOR kinase inhibitor to said subject. In one embodiment, the amount of p4EBPl is measured using flow cytometry. The methods provided herein are belived to have utility in following the inhibition of TOR kinase in a subject.

{0013] Further provided herein are methods for determining a dose-response relationship for the administration of a TOR kinase inhibitor to a subject, wherein said subject is administered varying doses of said TOR kinase inhibitor and the amount of TOR kinase activity inhibition in said subject resulting from each dose of said TOR kinase inhibitor is determined using a method provided herein.

[0014] Further provided herein are methods for determining whether a subject is sensitive to a TOR kinase inhibitor, comprising administering said subject said TOR kinase inhibitor and determining whether or not TOR kinase activity is inhibited in said subject using a method provided herein.

[0015] Further provided herein are methods for determining the effective amount of a TOR kinase inhibitor for the treatment or management of a disease in a subject, comprising administering said subject varying doses of said TOR kinase inhibitor and determining the amount of TOR kinase activity inhibition in said patient resulting from each dose of said TOR kinase inhibitor using a method provided herein.

[0016] Further provided herein are methods for treating or managing a disease associated with TOR kinase in a patient having a disease associated with TOR kinase, comprising administering to said patient an effective amount of a TOR kinase inhibitor, wherein the effective amount of said TOR inhibitor is determined using a method provided herein.

[0017] In certain embodiments, the methods provided herein are carried out by way of contacting a biological sample from a patient with a TOR kinase inhibitor ex vivo.

(0018] Further provided herein are kits comprising one or more containers filled with reagents for detecting p4EBPl using How cytometry and instructions for detecting p4EBPl using flow cytometry. [0019] The present embodiments can be understood more fully by reference to the detailed description and examples, which are intended to exemplify non-limiting embodiments.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG 1. Provides histograms representing cell populations of untreated cells

(shaded), D SO treated cells (dashed lined), specific blocking peptide treated cells (solid line), and Compound 1 treated cells (dotted line). (A) represents PC3 cells treated with 10 μΜ Compound 1. (B) Provides histograms representing CD91+ monocytes from whole blood of normal healthy volunteers treated ex vivo with 30 μΜ Compound 1. (C) Provides histograms representing CD91+ monocytes from whole blood of a lung cancer patient treated ex vivo with 30 μΜ Compound 1.

[0021] FIG 2. Illustrates reproducibility of p4EBPl assay with respect to day to day variation of p4EBPl in monocytes of the same healthy donors (blood was drawn from 3 healthy donors on 3 consecutive days). (A) Illustrates mean fluorescence intensity for all 3 donors on different days. (B) Illustrates inhibition by Compound 1. (C) Provides an illustrative histogram representing cell populations of untreated cells (shaded), DMSO treated cells (dashed lined), specific blocking peptide treated cells (solid line), and

Compound 1 treated cells (dotted line).

[0022] FIG 3. Illustrates the stability of p4EBPl signal in fixed frozen cells. All whole blood samples were treated with DMSO or Compound 1 at 37 °C for 2 hours and PBMCs were fixed and frozen without stabilization buffer. (A) Provides histograms of CD91 + monocytes from freshly prepared whole blood. (B) Provides a bar graph illustrating the stability of p4EBP l signals in the CD91+ monoctye population of frozen cells over a period of 1 month. (C) Provides a table summarizing the viability and mean fluorescence intensity (MF1) of treated cells.

[0023] FIG 4. (A) Illustrates ex vivo treatment of whole blood from healthy donor with Compound 1 for 2 hours at room temperature. Samples were processed in triplicate. Compound 1 treatment showed significant inhibition (p<0.005) at 5 μΜ and 0.5 μΜ. (B) Illustrates ex vivo treatment of whole blood from a healthy donor with Compound 1 at the indicated concentrations in triplicate for 2 hours at room temperature. Samples were processed in triplicate. Compound 1 treatment showed significant inhibition (p<0.005) at 5 μΜ and 0.5 μΜ.

[0024 J FIG 5. Illustrates that p4EBPl is inhibited in peripheral blood of patients.

Each line represents a patient.

[0025] FIG 6. Illustrates that p4EBPl is inhibited in peripheral blood of patients.

Each line represents a patient.

5. DETAILED DESCRIPTION

5.1 DEFINITIONS

[0026] The term "treating" as used herein means an alleviation, in whole or in part, of symptoms associated with a disorder or disease, or slowing, or halting of further progression or worsening of those symptoms.

[0027] The term "effective amount" in connection with an TOR kinase inhibitor means an amount capable of alleviating, in whole or in part, symptoms associated with a disease or disorder, or slowing or halting further progression or worsening of those symptoms. The effective amount of the TOR kinase inhibitor, for example in a

pharmaceutical composition, may be at a level that will exercise the desired effect; for example, about 0.005 mg/kg of a subject's body weight to about 100 mg/kg of a patient's body weight in unit dosage for both oral and parenteral administration. As will be apparent to those skilled in the art, it is to be expected that the effective amount of a TOR kinase inhibitor disclosed herein may vary depending on the severity of the indication being treated.

[0028] The terms "patient" and "subject" as used herein include an animal, including, but not limited to, an animal such as a cow, monkey, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, in one embodiment a mammal, in another embodiment a human. In one embodiment, a "'patient ^" or "subject" is a human having a disease provided herein, such as a disease associated with a TOR kinase.

[0029] The term "biological sample" as used herein includes blood samples and tissue samples, such as tumor samples. In one embodiment, the biological sample is a peripheral blood sample. In another embodiment the biological sample is plasma. In another embodiment, the biological sample is platelet rich plasma.

[0030] The term "TOR kinase inhibitor" as used herein means a compound (e.g. a small molecule) or a biologic (e.g., a protein) capable of inhibition of TOR kinase activity (i.e., in vitro or in vivo). In one embodiment, the TOR kinase inhibitor is a compound disclosed in WO 2008/023161 (see, e.g., page 5, line 5 to page 11, line 15), WO

2009/007751 (see, e.g., page 9, line 8 to page 26, line 8), WO 2009/007749 (see, e.g., page 9, line 21 to page 29, line 23), WO 2009/007750 (see, e.g., page 9, line 21 to page 32, line 22), WO 2009/007748 (see, e.g., page 9, line 6 to page 42, line 28), WO 2008/032028 (see, e.g., page 11, line 13 to page 21, line 13), WO 2008/032086 (see, e.g., page 10 line 21 to page 15, line 22), WO 2008/032072 (see, e.g., page 11, line 11 to page 16, line 13), WO 2008/032033 (see, e.g., page 11, line 3 to page 16, line 5), WO 2008/032089 (see, e.g., page 11. line 11 to page 16, line 13), WO 2008/032060 (see, e.g.. page 11, line 3 to page 16, line 6), WO 2008/032091 (see, e.g., page 11, line 11 to page 16, line 13), WO 2008/032036 (see, e.g., page 11, line 13 to page 21, line 13), WO 2008/032077 (see, e.g., page 10, line 21 to page 15, line 22), WO 2008/032064 (see, e.g., page 11, line 3 to page 16, line 5), WO 2008/032027 (see. e.g., page 10, line 21 to page 15. line 22). WO 2007/135398 (see. e.g., page 11, line 28 to page 16, line 6), WO 2007/129052 (see, e.g., page 10, line 8 to page 13, line 5), WO 2007/129044 (see, e.g., page 10. line 22 to page 13, line 20), WO 2007/080382 (see, e.g., page 9, line 20 to page 32, line 32), WO 2007/066102 (see, e.g., page 9, line 22 to page 14, line 17), WO 2007/066099 (see, e.g., page 9, line 22 to page 14, line 14), WO 2007/066103 (see, e.g., page 9, line 22 to page 14, line 16), WO 2007/060404 (see, e.g., 5, line 4 to page 7, line 25), WO 2006/090169 (see, e.g., page 4, lines 1-25), WO 2006/090167 (see, e.g., page 3, line 33 to page 6, line 23), WO 2008/115974 (see, e.g., page 4, paragraph [0012] to page 127, paragraph [0257]), WO 2009/052145 (see, e.g., page 5, paragraph

[0015] to page 81, paragraph [0082]), WO 2010/006072 (see, e.g., page 28, line 1 to page 34, line 1), WO 2007/044698 (see, e.g., page 3, paragraph [0010] to the bottom of page 7), WO 2007/044813 (see, e.g., page 3, paragraph [0010] to the middle of page 7), WO

2007/044729 (see, e.g., page 3, paragraph [0010] to the bottom of page 10), WO

2007/129161 (see, e.g., page 2, line 10 to page 9, line 19), WO 2006/046031 (see, e.g., page 2, line 15 to page 4, line 12), WO 2003/072557 (see, e.g., page 1, line 4 to page 2, line 27), WO 2004/048365 (see, e.g., page 1, line 4 to page 4, line 17). WO 2004/078754 (see, e.g., page 1 , line 4 to page 2, line 21), WO 2004/096797 (see, e.g., page 1 , line 4 to page 2, line 34). WO 2005/021519 (see, e.g. , page 1 , line 4 to page 4, line 17) or US 2007/1 12005 (see, e.g., page 2. paragraph [0012] to page 22, paragraph [0065]), each of which is incorporated by reference herein in its entirety. TOR kinase inhibitors can be obtained via standard, well- known synthetic methodology, see e.g., March, J. Advanced Organic Chemistry; Reactions Mechanisms, and Structure, 4th ed., 1992. Starting materials useful for preparing compounds of formula (III) and intermediates therefore, are commercially available or can be prepared from commercially available materials using known synthetic methods and reagents. Particular methods for preparing TOR kinase inhibitors are disclosed in U.S. Application No. 1 1/975,652, filed October 18, 2007, U.S. Application No. 1 1/975,657, filed October 18, 2007 and U.S. Application No. 12/605,791, filed October 26, 2009, each of which is incorporated by reference herein in its entirety. In a specific embodiment, the TOR kinase inhibitors do not include rapamycin or rapamycin analogs (rapalogs).

5.2 METHODS OF USE

[0031] Without being limited by theory, it is believed that because 4EBP 1 (4E- binding protein 1 , also referred to herein as "p4EBPl") is a direct substrates of TOR kinase (i.e. , TOR kinase catalyzes the phosphorylation of 4EBP1 to p4EBPl) the inhibition of TOR kinase activity in vivo can be measured by determining a subject ^' s p4EBPl levels (e.g., levels in a biological sample from a subject) pre- and post-treatment with a TOR kinase inhibitor.

[0032] Accordingly, provided herein are methods for detecting or measuring the inhibition of TOR kinase activity in a subject, comprising measuring the amount of p4EBPl in a biological sample from said subject prior to and after the administration of a TOR kinase inhibitor. In one embodiment, the amount of p4EBPl is measured using How cytometry.

[0033] In one embodiment, the amount of p4EBP l in whole blood from a subject is measured. In another embodiment, the amount of p4EBPl in peripheral blood mononuclear cells (PBMCs) from a subject is measured. In another embodiment, the amount of p4EBPl in a tissue sample from a subject is measured. In another embodiment, the amount of p4EBPl in a tumor sample from a subject is measured.

[0034J Further provided herein are methods for determining a dose-response relationship for the administration of a TOR kinase inhibitor to a subject, wherein said subject is administered varying doses of said TOR kinase inhibitor and the amount of TOR kinase activity inhibition in said subject resulting from each dose of said TOR kinase inhibitor is determined using a method provided herein.

[0035] Further provided herein are methods for determining whether a subject is sensitive to a TOR kinase inhibitor, comprising administering said subject said TOR kinase inhibitor and determining whether or not TOR kinase activity is inhibited in said subject using a method provided herein.

[0036] Further provided herein are methods for determining the effective amount of a TOR kinase inhibitor for the treatment or management of a disease in a subject, comprising administering said subject varying doses of said TOR kinase inhibitor and determining the amount of TOR kinase activity inhibition in said patient resulting from each dose of said TOR kinase inhibitor using a method provided herein.

[0037] Further provided herein are methods for treating or managing a disease associated with TOR kinase in a patient having a disease associated with TOR, comprising administering to said patient an effective amount of a TOR kinase inhibitor, wherein the effective amount of said TOR inhibitor is determined using a method provided herein.

[0038] In certain embodiments, the methods provided herein are carried out by way of contacting a biological sample from a patient with a TOR kinase inhibitor ex vivo. For example, instead of administration of a TOR kinase inhibitor to a subject, methods provided herein can comprise measuring the amount of p4EBPl in a biological sample from a subject, contacting said biological sample with a TOR kinase inhibitor ex vivo, followed by measurement of the amount of p4EBPl in said biological sample after said contacting. Accordingly, provided herein are methods for detecting or measuring the inhibition of TOR kinase activity in a biological sample from a subject, comprising measuring the amount of p4EBPl in said biological sample prior to and after contacting said biological sample with a TOR kinase inhibitor ex vivo. In one embodiment, the amount of p4EBPl is measured using flow cytometry.

[0039] In certain embodiments, an IC ₅ o value of about 250 μΜ or less, about 100 μΜ or less, about 10 μΜ or less, about 1 μΜ or less or about 0.10 μΜ or less indicates that a TOR kinase inhibitor is effective for treating a disease associated with TOR kinase activity, such as a disease provided herein.

[0040] Further provided herein are kits comprising one or more containers filled with reagents for detecting p4EBP l using flow cytometry and instructions for detecting p4EBPl using flow cytometry. In one embodiment, the kits comprise one or more containers filled with an anti-p4EBPl antibody. In certain embodiments, the anti-p4EBP l antibody is Alexa Fluor 647 mouse anti-p4EBP l or an anti-p4EBP l antibody conjugated to Alexa Fluor 488. In certain emobidments, kits provided herein further comprise one or more TOR kinase inhibitors, such as those provided herein, incorporated by reference herein or those known in the art.

[0041] In one embodiment, the patient has a solid tumor cancer. In another embodiment, the patient has a blood cancer. In a particular embodiment, the blood cancer is leukemia, such as chronic leukemia, acute leukemia, erythroleukemia, lymphocytic leukemia, myeloid leukemia or myelogenous leukemia, in another embodiment, the patient has a blood cancer other than lymphoblastic leukemia (e.g. , T-cell acute lymphoblastic leukemia).

5.3 ILLUSTRATIVE TOR KINASE INHIBITORS

[0042] Illustrative TOR kinase inhibitors include, but are not limited to, compounds having the following formula (I):

R ²

R ³

N . and pharmaceutically acceptable salts, clathrates, solvates, stereoisomers, tautomers, and prodrugs thereof, wherein:

R ^l is substituted or unsubstituted C j.g alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyi, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl;

R ² is H, substituted or unsubstituted C|.g alkyl, substituted or unsubstituted cycloalkyi, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkylalkyl; and

R ³ and R ⁴ are each independently H, substituted or unsubstituted C |.g alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyi, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkylalkyl, or R ³ and R ⁴ , together with the atoms to which they are attached, form a substituted or unsubstituted cycloalkyi or substituted or unsubstituted heterocyclyl;

or R ² and one of R ³ and R ⁴ , together with the atoms to which they are attached, form a substituted or unsubstituted heterocyclyl.

[0043 J in certain embodiments, the TOR kinase inhibitor is not a compound depicted below, namely:

6-(4-hydroxyphenyl)-4-(3-methoxybenzyl)-3,4-dihydropyrazino[ 2,3-b]pyrazin-

6-(4-(l H-l ,2,4-triazol-5-yl)phenyl)-3-(cyclohexylmethyl)-3,4-dihydropy razino[2,3- b]pyrazin-2( 1 H)-one;

or,

(R)-6-(4-(lH-l ,2,4 riazol-5-yl)phenyl)-3-(cyclohexylmethyl)-3,4-dihydropyrazino [2,3- b]pyrazin-2(lH)-one.

[0044] In some embodiments of compounds of formula (I), R ¹ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In one embodiment, R ^s is phenyl, pyridyl, pyrimidyl, benzimidazolyl, indolyl, indazolyl, l H-pyrrolo[2,3-b]pyridyl, 1 H-imidazo[4,5-b]pyridyl, lH-imidazo[4,5-b]pyridin-2(3H)-onyl, 3H-imidazo[4,5- b]pyridyl, or pyrazolyl, each optionally substituted. In some embodiments, R ¹ is phenyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C | . ₈ alkyl (for example, methyl), substituted or unsubstituted heterocyclvl (for example, substituted or unsubstituted triazolyl or pyrazolyl), halogen (for example, fluorine), aminocarbonyl, cyano, hydroxyalkyl (for example, hydroxypropyl), and hydroxy. In other embodiments, R ¹ is pyridyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C[.g alkyl, substituted or unsubstituted heterocyclyl (for example, substituted or unsubstituted triazolyl), halogen, aminocarbonyl, cyano, hydroxyalkyl, -OR, and -NR ₂ , wherein each R is independently H, or a substituted or unsubstituted C alkyl. In yet other embodiments, R ¹ is l H-pyrrolo[2,3-b]pyridyl or benzimidazolyl, each optionally substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C s alkyl, and -NR ₂ , wherein each R is independently H, or a substituted or unsubstituted C 1.4 alkyl.

(0045] In some embodiments of compounds of formula (I), R ¹ is

wherein R is at each occurrence independently H, or a substituted or unsubstituted C alkyl (for example, methyl); R' is at each occurrence independently a substituted or unsubstituted C alkyl, halogen (for example, fluorine), cyano, -OR, or -NR2; m is 0-3; and n is 0-3. It will be understood by those skilled in the art that any of the subsitutuents R' may be attached to any suitable atom of any of the rings in the fused ring systems. It will also be understood by those skilled in the art that the connecting bond of R ¹ (designated by the bisecting wavy line) may be attached to any of the atoms in any of the rings in the fused ring systems.

[00

- I J wherein R is at each occurrence independently H, or a substituted or unsubstituted C alkyl; R' is at each occurrence independently a substituted or

unsubstituted C M alkyl, halogen, cyano, -OR, or -NR ₂ ; m is 0-3 ; and n is 0-3.

[0047} In some embodiments of compounds of formula (I), R~ is H, substituted or unsubstituted Ci _-8 alkyl, substituted or unsubstituted cycloalkyi, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C alkyl-heterocyclyi, substituted or

unsubstituted C alkyl-aryl, or substituted or unsubstituted C M alkyl-cycloalkyl. For example, R ² is H, methyl, ethyl, n-propyl, isopropyl. n-butyl, .sec-butyl, isobutyl, (ert-butyi, n-pentyl, isopentyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl,

(C alkyl)-phenyl, (C M alkyl)-cyclopropyl, (C alkyl)-cyclobutyl,

(C M alkyl)-cyclopentyl, (C alkyl)-cyclohexyl, (C M aikyl)-pyrrolidyl,

(C alkyl)-piperidyl, (C alkyl )-piperazinyl, (C M alkyl)-morpholinyl,

(C|- alkyl)-tetrahydrofuranyl, or (C ₁ . alkyl)-tetrahydropyranyl, each optionally substituted.

[0048] In ot ,

wherein R is at each occurrence independently H, or a substituted or unsubstituted C M alkyl ( for example, methyl); R' is at each occurrence independently H, -OR, cyano, or a substituted or unsubstituted C M alkyl (for example, methyl); and p is 0-3.

[0049 J In some such embodiments, R ² is H, C alkyl, ( CY alkyl)(OR),

wherein R is at each occurrence independently H, or a substituted or unsubstituted Ci _-2 alkyl; R' is at each occurrence independently H, -OR, cyano, or a substituted or unsubstituted C| _-2 alkyl; and p is 0-1.

[0050] In some other embodiments of compounds of formula (I), R ² and one

R ³ and R ⁴ together with the atoms to which they are attached form a substituted or unsubstituted heterocyclyl. For example, in some embodiments, the compound of formula (I) is

wherein R is at each occurrence independently H, or a substituted or unsubstituted C | _ ₄ alkyl; R" is H, OR, or a substituted or unsubstituted C M alkyl; and R ¹ is as defined herein.

[0051 ] In some embodiments of compounds of formula (I), R ³ and R ⁴ are both H. In others, one of R ³ and R ⁴ is H and the other is other than H. In still others, one of R ³ and R ⁴ is C | -4 alkyl (for example, methyl) and the other is H. in still others, both of R ³ and R ⁴ are C M alkyl (for example, methyl). [0052] in some such embodiments described above, R ¹ is substituted or

unsubstituted aryl, or substituted or unsubstituted heteroaryl. For example, R ¹ is phenyl, pyridyl, pyrimidyl, benzimidazolyl, indolyl, indazolyl, l H-pyrrolo[2,3-b]pyridyl,

1 H-imidazo[4,5-b]pyridyl, 1 H-imidazo[4,5-b]pyridin-2(3H)-onyl,

3H-imidazo[4,5-b]pyridyl, or pyrazolyl, each optionally substituted. In some embodiments, R ¹ is phenyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted d _-8 alkyl, substituted or unsubstituted heterocyclyl, halogen, aminocarbonyl, cyano, hydroxyalkyl and hydroxy. In others, R ¹ is pyridyl substituted with one or more substituents independently selected from the group consisting of cyano, substituted or unsubstituted C | _-8 alkyl, substituted or unsubstituted heterocyclyl, hydroxyalkyl, halogen, aminocarbonyl, -OR, and -NR ₂ , wherein each R is independently H, or a substituted or unsubstituted C alkyl. In others, R ¹ is

lH-pyrrolo[2,3-b]pyridyl or benzimidazolyl, optionally substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C | -8 alkyl, and -NR ₂ , wherein R is independently H, or a substituted or unsubstituted C alkyl

[0053] In certain embodiments, the compounds of formula (I) have an R ¹ group set forth herein and an R ² group set forth herein.

[0054] In some embodiments of compounds of formula (I), the compound is

6-( lH-pyrrolo[2,3-b]pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-y l)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(4-methyl-6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4-yl )methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)-4-((/ra«i-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(l H)-one;

6-( 5-tluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl )phenyl)-4-((c»-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one;

6-(6-( l H-l ,2.4-triazol-3-yl)pyridin-3-yl)-4-((rr ns-4-methoxycyclohexyI)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-4-((ir m'-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2J-b]pyra2;in- 2( l H)-one;

6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((c 4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((rram-4-hydroxycyclohexyl )niethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(l H- l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(c/.v-4-hydroxycyclohexyl) -3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(lH- l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((cw-4-hydroxycyclohexyl)m ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-fluoro-2-methyl-4-(lH-l ,2,4-triazol-3-yl)phenyl)-4-(/ra¾y-4-methoxycyclohexyl)-3,4 - dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(lH-l ,2,4-triazol-3-yl)pyridm-3-yl)-4-(iram-4-methoxycyclohexyl)- 3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(lH-1.2,4-triazol-3-yl)pyridin-3-yl)-4-(/rara-4-hydroxy cyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)-4-((c/v-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one;

6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(cM-4-methoxycyclohexyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2f 1 H)-one;

6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(2-methoxyethy])-3,4-dihyd ropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-isopropyl-3,4-dihydropyraz ino[2,3-b]pyrazin- 2(l H)-one;

6-(5-fluoro-2-methyl-4-( l H-l ,2,4 riazol-3-yl)phenyl)-4-(c .v-4-hydroxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-iluoro-2-methyl-4-( l H- l ,2,4-triazol-3-yl)phenyl)-4-(cw-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(5-fluoro-2-methyl-4-( l H- L2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 6-(6 l H-l ,2,4-triazol-3-yl)pyridin-3-yI)-4-(tetrahydro-2H-pyran-4-yl) -3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-( 1 H-1 ,2,4-triazol-3-yl)pyridin-3-yl)-4-ethyl-3,4-dihydropyrazino[ 2,3-b]pyrazin-2( 1 H)- one;

6-(5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-4-(/ram-4-hydroxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-fluoro-2-methyl-4-( l H-l,2,4-triazol-3-yl)phenyl)-4-(tetrahydro-2H-pyran-4-yl)-3, 4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(5-fluoro-2-methyl-4-(l H-l ,2,4-triazol-3-yl)phenyl)-4-isopropyl-3,4-dihydropyrazino[2, 3- b]pyrazin-2( 1 H)-one;

4-ethyl-6-(5-fluoro-2-methyl-4-( l H-l ,2,4-triazol-3-yl)phenyl)-3,4-dihydi pyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(3-fluoro-2-methyl-4-(lH-l,2,4-triazol-3-yl)phenyI)-4-(tet rahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(3-fluoro-2-methyl-4-(l H-l ,2,4-triazol-3-yl)phenyl)-4-(cw-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(3-fluoro-2-methyl-4-( l H-l,2,4-triazol-3-yl)phenyl)-4-(/ ^l mm-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

4- (2-methoxyethyl)-6-(4-methyl-6-( l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(3-( l H-l ,2,4-triazol-5-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)eth yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

5- (8-(2-methoxyethyl)-6-oxo-5,6,7,8-tetrahydropyrazino[2,3-b]p yrazin-2-yl)-4- methyipicolinamide;

3-(6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetra hydropyrazino[2.3-b]pyrazin-2- yl)benzamide;

3-(6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetra hydropyrazino[2,3-b]pyrazin-2- y])benzonitrile;

5-(8-(/ram-4-methoxycyclohexyl)-6-oxo-5,6,7,8-tetrahydropyra zino[2,3-b]pyrazin-2-yl)-4- methylpicolinamide; 6-(lH-imidazo[4,5-b]pyridin-6-yl)-4-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(l H-indazol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-di hydropyrazino[2,3- bJpyrazin-2( 1 H)-one;

4-((l R,3S)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l,2,4-triazol- 3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

4-(( 1 S JR)-3-methoxycyciopentyl)-6-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

4-((l R,3R)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

4-((l S,3S)-3-methoxycyclopentyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3.4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

4-ethyl-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(l H-pyrrolo[2,3-b]pyridin-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl )ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

6-(lH-indol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(l H-indol-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethy])-3,4-dihy dropyrazino[2,3- b]pyrazin-2(lH)-one;

4-(((lR S)-3-methoxycyclopentyl)methyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

4-(((lS,3R)-3-methoxycyclopentyl)methyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yI)pyridin-3- yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(3-fluoro-2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

6-(3-fluoro-2-niethyI-4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one:

3J-dimethyl-6-(4-methyl-6-(4H-1.2,4-triazol-3-yl)pyridin-3-y l)-4-((tetrahydro-2H-^^ yl)methyl)-3,4-dihydropyrazino[2,3-bjpyrazin-2( l H)-one; 6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(( l R,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(( lS,3R)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((( l S,3S)-3-methoxycyclopentyl)methyl)-3,4- dihydropyr azino [2 , 3 -b]py razin-2( 1 H)-one ;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((l R,3R)-3-methoxycyclopeniyl)rnethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((l S,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((lR,3R)-3-methox ycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((( lR,3S)-3-methoxycyclopentyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(((lS,3R)-3-metho xycyclopentyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(3-fluoro-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4-dihydropyra zino[2,3- b]pyrazin-2( 1 H)-one;

6-(3-fluoro-4-(4H- l ,2,4-triazo]-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)eth yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3-yl)phenyl )- 1 '-((tetrahydro-2H-pyran-4-yl)methyl)- 1 Ή- spiro[cyclopentane-l ,2'-pyrazino[2,3-b]pyrazin]-3'(4'H)-one;

7'-(2-methy l-4-(4H- 1 ,2,4-triazol-3 -yl)pheny 1)- 1 '-((tetrahydro-2H-pyran-4-yl)methyl)- 1 Ή- spirofcyclobutane- 1 ,2'-pyrazino[2,3-b]pyrazin]-3'(4'H)-one;

4-(cyclopropylmethyi)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-y l)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3-yl)phenyl)- 1 Ή-spirofcyclopentane-l ,2'-pyrazino[2,3- b]pyrazin]-3'(4'H)-one;

7'-(2-methyl-4-(4H- 1 ,2,4-triazol-3-yl)phenyl)- 1 'H-spiro[cyclobutane- 1 ,2'-pyrazino[2,3- b]pyrazinj-3'(4'H)-one; 7'-(2-methyl-4-(4H-1.2,4-triazol-3-yl)phenyl)-l 'H-spiro[cyclopropane-1.2'-pyrazino[2,3- b]pyrazin]-3'(4'H)-one;

(R)-6-(4-(4H-L2.4-triazol-3-yl)phenyl)-4-((tetrahydrofuran-2 -yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

(S)-6-(4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-((tetrahydrofuran-2-yl)methyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-( l H-indazol-5-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-di hydropyrazino[2,3- bjpyrazin-2( 1 H)-one;

4-(6-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetra hydropyrazino[2 _! 3-b]pyrazin-2- yl)benzamide;

4-(2-methoxyethyl)-3,3-dimethyl-6-(2-methyl-4-(4H-l,2,4-tria zol-3-yl)phenyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

4-ethyl-3,3-dimethyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl phenyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(l H)-one; 33-dimethyl-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

(R)-6-(6-(l-hydroxyethyl)pyridin-3-yl)-4-(2-(tetrahydro-2H-p yran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

3.3- dimethyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

6-(6-(2-hydroxypropan-2-yl)-4-methylpyridin-3-yl)-4-(/ra/7.s *-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2 ydroxypropan-2-yl)-4-rnethylpyridin-3-yl)-4-((tetrahydro-2H- pyran-4-yl)methyl)-

3.4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

3 -dimethyl-6-(2-methyl-4-(4H- l ,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

3 J-dimethyl-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yi)-4-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 6-(6-(2-hydroxypropan-2-yl)-2-methylpyridin-3-yl)-4-((tetrah ydro-2H-pyran-4-yl)methyi)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)-2-methylpyridin-3-yl)-4-(tr «s-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

(S)-6-(6-(l-hydroxyethyl)pyridin-3-yl)-4-(2-(tetrahydro-2H-p yran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

3.3- dimethyl-6-(2-methyl-4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,3-dimethyl-4-(2-( tetrahydro-2H-pyran-4- yl)ethyI)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

6-(4-(2-hydroxypropan-2-yl)phenyl)-4-(ira«5-4-methoxycyc lohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(4-(2-hydroxypropan-2-yl)phenyl)-4-((/r«m-4-methoxycycloh exyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

4-(cM'-4-methoxycyclohexyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3.4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

4-(/ram-4-methoxycyclohexyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(4-(2-hydroxypropan-2-yl)phenyl)-4-((tetrahydro-2H-pyran-4 -yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

4- (2-methoxyethyl)-6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

9-(6-(4H-l ,2,4-triazol-3-yl)-3-pyridyl)-6,l l ,4a-trihydromorpholino[4,3-e]pyrazino[2,3- b]pyrazin-5-one;

6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-((tetrahydro-2H-pyran-4-yl )methyl)-

3.4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

5- (8-(m-4-methoxycyclohexyl)-6-oxo-5,6,7,8-tetrahydropyrazino[ 2,3-b]pyrazin-2-yl)-6- methylpicolinonitrile;

6- (6-(4H-l ,2,4 riazol-3-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4-yl )ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 9-(4-(4H-1.2.4-triazol-3-yl)-2-methylphenyl)-3-(2-methoxyace tyl)-6,l l ,4a- trihydropiperazino[ 1 ,2-e]pyrazino[2,3-b]pyrazin-5-one;

9-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)-6,l 1 ,4a-trihydropiperazino[ 1 ,2- e]pyrazino[2,3-b]pyrazin-5-one;

9-(4-(4H- 1 ,2,4-triazol-3-yl)-2-methylphenyl)-3-(2-methoxyethyl)-6, 1 1 ,4a- trihydropiperazino[l ,2-e]pyrazino[2,3-b]pyrazin-5-one;

4-(cyclopentylmethyl)-6-(2-methyl-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

9-(6-(4H- 1 ,2,4-triazoI-3-yl)-2-methyI-3-pyridyI)-6, 1 1 ,4a-trihydromorpholino[4,3- ejpyrazino[2,3-b]pyrazin-5-one;

4-(/raM5-4-hydroxycyclohexyl)-6-(6-(2-hydroxypropan-2-yl)pyr idin-3-yl)-3,4- dihydropyrazino [2 ,3 -b]pyrazin-2( 1 H)-one ;

4-(cw-4-hydroxycyclohexyl)-6-(6-(2-hydroxypropan-2-yl)pyridi n-3-yl)-3.4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydrofuran -3-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

4-(cyclopentylmethyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-y l)-3,4-dihydropyrazino b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-neopentyl-3,4-dih ydropyrazino[2,3-b]pyr 2(lH)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-isobutyl-3,4-dihy dropyrazino[2,3-b]pyraz 2(l H)-one;

3-methyl-6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(piperidin-4-yl)- 3,4-dihydropyrazino[2,3- b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-y )-4-(2-(tetrahydro-2H-pyran-3-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

8-(4-(4H- l ,2,4-triazol-3-yl)-2-methylphenyl)(3aS,2R)-2-methoxy-5, 10,3a- irihydropyrazino[2,3-b]pyrrolidino[ 1 ,2-eJpyrazin-4-one; 8-(4-(4H-l,2,4-triazol-3-yl)-2-methylphenyl)(2R,3aR)-2-metho xy-5, 10,3a- trihydropyrazino[2,3-b]pyrrolidino[ 1 ,2-e]pyrazin-4-one;

8-(4-(4H-l,2,4-triazol-3-yl)-2-methylphenyl)(2S,3aR)-2-me thoxy-5,10,3a- trihydropyrazino[2,3-b]pyrrolidino[ 1 ,2-ejpyrazin-4-one:

8- (4-(4H- l,2,4-triazol-3-yr)-2-methylphenyl)(2S,3aS)-2-methoxy-5,10,3 a- trihydropyrazino[2,3-b]pyrrolidino[l ,2-e]pyrazin-4-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(3-methoxypropyl) -3,4-dihydropyrazino[2,3- b]pyrazin-2(l H)-one;

(S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydrof uran-2-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

(R)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydrof uran-2-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

6-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one;

9- (4-(4H-l,2,4-triazol-3-yl)-2-methylphenyl)-3-methyl-6,l 1 ,4a-trihydropiperazino[l ,2- e] pyr azino [2,3 -b] py razin- 5 -one ;

9-(4-(4H-l,2,4-triazol-3-yl)phenyl)-6, l l ,4a-trihydromorpholino[4,3-e]pyrazino[2,3- b]pyrazin-5-one;

9-(4-(4H- 1 ,2,4-triazol-3-yl)-2-methylphenyl)-6, 1 1 ,4a-trihydropiperidino[l ,2-e]pyrazino[2,3- b]pyrazin-5-one;

6-(6-(2-hydroxypropan-2-yi)pyridin-3-yl)-4-(/ra«.s'-4-metho xycyclohexyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(c s-4-methoxycycIohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-rnorpholinoeth yl -3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-phenethyl-3,4-dih ydropyrazino[2,3-b]pyrazin- 2( l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydro-2H-py ran-4-yI)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 4-(cyclohexylmethyl)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl )-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((/ram-4-methoxyc yclohexyi)methyi)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((cw-4-methoxycyc lohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

(R)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydrofu ran-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

(S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(tetrahydrofu ran-3-yl)-3,4- dihydropyrazino [2,3 -b]pyrazin-2( 1 H)-one ;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-phenyl-3,4-dihydr opyrazino[2,3-b]pyrazin- 2(l H)-one;

(S)-6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3-methyl-4-(2-( tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

9-[6-(l-hydroxy-isopropyl)-3-pyridyl]-6,l l,4a-trihydromorpholino[4,3-e]pyrazino[2,3- b]pyrazin-5-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-((tetrahydro-2H-p yran-4-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-methoxyethyl)- 3.4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(2-amino-7-methyl-l H-benzo[d]imidazol-5-yl)-4-(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(3-(trifluorometh yl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

9-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyl)-6, l l ,4a rihydromorpholino[4,3- e]pyrazino[2.3-b]pyrazin-5-one;

6-(4-methyl-2-(methylamino)-l H-benzo[d]imidazol-6-yl)-4-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

8-(4-(4H-l ,2,4-triazol-3-yl)-2-methylphenyI)-5, 10,3a-trihydropyrazino[2,3- b]pyrrolidino[ 1 ,2-eJpyrazin-4-one; 6-(4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-ethyl-3,4-dihydropyrazino[2,3-b] pyrazin-2( lH)-one

6-(4-(4H- l ,2,4-triazol-3-yl)phenyl)-4-((tetrahydro-2H-pyTan-4-yl)methy l)-3,4- dihydropyrazino[2,3-b]pyrazin-2( lH)-one;

6-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-4-(2-(tetrahydro-2H -pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-methoxyethyl)-3,4-dihydropyra zino[2,3-b]pyrazin- 2(l H)-one;

6-(4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

6-(2-methyl-4-(4H-l ,2,4-triazol-3-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)eth yl)-3,4^ dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

6-(4-methyl- l H-benzo[d]imidazol-6-yl)-4-(2-(tetrahydro-2H-pyran-4-yl)ethy l)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

6-(4-(2-hydroxypropan-2-yl)phenyl)-4-(2-(tetrahydro-2H-pyran -4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

6-(4-( l H-l ,2,4-triazol-5-yl)phenyl)-4-(2-(tetrahydro-2H-pyran-4-yl)eth yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( lH)-one; or a pharmaceutically acceptable salt thereof.

[0055] Further illustrative TOR kinase inhibitors include, but are not limited to, compounds having the following formula (II):

(II)

and pharmaceutically acceptable salts, clathrates, solvates, stereoisomers, tautomers, and prodrugs thereof, wherein: R is substituted or unsubstituted Ci-8 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heterocyclylalkyl;

R ² is H, substituted or unsubstituted Ct-s alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocyclylalkyl, substituted or unsubstituted aralkyl, or substituted or unsubstituted cycloalkyialkyl; and

R ³ is H, or a substituted or unsubstituted C|.g alkyl.

[0056] In certain embodiments, the TOR kinase inhibitor is not 7-(4- hydroxyphenyl)-l -(3-methoxybenzyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one, depicted below:

[0057] In some embodiments of compounds of formula (II), R ¹ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryi. For example, R ¹ is phenyl, pyridyl, pyrimidyl, benzimidazolyl, l H-pyrrolo[2,3-b]pyridyl, indazolyl, indolyl, l H-imidazo[4,5-b]pyridyl, lH-imidazo[4,5-b]pyridin-2(3H)-onyl,

3H-imidazo[4,5-b]pyridyl, or pyrazolyl, each optionally substituted. In some embodiments, R ¹ is phenyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C).g alkyl (for example, methyl), substituted or unsubstituted heterocyclyl (for example, a substituted or unsubstituted triazolyl or pyrazolyl), aminocarbonyl, halogen (for example, fluorine), cyano, hydroxyalkyl and hydroxy. In other embodiments, R ^f is pyridyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C i.g alkyl ( for example, methyl), substituted or unsubstituted heterocyclyl ( for example, a substituted or unsubstituted triazolyl), halogen, aminocarbonyl , cyano, hydroxyalkyl (for example, hydroxypropyl), -OR, and -NR ₂ , wherein each R is independently H, or a substituted or unsubstituted C M alkyl. In some embodiments, R ¹ is l H-pyrrolo[2,3-b]pyridyl or benzimidazolyl, optionally substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted Cj.g alkyl, and -NR ₂ , wherein R is independently H, or a substituted or unsubstituted C alkyl.

[0058 J In some embodiments, R ^! is

wherein R is at each occurrence independently H, or a substituted or unsubstituted C alkyl (for example, methyl); R' is at each occurrence independently a substituted or unsubstituted C alkyl (for example, methyl), halogen (for example, fluoro), cyano, -OR, or -NR ₂ ; m is 0-3; and n is 0-3. It will be understood by those skilled in the art that any of the subsitutuents R' may be attached to any suitable atom of any of the rings in the fused ring systems.

[0059] In some embodiments of compounds of formula (II), R ¹ is

wherein R is at each occurrence independently H, or a substituted or unsubstituted C alkyl; R' is at each occurrence independently a substituted or

unsubstituted C alkyl, halogen, cyano, -OR or -NR ₂ ; m is 0-3; and n is 0-3.

[0060] In some embodiments of compounds of formula (II), R ² is H, substituted or unsubstituted C | . ₈ alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted C M alkyl-heterocyclyl, substituted or

unsubstituted C alkyl-aryl, or substituted or unsubstituted C alkyl-cycloalkyl. For example, R ² is H, methyl, ethyl, n-propyl, isopropyl, n-butyi, sec-butyl, isobutyl, /cr/-butyl, n-pentyl, isopentyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl,

(C M alkyl)-phenyl, (C alkyl)-cyclopropyl, (C M alkyI)-cyclobutyl,

(C alkyl)-cyclopentyl, (C alkyl)-cyclohexyl, (C alkyl)-pyrrolidyl,

(CM alkyl)-piperidyl, (C alkyl)-piperazinyl, (C M alkyl)-mo holinyl,

(C M alkyl)-tetrahydrofuranyl, or (C ₁ -4 alkyl)-tetrahydropyranyl, each optionally substituted.

[00 11 In oth

wherein R is at each occurrence independently H, or a substituted or unsubstituted C alkyl (for example, methyl); R' is at each occurrence independently H, -OR, cyano,or a substituted or unsubstituted C alkyl (for example, methyl); and p is 0-3. f0062] In other embodiments of compounds of formula (II), R ² is H, C alkyl,

(C i-4alkyl)(OR),

wherein R is at each occurrence independently H, or a substituted or unsubstituted Ci _-2 alkyl; R' is at each occurrence independently H, -OR, cyano, or a substituted or unsubstituted C | _-2 alkyl; and p is 0- 1 .

[0063] In other embodiments of compounds of formula (II), R ³ is H.

[0064] In some such embodiments described herein, R ¹ is substituted or

unsubstituted aryl, or substituted or unsubstituted heteroaryl. For example, R ¹ is phenyl, pyridyl, pyrimidyl, benzimidazolyl, l H-pyrrolo[2,3-b]pyridyl, indazolyl, indolyl,

1 H-imidazo[4,5-b]pyridine, pyridyl, 1 H-imidazo[4,5-b]pyridin-2(3H)-onyl,

3H-imidazo[4,5-b]pyridyl, or pyrazolyl, each optionally substituted. In some embodiments, R ¹ is phenyl substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted C _\ . _% alkyl, substituted or unsubstituted heterocyclyl, aminocarbonyl, halogen, cyano, hydroxyalkyl and hydroxy. In others, R ¹ is pyridyl substituted with one or more substituents independently selected from the group consisting of Ci _-8 alkyl, substituted or unsubstituted heterocyclyl, halogen, aminocarbonyl, cyano, hydroxyalkyl, -OR, and -NR ₂ , wherein each R is independently H, or a substituted or unsubstituted C M alkyl. In still others, R ¹ is l H-pyrrolo[2,3-b]pyridyl or benzimidazolyl, optionally substituted with one or more substituents independently selected from the group consisting of substituted or unsubstituted Ci _-8 alkyl, and -NRi, wherein R is independently H, or a substituted or unsubstituted C alkyl.

[0065] In certain embodiments, the compounds of formula (II) have an R ¹ group set forth herein and an R ² group set forth herein.

[0066] In some embodiments of compounds of formula (II), the compound is

7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -((trans-4- methoxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2( lH)-one;

7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(c/s-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(lH-pyrrolo[2,3-b]pyridin-3-yl)-l-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazoI-3-yI)phenyl)- 1 -((cw-4- methoxycyclohexyl)methyl)-3.4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one;

l-ethyl-7-(lH-pyrrolo[3,2-b]pyridin-5-yl)-3,4-dihydropyra zino[2,3-b]pyrazin-2(lH)-one;

7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-l -((c/.y-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(lH-benzo[d]imidazol-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl) ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(lH-pyrrolo[2,3-b]pyridin-4-yl)-l-(2-(tetrahydro-2H-pyran- 4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-(lH-1.2,4-triazol-3-yl)pyridin-3-yl)-l-((/ ^, ram-4-methoxycyclohexyr)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)-l-((fr n«-4-hydroxycyclohexyl)methyI)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-( 1 H-l ,2,4-triazol-3-yl)pyridin-3-yl)-l -(c.v-4-hydroxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(5-fluoro-2-methyl-4-(lH-l,2,4-triazol-3-yr)phenyl)-l-( «-4-hydroxycyclohexyi)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)-l-(tetrahydro-2H-p yran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-y t)- 1 -(2-methoxyethyl)-3 ,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(6-(lH-l,2^-triazol-3-\1)pyridin-3-yl)-l-ethyl-3,4-dihydro pyrazino[2,3-b]pyrazin-2(lH)- one;

7-(5-fluoro-2-methyl-4-(lH-l,2,4 riazol-3-yl)phenyl)-l-((m-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one;

7-(5-fluoro-2-methyl-4-( 1 H- 1.2,4-triazol-3 -yl)phenyl)- 1 -(tetrahydro-2H-pyran-4-yl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(lH-indol-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(5-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -((/r m-4- hydroxycyclohexyl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2(lH)-one;

7-(6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)-l-((c^-4-hydrox ycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(6-(lH-l,2,4-triazol-3-yl)pyridin-3-yl)-l-(/7W?.y-4-hydrox ycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(/ra¾y-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(6-(lH-l,2,4-triazol-3-y])pyridin-3-yl)-l-isopropy]-3,4-di hydropyrazino[2,3-b]pyrazin- 2(lH)-one;

7-(5-fluoro-2-methyl-4-(lH-l,2,4-triazol-3-yl)phenyl)-l-(/ra m-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(5-fluoro-2-methyl-4-(lH-l,2,4-triazol-3-yl)phenyl)-l-( ran5-4-hydroxycyc[ohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(5-fluoro-2-methyl-4-(lH-l,2,4-triazol-3-yl)phenyl)-l-(2-m ethoxyethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2(lH)-one;

7-(5-fluoro-2-methyl-4-( 1 H-l ,2.4-triazol-3-yl)phenyl)-l -isopropyl-3,4-dihydropyrazino[2,3- b]pyrazin-2(lH)-one;

l-ethyl-7-(5- Iuoro-2-methyl-4-(lH-l,2,4-triazol-3-yl)phenyl)-3,4-dihydrop yrazino[2,3- b]pyrazin-2( 1 H)-one; 7-(2-hydroxypyridin-4-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

l -isopropyl-7-(4-methyl-6-(l H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

5-(8-isopropyl-7-oxo-5,6J,84etrahydropyrazino[2,3-b]pyrazin- 2-yl)-4-methylpicolinamide; 7-(lH-indazol-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3, 4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(2-aminopyrimidin-5-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

7-(2-aminopyridin-4-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl )-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(6-(methylamino)pyridin-3 -yl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(6-hydroxypyridin-3-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

7-(4-(l H-pyrazol-3-yl)phenyl)-l-(2-methoxyethyl)-3,4-dihydropyrazin o[2,3-b]pyrazin- 2( l H)-one;

7-(pyridin-3-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-d ihydropyrazino[2,3-b]pyrazin- 2(l H)-one;

7-f l H-indazol-4-yl)-l-(2-methoxyethyl)-3,4-dihydropyrazino[2,3-b ]pyrazin-2(l H)-one; 7-(lH-indazol-6-yl)-l-(2-methoxyethyl)-3,4-dihydropyrazino[2 ,3-b]pyrazin-2(l H)-one; 7-(pyrimidin-5-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

7-(6-raethoxypyridin-3-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

1 -(2-methoxyethyl)-7-( 1 H-pyrrolo[2,3-b]pyridin-5-yl)-3,4-dihydropyrazino[2,3-b]pyra zin- 2( l H)-one;

1 -ethyl-7-( 1 H-pyrrolo[2,3-b]pyridin-5-yl)-3.4-dihydropyrazino[2,3-bJpyra zin-2( 1 Fi)-one; l-ethyl-7-( l H-indazol-4-yl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one; 7-(pyridin-4-y 1)- 1 -(2-(tetrahydro-2H-pyran-^

2(l H)-one;

7-(6-aminopyridin-3-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( I H)-one;

1 - methyl-7-(2-methyl-6-( H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

2- (2-hydroxypropan-2-yl)-5-(8-(/ram-4-methoxycyclohexyl)-7-oxo -5, 6,7,8- tetrahydropyrazino[2,3-b]pyrazin-2-yl)pyridine 1 -oxide;

4- methyl-5-(7-oxo-8-((tetrahydro-2H-pyran-4-yl)methyl)-5,6,7,8 -tetrahydropyrazino[2,3- b]pyrazin-2-yl)picolinamide;

5- (8-((c/5-4-methoxycyclohexyl)methyl)-7-oxo-5,6,7.8-tetrahydr opyrazino[2,3-b]pyrazin-2- yl)-4-methylpicolinamide;

7-(l H-pyrazol-4-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2,3 - b]pyrazin-2( 1 H)-one;

1 -(/ra/75-4-methoxycyclohexyl)-7-(4-methyl-6-( 1 H- 1 ,2,4-triazol-3 -yl)pyridin-3-y l)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

3 -((7-(2-methyl-6-(4H- 1 ,2,4-triazol-3 -yl)pyridin-3 -yl)-2-oxo-3 ,4-dihydropyrazino[2,3- b]pyrazin- 1 (2H)-yl)methyl)benzonitrile;

1 -((/ri/m-4-methoxycyclohexy])methy])-7-(4-meihy]-6-(l H- 1 ,2,4-triazol-3-y])pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

3- (7-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetrahy dropyrazino[2,3-bjpyrazin-2- yl)benzamide;

5-(8-((iram'-4-methoxycyclohexyl)methyl)-7-oxo-5,6,7,8-tetra hydropyrazino[2,3-b]pyrazin-

2- yl)-4-methylpicolinamide;

3- ((7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-2-oxo-3,4-dihydro pyrazino[2,3-b]pyrazin- 1 (2H)-yl)methyl)benzonitrile;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(( 1 R,3R)-3-methoxycyclopentyl)-3,4- dihydropyrazinof 2.3-b]pyrazin-2( 1 H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -((l S,3R)-3-methoxycyclopentyl)-3,4- dihydropyrazino 2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- l-((l S,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)- l -({ l R,3S)-3-methoxycyclopentyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-( l H-indazoI-6-yl)- l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihydropyrazino[2, 3- b]pyrazin-2( 1 H)-one;

7-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(2-morpholinoethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

l-(/rara-4-hydroxycyclohexyl)-7-(2-methyl-6-(4H-1.2,4-iri azol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3 -b] pyrazin-2( 1 H)-one;

l -(d5-4-hydroxycyclohexyl)-7-(2-methyi-6-(4H-l,2,4-triazol-3- yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(2-morpholinoethy l)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

l-isopropyl-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-( l H-imidazo[4,5-b]pyridin-6-yl)-l-(2-(tetrahydro-2H-pyran-4-yl )ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

1 -((c«-4-methoxycycIohexyl)methyl)-7-(2-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

l-(/ram-4-hydroxycycLohexyl )-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

l -(t7.s'-4-hydroxycyclohexyl)-7-(6-(2-hydroxypropan-2-yl)pyri din-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

4-(7-oxo-8-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-5,6,7,8-tetra hydropyrazino[2,3-b]pyrazin-2- yl)benzamide;

7-( l H-indazol-5-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3.4-dihydropyrazino[2.3 - b]pyrazin-2( 1 H)-one;

7-(l H-pyrrolo[2,3-b]pyridin-5-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(2-methyl-6-(4H- 12^triazol-3-yl)pyridin-3-yl)-l-(tetrahydro-2H-pyran-4-yl)-3 ,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

1 -(( 1 S,3R)-3-methoxycyclopentyl)-7-(2-methyi-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

l -(( lR R)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

l -(( lR,3S)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

l-((l S,3S)-3-methoxycyclopentyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-( H-indol-5-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dihy dropyrazino[2,3- b ]pyrazin-2( 1 H)-one;

l-ethyl-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-( lH-indol-6-yl)-l-(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4-dih ydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(4-(2-hydroxypropan-2-yl)phenyl)-l -(/ram-4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-y])-l -(tetrahydro-2H-pyran-4-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

1 -((iram-4-methoxycyclohexyl)methyl)-7-(2-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one;

7-(6-(2-hydroxypropan-2-yI)pyridin-3-y[)- l- (cM-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

l-(2-methoxyethyl)-7-(4-methyl-2-(methylamino)-l H-benzo[d]imidazol-6-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(7-methyl-2-oxo-2J-dihydro-lH-benzo[d]imidazol-5-yl)-l -((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

7-(2-methyl-4-(4H- 1 ,2,4-triazol-3-yl)phenyl)-3,4-dihydropyrazino[2,3-b]pyrazin- 2( 1 H)-one; l -(2-methoxyethyl)-7-(4-methyl-6-(l H- l ,2,4-triazol-3-yl)pyridin-3-yl)-3.4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

l -benzyl-7-(2-methyl-4-(4H-l,2,4-triazol-3-yl^

2( l H)-one;

7 3-fluoro-4-(4H-l ,2,4-triazol-3-yl)phenyl)-l -(2-methoxyethyl)-3,4-dihydropyrazino[2.3- b]pyrazin-2( 1 H)-one;

7-(3-fluoro-4-(4H-l _? 2,4-triazol-3-yl)phenyl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(3-fluoro-2-niethyl-4-(l H-l ,2,4-triazol-3-yl)phenyl)-l-(2-methoxyethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

l -(/ram-4-methoxycyclohexyl)-7-(2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(/r m ^, -4-methoxycyclohexyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( l H)-one*;

7-(5-fluoro-2-methyl-4-(4H-1 ,4-triazol-3-yl)phenyl)-l - 2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(3-fluoro-2-methyl-4-( 1 H- 1 ,2,4-triazol-3-yl)phenyl)- 1 -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

l -(2-methoxyethyl)-7-i2-methyl-6-(4H-l ,2,4-triazol-3-yl)pyridin-3-y])-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(6-(2-hydroxypropan-2-y])pyridin-3-yl)-l -((7ram'-4-methoxycyclohexyl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( lH)-one;

l-(cyclopentylmethyl)-7-(6-(2-hydroxypropan-2-yl)pyridin- 3-y[)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(4-(2-hydroxypropan-2-yl)phenyl)-l -(2-methoxyethyl)-3.4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

(S)-7-(6-( l-hydroxyethyl)pyridin-3-yl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2( 1 H -one;

(R)-7-(6-( 1 -hydroxyethyl)pyridin-3-yl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( ] H)-one; 7-(2-methyl-6-(4H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -((tetrahydro-2H-pyran-4-yl)methyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(4-(2-hydroxypropan-2-yl)phenyl)-l-(2-(tetrahydro-2H-pyran -4-yl)ethyl)-3,4- dihydropyrazino[2.3-b]pyrazin-2( 1 H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(4-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(lH)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(3-(trifluoromethyl)benzyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-(3-methoxypropyl) -3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(4-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)- 1 -(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2( lH)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l -(2-methoxyethyl)-3,4-dihydropyrazino[2,3- b]pyrazin-2( 1 H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-l-((tetrahydro-2H-p yran-4-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(4-methyl-2-(methylarnino)-l H-benzo[d]imidazol-6-yl)-l-((tetrahydro-2H-pyran-4- yl)methyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(2-amjno-4-methyl-l H-benzo[d]imidazol-6-yl)-l -((tetrahydro-2H-pyran-4-yl)methyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(2-methy[-6-(4H- l ,2,4-triazol-3-yl)pyridin-3-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)- 3,4-dihydropyrazino[2,3-b]pyrazin-2(l H)-one;

(R)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-y[)-3-methy[- l-(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

(S)-7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3-methyl-l-(2-( tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2,3-b]pyrazin-2( l H)-one;

7-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-3.3 -dimethyl- l -(2-(tetrahydro-2H-pyran-4- yl)ethyl)-3,4-dihydropyrazino[2.3-b]pyrazin-2( 1 H)-one;

7-(2-amino-4-methyl- l H-benzo[dJimidazol-6-yl)- l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one; 7-(6-(2-hydroxypropan-2-y{)pyridin-3-y!)- I -(2-(tetrahydro-2H-pyran-4-yl)ethyi)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(2-methyl-4-( lH-l ,2,4-triazol-3-yl)phenyl) -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

7-(4-( l H-l ,2,4-triazol-5-yl)phenyl)-l -(2-(tetrahydro-2H-pyran-4-yl)ethyl)-3,4- dihydropyrazino[2,3-b]pyrazin-2( 1 H)-one;

1 -( 1 -hydroxypropan-2-yl)-7-(2-methyl-6-( 1 H- 1 ,2,4-triazol-3-yl)pyridin-3-yl)-3,4- dihydropyrazino[2,3-b ]pyrazin-2( 1 H)-one;

l-(2-hydroxyethyl)-7-(2-methyl-6-(lH-l,2,4-triazol-3-yl)p yridin-3-yl)-3,4- dihydropyrazino[2,3-b]pyrazin-2(l H)-one; or a pharmaceutically acceptable salt thereof.

6. EXAMPLES

6.1 FLOW CYTOMETRY ASSAY

{0067] Antibodies

[0068] The following antibodies were used: (i) Phospho-4EBP 1 (T37/46), Alexa

Fluor 647 Conjugate (Cell Signaling, cat# 5123); (ii) Mouse Anti-human CD3 (FITC)(BD Biosciences, cat# 555332); and (iii) Mouse Anti-human CD91 (PE) (BD Biosciences, cat# 550497).

[0069\ Reagents

[0070] The following reagents were used: (i) Phospho-4EBPl blocking peptide

( lOO^ig @1 mg/ml), (Cell Signaling Technology, cat# 1052) (store at -20 °C); (ii) Phospho- 4EBP1 blocking peptide buffer, containing of 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA and 5% glycerol (store at -20 °C); (iii) BD Stain Buffer (BSA), Cat# 554657, BD Biosciences, containing Dulbeccos Phosphate Buffered Saline (DPBS) pH 7.4, 0.2% (w/v) bovine serum albumin (BSA) and 0.09% sodium azide (NaN ₃ ) (store at 4 °C); (iv) BD PhosFlow Lyse/fix buffer (5x), Cat# 558049, BD Biosciences, a buffered solution containing <40% formaldehyde and <50% diethylene glycol (store at room temperature); (v) BD PhospFlow Perm Buffer I I, cat# 558050, BD Biosciences, a buffered solution containing 90% methanol (store at room temperature); (vi) BD Stabilizing Fixative (3x), cat# 338036, BD Biosciences (store at room temperature); (vii) 96 well icrotest U-Bottom plates; cat# 353077, Falcon; (viii) BD vacutainer tubes (heparin), cat# 367874 (plastic), cat# 366480 (glass); (ix) Compound 1 , an illustrative TOR kinase inhibitor found in International Patent Publication No. WO 2010/062571 , published on June 3, 2010, the contents of which are incorporated herein by reference in their entirety (see, e.g. , Section 4.2, pages 1 -43 and Table 1 ); (x) normal healthy volunteer whole blood obtained from TSRI Normal Blood Donor Service, La Jolla, CA; and (xi) patient whole blood obtained from Conversant Biologies.

[0071] Treatment of Whole Blood

[0072] Whole blood was collected from healthy donors/patients into vacutainer tubes containing sodium heparin (BD, cat# 367874 (plastic), cat# 366480 (glass)). Care was taken to mix the blood thoroughly in the tube to prevent clotting.

[0073] Compound dilutions were prepared as follows: Compound 1 was dissolved in 100% DMSO to make 30 mM or 10 mM stock concentration, the 30 ni or 10 niM stock concentrations of Compound 1 were diluted in 100% DMSO to make 5 mM, 0.5 mM and 0.1 mM stock concentrations and 1 : 100 dilutions of the 5 mM, 0.5 mM and 0.1 mM stock concentrations were made into media, with the final concentrations of Compound 1 in media being 50 μΜ, 5 μΜ and 1 μΜ, respectively. Dilutions were kept at room temperature before addition to the blood.

[0074] 1.8 mL of whole blood was transferred to a 50 mL conical tube and treated with 200 μΐ, of Compound 1 diluted in media for 2 hours in the dark at room temperature. The final Compound 1 concentration was 5 μΜ, 0.5 μΜ and 0.1 μΜ, respectively. The final DMSO concentration was be 0.1%. Each treatment was done in triplicate.

[0075] Fixing and Permeabilization

[0076] While waiting for blood treatments to finish, 5x BD Lyse/Fix Buffer (cat#

558049) was diluted with distilled (or deionized) water. The lx Lyse/Fix buffer was pre- warmed in a 37 °C water bath for 5- 10 minutes before use.

[0077] Cells were lysed/fixed immediately by mixing 1 volume of blood with 20 volumes of l x Lyse/fix buffer (for the 2 mL of blood + Compound 1 , 40 mL of l Lyse/fix buffer was added) and mixed thoroughly by inverting the tube several times. |0078] The Lyse/fix and blood mixture was incubated in a 37 °C water bath for 10 minutes.

[0079] Cells were pelleted by centrifugation at 800x g for 5 minutes and supernatant was removed by aspiration.

[0080J Cells were suspended with 1.3 mL of cold PBS and transferred to 1.5 ml eppendoff tube. Cells were pelleted by centrifugation at 800x g for 5 minutes and supernatant was removed by aspiration.

[00811 Cells were washed with 1 mL of cold PBS, spun at 800x g for 5 minutes and supernatant was removed by aspiration. (Cells can also be frozen at -80 °C directly at this step for later usage. If desired, cells from the 1.8 ml of blood can be split into 2 vials for 2 sets of staining reactions). For cells frozen at -80 °C, frozen tubes with cells are transferred onto ice and cells are suspended with 1 mL of cold PBS. Cells are pelleted by centrifugation at 800x g for 5 minutes and supernatant is removed by aspiration.

[0082] Cells were suspended and permeabilized by adding 1 mL of Perm Buffer III

( 1-10x10 ⁶ cells) and incubated on ice for 15 minutes (The cell pellet should be well resuspended without the presence of chunks. It is important that cells are treated for no more than 30 minutes. Over or under permeabilization of the cells can affect the overall phospho-epitope signals).

[0083] Cells were pelleted by centrifugation at 880x g for 5 minutes and supernatant was removed by aspiration.

[0084] Cells were washed with 1 mL of staining buffer, centrifuged at 880x g for 5 minutes and supernatant was removed by aspiration.

[0085] Antibody Addition and Data Collection

[0086] Cells were batch stained with surface antibodies, making sure that surface markers were compatible with Perm Buffer III system (if antibody is not compatible, surface labeling with antibody before treating cells with Perm Buffer ill should be tried. Becton

Dickinson recommends using 20 μΐ of each surface antibody for every 100 μ] reaction).

[0087] For PBMC cells from 1.8 mL whole blood, the cell pellet from above was resuspended in 30 of staining buffer (40 μL of staining buffer is used if only staining

CD91 cells). 10 μL of anti-CD3 and 10 μ] _^ of anri-CD91 were added to cells, mixed thoroughly and incubated at room temperature for 30 minutes in the dark (PC3 cells do not need to surface staining).

[0088] While waiting for the surface staining, 96 well U-bottom plates with phospho-antibodies and blocking peptide or buffer were prepared. p4EBP 1 staining solution, blocking peptide solution and control solution were also prepared during the waiting period, as follows: p4EBPl staining solution: 10 parts peptide buffer + 2 parts anti- p4EBPl antibody; blocking peptide solution: 10 parts blocking peptide + 2 parts anti- p4EBPl antibody; and control solution: 10 parts peptide buffer + 2 parts staining buffer.

[0089] 12 pL of staining solution, blocking peptide solution or control solution were added according to the following plate setting:

[0090] 88 μί of diluted cells were transferred to each well following the above plate layout. The plate was gently shaken to mix staining buffer with cells.

[0091 J The plate was shaken at 25 rpm in the dark at room temperature for 30 minutes. Cells were pelleted in the plate by centrifugation at 880x g for 5 minutes. The supernatant was removed quickly by inverting the plate and expelling the contents out.

[0092] The plate was shaken to loosen the cell pellet in the 96-well plate. Cells were washed with 200 μΐ of staining buffer per well. Cells were pelleted in the plate by centrifugation at 880x g for 5 minutes. Supernatant was removed by quickly inverting the plate and expelling contents out.

[0093] The plate was again shaken at 25 rpm in the dark at room temperature for 30 minutes. Cells were pelleted in the plate by centrifugation at 880x g for 5 minutes. The supernatant was removed quickly by inverting the plate and expelling the contents out.

[0094] During the centrifugation, the 3x Concentrate BD Stabilizing Fixative was diluted 1 :3 with deionized water at room temperature. [0095] The plate was shaken to loosen the cell pellet. Cells were resuspended in 200 μΐ of l BD Stabilizing Fixative.

[0096] The plate was read on a FACS Calibur flow cytometer equipped with a 96 well plate reader. Plates that cannot be read right away should be covered in foil to protect from light and stored at 4 °C. Plates should be read within 2-3 hours.

[0097] Data analysis was be carried out using FlowJo (flow cytometry analysis software) from Tree Star, Inc. Mean/median fluorescence intensity and percent inhibition of protein phosphorylation was assessed.

[0098] Results are illustrated in FIGS. 1 -4.

6.2 mTOR HTR-FRET ASSAY

[0001J The following is an example of an assay that can be used to determine the mTOR inhibitory activity of a test compound. Test compounds are dissolved in DMSO and prepared as 10 mM stocks and diluted appropriately for the experiments. Reagents are prepared as follows:

[0002] "Simple TOR buffer" (used to dilute high glycerol TOR fraction): 10 mM

Tris pH 7.4, 100 mM NaCl, 0.1% Tween-20, 1 mM DTT. Invitrogen mTOR

(cat#PR8683A) is diluted in this buffer to an assay concentration of 0.200 μg/mL.

[0003] ATP/Substrate solution: 0.075 mM ATP. 12.5 mM MnCl ₂ , 50 mM Hepes, pH 7.4, 50 mM β-GOP, 250 nM Microcystin LR, 0.25 mM EDTA, 5 mM DTT, and 3.5 μg/mL GST-p70S6.

[0004J Detection reagent solution: 50 mM HEPES, pH 7.4, 0.01% Triton X-100,

0.01% BSA, 0.1 mM EDTA, 12.7 _μ& /ι Τ Cy5-aGST Amersham (Cat#PA92002V), 9 ng/mL a-phospho p70S6 (Thr389) (Ceil Signaling Mouse Monoclonal #9206L),

627 ng mL a-mouse Lance Eu (Perkin Elmer Cat#AD0077).

[0099] To 20 of the Simple mTor buffer is added 0.5 μΐ, of test compound in

DMSO. To initiate the reaction 5 μL· of ATP/Substrate solution is added to 20 iL of the

Simple TOR buffer solution (control) and to the compound solution prepared above. The assay is stopped after 60 minutes by adding 5 iL of a 60 mM EDTA solution; 10 μL· of detection reagent solution is then added and the mixture is allowed to sit for at least 2 hours before reading on a Perkin-Elmer Envision Microplate Reader set to detect LANCE Eu TR- FRET (excitation at 320 nm and emission at 495/520 nm).

6.3 CLINICAL STUDY

[00100] The patient population was comprised of men and women, over 1 8 years old.

[00101] Compound 1 was administered orally, in an uninterrupted once-daily schedule. Each dose was taken in the morning, with the patient having fasted overnight (minimum of 6 hrs).

[00102] Each patient was administered a single dose of Compound 1 (Day -1 ), followed by a 24-hour wash out period and a 48-hour observation and pharmacokinetic sample collection period, which was then followed on Day 1 by daily dosing for 28 days.

[00103] The following initial doses were administered: Cohort 1 = 7.5 mg; Cohort 2 = 15 mg; Cohort 3 = 30 mg; Cohort 4 = 45 mg. Initial cohorts of one subject were given Compound 1 in dose increments of 100% (i.e., doubling the dose level each time) until the first instance of first-course grade 2 or higher toxicity suspected to be Compound 1 related.

[00104] Results are illustrated in FIGS. 5 and 6.

[00105] The embodiments disclosed herein are not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the disclosed embodiments and any embodiments that are functionally equivalent are encompassed by the present disclosure. Indeed, various modifications of the

embodiments disclosed herein are in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

[00106] A number of references have been cited, the disclosures of which are incorporated herein by reference in their entirety.

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