Substituted quinolinone piperazine compounds useful as T cell activators

Abstract

Disclosed are compounds of formula (I) or salts thereof, wherein: R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and m are defined herein. Also disclosed are methods of using such compounds to inhibit the activity of one or both of diacylglycerol kinase alpha (DGKα) and diacylglycerol kinase zeta (DGKζ) and pharmaceutical compositions comprising such compounds. These compounds can be used to treat viral infections and proliferative disorders, such as cancer. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Indian Provisional Application No. 201911053557 filed on December 23, 2019, which is incorporated herein in its entirety.

[0003] manual

[0004] The present invention generally relates to substituted quinolinone compounds that activate T cells, promote T cell proliferation and / or exhibit anti-tumor activity. Substituted quinolinone compounds, compositions comprising such compounds, and methods of use thereof are provided herein. The present invention further relates to pharmaceutical compositions comprising at least one compound according to the present invention, which can be used to treat proliferative disorders (such as cancer) and viral infections. Background Art

[0005] Human cancers have many genetic and epigenetic changes, producing new antigens that are potentially recognizable by the immune system (Sjoblom et al., (2006) Science 314: 268-74). The adaptive immune system, composed of T lymphocytes and B lymphocytes, has a strong anti-cancer potential, with a wide range of capabilities and precise specificity to respond to diverse tumor antigens. In addition, the immune system exhibits considerable plasticity and memory components. Successfully utilizing all these properties of the adaptive immune system will make immunotherapy unique among all cancer treatments. However, although endogenous immune responses to cancer have been observed in preclinical models and patients, this response is ineffective, and confirmed cancers are considered "self" and tolerated by the immune system. Tumors that contribute to this state of tolerance may actively destroy anti-tumor immunity using several different mechanisms. These mechanisms include dysfunctional T cell signaling (Mizoguchi et al., (1992) Science 258:1795-98), suppressive regulatory cells (Facciabene et al., (2012) Cancer Res. 72:2162-71), and the selection of endogenous "immune checkpoints" that serve to downregulate the magnitude of the adaptive immune response and protect normal tissues from collateral damage by tumors to evade immune destruction (Topalian et al., (2012) Curr. Opin. Immunol. 24:1-6; Mellman et al. (2011) Nature 480:480-489).

[0006] Diacylglycerol kinase (DGK) is a lipid kinase that mediates the conversion of diacylglycerol to phosphatidic acid to terminate the T cell function propagated by the TCR signal transduction pathway. Therefore, DGK is used as an intracellular checkpoint, and inhibition of DGK is expected to enhance T cell signal transduction pathways and T cell activation. Supportive evidence includes knockout mouse models of DGKα or DGKζ that show a highly reactive T cell phenotype and improved anti-tumor immune activity (Riese MJ et al., Journal of Biological Chemistry, (2011) 7: 5254-5265; Zha Y et al., Nature Immunology, (2006) 12: 1343; Olenchock BA et al., (2006) 11: 1174-81). Furthermore, tumor-infiltrating lymphocytes isolated from human renal cell carcinoma patients were observed to overexpress DGKa, which resulted in suppressed T cell function (Prinz, PU et al., J Immunology (2012) 12:5990-6000). Therefore, DGKα and DGKζ are considered as targets for cancer immunotherapy (Riese M.J. et al., Front Cell Dev Biol. (2016) 4:108; Chen, SS et al., Front Cell Dev Biol. (2016) 4:130; Avila-Flores, A. et al., Immunology and Cell Biology (2017) 95:549-563; Noessner, E., Front Cell Dev Biol. (2017) 5:16; Krishna, S., et al., Front Immunology (2013) 4:178; Jing, W. et al., Cancer Research (2017) 77:5676-5686).

[0007] There remains a need for compounds that are useful as inhibitors of one or both of DGKα and DGKζ. In addition, there remains a need for compounds that are useful as inhibitors of one or both of DGKα and DGKζ that have selectivity relative to other diacylglycerol kinases, protein kinases, and / or other lipid kinases.

[0008] Therefore, safe and effective agents that restore T cell activation, lower antigenic thresholds, enhance anti-tumor function, and / or overcome the inhibitory effects of one or more endogenous immune checkpoints (such as PD-1, LAG-3, and TGFβ) would be an important addition to the treatment of patients with proliferative disorders (such as cancer) as well as viral infections. Summary of the invention

[0009] Applicants have discovered compounds that are active as inhibitors of one or both of DGKα and DGKζ. In addition, applicants have discovered compounds that are active as inhibitors of one or both of DGKα and DGKζ and have selectivity relative to other diacylglycerol kinases, protein kinases, and / or other lipid kinases. These compounds are provided for use as medicaments having desirable stability, bioavailability, therapeutic index, and toxicity values ​​that are important for their pharmaceutical use.

[0010] The present invention provides substituted quinolinone-based compounds of formula (I) useful as inhibitors of DGKα, DGKζ, or both DGKα and DGKζ, including salts and prodrugs thereof.

[0011] The present invention also provides a pharmaceutical composition, which comprises a compound of formula (I) and / or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a method for treating a disease or disorder associated with the activity of DGKα, DGKζ, or both DGKα and DGKζ, comprising administering a compound of formula (I) and / or a pharmaceutically acceptable salt thereof to a mammalian patient.

[0013] The present invention also provides methods and intermediates for preparing the compound of formula (I) and / or its salt.

[0014] The present invention also provides a compound of formula (I) and / or a pharmaceutically acceptable salt thereof for use in therapy.

[0015] The present invention also provides the use of a compound of formula (I) and / or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating proliferative disorders such as cancer and viral infection.

[0016] The compounds of formula (I) and compositions comprising the compounds of formula (I) can be used to treat, prevent or cure viral infections and various proliferative disorders, such as cancer. Pharmaceutical compositions comprising these compounds can be used to treat, prevent or slow down the progression of diseases or disorders (such as viral infections and cancer) in a variety of therapeutic areas.

[0017] These and other features of the present invention will be set forth in expanded form as the disclosure proceeds. DETAILED DESCRIPTION

[0018] The first aspect of the present invention provides at least one compound of formula (I):

[0019]

[0020] or a salt thereof, wherein:

[0021] R1 is H, F, Cl, Br, -CN, zero to four R 1a Substituted C 1-3 Alkyl, with zero to four R 1a Substituted C 3-4 Cycloalkyl, 0 to 4 R 1a Substituted C 1-3 Alkoxy, -C(O)NR a R a 、-NR a R a 、-S(O) n R e , or -P(O)R e R e ;

[0022] Each R 1a are independently F, Cl, -CN, -OH, -OCH3, or -NR a R a ;

[0023] Each R a independently H or C 1-3 alkyl;

[0024] Each R e Independently C 3-4 Cycloalkyl or 0 to 4 R 1a Substituted C 1-3 alkyl;

[0025] R2 is H, zero to 4 R 2a Substituted C 1-3 Alkyl, with zero to four R 2a Substituted C 2-3 alkenyl, or 0 to 4 R 2a Substituted C 3-4 Cycloalkyl;

[0026] Each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 Alkyl), C 3-4 Cycloalkyl, C 3-4 Alkenyl, or C 3-4 Alkynyl;

[0027] R3 is H, F, Cl, Br, -CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, C 3-4 Fluorinated cycloalkyl, -NO2, or 0 to 2 R 3a substituted pyridyl;

[0028] Each R 3a It is halogen, -CN, C 1-3 Alkyl, or C 1-3 Alkoxy;

[0029] R4 is -CH2R 4a 、-CH2CH2R 4a 、-CH2CHR 4a R 4d 、-CHR 4a R 4b , or -CR 4a R 4b R 4c ;

[0030] R 4a and R 4b Independently are:

[0031] (i) C substituted with zero to four substituents independently selected from the following 1-6 Alkyl: F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy, -NR a R a 、-S(O)2R e , or -NR a S(O)2R e ;

[0032] (ii)C 3-6 Cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each substituted with zero to four substituents independently selected from the group consisting of F, Cl, Br, -CN, -OH, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-4 Hydroxyalkyl, -(CH2) 1-2 O(C 1-3 Alkyl), C 1-4 Alkoxy, -O(C 1-4 Hydroxyalkyl), -O(CH) 1-3 O(C 1-3 Alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-3 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 Alkyl), -NR c R c 、-NR a S(O)2(C 1-3 Alkyl), -NR aC(O)(C 1-3 Alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-3 Alkyl)2, -S(O)2(C 1-3 Alkyl), -O(CH2) 1-2 (C 3-6 Cycloalkyl), -O(CH2) 1-2 (morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and R d ;or

[0033] (iii) is selected from C 3-6 Cycloalkyl, heterocyclic, aryl and heteroaryl cyclic groups substituted with C 1-4 Alkyl, the cyclic group is substituted by zero to three substituents independently selected from the following: F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c 、-NR a S(O)2(C 1-3 Alkyl), -NR a C(O)(C 1-3 Alkyl), -NR a C(O)O(C 1-4 Alkyl) and C 3-6 Cycloalkyl;

[0034] or R 4a and R 4b Together with the carbon atom to which they are attached, they form C 3-6 Cycloalkyl or 3 to 6 membered heterocyclic group, each of which is substituted by zero to three R f replace;

[0035] Each R f are independently F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , or selected from C 3-6Cyclic groups of cycloalkyl, 3- to 6-membered heterocyclic groups, phenyl, monocyclic heteroaryl and bicyclic heteroaryl, each cyclic group being substituted by zero to three substituents independently selected from the group consisting of F, Cl, Br, -OH, -CN, C 1-6 Alkyl, C 1-3 Fluoroalkyl, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, and -NR c R c ;

[0036] R 4c It is C 1-6 Alkyl or C 3-6 cycloalkyl, each substituted by zero to 4 substituents independently selected from the group consisting of F, Cl, -OH, C 1-2 Alkoxy, C 1-2 Fluoroalkoxy, and -CN;

[0037] R 4d is -OCH3;

[0038] Each R c independently H or C 1-2 alkyl;

[0039] R d is phenyl substituted with zero to one substituent selected from the group consisting of F, Cl, -CN, -CH3, and -OCH3;

[0040] Each R5 is independently F, Cl, -CN, -OH, or any of zero to four R g Substituted C 1-6 Alkyl, with zero to four R g Substituted C 1-3 Alkoxy, with zero to four R g Substituted C 2-4 Alkenyl, 0 to 4 R g Substituted C 2-4 Alkynyl, 0 to 4 R g Substituted C 3-4 Cycloalkyl, substituted by zero to four R g Substituted phenyl, 0 to 3 R g Substituted oxadiazole, substituted with zero to four R g Substituted pyridyl, -(CH2) 1-2 (zero to 4 R g substituted heterocyclic group), -(CH2) 1-2 NR c C(O)(C 1-4 Alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4Alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 Cycloalkyl), -C(O)NR a R a , or -C(O)NR a (C 3-4 cycloalkyl), or two R5 are attached to the same carbon atom to form =0;

[0041] Each R g are independently F, Cl, -CN, -OH, C 1-3 Alkoxy, C 1-3 Fluoroalkoxy, -O(CH2) 1-2 O(C 1-2 Alkyl), C 3-5 Cycloalkyl, or -NR c R c ;

[0042] each R6 is H, F, Cl, -CN, -CH3, -CH2F, -CHF2, -CF3, or -OCH3;

[0043] m is zero, 1, 2, or 3; and

[0044] n is zero, 1, or 2.

[0045] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein: R1 is H, F, Cl, Br, -CN, 0 to 4 R 1a Substituted C 1-3 Alkyl, 0 to 3 R 1a Substituted cyclopropyl, substituted by zero to three R 1a Substituted C 1-3 Alkoxy, -C(O)NR a R a 、-NR a R a 、-S(O) n CH3, or -P(O)(CH3)2; each R 1a is independently F, Cl, or -CN; each R a independently H or C 1-3 Alkyl; R2 is H, with zero to two R 2a Substituted C 1-2 Alkyl, or zero to two R 2a Substituted C 2-3alkenyl; each R 2a are independently F, Cl, -CN, -OH, -O(C 1-2 alkyl), cyclopropyl, C 3-4 Alkenyl, or C 3-4 Alkynyl; R3 is H, F, Cl, Br, -CN, C 1-2 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, -NO2, or 0 to 2 R 3a Substituted pyridinyl; R 4a and R 4b are independently: (i) C substituted with zero to four substituents independently selected from the following 1-4 Alkyl: F, Cl, -CN, -OH, -OCH3, -SCH3, C 1-3 Fluoroalkoxy, and -NR a R a ; (ii) C 3-6 Cycloalkyl, heterocyclyl, phenyl, or heteroaryl, each substituted with zero to four substituents independently selected from the group consisting of F, Cl, Br, -CN, -OH, C 1-6 Alkyl, C 1-3 Fluoroalkyl, -CH2OH, -(CH2) 1-2 O(C 1-2 Alkyl), C 1-4 Alkoxy, -O(C 1-4 Hydroxyalkyl), -O(CH) 1-2 O(C 1-2 Alkyl), C 1-3 Fluoroalkoxy, -O(CH) 1-2 NR c R c , -OCH2CH=CH2, -OCH2C≡CH, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 Alkyl), -NR c R c 、-NR a S(O)2(C 1-3 Alkyl), -NR a C(O)(C 1-3 Alkyl), -NR a C(O)O(C 1-4 alkyl), -P(O)(C 1-2 Alkyl)2, -S(O)2(C 1-3 Alkyl), -O(CH2) 1-2 (C 3-4 Cycloalkyl), -O(CH2) 1-2(morpholinyl), cyclopropyl, cyanocyclopropyl, methylazetidinyl, acetylazetidinyl, (tert-butyloxycarbonyl)azetidinyl, triazolyl, tetrahydropyranyl, morpholinyl, thienyl, methylpiperidinyl, and R d ; or (iii) a person selected from C 3-6 Cycloalkyl, heterocyclic, phenyl, and heteroaryl cyclic groups substituted with C 1-3 Alkyl, the cyclic group is substituted by zero to three substituents independently selected from the following: F, Cl, Br, -OH, -CN, C 1-3 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c 、-NR a S(O)2(C 1-3 Alkyl), -NR a C(O)(C 1-3 Alkyl), -NR a C(O)O(C 1-4 alkyl), and C 3-4 Cycloalkyl; or R 4a and R 4b Together with the carbon atom to which they are attached, they form C 3-6 Cycloalkyl or 3 to 6 membered heterocyclic group, each of which is substituted by zero to three R f Replace; each R f are independently F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, -OCH2CH=CH2, -OCH2C≡CH, -NR c R c , or selected from C 3-6 Cyclic groups of cycloalkyl, 3- to 6-membered heterocyclyl, phenyl, monocyclic heteroaryl, and bicyclic heteroaryl, each cyclic group being substituted by zero to three substituents independently selected from the group consisting of F, Cl, Br, -OH, -CN, C 1-4 Alkyl, C 1-2 Fluoroalkyl, C 1-3 Alkoxy, C 1-2 Fluoroalkoxy, and -NR c R c ; R 4c It is C 1-4 Alkyl or C 3-6 cycloalkyl, each substituted by zero to 4 substituents independently selected from the group consisting of F, Cl, -OH, C 1-2Alkoxy, C 1-2 fluoroalkoxy, and -CN; each R5 is independently F, -CN, -OH, zero to four R g Substituted C 1-5 Alkyl, 0 to 3 R g Substituted C 1-2 Alkoxy, with zero to four R g Substituted C 2-3 Alkenyl, 0 to 4 R g Substituted C 2-3 Alkynyl, 0 to 4 R g Substituted C 3-4 Cycloalkyl, 0 to 3 R g Substituted phenyl, 0 to 3 R g Substituted oxadiazole, substituted by zero to three R g Substituted pyridyl, -(CH2) 1-2 (zero to 4 R g substituted heterocyclic group), -(CH2) 1-2 NR c C(O)(C 1-4 Alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 Alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 Cycloalkyl), -C(O)NR a R a , or -C(O)NR a (C 3-4 cycloalkyl), or two R5 are attached to the same carbon atom to form =0; each R6 is H, F, or -CH3; and m is zero, 1, 2, or 3.

[0046] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein: R1 is H, F, -CN, or -OCH3; R2 is H, -CH3, -CH2CN, -CH2CH2F, or -CH2CH=CH2; R3 is H, -CN, -CH2OH, -C(O)OCH2CH3, -NO2, or pyridyl; R4 is -CH2R 4a or -CHR 4a R 4b ; R 4ais phenyl, naphthyl, or indolyl, each substituted with zero to two substituents independently selected from the group consisting of F, -CH3, -CH2CH3, and -OCH3; R 4b is phenyl or fluorophenyl; each R5 is -CH3, or two R5 are attached to the same carbon atom to form =0; and m is zero, 1, or 2.

[0047] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R1 is H, F, Cl, Br, -CN, substituted by zero to four R 1a Substituted C 1-3 Alkyl, 0 to 3 R 1a Substituted cyclopropyl, substituted by zero to three R 1a Substituted C 1-3 Alkoxy, -C(O)NR a R a 、-NR a R a 、-S(O) n CH3, or -P(O)(CH3)2; This embodiment includes compounds wherein R1 is H, F, Cl, Br, -CN, -CH3, -CHF2, -CF3, cyclopropyl, or -OCH3. This embodiment also includes compounds wherein R1 is H, F, -CN, or -OCH3.

[0048] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R2 is H, substituted by zero to two R 2a Substituted C 1-2 Alkyl, or zero to two R 2a Substituted C 2-3 This embodiment includes compounds wherein R2 is H, -CH3, -CH2CN, -CH2CH2F, or -CH2CH=CH2. This embodiment also includes compounds wherein R2 is H, -CH3, -CH2CN, or -CH2CH2F.

[0049] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R3 is H, F, Cl, Br, -CN, C 1-2 Alkyl, C 1-2 Fluoroalkyl, C 3-4 Cycloalkyl, -NO2, or 0 to 2 R 3a This embodiment also includes compounds wherein R3 is H, -CN, -CH2OH, -C(O)OCH2CH3, -NO2, or pyridyl. This embodiment also includes compounds wherein R3 is H, -CN, -CH2OH, or -NO2.

[0050] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is -CH2R 4a or -CH2CH2R 4a This embodiment includes compounds wherein R4 is -CH2R 4a or-CD2R 4a .

[0051] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is -CH2R 4a This embodiment includes compounds wherein R4 is -CD2R 4a .

[0052] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is -CHR 4a R 4b or -CR 4a R 4b R 4c .

[0053] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is -CHR 4a R 4b .

[0054] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is -CH2R 4a or -CHR 4a R 4b .

[0055] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 1, 2, or 3; and each R5 is independently F, -CN, -OH, substituted by zero to 4 R g Substituted C 1-5 Alkyl, 0 to 3 R g Substituted C 1-2 Alkoxy, with zero to four R g Substituted C 2-3 Alkenyl, 0 to 4 R g Substituted C 2-3 Alkynyl, 0 to 4 R g Substituted C 3-4 Cycloalkyl, 0 to 3 R g Substituted phenyl, 0 to 3 R g Substituted oxadiazole, substituted by zero to three R g Substituted pyridyl, -(CH2) 1-2 (zero to 4 R g substituted heterocyclic group), -(CH2)1-2 NR c C(O)(C 1-4 Alkyl), -(CH2) 1-2 NR c C(O)O(C 1-4 Alkyl), -(CH2) 1-2 NR c S(O)2(C 1-4 alkyl), -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-4 alkyl), -C(O)O(C 3-4 Cycloalkyl), -C(O)NR a R a , or -C(O)NR a (C 3-4 cycloalkyl), or two R5 are attached to the same carbon atom to form =0. This embodiment includes compounds wherein each R5 is independently F, -CN, -OH, g Substituted C 1-2 Alkyl, 0 to 3 R g Substituted C 1-2 Alkoxy, with zero to two R g Substituted C 3-4 Cycloalkyl, -C(O)(C 1-4 alkyl), -C(O)OH, -C(O)O(C 1-2 alkyl), or -C(O)NR a R a , or two R5 are attached to the same carbon atom to form =0. Also included in this embodiment are compounds wherein each R5 is -CH3, or two R5 are attached to the same carbon atom to form =0.

[0056] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 2 and two R5 are attached to the same carbon to form =0.

[0057] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is zero.

[0058] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 1, 2, or 3. Included in this embodiment are compounds wherein m is 1 or 2. Also included in this embodiment are compounds wherein m is 1.

[0059] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 2 or 3. Included in this embodiment is a compound wherein m is 2.

[0060] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein m is 3.

[0061] In one embodiment, a compound of formula (I) or a salt thereof is provided, which has the structure of formula (II):

[0062]

[0063] Where R 5a , R 5b , R 5c , and R 5d One, two, or three of them are each R5 and R 5a , R 5b , R 5c , and R 5d In the remainder is each hydrogen. Such compounds are included in this embodiment, wherein each R is independently-CN ,-CH ,-CH CH ,-CH (CH ) ,-CHC (CH ) ,-CH F ,-C (CH ) F ,-CF (CH ) CH (CH ) ,-CH OH ,-C (CH ) OH ,-C (CH ) (OH) CH (CH ) ,-CH OCH ,-C (O) C (CH ) ,-C (O) OH ,-C (O) OCH ,-C (O) OC (CH ) ,-C (O) NH ,-C (O) NH (cyclopropyl ),-C (O) O (cyclopropyl), cyclopropyl, phenyl, methyl oxadiazolyl or picoline radicals.

[0064] In one embodiment, a compound of formula (I) or a salt thereof is provided, which has the structure of formula (III):

[0065]

[0066] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH3 and R 5c It is -CH3.

[0067] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH3 and R 5c It is -CH2CH3.

[0068] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH2CH3 and R 5c It is -CH3.

[0069] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH2CH3 and R 5c It is -CH2CH3.

[0070] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH3 and R 5c It is -CH2OH.

[0071] In one embodiment, a compound of formula (III) or a salt thereof is provided, wherein R 5a is -CH3 and R 5c It is -CH2OCH3.

[0072] In one embodiment, a compound of formula (I) or a compound of formula (III) or a salt thereof is provided, having the structure:

[0073]

[0074] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is:

[0075]

[0076] Included within this embodiment are compounds wherein R1 is H, Br, -CN, or -OCH3; and R2 is -CH3.

[0077] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein R4 is:

[0078] Included in this embodiment are compounds wherein R1 is H, Br, -CN, or -OCH3; and R2 is -CH3. Also included in this embodiment are compounds wherein R1 is -CN; and R2 is -CH3.

[0079] In one embodiment, a compound of formula (I) or a salt thereof is provided, wherein the compound is:

[0080] 6-Fluoro-4-{4-[(3-fluorophenyl)methyl]piperazin-1-yl}-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (1);

[0081] 6-Fluoro-4-{4-[(3-fluoro-5-methylphenyl)methyl]piperazin-1-yl}-1-methyl-3-(pyridin-4-yl)-1,2-dihydroquinolin-2-one (2);

[0082] 4-(4-((1H-indol-4-yl)methyl)piperazin-1-yl)-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one (3);

[0083] 4-{4-[(1-ethyl-1H-indol-4-yl)methyl]piperazin-1-yl}-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (4);

[0084] 4-(4-Benzyl-3-oxopiperazin-1-yl)-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (5);

[0085] 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile (6);

[0086] 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile (7);

[0087] 6-Methoxy-1-methyl-4-{4-[(naphthalen-1-yl)methyl]piperazin-1-yl}-3-nitro-1,2-dihydroquinolin-2-one (8);

[0088] 4-(4-Benzhydrylpiperazin-1-yl)-1-methylquinolin-2(1H)-one (9);

[0089] 4-[4-(Diphenylmethyl)piperazin-1-yl]-3-(hydroxymethyl)-1-methyl-1,2-dihydroquinolin-2-one (11);

[0090] 4-{4-[(2-methoxynaphthalen-1-yl)methyl]piperazin-1-yl}-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester (12);

[0091] 4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-3-nitroquinolin-2(1H)-one (13);

[0092] 4-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-1-(2-fluoroethyl)-3-nitro-1,2-dihydroquinolin-2-one (14);

[0093] 2-(4-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-3-nitro-2-oxo-1,2-dihydroquinolin-1-yl)acetonitrile (15); or

[0094] 4-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-3-nitro-1-(prop-2-en-1-yl)-1,2-dihydroquinolin-2-one (16).

[0095] The present invention can be implemented in other specific forms without departing from its spirit or basic attributes. The present invention encompasses all combinations of aspects of the present invention described herein and / or embodiments. It should be understood that any and all embodiments of the present invention can be combined with any one or more other embodiments to describe other embodiments. It should also be understood that each individual element in the embodiment is intended to be combined with any and all other elements from any embodiment to describe other embodiments.

[0096] definition

[0097] Those skilled in the art can more easily understand the features and advantages of the present invention after reading the following detailed description. It should be understood that certain features of the present invention described in different embodiments before and after for clarity reasons can also be combined into one embodiment. Conversely, the various features of the present invention described in one embodiment for simplicity reasons can also be combined to form its sub-combination. The embodiments identified herein as exemplary or preferred are intended to be illustrative rather than restrictive.

[0098] Unless otherwise specifically stated herein, when referring to the singular, the plural reference may also be included. For example, "a / an" may refer to one / a, or one or more / one or more.

[0099] As used herein, the phrase "compound and / or its salt" refers to at least one compound, at least one salt of the compound, or a combination thereof. For example, the compound of formula (I) and / or its salt includes a compound of formula (I); two compounds of formula (I); a salt of a compound of formula (I); a compound of formula (I) and one or more salts of a compound of formula (I); and two or more salts of a compound of formula (I).

[0100] Unless otherwise indicated, any atom with unsatisfied valences is assumed to have sufficient hydrogen atoms to satisfy the valences.

[0101] The definitions set forth herein take precedence over definitions set forth in any patent, patent application, and / or patent application publication incorporated herein by reference.

[0102] Listed below are definitions of various terms used to describe the present invention. These definitions apply to the terms as they are used throughout the specification, either individually or as part of a larger group (unless they are otherwise limited in specific instances).

[0103] Throughout the specification, groups and substituents thereof can be selected by one skilled in the art to provide stable moieties and compounds.

[0104] According to the convention used in the art, the structural formulas herein are used

[0105]

[0106] to depict the bond that is the point of attachment of a moiety or substituent to the core or backbone structure.

[0107] As used herein, the terms "halo" and "halogen" refer to F, Cl, Br, and I.

[0108] The term "cyano" refers to the group -CN.

[0109] The term "amino" refers to the group -NH2.

[0110] The term "oxo" refers to the group =0.

[0111] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups containing, for example, from 1 to 12 carbon atoms, from 1 to 6 carbon atoms, and from 1 to 4 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, and tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl. When a number appears in a subscript after the symbol "C", the subscript more specifically defines the number of carbon atoms that a particular group may contain. For example, "C 1-4 "Alkyl" refers to straight and branched chain alkyl groups having one to four carbon atoms.

[0112] As used herein, the term "fluoroalkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups substituted with one or more fluorine atoms. 1-4 "Fluoroalkyl" is intended to include C1, C2, C3 and C4 alkyl groups substituted with one or more fluorine atoms. Representative examples of fluoroalkyl groups include, but are not limited to, -CF3 and -CH2CF3.

[0113] The term "hydroxyalkyl" includes both branched and straight chain saturated alkyl groups substituted with one or more hydroxy groups. For example, "hydroxyalkyl" includes -CH2OH, -CH2CH2OH, and C 1-4 Hydroxyalkyl.

[0114] The term "alkenyl" refers to a straight or branched hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include vinyl or allyl. For example, "C 2-6 "Alkenyl" refers to straight-chain and branched alkenyl groups having two to six carbon atoms.

[0115] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond. Exemplary such groups include ethynyl. For example, "C 2-6 "Alkynyl" refers to straight-chain and branched alkynyl groups having two to six carbon atoms.

[0116] As used herein, the term "cycloalkyl" refers to a group derived from a non-aromatic monocyclic or polycyclic hydrocarbon molecule by removing a hydrogen atom from a saturated ring carbon atom. Representative examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. When a number appears in a subscript position after the symbol "C", the subscript more specifically defines the number of carbon atoms that a particular cycloalkyl group may contain. For example, "C 3-6 "Cycloalkyl" means a cycloalkyl group having three to six carbon atoms.

[0117] As used herein, the term "fluorocycloalkyl" is intended to include cycloalkyl groups substituted with one or more fluorine atoms.

[0118] As used herein, the term "alkoxy" refers to an alkyl group attached to the parent molecular moiety through an oxygen atom, such as methoxy (-OCH3). For example, "C 1-3 "Alkoxy" means an alkoxy group having one to three carbon atoms.

[0119] The terms "fluoroalkoxy" and "-O(fluoroalkyl)" refer to a fluoroalkyl group as defined above attached through an oxygen linkage (-O-). 1-4 "Fluoroalkoxy" is intended to include C1, C2, C3 and C4 fluoroalkoxy.

[0120] The terms "carbocycle," "carbocyclic," or "carbocyclyl" are used interchangeably and refer to a cyclic group having at least one saturated or partially saturated non-aromatic ring in which all atoms of all rings are carbon. The carbocyclyl ring may be unsubstituted or may contain one or more substituents where valence permits. Thus, the term includes non-aromatic rings such as cycloalkyl, cycloalkenyl, and cycloalkynyl rings. Exemplary bicyclic carbocyclyls include indanyl, indenyl, dihydronaphthyl, tetrahydronaphthyl, hexahydronaphthyl, octahydronaphthyl, decahydronaphthyl, bicycloheptyl, bicyclooctyl, and bicyclononyl.

[0121] The term "aryl" as used herein refers to a group of atoms derived from a molecule containing one or more aromatic rings by removing one hydrogen bonded to the aromatic rings. Representative examples of aryl include, but are not limited to, phenyl and naphthyl. Aryl rings may be unsubstituted or may contain one or more substituents where valence permits.

[0122] As used herein, "benzyl" refers to a methyl group in which one hydrogen atom is replaced by a phenyl group. The benzene ring may be unsubstituted or may contain one or more substituents as allowed by valence.

[0123] The term "heteroatom" refers to oxygen (O), sulfur (S) and nitrogen (N).

[0124] The terms "heterocycle", "heterocyclic", or "heterocyclyl" are used interchangeably and refer to a cyclic group having at least one saturated or partially saturated non-aromatic ring and wherein one or more of the rings have at least one heteroatom (O, S or N), the heteroatom-containing ring preferably having 1 to 3 heteroatoms independently selected from O, S and / or N. The ring of such a heteroatom-containing group may contain one or two oxygen or sulfur atoms and / or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. Nitrogen and sulfur atoms may be optionally oxidized, and nitrogen atoms may be optionally quaternized. Heterocyclic groups may be attached to any available nitrogen or carbon atom. Heterocycles may be unsubstituted or may contain one or more substituents where valence permits.

[0125] Exemplary monocyclic heterocyclyls include pyrrolidinyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane, tetrahydro-1,1-dioxothiphenyl, dihydroisoindolyl, and tetrahydroquinolinyl.

[0126] The term "heteroaryl" refers to substituted and unsubstituted aromatic 5 or 6-membered monocyclic groups and 9 or 10-membered bicyclic groups having at least one heteroatom (O, S or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2 or 3 heteroatoms independently selected from O, S and / or N. Each ring of the heteroaryl containing heteroatoms may contain one or two oxygen or sulfur atoms and / or from one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom. The fused rings constituting the bicyclic group are aromatic and may contain only carbon atoms. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. The bicyclic heteroaryl must contain only aromatic rings. The heteroaryl may be attached to any available nitrogen or carbon atom of any ring. The heteroaryl ring system may be unsubstituted or may contain one or more substituents.

[0127] Exemplary monocyclic heteroaryl groups include pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl.

[0128] Exemplary bicyclic heteroaryl groups include indolyl, benzothiazolyl, benzodioxolyl, benzoxazolyl, benzothiophenyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, and pyrrolopyridinyl.

[0129] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0130] The compound of formula (I) can form salts, which are also within the scope of the present invention. Unless otherwise indicated, reference to the compounds of the present invention should be understood to include reference to one or more salts thereof. The term "salt" refers to an acidic and / or basic salt formed with an inorganic and / or organic acid and base. In addition, the term "salt" can include zwitterions (inner salts), for example, when the compound of formula (I) contains a basic moiety such as an amine or pyridine or imidazole ring, and an acidic moiety such as a carboxylic acid. Pharmaceutically acceptable (i.e., nontoxic, physiologically acceptable) salts are preferred, such as acceptable metal salts and amine salts in which the cation does not significantly contribute to the toxicity or biological activity of the salt. However, other salts can be used, for example, in separation or purification steps that can be used during preparation, and are therefore covered within the scope of the present invention. The salt of the compound of formula (I) can be formed, for example, by reacting the compound of formula (I) with an amount (such as an equivalent amount) of an acid or base in a medium (such as a medium in which the salt is precipitated) or in an aqueous medium, followed by freeze drying.

[0131] Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acids (e.g., trifluoroacetic acid)), adipates, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), hydrobromide (formed with hydrogen bromide), hydroiodide, maleate (formed with

[0013] The invention also includes but is not limited to bis(hydroxy)amino acids, ...

[0132] Exemplary basic salts include ammonium salts; alkali metal salts such as sodium, lithium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; barium, zinc and aluminum salts; salts with organic bases (e.g., organic amines) such as trialkylamines (such as triethylamine), procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine (ephenamine), N,N'-dibenzylethylene-diamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines; and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., chlorides, bromides and iodides of methyl, ethyl, propyl and butyl), dialkyl sulfates (e.g., sulfates of dimethyl, diethyl, dibutyl and diamyl), long chain halides (e.g., chlorides, bromides and iodides of decyl, lauryl, myristyl and stearyl), aralkyl halides (e.g., bromides of benzyl and phenethyl), and other agents. Preferred salts include monohydrochlorides, bisulfates, methanesulfonates, phosphates or nitrates.

[0133] The compound of formula (I) may be provided as an amorphous solid or a crystalline solid. Lyophilization may be used to provide the compound of formula (I) as a solid.

[0134] It should also be understood that solvates (e.g., hydrates) of compounds of formula (I) are also within the scope of the present invention. The term "solvate" means a physical association of a compound of formula (I) with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid. "Solvate" encompasses both solution phase and separable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates, and ethyl acetate solvates. Solvation methods are known in the art.

[0135] Various forms of prodrugs are known in the art and are described in Rautio, J. et al., Nature Review Drug Discovery, 17, 559-587 (2018).

[0136] In addition, the compound of formula (I) can be isolated and purified (after its preparation) to obtain a composition containing an amount of the compound of formula (I) equal to or greater than 99% by weight ("substantially pure"), which is then used or formulated as described herein. Such "substantially pure" compounds of formula (I) are also considered herein as being part of the present invention.

[0137] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. The present invention is intended to encompass stable compounds.

[0138] A "therapeutically effective amount" is intended to include an amount of a compound of the invention alone or in combination with a plurality of compounds as claimed or in combination with other active ingredients that are effective in acting as DGKα and / or DGKζ inhibitors, or in the treatment or prevention of viral infections and proliferative disorders such as cancer.

[0139] As used herein, "treating" or "treatment" encompasses the treatment of a disease state in a mammal, particularly a human, and includes: (a) preventing the disease state from occurring in a mammal, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state; (b) inhibiting the disease state, i.e., arresting its development; and / or (c) relieving the disease state, i.e., causing regression of the disease state.

[0140] The compounds of the invention are intended to include all isotopes of atoms occurring in the compounds of the invention. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include 13 C and 14 C. Isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

[0141] The compound according to formula (I) and / or pharmaceutically acceptable salts thereof may be administered by any means suitable for the condition to be treated, which may depend on the need for site-specific treatment or the amount of compound of formula (I) to be delivered.

[0142] The present invention also includes a class of pharmaceutical compositions, the pharmaceutical compositions comprising a compound of formula (I) and / or a pharmaceutically acceptable salt thereof; and one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carrier" materials) and (if necessary) other active ingredients. The compound of formula (I) can be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for such a route, and at a dose effective for the intended treatment. For example, the compounds and compositions of the present invention can be administered orally, mucosally or parenterally (including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly and intrasternally) in the form of a dosage unit formulation containing conventional pharmaceutically acceptable carriers, excipients and vehicles. For example, the drug carrier may contain a mixture of mannitol or lactose and microcrystalline cellulose. The mixture may contain additional components such as lubricants (e.g., magnesium stearate) and disintegrants (such as crospovidone). The carrier mixture may be filled into a gelatin capsule or compressed into a tablet. The pharmaceutical composition may be administered, for example, in an oral dosage form or infusion form.

[0143] For oral administration, the pharmaceutical composition can be, for example, in the form of a tablet, capsule, liquid capsule, suspension or liquid. The pharmaceutical composition is preferably prepared into a dosage unit form containing a specific amount of active ingredient. For example, the pharmaceutical composition can be provided in tablets or capsules, and the tablets or capsules are included in the amount of active ingredient in the range of from about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, more preferably from about 0.5 to 100 mg. Depending on the patient's condition and other factors, the suitable daily dose for humans or other mammals may vary greatly, but can be determined using conventional methods.

[0144] Any pharmaceutical composition considered herein can be, for example, delivered orally via any acceptable and suitable oral formulation. Exemplary oral formulations include, but are not limited to, for example, tablets, lozenges, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups and elixirs. The pharmaceutical composition intended for oral administration can be prepared according to any method known in the art for making the pharmaceutical composition intended for oral administration. In order to provide pharmaceutically palatable preparations, the pharmaceutical composition according to the present invention can contain at least one agent selected from sweeteners, flavoring agents, coloring agents, demulcents, antioxidants and preservatives.

[0145] Tablets can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for making tablets. Exemplary excipients include, but are not limited to, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating agents and disintegrants such as microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, corn starch, and alginic acid; binders such as starch, gelatin, polyvinyl pyrrolidone, and gum arabic; and lubricants such as magnesium stearate, stearic acid, and talc. In addition, tablets can be uncoated or coated by known techniques to mask the unpleasant taste of drugs that taste unpleasant, or to delay the disintegration and absorption of active ingredients in the gastrointestinal tract, thereby maintaining the effect of the active ingredient for a longer period of time. Exemplary water-soluble taste-masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropyl cellulose. Exemplary time-delay materials include, but are not limited to, ethyl cellulose and cellulose acetate butyrate.

[0146] Hard gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one salt thereof with at least one inert solid diluent such as, for example, calcium carbonate, calcium phosphate and kaolin.

[0147] Soft gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier (such as, for example, polyethylene glycol) and at least one oil medium (such as, for example, peanut oil, liquid paraffin and olive oil).

[0148] Aqueous suspensions can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one excipient suitable for producing an aqueous suspension. Exemplary excipients suitable for producing an aqueous suspension include, but are not limited to, for example, suspending agents, such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents, such as, for example, naturally occurring phospholipids, such as lecithin; condensation products of alkylene oxides with fatty acids, such as, for example, polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, such as, for example, heptadecanedeylene-oxycetanol; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as, for example, polyoxyethylene sorbitol monooleate; and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as, for example, polyethylene sorbitan monooleate. The aqueous suspension may also contain at least one preservative, such as, for example, ethyl and n-propyl p-hydroxybenzoate; at least one coloring agent; at least one flavoring agent; and / or at least one sweetening agent, including, but not limited to, for example, sucrose, saccharin, and aspartame.

[0149] Oily suspensions can be prepared, for example, by suspending at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof in a vegetable oil (e.g., such as peanut oil, olive oil, sesame oil and coconut oil) or in a mineral oil (e.g., such as liquid paraffin). Oily suspensions can also contain at least one thickening agent, such as beeswax, hard paraffin and hexadecanol. In order to provide a palatable oily suspension, at least one of the above-mentioned sweeteners and / or at least one flavoring agent can be added to the oily suspension. Oily suspensions can further contain at least one preservative, including but not limited to, for example, an antioxidant, such as, for example, butylated hydroxyanisole and -tocopherol.

[0150] Dispersible powders and granules can be prepared, for example, by mixing at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof with at least one dispersant and / or wetting agent, at least one suspending agent and / or at least one preservative. Suitable dispersants, wetting agents and suspending agents are as described above. Exemplary preservatives include, but are not limited to, for example, antioxidants, such as ascorbic acid. In addition, dispersible powders and granules can also contain at least one excipient, including, but not limited to, for example, sweeteners, flavoring agents and coloring agents.

[0151] The emulsion of at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof can be prepared, for example, as an oil-in-water emulsion. The oil phase of the emulsion comprising the compound of formula (I) can be composed of known ingredients in a known manner. The oil phase can be provided by, for example, but not limited to, vegetable oils (e.g., such as olive oil and peanut oil), mineral oils (e.g., such as liquid paraffin) and mixtures thereof. Although the phase can contain only an emulsifier, it can contain a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Suitable emulsifiers include, for example, but are not limited to, natural phospholipids, such as soy lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as, for example, polyoxyethylene sorbitan monooleate. Preferably, a lipophilic emulsifier is used together with a hydrophilic emulsifier used as a stabilizer. It is also preferred to include both oil and fat. In addition, one or more emulsifiers form so-called emulsifying waxes with or without one or more stabilizers, and the wax forms so-called emulsifying ointment base together with oil and fat, which forms the oily dispersed phase of cream preparations. Emulsions can also contain sweeteners, flavorings, preservatives and / or antioxidants. Emulsifiers and emulsion stabilizers suitable for formulations of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, distearic acid glyceryl alone or with wax, or other materials well known in the art.

[0152] The compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof can also be delivered, for example, intravenously, subcutaneously and / or intramuscularly by any pharmaceutically acceptable and suitable injectable form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions containing acceptable vehicles and solvents, such as water, Ringer's solution, and isotonic sodium chloride solution; sterile oil-in-water microemulsions; and aqueous or oily suspensions.

[0153] Preparations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared by sterile powders or granules using one or more of the carriers or diluents mentioned in the preparations for oral administration or by using other suitable dispersants or wetting agents and suspending agents. The compound may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum and / or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical field. The active ingredient may also be administered by injection as a composition with a suitable carrier (including saline, dextrose or water) or with cyclodextrin (i.e. Captisol), co-solvent solubilization (i.e. propylene glycol) or micellar solubilization (i.e. Tween 80).

[0154] Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. The acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectables.

[0155] Sterile injectable oil-in-water microemulsions can be prepared, for example, by 1) dissolving at least one compound of formula (I) in an oil phase (such as, for example, a mixture of soybean oil and lecithin); 2) combining the oil phase containing formula (I) with a mixture of water and glycerol; and 3) processing the combination to form a microemulsion.

[0156] Sterile aqueous or oily suspensions can be prepared according to methods known in the art. For example, sterile aqueous solutions or suspensions can be prepared using nontoxic parenterally acceptable diluents or solvents (e.g., 1,3-butanediol); and sterile oily suspensions can be prepared using sterile nontoxic acceptable solvents or suspending media (e.g., sterile fixed oils (e.g., such as synthetic monoglycerides or diglycerides) and fatty acids (e.g., such as oleic acid)).

[0157] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-tocopheryl polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tweens, polyethoxylated castor oils such as CREMOPHOR surfactant (BASF), or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins such as α-, β- and γ-cyclodextrin or chemically modified derivatives such as hydroxyalkylcyclodextrins (including 2- and 3-hydroxypropylcyclodextrin) or other soluble derivatives may also be advantageously used to facilitate delivery of the compounds of the formulae described herein.

[0158] The pharmaceutically active compounds of the present invention can be processed according to conventional pharmaceutical methods to produce drugs for administration to patients (including humans and other mammals). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Such compositions may also contain adjuvants such as wetting agents, sweeteners, flavoring agents and aromatics.

[0159] The amount of the compound administered and the dosage regimen for treating the condition with the compound and / or composition of the present invention depend on various factors, including the age, weight, sex, medical condition, disease type, severity of the disease, route of administration and frequency, and the specific compound used of the subject. Therefore, the dosage regimen can vary widely, but can be routinely determined using standard methods. The daily dose of about 0.001 to 100 mg / kg body weight, preferably between about 0.0025 and about 50 mg / kg body weight, and most preferably between about 0.005 to 10 mg / kg body weight may be appropriate. The daily dose can be applied by one to four doses / day. Other dosing schedules include one dose per week and one dose every two days.

[0160] For therapeutic purposes, the active compound of the present invention is usually combined with one or more adjuvants suitable for the indicated route of administration. If administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose alkanoate, cellulose alkyl ester, talcum, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, gum arabic, sodium alginate, polyvinyl pyrrolidone and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. The above-mentioned capsule or tablet may include a controlled release formulation, which can be provided as a dispersion of the active compound in hydroxypropyl methylcellulose.

[0161] The pharmaceutical composition of the present invention comprises at least one compound of formula (I) and / or at least one pharmaceutically acceptable salt thereof and an additional agent optionally selected from any pharmaceutically acceptable carrier, adjuvant and vehicle. An alternative composition of the present invention comprises a compound of formula (I) as described herein or a prodrug thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0162] utility

[0163] The compounds of formula (I) are useful for treating cancer.

[0164] In another embodiment, the present invention provides a combination preparation of a compound of formula (I) and / or a pharmaceutically acceptable salt thereof, a stereoisomer or a tautomer thereof, and one or more additional therapeutic agents for simultaneous, separate or sequential use in the treatment and / or prevention of a variety of diseases or disorders associated with DGK target inhibition in T cells.

[0165] In another aspect, the present invention provides a method for treating a patient suffering from or susceptible to a medical condition associated with DGK target inhibition in T cells. A variety of medical conditions can be treated. The method comprises administering to the patient a therapeutically effective amount of a composition comprising a compound of formula (I) and / or a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a tautomer thereof. For example, the compounds described herein can be used to treat or prevent viral infections and proliferative diseases, such as cancer.

[0166] Compounds of formula (I) and pharmaceutical compositions comprising at least one compound of formula (I) can be used to treat or prevent any disease or condition associated with DGK target inhibition in T cells. They include viral infections and other infections (e.g., skin infections, GI infections, urinary tract infections, genitourinary infections, systemic infections) and proliferative diseases (e.g., cancer). Compounds of formula (I) and pharmaceutical compositions comprising at least one compound of formula (I) can be applied to animals, preferably mammals (e.g., domestic animals, cats, dogs, mice, rats), and more preferably humans. Any method of administration can be used to deliver the compound or pharmaceutical composition to the patient. In certain embodiments, a compound of formula (I) or a pharmaceutical composition comprising at least a compound of formula (I) is orally administered. In other embodiments, a pharmaceutical composition comprising at least a compound of formula (I) or a compound of formula (I) is administered parenterally.

[0167] The compounds of formula (I) can inhibit the activity of diacylglycerol kinase α and ζ (DGKα / ζ). For example, the compounds of formula (I) can be used to inhibit the activity of DGKα and DGKζ in cells or in individuals who need to regulate DGKα and DGKζ by administering an inhibitory amount of the compound of formula (I) or a salt thereof.

[0168] The present invention further provides a method for treating a disease associated with the activity or expression (including abnormal activity and / or overexpression) of DGKα and DGKζ in an individual (e.g., a patient) by administering a therapeutically effective amount or dose of a compound of formula (I) or a pharmaceutical composition thereof to an individual in need of such treatment. Examples of diseases may include any disease, disorder, or condition directly or indirectly associated with the expression or activity of DGKα and DGKζ enzymes (such as overexpression or abnormal activity). DGKα and DGKζ related diseases may also include any disease, disorder, or condition that can be prevented, improved, or cured by regulating the activity of DGKα and DGKζ enzymes. Examples of DGKα and DGKζ related diseases include cancer and viral infections, such as HIV infection, hepatitis B, and hepatitis C.

[0169] In one aspect, one or more compounds of formula (I) are administered sequentially prior to administration of an immuno-oncology agent. In another aspect, one or more compounds of formula (I) are administered simultaneously with an immuno-oncology agent. In yet another aspect, one or more compounds of formula (I) are administered sequentially after administration of an immuno-oncology agent.

[0170] In another aspect, the compounds of formula (I) may be co-formulated with an immuno-oncology agent.

[0171] Immuno-oncology agents include, for example, small molecule drugs, antibodies, or other biological or small molecules. Examples of biological immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In one aspect, the antibody is a monoclonal antibody. In another aspect, the monoclonal antibody is humanized or human.

[0172] In one aspect, the immuno-oncology agent is either (i) an agonist of a stimulatory (including co-stimulatory) receptor, or (ii) an antagonist of an inhibitory (including co-inhibitory) signal on T cells, both of which result in the amplification of antigen-specific T cell responses (often referred to as immune checkpoint modulators).

[0173] Some stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to homologous TNF receptor family members, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RAN KL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR 3. EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0174] In one aspect, T cell responses can be stimulated by a combination of a compound of formula (I) and one or more of the following: (i) antagonists (e.g., immune checkpoint inhibitors) of proteins that inhibit T cell activation (such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4), and (ii) agonists of proteins that stimulate T cell activation (such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H).

[0175] Other agents that can be combined with compounds of formula (I) for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, compounds of formula (I) can be combined with antagonists of KIR (such as lirilumab).

[0176] Still other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0177] In another aspect, the compounds of formula (I) can be used with one or more of the following: agonists that bind positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells, overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), deplete or inhibit Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes (such as IDO), or reverse / prevent T cell anergy or exhaustion) and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0178] In one aspect, the immuno-oncology agent is a CTLA-4 antagonist, such as an antagonist CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[0179] In another aspect, the immuno-oncology agent is a PD-1 antagonist, such as an antagonist PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). Immuno-oncology agents may also include pidilizumab (CT-011), although its specificity for PD-1 binding is questionable. Another approach to targeting the PD-1 receptor is a recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224.

[0180] In another aspect, the immuno-oncology agent is a PD-L1 antagonist, such as an antagonist PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[0181] In another aspect, the immuno-oncology agent is a LAG-3 antagonist, such as an antagonist LAG-3 antibody. Suitable LAG3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0182] In another aspect, the immuno-oncology agent is a CD137 (4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137 antibodies include, for example, Urelumab and PF-05082566 (WO12 / 32433).

[0183] In another aspect, the immuno-oncology agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683).

[0184] In another aspect, the immuno-oncology agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006 / 122150, WO07 / 75598, WO08 / 36653, WO08 / 36642), indoximod, BMS-986205, or NLG-919 (WO09 / 73620, WO09 / 1156652, WO11 / 56652, WO12 / 142237).

[0185] In another aspect, the immuno-oncology agent is an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469.

[0186] In another aspect, the immuno-oncology agent is an OX40L antagonist, such as an antagonist OX40 antibody. Suitable OX40L antagonists include, for example, RG-7888 (WO06 / 029879).

[0187] In another aspect, the immuno-oncology agent is a CD40 agonist, such as an agonistic CD40 antibody. In yet another embodiment, the immuno-oncology agent is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab or dacetuzumab.

[0188] In another aspect, the immuno-oncology agent is a CD27 agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab.

[0189] In another aspect, the immuno-oncology agent is MGA271 (directed against B7H3) (WO 11 / 109400).

[0190] Combination therapy is intended to include the administration of these therapeutic agents in a sequential manner (i.e., wherein each therapeutic agent is administered at different times) and the administration of these therapeutic agents or at least two of these therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dosage form of each therapeutic agent with a fixed ratio or with multiple single dosage forms of each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption by mucosal tissue. The therapeutic agent can be administered by the same route or by different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally or all therapeutic agents can be administered by intravenous injection. Combination therapy can also include the further administration of the therapeutic agent as described above in combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiotherapy). In the case where combination therapy further includes non-drug therapy, the non-drug therapy can be performed at any appropriate time, as long as the beneficial effect is achieved from the combined action of the therapeutic agent and the combination of non-drug therapy. For example, in appropriate circumstances, when the non-drug therapy is temporarily removed from the administration of the therapeutic agent (perhaps for days or even weeks), the beneficial effects are still achieved.

[0191] As used herein, the term "cell" is intended to refer to a cell in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell can be a part of a tissue sample excised from an organism (such as a mammal). In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism (such as a mammal).

[0192] As used herein, the term "contacting" refers to bringing the indicated parts together in an in vitro system or an in vivo system. For example, "contacting" the DGKα and DGKζ enzymes with a compound of formula (I) includes administering a compound of the invention to an individual or patient (such as a human) having DGKα and DGKζ, and, for example, introducing a compound of formula (I) into a sample containing cells or purified preparations containing DGKα and DGKζ enzymes.

[0193] The term "DGKα and DGKζ inhibitor" refers to an agent that is capable of inhibiting the activity of diacylglycerol kinase α and / or diacylglycerol kinase ζ (DGKα and DGKζ) in T cells, thereby resulting in T cell stimulation. DGKα and DGKζ inhibitors can be reversible or irreversible DGKα and DGKζ inhibitors. "Reversible DGKα and DGKζ inhibitors" are compounds that reversibly inhibit the enzymatic activity of DGKα and DGKζ at the catalytic site or the non-catalytic site, and "irreversible DGKα and DGKζ inhibitors" are compounds that irreversibly destroy the enzymatic activity of DGKα and DGKζ by forming a covalent bond with the enzyme.

[0194] The types of cancers that can be treated with compounds of formula (I) include, but are not limited to, brain cancer, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, blood cancer, lung cancer, and bone cancer. Examples of such cancer types include neuroblastoma, intestinal cancer (such as rectal cancer, colon cancer, familiar adenomatous polyposis and hereditary non-polyposis colorectal cancer), esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumors (such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumor), Hodgkin's lymphoma, Non-Hodgkin lymphoma, Burkeley lymphoma, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma.

[0195] One or more additional agents or treatments (e.g., such as antiviral agents, chemotherapeutic agents or other anticancer agents, immunopotentiators, immunosuppressants, radiation, anti-tumor and antiviral vaccines, cytokine therapy (e.g., IL2 and GM-CSF), and / or tyrosine kinase inhibitors) may optionally be combined with compounds of formula (I) for the treatment of DGKα and DGKζ-related diseases, disorders or conditions. The agents may be combined with the compounds of the invention in a single dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms.

[0196] Suitable chemotherapeutic or other anticancer agents include, for example, alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as uracil mustard, chlormethine, cyclophosphamide, Ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, and temozolomide.

[0197] In the treatment of melanoma, suitable agents for use in combination with the compounds of formula (I) include: dacarbazine (DTIC), optionally together with other chemotherapy drugs such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen", consisting of DTIC, BCNU, cisplatin and tamoxifen; cisplatin, vinblastine and DTIC, temozolomide or YERVOY TM In the treatment of melanoma, the compounds of formula (I) may also be combined with immunotherapy drugs, including cytokines, such as interferon alpha, interleukin 2 and tumor necrosis factor (TNF).

[0198] Compounds of formula (I) may also be used in combination with vaccine therapy to treat melanoma. Anti-melanoma vaccines are similar in some respects to antiviral vaccines used to prevent diseases caused by viruses (polio, measles and mumps). Weakened melanoma cells or parts of melanoma cells (called antigens) may be injected into a patient to stimulate the body's immune system to destroy melanoma cells.

[0199] Melanoma confined to the arms or legs can also be treated with a combination of agents including one or more compounds of formula (I) using a hyperthermic isolated limb perfusion technique. This treatment regimen temporarily separates the circulation of the affected limb from the rest of the body, and high doses of chemotherapy are injected into the arteries that supply blood to the limb, thereby providing high doses to the tumor area without exposing the internal organs to these doses that otherwise cause serious side effects. Typically, the fluid is warmed to 38.9°C to 40°C. Melphalan is the most commonly used drug in this chemotherapy. This can be administered together with another agent called tumor necrosis factor (TNF).

[0200] Suitable chemotherapeutic or other anticancer agents include, for example, antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors) such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine.

[0201] Suitable chemotherapeutic or other anticancer agents further include, for example, certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (Taxol), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (particularly IFN-a), etoposide and teniposide.

[0202] Other cytotoxic agents include navelbene, CPT-11, anastrazole, letrozole, capecitabine, and droloxafine.

[0203] Also suitable are cytotoxic agents such as podophyllotoxin; antitumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitory agents; antihormonal therapeutics; folinic acid; tegafur; and hematopoietic growth factors.

[0204] One or more other anti-cancer agents including antibody therapeutics such as trastuzumab Antibodies against co-stimulatory molecules (such as CTLA-4, 4-1BB and PD-1), or antibodies against cytokines (IL-10 or TGF-β).

[0205] Other anticancer agents also include those that block the migration of immune cells, such as antagonists of chemokine receptors, including CCR2 and CCR4.

[0206] Other anti-cancer agents also include those that enhance the immune system such as helper or adoptive T cell transfer.

[0207] Anticancer vaccines include dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

[0208] The pharmaceutical composition of the present invention may optionally contain at least one signal transduction inhibitor (STI). A "signal transduction inhibitor" is an agent that selectively inhibits one or more important steps in the signal transduction pathway (in the normal function of cancer cells), thereby leading to apoptosis. Suitable STIs include, but are not limited to: (i) bcr / abl kinase inhibitors, such as STI 571 (ii) epidermal growth factor (EGF) receptor inhibitors, such as kinase inhibitors ( SSI-774) and antibodies (Imclone: ​​C225 [Goldstein et al., Clin. Cancer Res., 1: 1311-1318 (1995)] and Abgenix: ABX-EGF); (iii) her-2 / neu receptor inhibitors, such as farnesyl transferase inhibitors (FTIs), such as, for example, L-744,832 (Kohl et al., Nat. Med., 1(8): 792-797 (1995)); (iv) inhibitors of Akt family kinases or Akt pathways, such as, for example, rapamycin (see, e.g., Sekulic et al., Cancer Res., 1(8): 792-797 (1995)); Res., 60:3504-3513 (2000)); (v) cell cycle kinase inhibitors, such as, for example, flavopiridol and UCN-O1 (see, for example, Sausville, Curr. Med. Chem. Anti-Canc. Agents, 3:47-56 (2003)); and (vi) phosphatidylinositol kinase inhibitors, such as, for example, LY294002 (see, for example, Vlahos et al., J. Biol. Chem., 269:5241-5248 (1994)). Alternatively, at least one STI and at least one compound of formula (I) may be in separate pharmaceutical compositions. In a specific embodiment of the present invention, at least one compound of formula (I) and at least one STI may be administered to a patient simultaneously or sequentially. In other words, at least one compound of formula (I) may be administered first, at least one STI may be administered first, or at least one compound of formula (I) and at least one STI may be administered simultaneously. Additionally, when more than one compound of formula (I) and / or STI is used, the compounds may be administered in any order.

[0209] The present invention further provides a pharmaceutical composition for treating a chronic viral infection in a patient, the pharmaceutical composition comprising at least one compound of formula (I), optionally at least one chemotherapeutic agent, and optionally at least one antiviral agent in a pharmaceutically acceptable carrier.

[0210] Also provided is a method for treating chronic viral infection in a patient by administering an effective amount of the above pharmaceutical composition.

[0211] In a specific embodiment of the invention, at least one compound of formula (I) and at least one chemotherapeutic agent may be administered to a patient simultaneously or sequentially. In other words, at least one compound of formula (I) may be administered first, at least one chemotherapeutic agent may be administered first, or at least one compound of formula (I) and at least one STI may be administered simultaneously. In addition, when more than one compound of formula (I) and / or chemotherapeutic agent is used, the compounds may be administered in any order. Similarly, any antiviral agent or STI may also be administered at any point compared to the administration of the compound of formula (I).

[0212] Chronic viral infections that can be treated using the combination therapy of the present invention include, but are not limited to, diseases caused by: Hepatitis C virus (HCV), human papillomavirus (HPV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella zoster virus, coxsackie virus, human immunodeficiency virus (HIV). Notably, parasitic infections (e.g., malaria) can also be treated by the above method, wherein compounds known to treat parasitic disorders are optionally added in place of antiviral agents.

[0213] Suitable antiviral agents contemplated for use in combination with the compounds of formula (I) may include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and other antiviral drugs.

[0214] Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil [bis(POM)-PMEA]; lobucavir; BCH-I0652; emtricitabine [(-)-FTC]; β-L-FD4 (also known as β-L-D4C and designated β-L-2',3'-dideoxy-5-fluoro-cytidine (cytidene)); DAPD ((-)-β-D-2,6,-diamino-purine dioxolane); and lodenosine (FddA). Typical suitable NNRTIs include nevirapine (BI-RG-587); delavirdine (BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione); and (+)-calendula lactone A (NSC-675451)) and B. Typical suitable protease inhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK-639); nelfinavir (AG-1343); amprenavir (141W94); lasinavir; DMP-450; BMS-2322623; ABT-378; and AG-1549. Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside, and Yissum Project No. 11607.

[0215] The present invention also includes a pharmaceutical kit that can be used, for example, to treat or prevent DGKα and DGKζ related diseases or disorders and other diseases mentioned herein, the pharmaceutical kit comprising one or more containers containing a pharmaceutical composition, the pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I). If desired, such a kit may further include one or more of the following: various conventional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, which will be readily apparent to those skilled in the art. The kit may also include instructions as inserts or as labels, indicating the amount of the components to be administered, administration instructions, and / or instructions for mixing the components.

[0216] Combination therapy is intended to include the administration of these therapeutic agents in a sequential manner (i.e., wherein each therapeutic agent is administered at different times) and the administration of these therapeutic agents or at least two of these therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dosage form of each therapeutic agent with a fixed ratio or with multiple single dosage forms of each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route, including but not limited to oral routes, intravenous routes, intramuscular routes, and direct absorption by mucosal tissue. The therapeutic agent can be administered by the same route or by different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while the other therapeutic agents of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally or all therapeutic agents can be administered by intravenous injection. Combination therapy can also include the further administration of the therapeutic agent as described above in combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiotherapy). In the case where combination therapy also includes non-drug therapy, the non-drug therapy can be performed at any appropriate time, as long as the beneficial effect is achieved from the combined action of the therapeutic agent and the combination of non-drug therapy. For example, in appropriate circumstances, when the non-drug therapy is temporarily removed from the administration of the therapeutic agent (perhaps for days or even weeks), the beneficial effects are still achieved.

[0217] The present invention also provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more compounds of formula (I) formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents, and optionally one or more additional therapeutic agents as described above.

[0218] The compounds of the invention may be administered by any suitable means for any of the uses described herein: for example, orally, such as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions (including nanosuspensions, microsuspensions, spray-dried dispersions), syrups and emulsions; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension); nasally, including administration to the nasal membrane, such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally, such as in the form of a suppository. They may be administered alone, but will generally be administered with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice.

[0219] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, which participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient, the other ingredients including: that is, adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricants and dispensing agents, depending on the mode of administration and dosage form.

[0220] The term "pharmaceutical composition" means a composition comprising a compound of the present invention in combination with at least one additional pharmaceutically acceptable carrier.

[0221] Pharmaceutically acceptable carriers are formulated according to many factors within the scope of knowledge of those of ordinary skill in the art. These include, but are not limited to: the type and nature of the active agent being formulated; the subject to whom the composition containing the agent is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as various solid and semisolid dosage forms. Such carriers may also include many different ingredients and additives in addition to the active agent, and such additional ingredients are included in the formulation for a variety of reasons well known to those of ordinary skill in the art (e.g., stabilization of active agents, adhesives, etc.). Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection are found in a variety of readily available sources, such as Allen, LVJr. et al. Remington: The Science and Practice of Pharmacy (Volume 2), 22nd Edition (2012), Pharmaceutical Press.

[0222] Of course, the dosage regimen of the compounds of this invention will vary according to known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition and weight of the recipient; the nature and extent of the symptoms; kind of concurrent treatment; frequency of treatment; route of administration, the patient's renal and hepatic function and the desired effect.

[0223] As a general guide, the daily oral dosage range for each active ingredient, when used for the indicated effects, will be between about 0.001 to about 5000 mg / day, preferably between about 0.01 to about 1000 mg / day, and most preferably between 0.1 to about 250 mg / day. Intravenously, during a constant rate infusion, the most preferred dosage range will be from about 0.01 to about 10 mg / kg / minute. The compounds of the invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily.

[0224] The compounds are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as pharmaceutical carriers) appropriately selected with respect to the intended form of administration (e.g., oral tablets, capsules, elixirs and syrups), and in accordance with conventional pharmaceutical practice.

[0225] Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 mg to about 2000 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient will generally be present in an amount of about 0.1%-95% by weight based on the total weight of the composition.

[0226] A typical capsule for oral administration contains at least one compound of the invention (250 mg), lactose (75 mg) and magnesium stearate (15 mg).The mixture is passed through a 60 mesh screen and packaged into size L gelatin capsules.

[0227] A typical injectable formulation is produced by aseptically placing at least one compound of the invention (250 mg) into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of normal saline to produce an injectable formulation.

[0228] The present invention includes within its scope a pharmaceutical composition comprising a therapeutically effective amount of at least one compound of the present invention (alone or in combination with a pharmaceutical carrier) as an active ingredient. Optionally, the compounds of the present invention can be used alone, in combination with other compounds of the present invention, or in combination with one or more other therapeutic agents (e.g., anticancer agents or other pharmaceutically active materials).

[0229] Regardless of the administration route selected, the compound of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical composition of the present invention can be formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0230] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve a therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0231] The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0232] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to achieve a therapeutic effect, and gradually increase the dosage until the effect is achieved.

[0233] Typically, the appropriate daily dose of the compounds of this invention will be the amount of the compound that effectively produces the lowest dose of the therapeutic effect. Typically, such effective doses will depend on the factors described above. Typically, the range of oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for patients will be from about 0.01 mg to about 50 mg / kg body weight / day.

[0234] If desired, the effective daily dose of active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day (optionally in unit dosage form). In certain aspects of the invention, administration is once daily.

[0235] While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0236] When used in combination with the compounds of the present invention, the above other therapeutic agents can be used, for example, in the amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art. In the methods of the present invention, one or more such other therapeutic agents can be administered before, simultaneously with, or after the administration of the compounds of the present invention.

[0237] Preparation method

[0238] The compounds of the present invention can be synthesized by a variety of methods available to those skilled in the art of organic chemistry. General synthetic schemes for preparing the compounds of the present invention are described below. These schemes are illustrative and are not intended to limit the possible techniques that a person skilled in the art can use to prepare the compounds disclosed herein. The different methods for preparing the compounds of the present invention are clear to those skilled in the art. Examples of the compounds of the present invention prepared by the methods described in the general schemes are given in the Examples section listed below. The preparation of homochiral embodiments can be carried out by techniques known to those skilled in the art. For example, homochiral compounds can be prepared by separating racemic products or diastereomers via chiral phase preparative HPLC. Alternatively, the example compounds can be prepared by known methods to obtain products enriched in enantiomers or diastereomers.

[0239] The reactions and techniques described in this section are carried out in a solvent suitable for the reagents and materials used, and are applicable to the conversions performed. In addition, in the description of the synthetic method given below, it should be understood that all proposed reaction conditions (including the selection of solvent, reaction atmosphere, reaction temperature, duration of the experiment and post-processing procedures) are selected as standard conditions for the reaction, and those skilled in the art should easily recognize this. It should be understood by those skilled in the art of organic synthesis that the functional groups present in each part of the molecule must be compatible with the proposed reagents and reactions. Such restrictions on substituents compatible with the reaction conditions should be easily clear to those skilled in the art, wherein alternatives are required when there are incompatible substituents. This will sometimes require judgment to modify the order of the synthetic steps or select a specific method scheme rather than another, in order to obtain the compounds of the present invention. It should also be recognized that another major consideration in the planning of any synthetic route in this field is to judiciously select the protecting group for protecting the reactive functional groups present in the compounds described in the present invention. An authoritative explanation describing many alternatives for the trained practitioner is Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4th ed., Wiley and Sons (2007).

[0240] Methods that can be used to synthesize intermediates useful in preparing the examples of the present invention are shown in the following schemes.

[0241] Benzoxazine-2,4(1H)-dione of the type shown can be treated with a strong base and a methylating agent such as iodomethane to give 1-methyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione. These can be treated with activated acetates such as nitroacetate, malonate, and cyanoacetate to produce 1-methyl-3-nitroquinoline-2,4(1H,3H)-dione, 1-methyl-3-carboxyethylquinoline-2,4(1H,3H)-dione, and 1-methyl-3-cyanoquinoline-2,4(1H,3H)-dione, respectively. The 3-ester derivative can be further transformed by alkaline hydrolysis and acidic decarboxylation to provide 6-fluoro-4-hydroxy-1-methylquinoline-2(1H)-one.

[0242] Solution 1

[0243]

[0244] Alternatively, quinolinediones can be prepared from substituted 2-aminobenzoic acids. For example, methyl 2-amino-5-bromobenzoate can be converted to 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile in four steps.

[0245] Solution 2

[0246]

[0247] Such quinolinediones can in turn be converted to the related 4-chloro or 4-trifluoromethanesulfonate derivatives, which can be reacted with a variety of functionalized piperazines to give protected compounds such as tert-butyl 4-(6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate or Examples of the present invention.

[0248] Solution 3

[0249]

[0250] Protected piperazines such as tert-butyl 4-(6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate can be further transformed by deprotecting the piperazine with, for example, TFA in dichloromethane and reacting with an electrophile such as 3-fluorobenzyl bromide to provide 6-fluoro-4-{4-[(3-fluorophenyl)methyl]piperazin-1-yl}-1-methyl-3-nitro-1,2-dihydroquinolin-2-one.

[0251] Solution 4

[0252]

[0253] The compounds of the present invention can also be prepared by reacting a quinolinedione or an aminoquinolone with an electrophilic reagent such as phosphorus oxychloride in DMF, nitric acid in acetic acid or N-bromosuccinimide in DMF to give, for example, 4-(4-benzhydrylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbaldehyde, 4-hydroxy-1-methyl-3-nitroquinolin-2(1H)-one and tert-butyl 4-(3-bromo-6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate, respectively. These compounds can be further transformed as desired to give the compounds of the present invention. Examples of the present invention can be prepared from 4-hydroxy-1-methyl-3-nitroquinolin-2(1H)-one by following the steps outlined in Scheme 3.

[0254] Solution 5

[0255]

[0256] 4-(3-bromo-6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester can be reacted with aryl and heteroaryl boronic acids or statins in the presence of a suitable catalyst to provide, for example, 4-(6-fluoro-1-methyl-2-oxo-3-(pyridin-4-yl)-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester. These embodiments can be further transformed by deprotection and alkylation of piperazine, as shown in Scheme 3.

[0257] Solution 6

[0258]

[0259] Example

[0260] The following examples illustrate specific and preferred embodiments of the present invention, and do not limit the scope of the present invention. Unless otherwise specified, chemical abbreviations and symbols and scientific abbreviations and symbols have their usual and customary meanings. The above defines other abbreviations used in the examples and other places in the application. Common intermediates can usually be used to prepare more than one example, and are identified in order (e.g., intermediate 1, intermediate 2, etc.) and abbreviated as Int.1 or I1, Int.2 or I2, etc. The compounds of the examples are identified by the examples and steps for preparing them (e.g., "1-A" represents Example 1, Step A) or only in the case where the compound is the title compound of the example (e.g., "1" represents the title compound of Example 1). In some cases, alternative preparations of intermediates or examples are described. Often, chemical technicians in the field of synthesis can design alternative preparations that may be desired based on one or more considerations, such as shorter reaction times, cheaper starting materials, easy to operate or separate, improve yields, be suitable for catalysis, avoid toxic reagents, the availability of special instruments, and reduce the number of linear steps, etc. The purpose of describing alternative preparation methods is to further enable the preparation of embodiments of the present invention. In some cases, some of the functional groups in the outlined examples and claims may be replaced by well-known bioisosteric substitutes known in the art, such as replacement of a carboxylic acid group with a tetrazole or phosphate moiety. 1 H NMR data were processed using water suppression. The reported spectra were not corrected for water suppression. Protons adjacent to the water suppression frequency of 3.35 ppm exhibit reduced signal intensity.

[0261] abbreviation

[0262] Ac Acetyl

[0263] AcOH Acetic acid

[0264] Ac2O Acetic anhydride

[0265] anhyd. anhyd

[0266] aq. Aqueous solution

[0267] Boc tert-Butyloxycarbonyl

[0268] BOP Benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate

[0269] Bu Butyl

[0270] DCM Dichloromethane

[0271] DEA Diethylamine

[0272] DIEA or DIPEA Diisopropylethylamine

[0273] DMF Dimethylformamide

[0274] DMSO Dimethyl sulfoxide

[0275] dppf 1,1'-Bis(diphenylphosphino)ferrocene

[0276] EDTA Ethylenediaminetetraacetic acid

[0277] Et Ethyl

[0278] Et3N Triethylamine

[0279] EtOAc Ethyl acetate

[0280] EtOH

[0281] h, hour or hr hour

[0282] HCl

[0283] HPLC High Pressure Liquid Chromatography

[0284] LC Liquid Chromatography

[0285] LCMS liquid chromatography-mass spectrometry

[0286] M Moore

[0287] M +1 (M+H) +

[0288] Me Methyl

[0289] MeI Methyl iodide

[0290] MeOH Methanol

[0291] Mesyl-Cl Methanesulfonyl chloride

[0292] MHz Megahertz

[0293] mins minutes

[0294] mM millimole

[0295] MS mass spectrometry

[0296] n or N Normal

[0297] NBS N-bromosuccinimide

[0298] NH4OAc Ammonium acetate

[0299] KHMDS Potassium bis(trimethylsilyl)amide

[0300] nM nanomolar

[0301] NMP N-Methylpyrrolidone

[0302] Pd2(dba)3 tris(dibenzylideneacetone)dipalladium

[0303] pet ether petroleum ether

[0304] Ph Phenyl

[0305] POCl3 Phosphorus oxychloride

[0306] rt or Ret time retention time

[0307] sat. saturated

[0308] TEA Triethylamine

[0309] Tetrakis Tetrakis(triphenylphosphine)palladium(0)

[0310] TFA Trifluoroacetic acid

[0311] Tf2O trifluoromethanesulfonic anhydride

[0312] THF Tetrahydrofuran

[0313] Intermediate 1

[0314] 6-Fluoro-1-methyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

[0315]

[0316] To a solution of 6-fluoro-1H-benzo [d] [1,3] oxazine-2,4-dione (10.2 g, 56.3 mmol) in DMF (60 mL) was added a 60% dispersion of sodium hydride (2.70 g, 67.6 mmol) in mineral oil at 0 ° C. The reaction mixture was warmed to room temperature and stirred for 30 min. Then iodomethane (4.23 mL, 67.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature overnight. The reaction was quenched with water. Ether was added to the reaction mixture. A light yellow solid was collected as 6-fluoro-1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (9.55 g, 48.9 mmol, 87% yield). LC / MS LC / MS column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8mL / min; Detection: UV at 220nm. Retention time 1.982min, 196.0 (M+H + ). 1 HNMR (400MHz, DMSO-d6) δ7.82-7.73(m,2H),7.56-7.46(m,1H),3.47(s,3H).

[0317] Intermediate 2

[0318] 6-Fluoro-1-methyl-3-nitroquinoline-2,4(1H,3H)-dione

[0319]

[0320] To a solution of ethyl 2-nitroacetate (3.38 g, 25.4 mmol) in NMP (15 mL) in a round-bottom flask at 0 ° C, sodium hydride (1.107 g, 27.7 mmol) was added in batches in a 60% dispersion in mineral oil. The reaction mixture was stirred at 0 ° C for 5 minutes, and then stirred at room temperature for 15 minutes. Next, 6-fluoro-1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (4.5 g, 23.06 mmol) was added and the reaction mixture was heated at 120 ° C for 2 h. LC / MS shows that the reaction is complete. The reaction is quenched with ice water. The reaction mixture is acidified with 1N HCl solution. Ether is added and a yellow solid is collected, which is 6-fluoro-1-methyl-3-nitroquinoline-2,4 (1H, 3H)-dione (2.69 g, 11.29 mmol, 49.0% yield). LC / MS LC / MS column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); temperature: 40°C; gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; flow rate: 0.8mL / min; detection: UV at 220nm. Retention time 1.488min, 237.1 (MH-). 1 H NMR (400MHz, DMSO-d6) δ7.85 (dd, J = 9.2, 2.6Hz, 1H), 7.72-7.47 (m, 2H), 6.73 (s, br, 1H), 3.58 (s, 3H).

[0321] Intermediate 3

[0322] 4-Chloro-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one

[0323]

[0324] In sealed tube, add 6-fluoro-1-methyl-3-nitroquinoline-2,4 (1H, 3H)-dione (1.4g, 5.88mmol) and phosphorus oxychloride (10mL, 107mmol).Reactant mixture is heated at 95 DEG C for 4h.Reactant mixture is concentrated and residue is poured into ice water and neutralized with saturated NaHCO solution.Mixture is extracted with dichloromethane (2x40mL) and organic layer is merged, dried (MgSO4) and concentrated to obtain 4-chloro-6-fluoro-1-methyl-3-nitroquinoline-2 (1H)-one (1.28g, 4.99mmol, 85% yield) in orange solid. LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 40° C.; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 2.648 min, 257.0 (M+H + ). 1 H NMR (500MHz, DMSO-d6) δ7.93 (d, J = 9.5Hz, 1H), 7.86-7.79 (m, 2H), 3.72 (s, 3H).

[0325] Intermediate 4

[0326] tert-Butyl 4-(6-fluoro-1-methyl-3-nitro-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate

[0327]

[0328] To a solution of 4-chloro-6-fluoro-1-methyl-3-nitroquinoline-2(1H)-one (600mg, 2.338mmol) in DMF (8mL) was added tert-butyl piperazine-1-carboxylate (435mg, 2.338mmol) and triethylamine (0.978mL, 7.01mmol). The reaction mixture was stirred at room temperature overnight. LC / MS showed that the reaction was complete. The reaction was quenched with water. A yellow solid was collected as the product tert-butyl 4-(6-fluoro-1-methyl-3-nitro-2-oxo-1,2-dihydroquinoline-4-yl) piperazine-1-carboxylate (765mg, 1.882mmol, 81% yield). LC / MS LC / MS column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8mL / min; Detection: UV at 220nm. Retention time 3.0min, 407.1 (M+H + ). 1 H NMR (500MHz, DMSO-d6) δ7.79-7.63(m,3H),3.65(s,3H),3.58(br.s.,4H),3.07(br.s.,4H),1.43(s,9H).

[0329] Intermediate 5

[0330] 6-Fluoro-1-methyl-3-nitro-4-(piperazin-1-yl)quinolin-2(1H)-one, TFA

[0331]

[0332] To a solution of tert-butyl 4-(6-fluoro-1-methyl-3-nitro-2-oxo-1,2-dihydroquinoline-4-yl)piperazine-1-carboxylate (2.1 g, 5.17 mmol) in CH2Cl2 (10 mL) was added TFA (5 mL, 64.9 mmol). The reaction mixture was stirred at room temperature overnight. LC / MS showed that the reaction was complete. The reaction mixture was concentrated and ethyl acetate was added. A yellow solid was obtained as the product 6-fluoro-1-methyl-3-nitro-4-(piperazine-1-yl)quinoline-2(1H)-one, TFA (1.72 g, 4.09 mmol, 79% yield). LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; Mobile phase A: 10:90 methanol:water (containing 0.1% TFA); Mobile phase B: 90:10 methanol:water (containing 0.1% TFA); Gradient: 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 1.62 min, 307.2 (M+H + ). 1 HNMR (400MHz, DMSO-d6) δ7.73-7.67(m,2H),7.64(d,J=10.0Hz,1H),3.65(s,3H),3.11-3.03(m,4H),2.93(br.s.,4H).

[0333] Example 1

[0334] 6-Fluoro-4-{4-[(3-fluorophenyl)methyl]piperazin-1-yl}-1-methyl-3-nitro-1,2-dihydroquinolin-2-one

[0335]

[0336] Prepare 6-fluoro-1-methyl-3-nitro-4-(piperazine-1-yl)quinoline-2(1H)-one, a solution of TFA (340mg, 816μmol) and DIPEA (646mL, 3.6mmol) in DMF (17.0mL). Add 500μL of 6-fluoro-1-methyl-3-nitro-4-(piperazine-1-yl)quinoline-2(1H)-one, TFA / DIPEA solution to the 3-fluorobenzyl bromide weighed into a 16x100mm threaded vial. The vial is capped and allowed to shake at room temperature until the reaction is complete, as measured by LCMS analysis (approximately 24 hours). A 500 μL aliquot of DMF was added to a vial and the crude material was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19×200 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 45%-85% B over 15 minutes, then hold at 100% B for 5 minutes; Flow rate: 20 mL / min. Fractions containing product were combined and dried via centrifugal evaporation. LC\MS conditions: Column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; Mobile phase A: 10:90 methanol:water (containing 0.1% TFA); Mobile phase B: 90:10 methanol:water (containing 0.1% TFA); Gradient: 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. MS ion 415.1 was observed, retention time 1.23 minutes.

[0337] Intermediate 6

[0338] Ethyl 6-fluoro-1-methyl-2,4-dioxo-1,2,3,4-tetrahydroquinoline-3-carboxylate

[0339]

[0340] To a solution of diethyl malonate (0.903g, 5.64mmol) in NMP (10mL) was added a 60% dispersion of sodium hydride (0.246g, 6.15mmol) in mineral oil in a round-bottom flask at 0°C. The reaction mixture was stirred at 0°C for 5 minutes and then at room temperature for 15 minutes. Next, 6-fluoro-1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (1g, 5.12mmol) was added and the reaction mixture was heated at 120°C for 2h. LC / MS showed that the reaction was complete. The reaction was quenched with ice water and acidified with 1N HCl solution. Ether was added and a beige solid was collected, which was the product 6-fluoro-1-methyl-2,4-dioxo-1,2,3,4-tetrahydroquinoline-3-ethyl carboxylate (245mg, 0.924mmol, 18.03% yield). LC / MS column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8mL / min; Detection: UV at 220nm. Retention time 1.660min, 264.1 (MH-). 1 H NMR (400MHz, DMSO-d6) δ7.77(dd,J=9.3,2.9Hz,1H),7.68-7.54(m,2H),4.32(q,J=7.1Hz,2H),3.56(s,3H),1.30(t,J=7.1Hz,3H).

[0341] Intermediate 7

[0342] 6-Fluoro-4-hydroxy-1-methylquinolin-2(1H)-one

[0343]

[0344] A suspension of 6-fluoro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester (6.5 g, 24.51 mmol) in 2M sodium hydroxide (150 mL, 300 mmol) was heated to 100 ° C for 12 hours. The reaction mixture was cooled to room temperature and 50 mL of 6N HCl was added dropwise with stirring. After about 30 min, the solid was collected by filtration. The filtrate was washed with water and then dried overnight under vacuum. The solid was further dried by suspending in toluene and evaporated to dryness (2x) to obtain the product (4.93 g, 24.51 mmol, 100% yield) as an off-white solid. m / z; 194 (M+H)+ .

[0345] Intermediate 8

[0346] 4-Chloro-6-fluoro-1-methylquinolin-2(1H)-one

[0347]

[0348] In a dry vial equipped with a magnetic stirrer, 6-fluoro-1-methylquinoline-2,4 (1H, 3H)-dione (1000mg, 5.18mmol) was added, followed by phosphorus oxychloride (5790μL, 62.1mmol). The mixture was maintained under a nitrogen atmosphere and placed in an oil bath at 100°C for a period of 1h. The reaction mixture was taken out from the oil bath and allowed to cool to room temperature, then evaporated under vacuum. The residue was dissolved / suspended in acetonitrile and placed in an ice bath. Ice crystals were added and the reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was concentrated in a vacuum to remove acetonitrile. The resulting suspension was filtered and the product was collected by filtration. The tan solid was dried at 50°C under vacuum to obtain the title compound (974mg) as a reddish brown solid. 1 H NMR (400MHz, DMSO-d6) δ7.82-7.58(m,3H),7.05(s,1H),3.64(s,3H).

[0349] Intermediate 9

[0350] tert-Butyl 4-(6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate

[0351]

[0352] 4-Chloro-6-fluoro-1-methylquinoline-2(1H)-one (940mg, 4.44mmol) and tert-butyl piperazine-1-carboxylate (910mg, 4.89mmol) are dissolved in a mixture of DMA (20mL) and t-BuOH (5.00mL). Cesium carbonate (4342mg, 13.33mmol) and (2-dicyclohexylphosphino-2', 6'-diisopropyl-1,1'-biphenyl) [2-(2'-amino-1,1'-biphenyl)] palladium (ii) methanesulfonate (186mg, 0.222mmol) are added. The reaction mixture is degassed and then flushed with nitrogen and then heated at 85°C overnight. The reaction mixture is diluted with 0.1M EDTA solution and EtOAc is added. The reaction mixture is filtered through diatomaceous earth to remove the palladium of the colloidal precipitation. The organic layer is then separated, and the aqueous mixture is re-extracted with EtOAc (2x). The combined extracts were washed once with brine, then dried over MgSO4, filtered and concentrated in vacuo to give a crude product in the form of a dark purple viscous oil. The residue was dissolved in DCM and absorbed on silica gel. The material was then graded using flash chromatography on silica gel (eluted with 90%-100% EtOAc in hexane). The uniform fractions were combined to give a product in the form of a colorless oil, 960 mg. 1 H NMR (400MHz, DMSO-d6) δ7.63-7.44 (m, 3H), 6.13 (s, 1H), 3.57 (s, 7H), 2.99 (br t, J = 4.6Hz, 4H), 1.44 (s, 9H).

[0353] Intermediate 10

[0354] tert-Butyl 4-(3-bromo-6-fluoro-1-methyl-2-oxo-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate

[0355]

[0356] 4-(6-fluoro-1-methyl-2-oxo-1,2-dihydroquinoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester (98mg, 0.271mmol) was dissolved in DMF (2mL) in a round-bottom flask maintained under a nitrogen atmosphere. The solution was cooled to 0 ° C, and N-bromosuccinimide (43.9mg, 0.247mmol) was added in a single portion. The mixture was then taken out from the ice bath and allowed to warm slightly for 15min. The solvent was then removed under high vacuum and the resulting residue was dissolved in DCM, absorbed on SiO2 and then chromatographed using 40% EtOAc in hexane. The uniform fractions were combined and evaporated in vacuo to obtain the product (98mg, 88% yield) as a colorless film.1 H NMR (400 MHz, chloroform-d) δ 7.80-7.73 (m, 1H), 7.39-7.30 (m, 2H), 3.78 (s, 3H), 1.51 (s, 9H). A DCM signal was observed at 5.3 ppm.

[0357] Intermediate 11

[0358] tert-Butyl 4-(6-fluoro-1-methyl-2-oxo-3-(pyridin-4-yl)-1,2-dihydroquinolin-4-yl)piperazine-1-carboxylate

[0359]

[0360] 4-(3-bromo-6-fluoro-1-methyl-2-oxo-1,2-dihydroquinoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester (562mg, 1.276mmol) and 4-(tributylstannyl)pyridine (0.392mL, 1.276mmol) are dissolved in anhydrous DMF (10mL). The reaction flask is evacuated and flushed with nitrogen three times. Tetrakis (147mg, 0.128mmol) is then added, and the evacuation / flushing procedure is repeated twice. The reaction mixture is heated in a sealed tube at 100°C under nitrogen for 48h. The reaction mixture is then graded using flash chromatography using 5% MeOH in DCM. The uniform fractions are combined and evaporated in vacuo to obtain the product, 928mg, as a yellow oil.

[0361] Intermediate 12

[0362] 6-Fluoro-1-methyl-4-(piperazin-1-yl)-3-(pyridin-4-yl)quinolin-2(1H)-one

[0363]

[0364] Trifluoroacetic acid (8.15mL, 106mmol) is added to a solution of 4-(6-fluoro-1-methyl-2-oxo-3-(pyridin-4-yl)-1,2-dihydroquinoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester (928mg, 2.116mmol) in DCM (8mL). The reaction mixture is stirred at room temperature for a period of 1h. The mixture is then diluted with water and the strongly acidic aqueous layer is separated from the dichloromethane solution. The organic layer is re-extracted with other water (the aqueous extract remains highly acidic). The combined aqueous extracts are combined and then alkalized (pH about 14) by adding solid KOH. The resulting mixture is extracted (3x) using DCM and the combined extracts are dried over MgSO4, filtered and evaporated in a vacuum to obtain an oil (standing to solidify overnight) (243mg).

[0365] Example 2

[0366] 6-Fluoro-4-{4-[(3-fluoro-5-methylphenyl)methyl]piperazin-1-yl}-1-methyl-3-(pyridin-4-yl)-1,2-dihydroquinolin-2-one

[0367]

[0368] To a solution of 6-fluoro-1-methyl-4-(piperazin-1-yl)-3-(pyridin-4-yl)quinolin-2(1H)-one (22 mg, 0.065 mmol) in DMF (1.5 mL) was added 4-formyl-3-hydroxybenzonitrile (14.35 mg, 0.098 mmol). The reaction mixture was stirred at room temperature for 1 h, then sodium cyanoborohydride (8.18 mg, 0.130 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction product was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 35%-75% B over 20 minutes, then hold at 100% B for 5 minutes; Flow rate: 20 mL / min. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 13.5 mg and its purity estimated by LCMS analysis was 100%. Analytical LC / MS was used to determine the final purity. Injection 1 conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 mm particles; Mobile phase A: 5:95 acetonitrile: water (with 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile: water (with 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). Injection 1 results: Purity: 100.0%; Observed mass: 461.22; Retention time: 1.48 min. Injection 2 conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). Injection 2 results: Purity: 100.0%; Observed mass: 461.2; Retention time: 2.26 min. 1HNMR(500MHz,DMSO-d6)δ8.74(d,J=6.6Hz,2H),7.79(d,J=6.6Hz,2H),7.70-7.63(m,2H),7.62-7.53(m,1H),7.00-6.92(m,1H),6.89(br d,J=8.8Hz,2H),3.63(s,3H),3.49(s,2H),2.82(br s,4H),2.50-2.44(m,4H),2.31(s,3H).

[0369] Intermediate 13

[0370] 4-(Piperazin-1-ylmethyl)-1H-indole, 2TFA

[0371]

[0372] 1H-indole-4-carboxaldehyde (250mg, 1.722mmol) is added to a solution of piperazine-1-tert-butyl formate (481mg, 2.58mmol) in DMF (1.5mL). The reaction mixture is stirred at room temperature for 1h. Then sodium cyanoborohydride (325mg, 5.17mmol) is added and the reaction mixture is stirred at room temperature for 6 days. The reaction mixture is then added to methanol, filtered and the filtrate is purified by preparative HPLC using CH3CN-H2O-TFA buffer system. The fractions are collected and concentrated overnight under speed-vac. LC / MS shows that most of the material does not contain a boc group. The material is dissolved in dichloromethane (5mL) and 1mL of TFA is added. The reaction mixture is stirred at room temperature for 3h and concentrated to obtain 4-(piperazine-1-ylmethyl)-1H-indole, 2TFA (420mg, 0.947mmol, 55.0% yield) as a white solid. LC / MS column: Phenomenex LUNAC18, 2.0X50mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 1.577 min, 216.1 (MH + ).

[0373] Example 3

[0374] 4-(4-((1H-indol-4-yl)methyl)piperazin-1-yl)-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one

[0375]

[0376] To a solution of 4-chloro-6-fluoro-1-methyl-3-nitroquinoline-2(1H)-one (150mg, 0.585mmol) in DMF (5mL) was added 4-(piperazine-1-ylmethyl)-1H-indole, 2TFA (285mg, 0.643mmol) and triethylamine (0.326mL, 2.338mmol). The reaction mixture was stirred at room temperature over the weekend. LC / MS showed that the reaction was complete. The crude product was purified by preparative HPLC using a CH3CN-H2O-TFA buffer system. The fractions were collected and concentrated overnight under speed-vac. The product was purified again by preparative HPLC using a CH3CN-H2O-ammonium acetate buffer system. The fractions were collected and concentrated overnight under speed-vac. A yellow solid was obtained as the final product 4-(4-((1H-indol-4-yl)methyl)piperazin-1-yl)-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one (180 mg, 0.413 mmol, 70.7% yield). LC / MS column: Phenomenex LUNA C18, 2.0X50 mm, 3-mm particles; mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); temperature: 40°C; gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; flow rate: 0.8 mL / min; detection: UV at 220 nm. Retention time 3.1 min, 436.1 (MH + ).

[0377] Example 4

[0378] 4-{4-[(1-ethyl-1H-indol-4-yl)methyl]piperazin-1-yl}-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one

[0379]

[0380] To a solution of 4-(4-((1H-indol-4-yl)methyl)piperazine-1-yl)-6-fluoro-1-methyl-3-nitroquinoline-2(1H)-one (15 mg, 0.034 mmol) in DMF (1.5 mL) was added a 60% dispersion of sodium hydride (2.76 mg, 0.069 mmol) in mineral oil. The reaction mixture was stirred at room temperature for 15 min. Iodoethane (10.75 mg, 0.069 mmol) was then added. The reaction mixture was stirred at room temperature overnight. LC / MS showed that the reaction was complete. The reaction was quenched with methanol. The reaction mixture was filtered and the filtrate was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10-mM ammonium acetate); Gradient: 20%-100% B over 15 minutes, then hold at 100% B for 5 minutes; Flow rate: 20 mL / min. Fractions containing the product were combined and dried via centrifugal evaporation. LC\MS conditions Column: Phenomenex LUNA C18, 2.0X50mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8mL / min; Detection: UV at 220nm: observed MS ion 464.1, retention time 2.28 minutes. 1 H NMR(500MHz,DMSO-d6)δ7.80-7.65(m,2H),7.63-7.49(m,1H),7.45-7.32(m,2H),7.17-7.06(m,1H),7.03-6.94(m,1H),6.73 -6.51(m,1H),4.20(q,J=9.3Hz,2H),3.83(s,2H),3.64(s,3H),3.16-3.09(m,4H),2.71-2.59(m,4H),1.37(t,J=9.3Hz,3H).

[0381] Example 5

[0382] 4-(4-Benzyl-3-oxopiperazin-1-yl)-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one

[0383]

[0384] 4-Chloro-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one (25 mg, 0.097 mmol), 1-benzylpiperazin-2-one (20.39 mg, 0.107 mmol) and Hunig's base (0.037 mL, 0.214 mmol) were dissolved in DMF (2 mL). The reaction mixture was stirred at room temperature overnight. The solution containing the product was fractionated using preparative LC / MS under the following conditions: column: XBridge C18, 19x200 mm, 5-μm particles; mobile phase A: 5:95 acetonitrile: water (containing 10-mM ammonium acetate); mobile phase B: 95:5 acetonitrile: water (containing 10-mM ammonium acetate); gradient: 21%-61% B over 20 minutes, then held at 100% B for 4 minutes; flow rate: 20 mL / min. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 24.1 mg and its purity estimated by LCMS analysis was 100%. Analytical LC / MS was used to determine the final purity. Injection 1 conditions: Column: Waters XBridge C18, 2.1 mm x 50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). Injection 1 results: Purity: 100.0%; Observed mass: 411.2; Retention time: 1.6 min. Injection 2 conditions: Column: Waters XBridge C18, 2.1 mm x50 mm, 1.7 μm particles; Mobile phase A: 5:95 acetonitrile:water (with 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (with 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0% B to 100% B over 3 min, then hold at 100% B for 0.75 min; Flow rate: 1 mL / min; Detection: MS and UV (220 nm). Injection 2 results: Purity: 100.0%; Observed mass: 411.02; Retention time: 1.6 min. 1 H NMR (500MHz, DMSO-d6) δ7.77-7.61(m,3H),7.43-7.36(m,2H),7.35-7.27(m,3H),4.62(s,2H),3.88(s,2H),3.66(s,3H),2.56(s,4H).

[0385] Intermediate 14

[0386] 4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile

[0387]

[0388] In a round-bottom flask, methyl 2-cyanoacetate (4.95 g, 50 mmol) was dissolved in NMP (40 mL) and the mixture was cooled to 0 ° C. Sodium hydride (1.818 g, 50.0 mmol) was added and the reaction mixture was stirred at 0 ° C for 5 minutes and then at room temperature for 15 minutes. At room temperature, 1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (8.86 g, 50.0 mmol) was added and the reaction mixture was heated at 120 ° C for 2 hours. LCMS analysis showed that the reaction was complete. The reaction mixture was diluted with 60 mL of ether and the mixture was poured into 50 mL of ice and 100 mL of 1N HCl. The resulting solid was collected (6 g). As the ether evaporated, additional solids were formed in the filtrate and these solids were collected separately (2.2 g). The NMR and LCMS of the two solid samples were the same and consistent with the product. LC / MS 2.15 min, M +1 :201.2, accurate mass: 200.1. Starting %B=0, Final %B=100, Gradient time=4 minutes, Flow rate=0.8 mL / min, Wavelength=220 nm, Solvent pair=water-methanol-0.1% TFA, Solvent A=90% water-10% methanol-0.1% TFA, Solvent B=10% water-90% methanol-0.1% TFA, Column=Phenomenex-Luna 2.0X50 mm 3 μm. 1 H NMR (400MHz, DMSO-d6) δ8.11(dd,J=8.1,1.2Hz,1H),7.77(ddd,J=8.6,7.2,1.6Hz,1H),7.56(d,J=8.6Hz,1H),7.34(t,J=7.6Hz,1H),3.57(s,3H).

[0389] Intermediate 15

[0390] 4-Chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile

[0391]

[0392] In a round-bottom flask, 1-methyl-2,4-dioxo-1,2,3,4-tetrahydroquinoline-3-carbonitrile (6g, 30.0mmol) and phosphorus oxychloride (13.97ml, 150mmol) were combined at room temperature. The reaction mixture was heated at 110 ° C for 40 minutes. After cooling to room temperature, volatiles were removed on Rotovap. 50mL of ether was added to the crude material and volatiles were removed again on Rotovap. Water was slowly added under vigorous stirring and solids were collected by filtration. The residue was placed under vacuum overnight to provide the title compound (4.6g) as a light yellow solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.14 (dd, J = 8.2, 1.3 Hz, 1H), 7.82 (ddd, J = 8.6, 7.1, 1.5 Hz, 1H), 7.51-7.42 (m, 2H), 3.79 (s, 3H).

[0393] Example 6

[0394] 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile (homochiral)

[0395]

[0396] In a microwave vial were placed 4-chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile (25 mg, 0.114 mmol), (2R,5S)-1-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazine (36.2 mg, 0.114 mmol) and Hunig's base (0.040 mL, 0.229 mmol). The mixture was dissolved in NMP (2 mL). The reaction mixture was heated at 150 °C for 14 h and then cooled to room temperature. The mixture was filtered through a 4 μm frit, concentrated and the residue was purified by preparative HPLC: Column: XBridge C18, 200 mm x 19 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 41% B for 0 min, 41%-81% B over 22 min, then 100% B for 6 min; Flow rate: 20 mL / min; Column temperature: 25°C. Fraction collection was triggered by MS and UV signals. Fractions containing the product were combined and dried via centrifugal evaporation. LC\MS conditions QC-ACN-AA-XB: LCMS: m / z=499.2 (M+H); Retention time 2.58 min.

[0397] Example 7

[0398] 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile (homochiral)

[0399]

[0400] To a stirred solution of 4-((2S,5R)-2,5-dimethylpiperazine-1-yl)-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile (0.08 g, 0.270 mmol) in acetonitrile (5 mL) at room temperature was added DIPEA (0.141 mL, 0.810 mmol) and 4,4'-(bromomethylene)bis(fluorobenzene) (0.115 g, 0.405 mmol). The reaction mixture was heated at 85 °C for 16 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure to yield a crude product, which was purified by reverse phase preparative HPLC. Method: Column: Waters XBridge C18, 150 mm x 19 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 15% B for 0 min, 15%-65% B over 25 min, then 100% B for 5 min; Flow rate: 15 mL / min. Fractions were concentrated under reduced pressure to purify 4-((2S,5R)-4-(bis(4-fluorophenyl)methyl)-2,5-dimethylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile (50 mg, 0.100 mmol, 37.2% yield). LCMS: m / z=499.2 (M+H); rt 2.426 min. LCMS method Column: XBridge BEH XP C18 (50x2.1) mm, 2.5 μm Mobile phase A: 95% water: 5% acetonitrile; 10 mM NH4OAC, Mobile phase B: 5% water: 95% acetonitrile; 10 mM NH4OAC Flow rate: 1.1 mL / min, Temperature: 50°C, 1H NMR(400MHz,DMSO-d6)δppm 8.12(d,J=1.71Hz,1H)7.97(dd,J=8.80,1.71Hz,1H)7.66(d,J=8.80Hz,1H)7.51 -7.62(m,4H)7.15(dt,J=17.61,8.80Hz,4H)6.06(s,1H)4.82(s,1H)3.75-3.83(m ,1H)3.55-3.65(m,1H)3.33(s,3H)3.01-3.11(m,2H)2.65(dd,J=11.86,2.57Hz, 1H) 2.27 (dd, J=11.49, 1.96Hz, 1H) 1.09 (d, J=6.60Hz, 3H) 1.04 (d, J=6.36Hz, 3H).

[0401] Intermediate 16

[0402] 6-Methoxy-1-methyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione

[0403]

[0404] To a solution of 6-methoxy-1H-benzo [d] [1,3] oxazine-2,4-dione (3 g, 15.53 mmol) in DMF (20 mL) was added a 60% dispersion of sodium hydride (0.745 g, 18.64 mmol) in mineral oil at 0 ° C. The reaction mixture was warmed to room temperature and stirred for 30 min. Then iodomethane (1.165 mL, 18.64 mmol) was added dropwise. The reaction mixture was then stirred at room temperature overnight. The reaction was quenched with water. Ether was added to the mixture. The product 6-methoxy-1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (3.11 g, 15.01 mmol, 97% yield) was collected as a beige solid. LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 40° C.; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 2.032 min, 208.0 (M+H + ). 1 H NMR (400MHz, DMSO-d6) δ7.68-7.23(m,3H),3.85(s,3H),3.46(s,3H).

[0405] Intermediate 17

[0406] 6-Methoxy-1-methyl-3-nitroquinoline-2,4(1H,3H)-dione

[0407]

[0408] To a solution of ethyl 2-nitroacetate (2.120g, 15.93mmol) in NMP (10mL) was added a 60% dispersion of sodium hydride (0.695g, 17.38mmol) in mineral oil in a round-bottom flask at 0°C. The reaction mixture was stirred at 0°C for 5 minutes and then at room temperature for 15 minutes. Then 6-methoxy-1-methyl-1H-benzo [d][1,3]oxazine-2,4-dione (3g, 14.48mmol) was added and the reaction mixture was heated at 120°C for 3h. LC / MS shows that the reaction is complete. The reaction is quenched with ice water. The mixture is acidified with 1N HCl solution. Ether is added and 6-methoxy-1-methyl-3-nitroquinoline-2,4 (1H, 3H)-dione (1.66g, 6.63mmol, 45.8% yield) as a yellow solid is obtained. LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 40° C.; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 1.613 min, 251.1 (M+H + ). 1 HNMR (400MHz, DMSO-d6) δ7.61 (d, J = 2.7Hz, 1H), 7.54 (d, J = 9.3Hz, 1H), 7.39 (dd, J = 9.2, 2.8Hz, 1H), 3.85 (s, 3H), 3.59 (s, 3H).

[0409] Intermediate 18

[0410] 4-Chloro-6-methoxy-1-methyl-3-nitroquinolin-2(1H)-one

[0411]

[0412] In sealed tube, add 6-methoxy-1-methyl-3-nitroquinoline-24 (1H3H)-dione (1.65g, 6.59mmol) and phosphorus oxychloride (10mL, 107mmol).Reactant mixture is heated at 95 DEG C for 4h.Then reactant mixture is concentrated and residue is poured into ice water and with NaHCO3solid neutralization.Mixture is extracted with dichloromethane (2X40mL) and organic layer is merged, dry (MgSO4) and concentrate to obtain 4-chloro-6-methoxy-1-methyl-3-nitroquinoline-2 (1H)-one (1.58g, 5.88mmol, 89% yield) in orange solid. LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 40° C.; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 2.703 min, 269.1 (M+H + ). 1 H NMR (500MHz, DMSO-d6) δ7.76 (d, J = 9.2 Hz, 1H), 7.57 (d, J = 9.5 Hz, 1H), 7.47 (s, 1H), 3.91 (s, 3H), 3.72 (s, 3H).

[0413] Example 8

[0414] 6-Methoxy-1-methyl-4-{4-[(naphthalen-1-yl)methyl]piperazin-1-yl}-3-nitro-1,2-dihydroquinolin-2-one

[0415]

[0416] To a solution of 4-chloro-6-methoxy-1-methyl-3-nitroquinolin-2(1H)-one (15 mg, 0.056 mmol) in DMF (1 mL) was added 1-(naphthalen-1-ylmethyl)piperazine (12.64 mg, 0.056 mmol) and triethylamine (0.023 mL, 0.168 mmol). The reaction mixture was stirred at room temperature overnight. LC / MS showed that the reaction was complete. CH3CN was then added to the reaction mixture. The reaction mixture was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10-mM ammonium acetate); Gradient: 50%-90% B over 15 minutes, then held at 100% B for 5 minutes; Flow rate: 20 mL / min. Fractions containing product were combined and dried via centrifugal evaporation. LC\MS: Column: Phenomenex LUNA C18, 2.0X50 mm, 3-mm particles; Mobile phase A: 5:95 acetonitrile:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10 mM ammonium acetate); Temperature: 40°C; Gradient: 0% B, 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Observed MS ion 459.2, retention time 2.34 minutes. 1 H NMR(500MHz,DMSO-d6)δ8.31(d,J=8.4Hz,1H),7.95-7.89(m,1H),7.88-7.83(m,1H),7.61-7.37(m ,6H),7.36-7.31(m,1H),4.00(s,2H),3.88(s,3H),3.60(s,3H),3.13(br,s,4H),2.69(br,s,4H).

[0417] Intermediate 19

[0418] 4-Chloro-1-methylquinolin-2(1H)-one

[0419]

[0420] At room temperature, 4-hydroxy-1-methylquinoline-2 (1H) -one (1.4g, 7.99mmol) and phosphorus oxychloride (7.45ml, 80mmol) are combined in a 100mL round-bottomed flask equipped with a stirring rod and a reflux condenser. The round-bottomed flask is placed in a 95°C oil bath. After 2.5 hours, the reaction mixture is allowed to cool to room temperature. The reaction mixture is slowly added to 300mL ice water. The slurry is extracted with 200mL dichloromethane, and owing to the presence of very little material after rotary evaporation, it is discarded. The aqueous portion is slowly added to excessive solid sodium bicarbonate. Dichloromethane (300mL) is added and residual solids are removed by filtration. The organic portion is dried over magnesium sulfate. The solvent is removed by rotary evaporation to obtain an orange solid (1.1g).

[0421] Starting Materials: 1 H NMR (400MHz, DMSO-d6) δ11.30 (s, 1H), 7.90 (dd, J = 7.8, 1.5Hz, 1H), 7.68-7.59 ( m, 1H), 7.48 (d, J = 8.6Hz, 1H), 7.25 (t, J = 7.6Hz, 1H), 5.88 (s, 1H), 3.54 (s, 3H).

[0422] product: 1 H NMR (400MHz, DMSO-d6) δ7.98(dd,J=8.1,1.2Hz,1H),7.81-7.72(m,1H),7.65(d,J=8.3Hz,1H),7.46-7.37(m,1H),6.96(s,1H),3.64(s,3H). LC / MS 2.32 min, M+1: 194.0, Exact mass: 193.03, Start %B=0, Final %B=100, Gradient time=4 min, Flow rate=0.8 ml / min, Wavelength=220, Solvent pair=ACN:water:ammonium acetate, Solvent A=5%ACN:95%water:10 mM ammonium acetate, Solvent B=95%ACN:5%water:10 mM ammonium acetate, Column=Phenomenex LUNA C18, 30x2, 3 μm, Oven temp. =40°C, LC / MS 3.02 min, M+1: 194.1, exact mass: 193.03, starting %B=0, final %B=100, gradient time=4 min, flow rate=0.8 mL / min, wavelength=220 nm, solvent pair=water-methanol-0.1% TFA, solvent A=90% water-10% methanol-0.1% TFA, solvent B=10% water-90% methanol-0.1% TFA, column=Phenomenex-Luna 2.0X50 mm 3μm.

[0423] Example 9

[0424] 4-(4-Benzhydrylpiperazin-1-yl)-1-methylquinolin-2(1H)-one

[0425]

[0426] In a microwave reaction vial, 1-benzhydrylpiperazine (0.555 g, 2.200 mmol) and 4-chloro-1-methylquinolin-2(1H)-one (0.387 g, 2 mmol) were dissolved in NMP (8 mL). The reaction vial was sealed under argon and heated at 200° C. for 30 minutes. The crude reaction mixture was directly chromatographed by reverse phase HPLC. Initial %B=30 to final %B=80, gradient time=10 minutes, flow rate=35 mL / minute, wavelength=220 nm, solvent A=10% methanol, 90% water (containing 0.1% TFA), solvent B=90% methanol, 10% water (containing 0.1% TFA), column=Phenomenex-Luna 30X50 mm S10. The product (250 mg) was obtained as a white solid. 1 H NMR (500MHz, DMSO-d6) δ7.74(d,J=7.7Hz,1H),7.62-7.55(m,1H),7.53-7.45(m,5H),7.33(t,J=7.5Hz,4H),7.22( q,J=6.8Hz,3H),6.01(s,1H),4.42(s,1H),3.55(s,3H),3.40(d,J=3.7Hz,2H),3.09(br.s.,3H),2.56(br.s.,3H).

[0427] Example 10

[0428] 4-(4-Benzhydrylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbaldehyde

[0429]

[0430] DMF (1mL) was cooled in ice water and phosphorus oxychloride (140 μL, 1.500mmol) was added. After stirring for 10 minutes, 4-(4-benzhydrylpiperazine-1-yl)-1-methylquinoline-2(1H)-one (205mg, 0.5mmol) in 1mL DMF was added and the reaction mixture was stirred to room temperature. After 20 minutes, LC\MS analysis showed product substance and no starting material substance. Excess ice was added and the reaction mixture was stirred until the ice melted. The pH was adjusted to 10 with 1N NaOH aqueous solution. The yellow precipitate (108mg) was collected using a Buchner funnel and dried overnight under vacuum. Proton NMR was consistent with the desired aldehyde. 1 H NMR (400 MHz, CHLOROFORM-d) δ 10.44 (s, 1H), 8.16-7.89 (m, 5H), 7.39 (br. s., 10H), 5.22-5.03 (m, 1H), 4.64-4.29 (m, 2H), 3.66-3.35 (m, 5H).

[0431] Embodiment 11

[0432] 4-[4-(Diphenylmethyl)piperazin-1-yl]-3-(hydroxymethyl)-1-methyl-1,2-dihydroquinolin-2-one

[0433]

[0434] In a reaction vial, sodium borohydride (0.011 g, 0.300 mmol) and 4-(4-benzhydrylpiperazin-1-yl)-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbaldehyde (0.044 g, 0.1 mmol) were combined in methanol (0.500 mL). Gas evaporation and a change in solution from yellow to colorless were observed. The crude material was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 45%-85% B over 20 minutes, then hold at 100% B for 5 minutes; Flow rate: 20 mL / min. Fractions containing product were combined and dried via centrifugal evaporation. The yield of the product was 6.3 mg, and its purity estimated by LCMS analysis was 100%. Two analytical LC / MS injections were used to determine the final purity. Injection 1 conditions: column: Waters Acquity UPLC BEH C18, 2.1x50mm, 1.7-μm particles; mobile phase A: 5:95 acetonitrile: water (containing 10mM ammonium acetate); mobile phase B: 95:5 acetonitrile: water (containing 10mM ammonium acetate); temperature: 50°C; gradient: 0-100% B over 3 minutes, then hold at 100% B for 0.75 minutes; flow rate: 1.0mL / min; detection: UV at 220nm. LC\MS: 2.2 minutes, M+1=440.1. Injection 2 conditions: Column: Waters Acquity UPLC BEH C18, 2.1x50mm, 1.7-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 0.1% trifluoroacetic acid); Mobile phase B: 95:5 acetonitrile:water (with 0.1% trifluoroacetic acid); Temperature: 50°C; Gradient: 0-100% B over 3 minutes, then hold at 100% B for 0.75 minutes; Flow rate: 1.0 mL / min; Detection: UV at 220 nm. LC\MS: 1.4 minutes, M+1=440.3. Proton NMR was obtained in deuterated DMSO using water suppression. No piperazine hydrogens were observed and the integrated methyl peak was consistent with low water suppression. 1 H NMR(500MHz,DMSO-d6)δ7.96(d,J=8.1Hz,1H),7.60-7.54(m,1H),7.53-7.47(m,5H),7.33(t,J=7.5Hz,4H),7 .26(t,J=7.7Hz,1H),7.23-7.18(m,2H),4.78-4.71(m,1H),4.61(d,J=5.1Hz,2H),4.43(s,1H),3.61(s,2H).

[0435] Intermediate 20

[0436] 4-Hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester

[0437]

[0438] In a round-bottom flask, diethyl malonate (6.8g, 42.5mmol) was dissolved in NMP (40mL) and cooled to 0°C. Sodium hydride (1.08g, 29.7mmol) was added and the reaction mixture was stirred at 0°C for 5 minutes, then at room temperature for 15 minutes. At room temperature, 1-methyl-1H-benzo [d] [1,3] oxazine-2,4-dione (5.1g, 28.8mmol) was added and the reaction mixture was heated at 120°C for 3 hours. Ether (60mL) was added and the solid was collected on the filter. The solid was washed with another 30mL of ether. The solid was resuspended in 25mL methanol and 50mL water. Hydrochloric acid (6mL, 5M aqueous solution) was added and the mixture was extracted three times with 80mL portions of ethyl acetate. The combined organic matter was extracted with 1x20mL brine. The organic portion was dried over magnesium sulfate. The solvent was removed by rotary evaporation to obtain the title compound (5.3g, 51%). 1 H NMR (400 MHz, CHLOROFORM-d) δ 14.23 (s, 1H), 8.21 (dd, J = 8.0, 1.5 Hz, 1H), 7.74-7.65 (m, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.30-7.25 (m, 1H), 4.52 (q, J = 7.2 Hz, 2H), 3.67 (s, 3H), 1.50 (t, J = 7.2 Hz, 3H).

[0439] Intermediate 21

[0440] 4-Chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester

[0441]

[0442] In a round-bottom flask, 1-methyl-2,4-dioxo-1,2,3,4-tetrahydroquinoline-3-ethyl formate (5g, 20.22mmol) and phosphorus oxychloride (5ml, 53.6mmol) were added. The reaction mixture was heated at 110°C for 40 minutes. Volatiles were removed under Rotovap. 50mL of ice was added in batches over 15 minutes to the crude residue, and then 150mL of water was added. The mixture was stirred for 15 minutes and ethyl acetate was added. The organic portion was extracted with 20mL of 1x water, 3x1N aqueous sodium hydroxide solution, 1x water and 1x brine. The organic portion was dried over magnesium sulfate and the solvent was removed by rotary evaporation. The residue was placed under vacuum overnight to obtain 3.9g of light yellow solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.09 (dd, J = 8.2, 1.1 Hz, 1H), 7.73-7.68 (m, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 4.49 (q, J = 7.0 Hz, 2H), 3.75 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).

[0443] Intermediate 22

[0444] Ethyl 6-fluoro-1-methyl-2-oxo-4-(((trifluoromethyl)sulfonyl)oxy)-1,2-dihydroquinoline-3-carboxylate

[0445]

[0446] 1,1,1-trifluoro-N-phenyl-N-(trifluoromethyl)sulfonylmethanesulfonamide (404mg, 1.131mmol) is added to a solution of 6-fluoro-1-methyl-2,4-dioxo-1,2,3,4-tetrahydroquinoline-3-carboxylic acid ethyl ester (100mg, 0.377mmol) and DIEA (0.263mL, 1.508mmol) in THF (3mL). The resulting solution is stirred at room temperature over the weekend. LC / MS shows that the reaction is complete. The reaction mixture is concentrated. The crude product is purified by Biotage column (using hexane to 40% ethyl acetate in hexane as eluent). The product appears with about 30% ethyl acetate in hexane. Fractions were collected and concentrated to give ethyl 6-fluoro-1-methyl-2-oxo-4-(((trifluoromethyl)sulfonyl)oxy)-1,2-dihydroquinoline-3-carboxylate (71 mg, 0.179 mmol, 47.4% yield) as a light yellow solid. LC / MS column: Phenomenex LUNA C18, 2.0×50 mm, 3-mm particles; mobile phase A: 10:90 methanol:water (with 0.1% TFA); mobile phase B: 90:10 methanol:water (with 0.1% TFA); gradient: 0-100% B over 4 minutes, then hold at 100% B for 1 minute; flow rate: 0.8 mL / min; detection: UV at 220 nm. Retention time 3.9 min, 397.9 (M+H + ). 1 H NMR (400 MHz, CHLOROFORM-d) δ 7.60 (dd, J = 8.3, 2.7 Hz, 1H), 7.55-7.44 (m, 2H), 4.48 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).

[0447] Intermediate 23

[0448] 1-((2-methoxynaphthalen-1-yl)methyl)piperazine

[0449]

[0450] DMF (10mL) solution of 1-BOC-piperazine (2.8g, 15.03mmol) and 2-methoxy-1-naphthaldehyde (2.80g, 15.03mmol) was stirred at room temperature for 1 hour. Sodium cyanoborohydride (1.417g, 22.55mmol) was added and the reaction mixture was stirred at room temperature overnight. LC / MS analysis showed that the reaction was complete. The solvent was removed and the residue was purified using 1:1 Hex / ethyl acetate on 40g silica gel column. The fraction containing 4-((2-methoxynaphthalene-1-yl) methyl) piperazine-1-tert-butyl formate was merged and a rotary evaporator was used to remove the solvent. The solid material was dissolved in dichloromethane (20mL) and TFA (5mL, 64.9mmol) was added. The reaction mixture was stirred for 1 hour and the solvent was removed to obtain a white solid (3.85g). LCMS: Column: Phenomenex LUNA C18, 2.0X50 mm, 3-mm particles; Mobile phase A: 10:90 methanol:water (containing 0.1% TFA); Mobile phase B: 90:10 methanol:water (containing 0.1% TFA); Gradient: 0-100% B over 4 minutes, then hold at 100% B for 1 minute; Flow rate: 0.8 mL / min; Detection: UV at 220 nm. Retention time 0.8 minutes, M+1=257.3.

[0451] Example 12

[0452] 4-{4-[(2-methoxynaphthalen-1-yl)methyl]piperazin-1-yl}-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester

[0453]

[0454] 1-((2-methoxynaphthalen-1-yl)methyl)piperazine (1.55 g, 6.05 mmol), 4-chloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid ethyl ester (1.607 g, 6.05 mmol) and triethylamine (0.843 mL, 6.05 mmol) were combined in DMF (12 mL) in a microwave reaction vessel. The reaction mixture was heated at 100 ° C in a microwave reactor for 1 hour and then allowed to stand at room temperature over the weekend. A crystalline precipitate was formed over the weekend, which was consistent with triethylammonium chloride by NMR. The liquid portion was poured into 100 mL of water and the precipitated solid was collected. NMR and LCMS of the dried solid showed that the product was contaminated by trace amounts of starting material and DMF. 1H NMR (400 MHz, CHLOROFORM-d) δ 8.27 (d, J = 8.6 Hz, 1H), 8.01 (dd, J = 8.1, 1.5 Hz, 1H), 7.82 (dd, J = 11.4, 8.7 Hz, 2H), 7.64-7.48 (m, 2H), 7.47-7.23 (m, 5H, CHCl3 overlap), 4.39 (q, J = 7.2 Hz, 2H), 4.09 (s, 2H), 4.00 (s, 3H), 3.67 (s, 3H), 3.22 (br t, J = 4.5 Hz, 4H), 2.77 (br s, 3H), 1.38 (t, J = 7.1 Hz, 3H). The crude material was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10-mM ammonium acetate); Gradient: 50%-90% B over 15 minutes, then held at 100% B for 5 minutes; Flow rate: 20 mL / min. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 22.3 mg, and its purity estimated by LCMS analysis was 100%. Two analytical LC / MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters BEH C18, 2.0x50 mm, 1.7-μm particles; Mobile phase A: 5:95 acetonitrile:water (containing 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (containing 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B, 0-100% B over 3 minutes, then hold at 100% B for 0.5 minutes; Flow rate: 1 mL / min; Detection: UV at 220 nm. LCMS: 2.07 minutes, M+1=486.23, M-1=484.23. Injection 2 conditions: Column: Waters BEH C18, 2.0x50 mm, 1.7-um particles; Mobile phase A: 5:95 methanol:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 methanol:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B, 0-100% B over 3 minutes, then hold at 100% B for 0.5 minutes; Flow rate: 0.5 mL / min; Detection: UV at 220 nm. LCMS: 3.22 minutes, M+1=486.23, M-1=484.23. 1H NMR (500MHz, DMSO-d6) δ8.19(d,J=8.8Hz,1H),7.99-7.89(m,2H),7.85(d,J=8.1Hz,1H),7.73-7.61(m,1H),7.57-7.49(m ,2H),7.46(d,J=9.2Hz,1H),7.36(q,J=7.8Hz,2H),4.22(q,J=7.0Hz,2H),4.01(s,2H),3.94(s,3H),3.56(s,3H),3.03(br s, 4H), 2.69 (br s, 4H), 1.23 (t, J = 7.0Hz, 3H).

[0455] Intermediate 24

[0456] 2,4-Dichloro-3-nitroquinoline

[0457]

[0458] 3-nitroquinoline-2,4-diol (2g, 9.70mmol) and phosphorus trichloride (9.04ml, 97mmol) were added to a 100mL round-bottom flask. N,N-dimethylaniline (2.485ml, 19.40mmol) was added to this yellow slurry and the reaction mixture was stirred at 120°C for 1.5 hours. The reaction mixture was cooled and concentrated. The brown residue was absorbed in toluene (10mL) and concentrated to remove any unreacted POCl. The residue was treated with ice (25g) for 20min and neutralized with solid NaCO. The mixture was extracted with ether (3X20mL). The combined organic layer was dried to obtain a light brown product.

[0459] Intermediate 25

[0460] 4-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-2-chloro-3-nitroquinoline

[0461]

[0462] 1-(bis(4-fluorophenyl)methyl)piperazine (1.186 g, 4.11 mmol) and triethylamine (1.147 mL, 8.23 ​​mmol) were added to a DMF (10 mL) solution of 2,4-dichloro-3-nitroquinoline (1 g, 4.11 mmol). The reaction mixture was stirred at room temperature overnight. LC / MS analysis showed that the reaction was complete. The mixture was diluted with ethyl acetate and washed with water, brine and dried. The residue was purified via silica gel chromatography (1:2 hexane:ethyl acetate; 24 g column). The fractions were collected to obtain the product (2 g) as a yellow solid. The product (0.1 mmol) was purified via preparative LC / MS using the following conditions: column: XBridge C18, 19x200 mm, 5-μm particles; mobile phase A: 5:95 acetonitrile: water (containing 10-mM ammonium acetate); mobile phase B: 95:5 acetonitrile: water (containing 10-mM ammonium acetate); gradient: 80%-100% B over 15 minutes, then held at 100% B for 5 minutes; flow rate: 20 mL / min. The fractions containing the product were combined and dried via centrifugal evaporation. The yield of the product was 5.0 mg, and its purity estimated by LCMS analysis was 100%. Two analytical LC / MS injections were used to determine the final purity. Injection 1 conditions: Column: Waters BEH C18, 2.0x50 mm, 1.7-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0-100% B over 3 minutes, then hold at 100% B for 0.5 minutes; Flow rate: 1.0 mL / min; Detection: UV at 220 nm. Injection 2 conditions: Column: Waters BEH C18, 2.0x50 mm, 1.7-μm particles; Mobile phase A: 5:95 methanol:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 methanol:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0-100% B over 3 minutes, then hold at 100% B for 0.5 minutes; Flow rate: 0.5 mL / min; Detection: UV at 220 nm. 1 H NMR(500MHz,DMSO-d6)δ8.11(d,J=8.4Hz,1H),8.00-7.95(m,1H),7.94-7.88(m,1H),7.73 (t,J=7.7Hz,1H),7.51(dd,J=8.1,5.9Hz,4H),7.16(t,J=8.6Hz,4H),4.53(s,1H),3.31(br s,4H),2.57(br s,4H).

[0463] Embodiment 13

[0464] 4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-3-nitroquinolin-2(1H)-one

[0465]

[0466] AcOH (10 mL, 175 mmol) and water (2.5 mL) were added to 4-(4-(bis(4-fluorophenyl)methyl)piperazine-1-yl)-2-chloro-3-nitroquinoline (600 mg, 1.212 mmol). The reaction mixture was heated at 100 °C for 5 h. The solvent was removed and dichloromethane was added to give 450 mg of a yellow solid (3:1 product to starting material). The remaining material was purified via Biotage (1:4 to 1:1 Hex / EtOAc; 40 g silica gel column). The fractions were collected to give the product (190 mg) as a yellow solid.

[0467] Embodiment 14

[0468] 4-{4-[Bis(4-fluorophenyl)methyl]piperazin-1-yl}-1-(2-fluoroethyl)-3-nitro-1,2-dihydroquinolin-2-one

[0469]

[0470] To a solution of Example 13 (20 mg, 0.042 mmol) in DMF was added sodium hydride (2.015 mg, 0.084 mmol) at 0°C. After 5 min, 1-fluoro-2-iodoethane (14.60 mg, 0.084 mmol) was added and the reaction vessel was shaken overnight at room temperature under N2. LC / MS indicated conversion to product. The crude material was purified via preparative LC / MS using the following conditions: Column: XBridge C18, 19x200 mm, 5-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10-mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10-mM ammonium acetate); Gradient: 60%-100% B over 15 min, then hold at 100% B for 5 min; Flow rate: 20 mL / min. Fractions containing the desired product were combined and dried via centrifugal evaporation. Calculated molecular weight 522.528. LC\MS conditions; Waters BEH C18, 2.0x50mm, 1.7-μm particles; Mobile phase A: 5:95 acetonitrile:water (with 10 mM ammonium acetate); Mobile phase B: 95:5 acetonitrile:water (with 10 mM ammonium acetate); Temperature: 50°C; Gradient: 0% B, 0-100% B over 3 minutes, then hold at 100% B for 0.5 minutes; Flow rate: 1 mL / min; Detection: UV at 220 nm. MS ion 523.1 observed, retention time 2.41 minutes.

[0471] The following compounds were prepared in library format from the appropriate alkyl halides using the above procedure. The analytical LCMS conditions used to confirm the products were the same as in the above procedure except when methanol was substituted for acetonitrile in the mobile phase as indicated.

[0472]

[0473]

[0474] Biological assays

[0475] The pharmacological properties of the compounds of the invention can be demonstrated by a number of biological assays. The following exemplary biological assays have been performed with the compounds of the invention.

[0476] Assay 1: In vitro DGK inhibition assay - Method A

[0477] DGKα and DGKζ reactions were performed using extruded liposomes (DGKα and DGKζ LIPGLO assays). The reactions were performed in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). For the extruded liposome reactions, the lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC. The reactions were performed in 150 μM ATP. The enzyme concentration for DGKα and DGKζ was 5 nM.

[0478] Compound inhibition study was carried out as follows: 50nL droplets of each test compound dissolved in DMSO (for each compound, the highest concentration 10mM, 11 points of continuous 3-fold dilution multiples) were transferred to the holes of white 1536-well plates (Corning3725). 5mL enzyme / substrate solution (2x final reaction concentration) was prepared by merging 2.5mL 4x enzyme solution (20nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5mL 4x liposome solution (composition described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2x enzyme / substrate solution was added to the hole containing the test compound and the reaction was started by adding 1 μL 300uM ATP. Allow the reaction to proceed for 1h, after which 2 μL Glo reagents (Promega V9101) were added and incubated for 40 minutes. Next, 4 μL kinase detection reagents were added and incubated for 30 minutes. EnVision microplate reader was used to record luminescence. Percent inhibition was calculated from the ATP conversion produced by the no enzyme control reaction (100% inhibition) and the ATP conversion produced by the vehicle only reaction (0% inhibition). Compounds were evaluated at 11 concentrations to determine the IC 50 .

[0479] 4x Liposome Preparation

[0480] Lipid composition is 5mol% DAG (Avanti 800811O), 40mol% PS (Avanti 840035P) and 55mol% PC (Avanti 850457), and the total lipid concentration for 4x liposome solution is 15.2mg / mL. PC, DAG and PS are dissolved in chloroform, merged, and dried into a film in a vacuum. Lipid is hydrated to 20mM in 50mM MOPS pH 7.5, 100mM NaCl, 5mM MgCl2, and freeze-thawed five times. The lipid suspension is extruded eleven times through a 100nm polycarbonate filter. Dynamic light scattering is carried out to confirm the size of the liposome (radius is 50-60nm). The liposome preparation is stored at 4°C for up to four weeks.

[0481] Baculovirus expression of human DGKα and DGKζ

[0482] Human DGK-α-TVMV-His-pFBgate and human DGK-ζ-transcription variant-2-TVMV-His-pFBgate baculovirus samples were generated using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. The DNA used to express DGK-α and DGK-ζ has SEQ ID NO: 1 and 3, respectively. Baculovirus amplification was achieved using infected Sf9 cells at a virus / cell ratio of 1:1500 and grown at 27°C for 65 hours after transfection.

[0483] Amplification of expression of each protein was performed in a Cellbag 50L WAVE-Bioreactor system 20 / 50 from GE Healthcare Bioscience. 12L of 2 × 10 6 Sf9 cells (Expression System, Davis, CA) grown in ESF921 insect medium (Expression System) at 1:200 virus / cell ratio were infected with the virus stock and grown at 27°C for 66-68 hours post infection. Infected cell cultures were harvested by centrifugation at 2000 rpm for 20 min at 4° C. in a RC12BP centrifuge. Cell pellets were stored at −70° C. until purification.

[0484] Purification of human DGK-α and DGK-ζ

[0485] Full-length human DGKα and DGKζ (each expressed with a TVMV-cleavable C-terminal Hexa-His tag sequence (SEQ ID NOs: 2 and 4, respectively) and produced as described above) were purified from insect cell paste infected with Sf9 baculovirus. Cells were lysed by nitrogen cavitation using a nitrogen bomb (Parr Instruments) and the lysate was clarified by centrifugation. The clarified lysate was purified to about 90% homogeneity using three consecutive column chromatography steps on the Purifier Plus system. This three-step column chromatography includes nickel affinity resin capture (i.e., HisTrap FF crude, GE Healthcare), followed by size exclusion chromatography (i.e., HiLoad 26 / 600 Superdex 200 preparative grade for DGK-α, GE Healthcare, and HiPrep 26 / 600 Sephacryl S 300_HR for DGK-ζ, GE Healthcare). The third step is ion exchange chromatography, and is different for the two isotypes. DGKα was refined using Q-Sepharose anion exchange chromatography (GE Healthcare). DGKζ was refined using SP Sepharose cation exchange chromatography (GE Healthcare). Protein was delivered at a concentration of ≥2 mg / mL. The formulation buffer was identical for both proteins: 50 mM Hepes, pH 7.2, 500 mM NaCl, 10% v / v glycerol, 1 mM TCEP, and 0.5 mM EDTA.

[0486] Assay 2: In vitro DGK inhibition assay - Method B

[0487] DGKα and DGKζ reactions were performed using extruded liposomes (DGKα and DGKζ LIPGLO assays). The reactions were performed in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). For extruded liposome reactions (5 mM total lipids), the lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC. The reactions were performed in 150 μM ATP. The enzyme concentration for DGKα and DGKζ was 5 nM.

[0488] Compound inhibition study was carried out as follows: 25nL droplets of each test compound dissolved in DMSO (for each compound, the highest concentration 10mM, 11 points of continuous 3-fold dilution multiples) were transferred to the holes of white 1536-well plates (Corning3725). 5mL enzyme / lipid substrate solution (2x final reaction concentration) was prepared by merging 2.5mL 4x enzyme solution (20nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5mL 4x detergent / lipid micelle solution (composition described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2x enzyme / lipid substrate solution was added to the hole containing the test compound and the reaction was started by adding 1 μL 300uM ATP. Allow the reaction to proceed for 2h, after which 2 μL Glo reagents (Promega V9101) were added and incubated for 40 minutes. Next, 4 μL kinase detection reagents were added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. Percent inhibition was calculated from ATP conversion produced by no enzyme control reactions (100% inhibition) and by vehicle-only reactions (0% inhibition). Compounds were evaluated at 11 concentrations to determine IC 50 .

[0489] 2x Liposome Preparation

[0490] Lipid composition is 5mol% DAG (Avanti 800811O), 40mol% PS (Avanti 840035P) and 55mol% PC (Avanti 850457), and the total lipid concentration for the liposome solution is 7-8mg / mL. PC, DAG and PS are dissolved in chloroform, merged, and dried into a film in a vacuum. Lipid is hydrated to 20mM in 50mM MOPS pH 7.5, 100mM NaCl, 5mM MgCl2, and freeze-thawed five times. The lipid suspension is extruded through a 100nm polycarbonate filter 10-12 times. Dynamic light scattering is carried out to confirm the size of the liposome (radius is 50-60nm). The liposome preparation is stored at 4°C for up to four weeks.

[0491] Baculovirus expression of nearly full-length human DGKα and full-length DGKζ

[0492] Human MA-hDGKα-(S9-S727)-Ct-TVMV-His-pFBgate and full-length human DGK-ζ-transcript variant-2-TVMV-His-pFBgate baculovirus samples were generated using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol (Note: the MA- in the DGKα reagent name indicates that two additional amino acids were added before Ser-9). The DNA used to express DGK-α(9-727) and DGK-ζ has SEQ ID NOs: 5 and 3, respectively. Baculovirus amplification was achieved using infected Sf9 cells at a virus / cell ratio of 1:1500 and grown at 27°C for 65 hours after transfection.

[0493] The expression of nearly full-length DGK-α(9-727) protein was scaled up in 2L flasks using the Cellbag 50L WAVE-bioreactor system 20 / 50 from GE Healthcare Bioscience, and the expression of full-length DGKζ protein was scaled up. The proteins were expressed in different volumes using similar conditions. For the expression of DGKα(9-727), 2x2L flasks (each containing 0.8L final volume of medium) were used, and DGKζ was grown at a scale of 12L in a 50L Cellbag. For each, an initial density of 2×10 6 Sf9 cells (Expression System, Davis, CA) at 10 cells / mL were inoculated in ESF921 insect medium (Expression System), infected with virus stock at a virus / cell ratio of 1:200, and grown at 27°C for 66-68 hours post infection. Infected cell cultures were harvested by centrifugation at 2000 rpm for 20 min at 4° C. in a RC12BP centrifuge. Cell pellets were stored at −80° C. until purification.

[0494] Purification of human DGK-α and DGK-ζ

[0495] Human DGKα(9-727) and full-length DGKζ (each expressed with a TVMV-cleavable C-terminal Hexa-His tag sequence (SEQ ID NO: 2 and 4, respectively) and produced as described above) were purified from insect cell paste infected with Sf9 baculovirus. The cell paste was thawed and suspended in buffer (50 mM HEPES, pH 7.2, 300 mM NaCl, 10% v / v glycerol, 1 mM TCEP containing nuclease (benzonase) and protease inhibitors) to a 1:10 v / v original culture volume. Lysis was achieved using nitrogen cavitation with a nitrogen bomb (Parr Instruments), and the lysate was clarified by high-speed centrifugation. The clarified lysate was purified to about 90% homogeneity using two or three consecutive column chromatography steps on the Purifier Plus system. Both isoforms were purified by nickel affinity purification and imidazole gradient elution (i.e. HisTrap FF, GE Healthcare), followed by size exclusion chromatography (i.e. for DGKα (9-727), HiLoad26 / 600Superdex 200prep grade, GE Healthcare; and for DGKζ, HiPrep 26 / 600Sephacryl S 300 HR, GE Healthcare). These two steps yielded >90% pure DGKα (9-727). Achieving similar purity for full-length DGKζ requires a third step, using cation exchange chromatography (SP Sepharose FF, GE Healthcare), and eluting with a NaCl gradient. Final formulation buffers were similar for both proteins, with DGKα(9-727) prepared in 50 mM Hepes, pH 7.3, 300 mM NaCl, 10% v / v glycerol, and 1 mM TCEP, and full-length DGKζ prepared in 50 mM Hepes, pH 7.3, 500 mM NaCl, 5% v / v glycerol, and 1 mM TCEP. Proteins were concentrated to 1-2 mg / mL, snap frozen, and kept at -80°C for long-term storage.

[0496] Assay 3: Raji CD4 T cell IL2 assay

[0497] 1536-well IL-2 assay was performed using pre-activated CD4 T cells and Raji cells in a 4 μL volume. Before the assay, CD4 T cells were pre-activated by treatment with 1.5 μg / mL, 1 μg / mL and 10 μg / mL of α-CD3, α-CD28 and PHA, respectively. Raji cells were treated with 10,000 ng / mL of staphylococcal enterotoxin B (SEB). First, the serially diluted compounds were transferred to a 1536-well assay plate (Corning, #3727), followed by addition of 2 μL pre-activated CD4 T cells (final density of 6000 cells / well) and 2 μL of SEB-treated Raji cells (2000 cells / well). After culturing in a 37°C / 5% CO2 incubator for 24 hours, 4 μl IL-2 detection reagents were added to an assay plate (Cisbio, #64IL2PEC). The assay plate was read on an Envision reader. To evaluate the cytotoxicity of the compounds, Raji or CD4 T cells were incubated with serially diluted compounds. After 24 h of incubation, 4 μL of Cell Titer Glo (Promega, #G7572) was added and the plate was read on an Envision reader. The 50% effective concentration (IC) was calculated using the four-parameter logistic formula y = A + ((BA) / (1 + ((C / x)^D))) 50 ), where A and B represent the minimum and maximum activation or inhibition, respectively, and C is the IC 50 , D is the slope and x represents the compound concentration.

[0498] Assay 4: CellTiter-Glo CD8 T Cell Proliferation Assay

[0499] Frozen naive human CD8 T cells were thawed in RPMI+10% FBS, incubated at 37°C for 2h, and counted. 384-well tissue culture plates were coated with 20μl of 0.1μg / mL anti-human CD3 in ordinary RPMI at 4°C overnight, removed from the plate, and then 20k / 40μL CD8 T cells (with 0.5μg / ml soluble anti-human CD28) were added to each well. Immediately after cell plating, the compound was added (echoed) to the cell plate with an echo instrument. After 72h of culture in a 37°C incubator, 10μL CellTiter-glo reagent (Promega catalog number G7570) was added to each well. The plate was shaken vigorously for 5min, incubated for another 15min at room temperature, and CD8 T cell proliferation was read on Envision. In the analysis, 0.1μg / mL anti-CD3 and 0.5μg / mL anti-CD28 stimulated CD8 T cell signals were background. 3 μM of the reference compound 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile was used to set the 100% range, and the EC 50 Set to absolute 50% to normalize data.

[0500] Assay 5: DGK AP1-reporter assay

[0501] The Jurkat AP1-luciferase reporter was generated using the Cignal Lenti AP1 Reporter reporter (luc) kit from SABiosciences (CLS-011L).

[0502] Compounds were transferred from the Echo LDV plate to individual wells of a 384-well plate (white, solid bottom, opaque PE CulturPlate 6007768) using an Echo550 instrument. Sample volume was 30 nL / well; and one destination plate per source plate. Cell suspensions were prepared by transferring 40 mL of cells (2x20 mL) to a clean 50 mL conical tube. Cells were concentrated by centrifugation (1200 rpm; 5 min; ambient temperature). The supernatant was removed and all cells were suspended in RPMI (Gibco 11875) + 10% FBS to a concentration of 1.35 x 10 6 Cells (30 μL cell suspension / well) were manually added to the 384-well TC plate containing the compound using a multichannel pipette, 4.0x10 4 The cell plate was incubated at 37°C and 5% CO2 for 20 minutes.

[0503] During the incubation, an anti-CD3 antibody (αCD3) solution was prepared by mixing 3 μL aCD3 (1.3 mg / mL) with 10 mL of culture medium [final concentration = 0.4 μg / mL]. Next, 1.5 μl aCD3 (1.3 mg / mL) was mixed with 0.5 mL of culture medium [final concentration = 4 μg / ml]. After 20 minutes, 10 μL of culture medium was added to all wells in column 1 (wells A to M), and 10 μL of αCD3 (4 ug / mL) was added to each well in column 1 (rows N to P) for reference. 10 μL of αCD3 (0.4 ug / mL) was then added to each well using a multichannel pipette. αCD3 stimulated + / - compound treated cells were incubated at 37°C 5% CO2 for 6 hours.

[0504] During this incubation period, the Steady-Glo (Promega E2520) reagent was slowly thawed to ambient temperature. Next, 20 μL of Steady-Glo reagent / well was added using a multi-drop Combi-dispenser. Bubbles were removed by centrifugation (2000 rpm, ambient temperature, 10 seconds). The cells were incubated at room temperature for 5 minutes. The samples were characterized by measuring relative light units (RLU) according to the luminescence protocol using an Envision plate reader. The data were analyzed using the reference compound 8-(4-(bis(4-fluorophenyl)methyl)piperazine-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile and normalized to 100% inhibition. Assay 6: Murine Cytotoxic T Lymphocyte Assay

[0505] An antigen-specific cytolytic T cell (CTL) assay was developed to functionally evaluate the ability of DGKα and DGKζ inhibitors to enhance effector T cell-mediated tumor cell killing activity. CD8+ T cells isolated from OT-1 transgenic mice recognized antigen-presenting cells MC38, which presented the ovalbumin-derived peptide SIINFEKL. Recognition of cognate antigen initiated the cytolytic activity of OT-1 antigen-specific CD8+ T cells.

[0506] Functionalized CTL cells were generated as follows: OT-1 splenocytes were isolated from 8-12 week old mice and amplified in the presence of 1 μg / mL of SIINFEKL peptide and 10 U / mL of mIL2. Three days later, fresh culture medium with mIL2 U / ml was added. On the 5th day of amplification, CD8+T cells were isolated and ready for use. Activated CTL cells can be stored frozen for 6 months. Separately, one million MC38 tumor cells were pulsed with 1 μg / mL of SIINFEKL-OVA peptide at 37°C for 3 hours. The cells were washed with fresh culture medium (3X) to remove excess peptides. Finally, CTL cells pretreated with DGK inhibitor for 1 hour in a 96-well U-bottom plate were merged with MC38 tumor cells loaded with antigen at a ratio of 1:10. The cells were then rotated at 700 rpm for 5 min and placed in an incubator at 37°C overnight. After 24 hours, supernatants were collected to analyze IFN-γ cytokine levels by AlphaLisa purchased from Perkin Elmer.

[0507] Assay 7: PHA proliferation assay

[0508] Phytohemagglutinin (PHA) stimulated embryonic cells from frozen stocks were incubated in RPMI medium (Gibco, ThermoFisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (SigmaAldrich, St. Louis, MO) for one hour and then added to individual wells (10,000 cells / well) of a 384-well plate. Compounds were transferred to individual wells of a 384-well plate and treated cells were maintained in medium containing human IL2 (20 ng / mL) at 37°C 5% CO2 for 72 h before growth was measured using MTS reagent [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethyloxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] according to the manufacturer's instructions (Promega, Madison, WI). Percent inhibition was calculated by comparing values ​​between IL2 stimulation (0% inhibition) and unstimulated controls (100% inhibition). The inhibitory concentration (IC) was calculated based on the 50% inhibition of the fold induction between IL2-stimulated and unstimulated treatments. 50 ) determination.

[0509] Assay 8: Human CD8 T cell IFN-γ assay

[0510] Frozen naive human CD8 T cells were thawed in AIM-V medium, incubated at 37°C for 2h, and counted. 384-well tissue culture plates were coated overnight at 4°C with 20 μL of 0.05 μg / mL anti-human CD3 in PBS, removed from the plate, and 40,000 cells / 40 microliters of CD8 T cells (with 0.1 μg / mL soluble anti-human CD28) were added to each well. After cell plating, the compound was immediately transferred to the cell plate using an Echo liquid handler. After incubation in a 37°C incubator for 20h, 3 microliters / well of supernatant was transferred to a new 384-well white assay plate for cytokine measurement.

[0511] Interferon-γ (IFN-γ) was quantified using the AlphLISA kit (Catalog No. AL217) as described in the manufacturer's manual (Perkin Elmer). The counts from each well were converted to IFN-γ concentrations (pg / mL). Compound EC was determined by setting 0.05 μg / mL anti-CD3 plus 0.1 μg / mL anti-CD28 as the baseline and 3 μM of the reference compound 8-(4-(bis(4-fluorophenyl)methyl)piperazine-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile with co-stimulation of anti-CD3 plus anti-CD28 as 100% activation. 50 value.

[0512] Assay 9: Human CD8 T cell pERK assay

[0513] Frozen naive human CD8 T cells were thawed in AIM-V medium, incubated at 37°C for 2h, and counted. CD8 positive T cells were added to 384-well tissue culture plates at 20,000 cells / well in AIM-V medium. A compound was added to each well, and then bead-bound anti-human CD3 and anti-CD28 mAbs were added at a final concentration of 0.3 μg / mL. The cells were incubated at 37°C for 10 minutes. The reaction was terminated by adding lysis buffer from the AlphaLISA Surefire kit. (Perkin Elmer, catalog number ALSU-PERK-A). The lysate (5 μL / well) was transferred to a new 384-well white assay plate for pERK activation measurement.

[0514] Compound EC was determined by setting anti-CD3 plus anti-CD28 as baseline and costimulation of 3 μM 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile with anti-CD3 plus anti-CD28 as 100% activation. 50 value.

[0515] Assay 10: Human whole blood IFN-γ assay

[0516] Human venous whole blood (22.5 μL / well) obtained from healthy donors was pretreated with compounds for one hour at 37° C. in a humidified 95% air / 5% CO2 incubator. The blood was stimulated with 2.5 μL of anti-human CD3 and anti-CD28 mAbs, each at a final concentration of 1 μg / mL, at 37° C. for 24 hours. IFN-γ in the supernatant was measured using the AlphLISA kit (Catalog No. AL217).

[0517] Compound EC was determined by setting anti-CD3 plus anti-CD28 as baseline and co-stimulation of the reference compound 8-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-5-methyl-7-nitro-6-oxo-5,6-dihydro-1,5-naphthyridine-2-carbonitrile with anti-CD3 plus anti-CD28 at 3 μM as 100% activation. 50 value.

[0518] Assay 11: DGK Human Whole Blood pERK Assay

[0519] Human whole blood ERK phosphorylation assay was performed with human venous whole blood obtained from healthy donors (drawn with heparin as anticoagulant). Compounds serially diluted in DMSO (11 points, 3-fold) were added to a 384-well plate at 20 nL / well using an ECHO 550 acoustic dispenser (Labcyte) to reach a final starting concentration of 20 μM in the assay. Heparinized human whole blood was added to the compound plate at 9 μL / well and incubated at 37°C for one hour in a humidified 95% air / 5% CO2 incubator. After one hour of compound incubation, 1 μL of human anti-CD3 antibody (in-house) in the presence of cross-linked antibody goat anti-mouse IgG (4 μg / mL) was added to the wells at a final concentration of 1 μg / mL for stimulating the pathway and incubated for an additional 15 minutes at 37°C. Stimulation was stopped by adding 90 μL of fixation / lysis buffer (BD 558049). The cells were washed and stained with anti-CD8 PE (BD 555635) antibody for 60 minutes at room temperature, washed again, and permeabilized using Perm III buffer (BD558050) on ice for 30 minutes. 647 anti-ERK1 / 2 Phospho (Thr202 / Tyr204) antibody (Bioleged 675504) was used for staining at a dilution of 1:50 for 60 minutes. The samples were washed and resuspended in dPBS containing 1% BSA (dPBS, Gibco 14190136; BSA, Sigma-Aldrich A9205). Samples were analyzed by iQue Screener PLUS. pERK activation was quantified by the percentage of pERK positive population in the CD8 positive population. Compound potency was calculated based on 20 μM concentration of internal compound as 100% activation and anti-CD3 control as 0% activation.

[0520] Table A

[0521] In vitro DGK inhibition IC 50 Activity value

[0522]

[0523] Table A lists the in vitro DGK inhibition IC measured in the DGKα and DGKζ liposome (LIPGLO) assays. 50 Activity value.

[0524] The compounds of the present invention have activity as one or more inhibitors of one or both of the DGKα and DGKζ enzymes and are therefore useful in treating diseases associated with inhibition of DGKα and DGKζ activity.

[0525] Nucleotide sequence encoding hDGKα-(M1-S735)-Ct-TVMV-His:

[0526]

[0527]

[0528] The amino acid sequence of hDGKα-(M1-S735)-Ct-TVMV-His:

[0529]

[0530] Nucleotide sequence encoding hDGKζ-(M1-A928)-transcription variant-2Ct-TVMV-His:

[0531]

[0532]

[0533] The amino acid sequence of hDGKζ-(M1-A928)-transcription variant-2Ct-TVMV-His:

[0534]

[0535] Nucleotide sequence encoding MA-hDGKα-(S9-S727)-Ct-TVMV-His:

[0536]

[0537] AND -Amino acid sequence of hDGKα-(S9-S727)-Ct-TVMV-His:

[0538]

Claims

1. A compound of formula (I): or a salt thereof, wherein: R1 is F, -CN, or -OCH3; R2 is H, -CH3, -CH2CN, -CH2CH2F, or -CH2CH=CH2; R3 is H, -CN, -CH2OH, -C(O)OCH2CH3, -NO2, or pyridyl; R4 is -CH2R 4a or -CHR 4a R 4b ; R 4a is phenyl, naphthyl, or indolyl, each substituted with zero to two substituents independently selected from the group consisting of F, -CH3, -CH2CH3, and -OCH3; R 4b is phenyl or fluorophenyl; Each R5 is -CH3, or two R5 are attached to the same carbon atom to form =0; each R6 is H; and m is zero, 1, or 2.

2. The compound or salt thereof according to claim 1, wherein: R1 is F; and R2 is -CH3.

3. The compound or salt thereof according to claim 1, wherein the compound or salt thereof has the structure: in: R 5a and R 5b is independently -CH3.

4. The compound or salt thereof according to claim 1, wherein the compound or salt thereof has the structure:

5. The compound or salt thereof according to claim 1, wherein R4 is -CH2R 4a .

6. The compound or salt thereof according to claim 1, wherein R4 is -CHR 4a R 4b .

7. A compound or a salt thereof, wherein the compound is: 6-Fluoro-4-{4-[(3-fluorophenyl)methyl]piperazin-1-yl}-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (1); 6-Fluoro-4-{4-[(3-fluoro-5-methylphenyl)methyl]piperazin-1-yl}-1-methyl-3-(pyridin-4-yl)-1,2-dihydroquinolin-2-one (2); 4-(4-((1H-indol-4-yl)methyl)piperazin-1-yl)-6-fluoro-1-methyl-3-nitroquinolin-2(1H)-one (3); 4-{4-[(1-ethyl-1H-indol-4-yl)methyl]piperazin-1-yl}-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (4); 4-(4-Benzyl-3-oxopiperazin-1-yl)-6-fluoro-1-methyl-3-nitro-1,2-dihydroquinolin-2-one (5); 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbonitrile (6); 4-[(2S,5R)-4-[Bis(4-fluorophenyl)methyl]-2,5-dimethylpiperazin-1-yl]-1-methyl-2-oxo-1,2-dihydroquinoline-6-carbonitrile (7); 6-Methoxy-1-methyl-4-{4-[(naphthalen-1-yl)methyl]piperazin-1-yl}-3-nitro-1,2-dihydroquinolin-2-one (8).

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

9. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer or viral infection.

10. The use according to claim 9, wherein the cancer is selected from the group consisting of colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, kidney cancer, head and neck cancer, lymphoma, leukemia and melanoma.

11. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of at least one diacylglycerol kinase selected from diacylglycerol kinase α (DGKα) and diacylglycerol kinase ζ (DGKζ).

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