Substituted pyrrolo[1,2-a]pyrazines and pyrrolo[1,2-a][1,4]diazepines as TREX1 inhibitors

The present invention provides compounds of Formula I wherein  X, R1, R2, R3 and R4 are as defined herein, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug ester or solvate form thereof, wherein all of the variables are as defined herein. These compounds are effective at modulating the TREX1 protein and thus can be used as medicaments for treating or preventing disorders affected by the inhibition of TREX1.

FIELD OF THE INVENTION

The present invention provides novel cyclic compounds, and analogues thereof, which are three prime repair exonuclease 1 (TREX1) inhibitors and are useful in preventing or treating disorders associated with TREX1. This invention also relates to pharmaceutical compositions containing these compounds and methods of using the same.

BACKGROUND OF THE INVENTION

Three-prime repair exonuclease 1 (gene name TREX-1), also called Deoxyribonuclease III or DNase III, is an enzyme that degrades deoxyribonucleic acid by cleaving the 3′ terminal base of a DNA polymer. TREX1 can digest single-stranded and double-stranded DNA containing a mismatched 3′ overhang. TREX1 is an endoplasmic reticulum-associated cytosolic protein and can also be found in the nucleus.

TREX1 functions to prevent cell-intrinsic initiation of autoimmunity by degrading cytosolic self DNA from endogenous retroelements (Stetson, D. B et al. (2008) Cell. 134(4). 587-598). TREX 1 prevents chronic ATM-dependent checkpoint activation by processing ssDNA polynucleotide species arising from the processing of aberrant DNA replication intermediates (Yang Y. G. et. al. (2007) Cell 131(5), 873-86). Cytosolic DNA fragments and retroelements are sensed by pattern recognition receptors, such as cyclic GMP-AMP synthase (gene name cGAS). When cGAS binds DNA its enzyme activity is greatly enhanced and it produces cyclic GMP-AMP which serves as a secondary messenger that binds to and activates Stimulator of Interferon Genes (STING) and thereby initiating a type 1 interferon immune response (Wu J. et al., (2012) Science. 339(6121), 826-30; Sun L. et al., (2012). Science. 339(6121). TREX1 DNA degrading activity can attenuate such responses as a check to prevent excessive type 1 interferon responses.

Defects in TREX1 have numerous biological consequences. Defects in TREX1 function are associated with type 1 interferon driven systemic inflammatory and autoimmune conditions. These include Familial Chilblain Lupus, Aicardi-Goutie'res syndrome (AGS), Retinal Vasculopathy and Cerebral leukodystrophy (RVCL) (Ablasser, A. et al. (2014) J. Immun. 192, 5993-5997). Similar to activating mutations of STING (Jeremiah, N. et al. (2014) JCI, 124(12), 5516-5520), inactivating mutations in TREX1 (Yan, N. et al. (2017) J. Interfer. Cyt. Res. 2017 37(5), 198-206) are associated with type 1 interferon-driven systemic inflammatory and autoimmune conditions. It follows then that TREX1 inhibitors should have the same therapeutic consequence as STING agonists in the context of cancer immunotherapy. TREX1 inhibitors can be used in combinatorial strategies to maximizing the immunogenicity of radiation therapy (Vanpouille-Box (2017) Nature Commun. 8, 81658). Inactivating mutations in TREX1 confers resistance to RNA viruses, including HIV, VSV, influenza, West Nile and Sendai viruses. Therefore, TREX1 inhibition could be used in antiviral therapy as well.

The potential therapeutic benefits of enhancing both innate and adaptive immunity make TREX1 an attractive therapeutic target that demonstrates impressive activity by itself and can also be combined with other immunotherapies.

SUMMARY OF THE INVENTION

It has been found that cyclic compounds in accordance with the present invention are effective at inhibiting TREX1 in assays. Accordingly, the present invention provides novel cyclic analogues which are TREX1 inhibitors and are useful as selective immunotherapies, including stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.

The present invention also provides processes and intermediates for making the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.

The present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one of the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.

The present invention also provides a method for the treatment or prophylaxis of disorders, diseases, syndromes, or conditions affected by the inhibition of TREX1 comprising administering to a patient in need of such treatment or prophylaxis a therapeutically effective amount of at least one of the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.

The present invention also provides the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof, for use in therapy.

The present invention also provides the use of the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof, for the manufacture of a medicament for the treatment or prophylaxis of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1.

DETAILED DESCRIPTION

In one embodiment, the present invention provides cyclic compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, of Formula I having the structure:

A is a 6 to 7-membered monocyclic heterocyclyl or a 8- to 12-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms;

X is independently selected from —C(═O)—NR5—; —C(═O)—NR5—S(═O)2—; —C1-C6alkyl-NR5—S(═O)2—; —C1-C6alkyl-NR5—C(═O)—; and a 5-membered nitrogen containing heteroaryl;

R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, C6-C10aryl-S—C1-C6-alkyl, C6-C10aryl-S(═O)—C1-C6-alkyl, C6-C10aryl-S(═O)2—C1-C6-alkyl, C6-C10aryl-C1-C6-alkyl, a 5- to 10-membered heteroaryl-oxy-C1-C6-alkyl, a 5- to 10-membered heteroaryl-S—C1-C6-alkyl, a 5- to 10-membered heteroaryl-S(═O)—C1-C6-alkyl, a 5- to 10-membered heteroaryl-S(═O)2—C1-C6-alkyl and a 5- to 10-membered heteroaryl-C1-C6-alkyl, wherein any aryl and heteroaryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R3is independently selected from C1-C6alkyl, C6-C10aryl, C6-C10aryl-C1-C6-alkyl, a 5- to 10-membered heteroaryl-C1-C6-alkyl, and C3-C6cycloalkyl-C1-C6-alkyl, wherein any cycloalkyl, aryl and heteroaryl, may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, CN, —C(═O)OH, —C(═O)NR8R9; —NR8R9; —O—NR8R9; —S(═O)2OH; —S(═O)2NR8R9; —NR8—S(═O)2—R9; —C(═O)NR8—S(═O)2—R9; —C(═O)NR8—S(═O)2—NR28R29; —NR28—C(═O)NR8—S(═O)2—R9; —NR28—C(═O)NR8R9; —S(═O)2—R9; —S(═O)—R9; —S—R9; C6-C10aryl, or a 5-membered nitrogen containing heteroaryl, wherein the aryl and heteroaryl may be optionally substituted with one or more substituents selected from the group consisting of OH, ═O, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R5, at each occurrence, is independently selected from H, C1-C6alkyl and halo C1-C6alkyl;

R7is independently selected from C1-C6alkyl and C1-C6alkoxy;

R9is independently selected from H, OH, C1-C4alkyl, C1-C4alkoxy, C3-C6cycloalkyl and C6-C10aryl, wherein the alkyl, alkoxy, cycloalkyl and aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, ═O, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

In another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein A is a 8- to 11-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein A is a 8- to 10-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms.

In still yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein A is

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein the compound is a compound of Formula Ia or Ib:

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein A is a 6- to 7-membered monocyclic heterocyclyl, wherein the heterocyclyl contains at least two N atoms.

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein X is independently selected from —C(═O)—NR5—; —C(═O)—NR5—S(═O)2—; —C1-C6alkyl-NR5—C(═O)—; and a 5-membered nitrogen containing heteroaryl.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein X is independently selected from —C(═O)—NR5—; —C1-C6alkyl-NR5—C(═O)—; and a 5-membered nitrogen containing heteroaryl.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein X is independently selected from —C(═O)—NR5—; and —C1-C6alkyl-NR5—C(═O)—.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein X is —C(═O)—NR5—.

In still yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, C6-C10aryl-S—C1-C6-alkyl, C6-C10aryl-S(═O)2—C1-C6-alkyl and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy.

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, C6-C10aryl-S—C1-C6-alkyl and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy.

In another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R2is independently selected from C1-C6alkyl and C6-C10aryl-C1-C6-alkyl, wherein the aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R3is independently selected from C1-C6alkyl, C6-C10aryl, C6-C10aryl-C1-C6-alkyl, and a 5- to 10-membered heteroaryl-C1-C6-alkyl, wherein any C6-C10aryl and heteroaryl, may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R3is independently selected from C1-C6alkyl, C6-C10aryl, C6-C10aryl-C1-C6-alkyl, a 5-membered heteroaryl-C1-C6-alkyl, and a 6-membered heteroaryl-C1-C6-alkyl, wherein any aryl and heteroaryl, may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy.

In yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein R3is independently selected from C1-C6alkyl, C6-C10aryl, and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy.

In still yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein:

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, CN, —C(═O)OH, —C(═O)NR8R9; C6-C10aryl, or a 5-membered nitrogen containing heteroaryl, wherein the aryl and heteroaryl may be optionally substituted with OH, ═O, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein the 5-membered nitrogen containing heteroaryl in the definition of R4is selected from the group consisting of

wherein Y is O, S, N or C and Z is O, S, or N(CH3).

A is a 8- to 11-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms;

X is independently selected from —C(═O)—NR5—; —C(═O)—NR5—S(═O)2—; —C1-C6alkyl-NR5—C(═O)—; and a 5-membered nitrogen containing heteroaryl;

R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, C6-C10aryl-S—C1-C6-alkyl, C6-C10aryl-S(═O)—C1-C6-alkyl, C6-C10aryl-S(═O)2—C1-C6-alkyl and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R3is independently selected from C1-C6alkyl, C6-C10aryl, C6-C10aryl-C1-C6-alkyl, and a 5- to 10-membered heteroaryl-C1-C6-alkyl, wherein any C6-C10aryl and heteroaryl, may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, CN, —C(═O)OH, —C(═O)NR8R9; C6-C10aryl, or a 5-membered nitrogen containing heteroaryl, wherein the aryl or heteroaryl may be optionally substituted with OH, ═O, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R5, at each occurrence, is independently selected from H, C1-C6alkyl and halo C1-C6alkyl;

R7is independently selected from C1-C6alkyl and C1-C6alkoxy;

R8is independently selected from H, C1-C4alkyl, C1-C4alkoxy, and halo-C1-C4-alkyl; and

A is a 8- to 10-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms;

X is independently selected from —C(═O)—NR5—; —C1-C6alkyl-NR5—C(═O)—; and a 5-membered nitrogen containing heteroaryl;

R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, C6-C10aryl-S—C1-C6-alkyl and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R3is independently selected from C1-C6alkyl, C6-C10aryl, C6-C10aryl-C1-C6-alkyl, and a 5-membered heteroaryl-C1-C6-alkyl, wherein any C6-C10aryl and heteroaryl, may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, CN, —C(═O)OH, —C(═O)NR8R9; C6-C10aryl, or a 5-membered nitrogen containing heteroaryl, wherein the aryl or heteroaryl may be optionally substituted with OH, ═O, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R5, at each occurrence, is independently selected from H, C1-C6alkyl and halo C1-C6alkyl;

R7is independently selected from C1-C6alkyl and C1-C6alkoxy;

R8is independently selected from H, C1-C4alkyl, C1-C4alkoxy, and halo-C1-C4-alkyl; and

A is a 8- to 10-membered bicyclic heterocyclyl, wherein the bicyclic heterocyclyl contains at least two N atoms;

X is independently selected from —C(═O)—NR5—; and —C1-C6alkyl-NR5—C(═O)—;

R2is independently selected from C1-C6alkyl, C6-C10aryloxy-C1-C6-alkyl, and C6-C10aryl-C1-C6-alkyl, wherein any aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R3is independently selected from C1-C6alkyl, C6-C10aryl and C6-C10aryl-C1-C6-alkyl, wherein any C6-C10aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, CN, —C(═O)OH, —C(═O)NR8R9; or C6-C10aryl, wherein the aryl may be optionally substituted with OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R5, at each occurrence, is independently selected from H, C1-C6alkyl and halo C1-C6alkyl;

R7is independently selected from C1-C6alkyl and C1-C6alkoxy;

R8is independently selected from H, C1-C4alkyl, C1-C4alkoxy, and halo-C1-C4-alkyl; and

X is independently selected from —C(═O)—NR5—; and —C1-C6alkyl-NR5—C(═O)—;

R2is independently selected from C1-C6alkyl and C6-C10aryl-C1-C6-alkyl, wherein the aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R3is independently selected from C1-C6alkyl, C6-C10aryl, and C6-C10aryl-C1-C6-alkyl, wherein any C6-C10aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C6alkoxy;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, —C(═O)OH, —C(═O)NR8R9; or C6-C10aryl, wherein the aryl may be optionally substituted with OH, CN, halo, C1-C4alkyl, C1-C4alkoxy, halo-C1-C4alkyl or halo-C1-C4alkoxy;

R5, at each occurrence, is independently selected from H and C1-C6alkyl;

R7is independently selected from C1-C6alkyl and C1-C6alkoxy;

R8is independently selected from H, C1-C4alkyl and halo-C1-C4-alkyl; and

In still yet another embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein:

X is —C(═O)—NR5—;

R2is independently selected from C1-C6alkyl and C6-C10aryl-C1-C6-alkyl, wherein the aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl or halo-C1-C4alkyl;

R3is independently selected from C1-C6alkyl, C6-C10aryl, and C6-C10aryl-C1-C6-alkyl, wherein any C6-C10aryl may be optionally substituted with one or more substituents selected from the group consisting of OH, CN, halo, C1-C4alkyl or halo-C1-C4alkyl;

R4is C1-C6alkyl, optionally substituted with one or more substituents selected from the group consisting of OH, —C(═O)OH, or C6-C10aryl, wherein the aryl may be optionally substituted with OH, CN, halo, C1-C4alkyl or halo-C1-C4alkyl;

R5, at each occurrence, is independently selected from H and C1-C6alkyl;

R6is independently selected from H, C1-C6alkyl and —C(═O)R7; and

R7is independently selected from C1-C6alkyl and C1-C6alkoxy.

In one embodiment, the present invention provides compounds, stereoisomers, tautomers, salts, solvates or prodrugs thereof, wherein the compound is selected from a compound set forth in the examples.

The compounds, stereoisomers, tautomers, salts, solvates or prodrugs of the invention have IC50values in the TREX1 exonuclease assay (described hereinafter) of about 100 μM or less, preferably 50 μM or less, and more preferably 25 μM or less, even more preferably 10 μM or less. Activity data for compounds, stereoisomers, tautomers, salts, solvates or prodrugs of the present invention are presented in Table 5.

In some embodiments, the present invention provides a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, alone or in combination with another therapeutic agent.

In some embodiments, the present invention provides a pharmaceutical composition which further includes another therapeutic agent(s). In a preferred embodiment, the present invention provides a pharmaceutical composition, wherein the additional therapeutic agent(s) are an anti-cancer agent, an anti-viral compound, an antigen, an adjuvant, a lipid, a liposome, a peptide, a cytotoxic agent, a chemotherapeutic agent, an immunomodulatory cell line, a checkpoint inhibitor, a biotherapeutic agent, an immunogenic agent, and cells transfected with genes encoding immune stimulating cytokines or a combination thereof. Preferably, the additional therapeutic agents are VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, ICOS, IL-2, IFNa2, GM-CSF, a STING agonist, another TREX1 inhibitor, a CTLA-4 pathway antagonist, a LAG-3 pathway antagonist, a PD-1 pathway antagonist, a PD-L1 antibody, a vascular endothelial growth factor (VEGF) receptor inhibitor, a topoisomerase II inhibitor, a smoothen inhibitor, an alkylating agent, an anti-tumor antibiotic, an anti-metabolite, a retinoid, Tim-3/gal9, CD73 inhibitors, adenosine A2A+/−A2B antagonists and an anti-cancer vaccine.

In one embodiment, the present invention provides a pharmaceutical composition which is utilized in combination with radiation therapy.

In some embodiments, the present invention provides a method for the treatment or prophylaxis of a disorder, disease, syndrome, or condition, wherein said disease, syndrome, or condition is affected by the inhibition of TREX1, which includes the step of administering to a subject (for example, a human) in need of such treatment or prophylaxis a therapeutically effective amount of at least one of the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof.

In some embodiments, the present invention provides methods for the treatment of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45, and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, wherein the disorder, disease, syndrome, or condition is selected cancer or a viral infection.

In some embodiments, the present invention provides methods for the treatment of a disease, syndrome, or condition affected by the inhibition of TREX1, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, wherein the disorder, disease, syndrome, or condition is cancer.

In some embodiments, the present invention provides methods for the treatment of cancer, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, in combination with radiation therapy.

In some embodiments, the present invention provides methods for the treatment of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, wherein the disorder, disease, syndrome, or condition is selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, bladder cancer and fibrosarcoma.

In some embodiments, the present invention provides methods for the treatment of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, wherein the disorder, disease, syndrome, or condition is a viral infection, preferably HIV.

In some embodiments, the present invention provides methods for the treatment of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1, which includes the steps of administering to a patient (for example, a human) in need thereof a therapeutically effective amount of a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, wherein the disorder, disease, syndrome, or condition is a HIV, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, bladder cancer or fibrosarcoma.

In yet another embodiment, the present invention provides uses a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, for the preparation of a medicament for treating a disorder, disease, syndrome, or condition selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, bladder cancer and fibrosarcoma, in a subject in need thereof.

In still yet another embodiment, the present invention provides uses a compound of Formula I, preferably, a compound selected from one of the examples, more preferably, Examples 1, 2B, 3, 4, 14B, 15, 16, 17, 18, 36, 38, 39, 45 and 48, or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrug esters, or solvates thereof, in a method for treating a disorder, disease, syndrome, or condition selected from the group consisting of viral infection, melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, bladder cancer and fibrosarcoma, in a subject in need thereof.

Other Embodiments of the Invention

In some embodiments, the present invention provides a process for making a compound of the present invention or a stereoisomer, tautomer, pharmaceutically acceptable salt, solvate or prodrug ester thereof.

In some embodiments, the present invention provides an intermediate for making a compound of the present invention or a stereoisomer, tautomer, pharmaceutically acceptable salt, solvate or prodrug ester thereof.

In some embodiments, the present invention provides a compound of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof, for use in therapy for the treatment or prophylaxis of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1.

In some embodiments, the present invention also provides the use of a compound of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrug esters thereof, for the manufacture of a medicament for the treatment or prophylaxis of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. This invention encompasses all combinations of preferred aspects of the invention noted herein. It is understood that any and all embodiments of the present invention may be taken in conjunction with any other embodiment or embodiments to describe additional embodiments. It is also to be understood that each individual element of the embodiments is its own independent embodiment. Furthermore, any element of an embodiment is meant to be combined with any and all other elements from any embodiment to describe an additional embodiment.

Chemistry

Compounds of this invention may have one or more asymmetric centers. Unless otherwise indicated, all chiral (enantiomeric and diastereomeric) and racemic forms of compounds of the present invention are included in the present invention. Many geometric isomers of olefins, C═N double bonds, and the like can also be present in the compounds, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. The present compounds can be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, (enantiomeric and diastereomeric) and racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomer form is specifically indicated. When no specific mention is made of the configuration (cis, trans or R or S) of a compound (or of an asymmetric carbon), then any one of the isomers or a mixture of more than one isomer is intended. Preferably, diastereomers are resolved prior to any type of in vitro or in vivo testing. The processes for preparation can use racemates, enantiomers, or diastereomers as starting materials. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention. When enantiomeric or diastereomeric products are prepared, they can be separated by conventional methods, for example, by chromatography or fractional crystallization. Compounds of the present invention, and salts thereof, may exist in multiple tautomeric forms, in which hydrogen atoms are transposed to other parts of the molecules and the chemical bonds between the atoms of the molecules are consequently rearranged. It should be understood that all tautomeric forms, insofar as they may exist, are included within the invention.

As used herein, the term “alkyl” or “alkylene”, alone or as part of another group, is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having from 1 to 10 carbons or the specified number of carbon atoms. For example, “C1-10alkyl” (or alkylene), is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10alkyl groups. Additionally, for example, “C1-C6alkyl” denotes alkyl having 1 to 6 carbon atoms. Alkyl groups can be unsubstituted or substituted with at least one hydrogen being replaced by another chemical group. Example 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, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), as well as chain isomers thereof, and the like as well as such groups which may optionally include 1 to 4 substituents such as halo, for example F, Br, Cl, or I, or CF3, alkyl, alkoxy, aryl, aryloxy, aryl(aryl) or diaryl, arylalkyl, arylalkyloxy, alkenyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkyloxy, amino, hydroxy, hydroxyalkyl, acyl, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, alkylthio, arylalkylthio, aryloxyaryl, alkylamido, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol, haloalkyl, trihaloalkyl, and/or alkylthio as well as (═O), OR18, SR18, (═S), —NR18R19, —N(alkyl)3+, —NR18SO2, —NR18SO2R20, —SO2R20, —SO2NR18R19, —SO2NR18C(═O)R19, SO3H, —PO(OH)2, —C(═O)R18, —CO2R18, —C(═O)NR18R19, —C(═O)(C1-C4alkylene)NR18R19, —C(═O)NR18(SO2)R19, —CO2(C1-C4alkylene)NR18R19, —NR18C(═O)R19, —NR18CO2R19, —NR18(C1-C4alkylene)CO2R19, ═N—OH, ═N—O-alkyl, wherein R18and R19are the same or different and are independently selected from hydrogen, alkyl, alkenyl, CO2H, CO2(alkyl), C3-C7cycloalkyl, phenyl, benzyl, phenylethyl, naphthyl, a 4- to 7-membered heterocyclo, or a 5- to 6-membered heteroaryl, or when attached to the same nitrogen atom may join to form a heterocyclo or heteroaryl, and R20is selected from same groups as R18and R19but is not hydrogen. Each group R18and R19when other than hydrogen, and each R20group optionally has up to three further substituents attached at any available carbon or nitrogen atom of R18, R19, and/or R20, said substituent(s) being the same or different and are independently selected from the group consisting of (C1-C6)alkyl, (C2-C6)alkenyl, hydroxy, halogen, cyano, nitro, CF3, O(C1-C6alkyl), OCF3, C(═O)H, C(═O)(C1-C6alkyl), CO2H, CO2(C1-C6alkyl), NHCO2(C1-C6alkyl), —S(C1-C6alkyl), —NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, N(CH3)3+, SO2(C1-C6alkyl), C(═O)(C1-C4alkylene)NH2, C(═O)(C1-C4alkylene)NH(alkyl), C(═O)(C1-C4alkylene)N(C1-C4alkyl)2, C3-C7cycloalkyl, phenyl, benzyl, phenylethyl, phenyloxy, benzyloxy, naphthyl, a 4- to 7-membered heterocyclo, or a 5- to 6-membered heteroaryl. When a substituted alkyl is substituted with an aryl, heterocyclo, cycloalkyl, or heteroaryl group, said ringed systems are as defined below and thus may have zero, one, two, or three substituents, also as defined below.

“Alkynyl” or “alkynylene”, alone or as part of another group, is intended to include hydrocarbon chains of either straight or branched configuration and having one or more carbon-carbon triple bonds that may occur in any stable point along the chain. For example, “C2-6alkynyl” (or alkynylene), is intended to include C2, C3, C4, C5, and C6alkynyl groups; such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and which may be optionally substituted with 1 to 4 substituents, namely, halogen, haloalkyl, alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, amino, heteroaryl, cycloheteroalkyl, hydroxy, alkanoylamino, alkylamido, arylcarbonylamino, nitro, cyano, thiol, and/or alkylthio.

The term “alkoxy” or “alkyloxy”, alone or as part of another group, refers to an —O-alkyl group, where alkyl is as defined above. “C1-6alkoxy” (or alkyloxy), is intended to include C1, C2, C3, C4, C5, and C6alkoxy groups. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and t-butoxy. Similarly, “alkylthio” or “thioalkoxy”, alone or as part of another group, represents an alkyl group or alkoxy group as defined above with the indicated number of carbon atoms attached through a sulphur bridge; for example methyl-S— and ethyl-S—.

“Halo” or “halogen”, alone or as part of another group, includes fluoro, chloro, bromo, and iodo.

“Halo-C1-C6-alkyl” or “Haloalkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 to 7 halogens, preferably 1 to 4 halogens, preferably F and/or Cl. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 1,1-difluoroethyl, 1-fluoroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Examples of haloalkyl also include “fluoroalkyl” that is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 to 7 fluorine atoms, preferably 1 to 4 fluorine atoms.

“Halo-C1-C4-alkoxy” or “haloalkyloxy” represents a haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. For example, “C1-6haloalkoxy”, is intended to include C1, C2, C3, C4, C5, and C6haloalkoxy groups. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, pentafluorothoxy, and the like. Similarly, “haloalkylthio” or “thiohaloalkoxy” represents a haloalkyl group as defined above with the indicated number of carbon atoms attached through a sulphur bridge; for example trifluoromethyl-S—, and pentafluoroethyl-S—.

Unless otherwise indicated, the term “cycloalkyl” as employed herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or 2 double bonds) cyclic hydrocarbon groups containing 1 to 3 rings, including monocyclic alkyl, bicyclic alkyl (or bicycloalkyl), and tricyclic alkyl, containing a total of 3 to 10 carbons forming the ring (C3-C10cycloalkyl), and which may be fused to 1 or 2 aromatic rings as described for aryl, which includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclohexenyl, cyclobutenyl, norbornyl,

any of which groups may be optionally substituted with 1 to 4 substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol, and/or alkylthio, and/or any of the substituents for alkyl, as well as such groups including 2 free bonds and thus are linking groups.

As used herein, the term “heterocycle,” “heterocyclo”, “heterocyclyl” or “heterocyclic” group is intended to mean a stable 4- to 14-membered monocyclic, bicyclic or tricyclic heterocyclic ring which is saturated or partially unsaturated and which consists of carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, NH, O and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, wherein p is 0, 1 or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or another substituent, if defined). The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. The heterocyclic rings described herein may optionally be substituted on carbon or on a nitrogen atom if the resulting compound is stable, with 1 to 3 groups selected from OH, OC1-C3alkoxy, Cl, F, Br, I, CN, NO2, NH2, N(CH3)H, N(CH3)2, CF3, OCF3, OCHF2, ═O, C(═O)CH3, SCH3, S(═O)CH3, S(═O)2CH3, C1-C3alkyl, CO2H and CO2CH3. A nitrogen in the heterocycle may optionally be quaternized. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1. Spiro and bridged rings are also included in the definition of heterocycle. A bridged ring occurs when one or more atoms (i.e., C, O, N, or S) link two non-adjacent carbon or nitrogen atoms. Examples of bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge. When the term “heterocycle” or “heterocyclyl” is used, it is not intended to include heteroaryl.

which optionally may be substituted.

As used herein, the term “aromatic heterocyclic group” or “heteroaryl” is intended to mean stable monocyclic and polycyclic aromatic hydrocarbons that include at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include, without limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane. Heteroaryl groups are unsubstituted or substituted with 1 to 3 groups selected from OH, OC1-C3alkoxy, Cl, F, Br, I, CN, NO2, NH2, N(CH3)H, N(CH3)2, CF3, OCF3, OCHF2, ═O, C(═O)CH3, SCH3, S(═O)CH3, S(═O)2CH3, C1-C3alkyl, CO2H and CO2CH3. The nitrogen atom is substituted or unsubstituted (i.e., N or NR wherein R is H or another substituent, if defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, wherein p is 0, 1 or 2). Bridged rings are also included in the definition of heteroaryl. A bridged ring occurs when one or more atoms (i.e., C, O, N, or S) link two non-adjacent carbon or nitrogen atoms. Examples of bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge.

and the like.

The designation “” or

attached to a ring or other group refers to a free bond or linking group.

Throughout the specification, groups and substituents thereof may be chosen by one skilled in the field to provide stable moieties and compounds and compounds useful as pharmaceutically-acceptable compounds and/or intermediate compounds useful in making pharmaceutically-acceptable compounds.

As referred to herein, the term “substituted” means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that normal valencies are maintained and that the substitution results in a stable compound. When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom are replaced. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

In cases wherein there are nitrogen atoms (e.g., amines) on compounds of the present invention, these may be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxides) to afford other compounds of this invention. Thus, shown and claimed nitrogen atoms are considered to cover both the shown nitrogen and its N-oxide (N→O) derivative. In cases in which there are quaternary carbon atoms in compounds of the present invention, these can be replaced by silicon atoms, provided they do not form Si—N or Si—O bonds.

When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 to 3 R10, then said group may optionally be substituted with up to three R10groups, and at each occurrence R10is selected independently from the definition of R10. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that 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, and/or other problem or complication, commensurate with a reasonable benefit/risk ratio.

In addition, compounds of formula I may have prodrug forms. Any compound that will be converted in vivo to provide the bioactive agent (i.e., a compound of formula I) is a prodrug within the scope and spirit of the invention. Various forms of prodrugs are well known in the art. For examples of such prodrug derivatives, see:a) Bundgaard, H., ed.,Design of Prodrugs, Elsevier (1985), and Widder, K. et al., eds.,Methods in Enzymology,112:309-396, Academic Press (1985);b) Bundgaard, H., Chapter 5, “Design and Application of Prodrugs,”A Textbook of Drug Design and Development, pp. 113-191, Krosgaard-Larsen, P. et al., eds., Harwood Academic Publishers (1991);c) Bundgaard, H.,Adv. Drug Deliv. Rev.,8:1-38 (1992);d) Bundgaard, H. et al.,J. Pharm. Sci.,77:285 (1988);e) Kakeya, N. et al.,Chem. Pharm. Bull.,32:692 (1984); andf) Rautio, J (Editor).Prodrugs and Targeted Delivery(Methods and Principles in Medicinal Chemistry), Vol 47, Wiley-VCH, 2011.

Isotopically labeled compounds of the present invention, i.e., wherein one or more of the atoms described are replaced by an isotope of that atom (e.g.,12C replaced by13C or by14C; and isotopes of hydrogen including tritium and deuterium), are also provided herein. Such compounds have a variety of potential uses, e.g., as standards and reagents in determining the ability of a potential pharmaceutical compound to bind to target proteins or receptors, or for imaging compounds of this invention bound to biological receptors in vivo or in vitro.

Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 98%, preferably 99%, compound of the present invention (“substantially pure”), which is then used or formulated as described herein. Such “substantially pure” compounds are also contemplated herein as part of the present invention.

The term “solvate” means a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art.

The compounds of the present invention can be prepared in a number of ways known to one skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are performed in a solvent or solvent mixture appropriate to the reagents and materials employed and suitable for the transformations being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent with the transformations proposed. This will sometimes require a judgment to modify the order of the synthetic steps or to select one particular process scheme over another in order to obtain a desired compound of the invention.

During the chemical syntheses, various protecting groups may be employed and subsequently removed in order to generate the compounds of the present invention. Exemplary protecting groups and conditions for their removal are described in Greene'sProtecting Groups in Organic SynthesisP. G. M. Nuts, T. W. Greene, Fourth Edition, Wiley, New York, 2006.

EXAMPLES

The following compounds of the invention have been prepared, isolated and characterized using the methods disclosed herein. They demonstrate a partial scope of the invention and are not meant to be limiting of the scope of the invention.

Compounds were analyzed on an Acquity Ultra Performance Liquid Chromatography system employing an Acquity UPLC BEH C18, 1.7 μm, 2.1×50 mm column. Detection was via an Aquity Ultra Performance LC PDA detector and an Acquity SQD single quadrupole mass spectrometer using H2O+0.1% formic acid (A) and ACN+0.1% formic acid (B) as eluents.

Compounds were analyzed on a Waters Alliance 2695 High-Performance Liquid Chromatography system employing a SunFire C18, 5 μm, 4.6×100 mm column. Detection was via a Waters 996 PDA detector using H2O+0.1% trifluoroacetic acid (A) and ACN+0.1% trifluoroacetic acid (B) as eluents.

1H NMR Spectroscopy was performed on a Bruker 400 MHz Avance II FTNMR Spectrometer. All1H NMR chemical shifts are reported in parts per million (ppm) and either referenced to the residual C—H signal from the deuterated solvent indicated or to tetramethylsilane.

To a mixture of Z-D-Glu-(Ot-Bu)-OH (1.00 g, 2.96 mmol) and 4-trifluoromethylbenzyl amine (0.42 mL, 3.0 mmol) in DCM (10 mL) was added EDAC (680 mg, 3.55 mmol) followed by HOBt H2O (480 mg, 3.55 mmol). The resultant reaction mixture was stirred at ambient temperature for 16 h. The mixture was then concentrated in vacuo and the crude residue was taken up in EtOAc (100 mL). This was washed successively with sat. NaHSO4(aq) (1×50 mL), sat. NaHCO3(aq) (1×50 mL) and brine (1×50 mL). The organic layer was then dried (Na2SO4), filtered and concentrated in vacuo to provide 1.39 g (95%) of (R)-tert-butyl 4-(((benzyloxy)carbonyl)amino)-5-oxo-5-((4-(trifluoromethyl)benzyl)amino)pentanoate which was used in the next step without further purification.

Following the methods described above for the preparation of Intermediates I.1-1.3, and substituting the corresponding reagents, the following intermediates were prepared as indicated in Table 1.

To a solution of the Weinreb amide obtained in step 1 above (0.20 g, 1.04 mmol) in anhydrous Et2O (5 mL) at 0° C. was added vinyl magnesium bromide (1.0 M in THF, 1.25 mL, 1.25 mmol). The reaction mixture was stirred at 0° C. for 30 min then allowed to warm to room temperature and was stirred an additional 2.5 h. The reaction mixture was then cooled to 0° C. and additional vinyl magnesium bromide (1.0 M in THF, 0.40 mL, 0.40 mmol) was added. The resultant mixture was warmed to room temperature and stirred an additional 1 h. The reaction mixture was then quenched with 2 N HCl (aq) and extracted with EtOAc (3×). The combined organic extracts were dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was then taken up in DCM and filtered through a 1 gram cartridge of SiO2, eluting with additional DCM. The filtrate was concentrated in vacuo to provide 0.13 g (78%) of Intermediate II.1.1H NMR (400 MHz, CDCl3): δ 7.31-7.19 (m, 5H), 6.37 (dd, 1H), 6.22 (dd, 1H), 5.84 (dd, 1H), 2.99-2.89 (m, 4H) ppm.

Using the synthetic method outlined above for the preparation of Intermediate II.1 the following intermediates were made from the corresponding reagent as indicated in Table 2.

To a solution of (2S,4R)-Boc-4-amino-Fmoc proline (330 mg, 0.729 mmol) in DCM (2 mL) was added DIPEA (0.35 mL, 2.0 mmol). This mixture was then added via pipette to a reaction vial containing Compound 1-1 (297 mg, 0.662 mmol). The vessel originally containing the proline reagent and DIPEA mixture was rinsed with DCM (1 mL) and this was also added via pipette to the reaction vial. BOP-Cl (96 mg, 0.38 mmol) was then added and the resultant reaction mixture was stirred at ambient temperature for 3 d. The reaction mixture was quenched with sat. NaHCO3(aq) and extracted with DCM (3×). The combined organic extracts were dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was purified by FCC (SiO2, elution with 0-50% EtOAc/hexanes) to provide 449 mg (77%) of Compound 1-2 as an off-white solid. LCMS (Method A): tR=1.83 min, m/z 883.6/885.6 (M+H)+.

To a solution of Compound 1-2 (445 mg; 0.503 mmol) in DCM (5 mL) was added dimethylamine (1.00 mL, 10.1 mmol) and this was stirred for 3 h at ambient temperature. This mixture was then concentrated in vacuo and the crude residue was taken up in MeOH (5 mL) and to this was added AcOH (0.10 mL) followed by NaCNBH3(1 M in THF, 0.65 mL, 0.65 mmol). The reaction mixture was stirred for 45 min. The mixture was then quenched with sat. NaHCO3(aq) and extracted with DCM (3×). The combined organic extracts were dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was purified by FCC (SiO2, elution with 0-50% EtOAc/hexanes) to provide 255 mg (78%) of Compound 1-3 as white solid. Analysis by LCMS indicated Compound 1-3 to be a mixture of two diastereomers in a ratio of 88:12. This mixture of diastereomers was carried directly into the next step. LCMS (Method A): tR=1.38 min (major diastereomer, 88%) and 1.45 min (minor diastereomer, 12%), m/z 645.5/647.5 (M+H)+.

Compound 1-3 (255 mg, 0.395 mmol) was taken up in 3N HCl in MeOH (2 mL) and stirred at 40° C. in a tightly capped reaction vial for 2 h. The reaction temperature was increased to 50° C. and stirring was continued for 30 min. The reaction mixture was then cooled, concentrated in vacuo and the crude residue was purified directly by mass-directed, preparative reverse phase HPLC (C18 column, elution with 5-95% ACN/H2O containing 0.25% formic acid). The desired fractions containing mainly the desired first eluting (major) diastereomer and a small amount of second eluting (minor) diastereomer were combined and partially concentrated in vacuo. To this was added 3N HCl (˜10 mL) and the volatiles were removed in vacuo. This treatment with 3N HCl was repeated to ensure formation of the hydrochloride salt. The mixture was then concentrated in vacuo and the residue was taken up in H2O and a small amount of ACN was added to provide a clear solution which was lyophilized to provide 96 mg (42%) of Example 1 as an off-white solid. Analysis by HPLC indicated Example 1 to be a mixture of diastereomers in a ratio of 96:4.1H NMR (400 MHz, CD3OD): δ 7.52 (d, 1H), 7.47 (d, 1H), 7.32-7.19 (m, 6H), 5.22 (dd, 1H), 5.04 (m, 1H), 4.35 (ABq, 2H), 4.15 (m, 1H), 3.96 (m, 1H), 3.85-3.62 (m, 3H), 3.37 (m, 1H), 3.20 (m, 1H), 2.77 (m, 1H), 2.59 (m, 1H), 2.38-2.26 (m, 2H), 2.16 (m, 1H), 1.98-1.66 (m, 4H), 1.50 (m, 1H), 0.98 (d, 3H), 0.92 (d, 3H) ppm; LCMS (Method A): tR=1.17 min, m/z 545.4/547.4 (M+H)+; HPLC: tR=4.866 min (96%, major diastereomer) and 5.207 min (4%, minor diastereomer).

Compound 3-3B (85 mg, 0.13 mmol) was treated with 3N HCl in MeOH (2 mL) and the resultant reaction mixture was heated to 85° C. in a tightly capped reaction vial for 2 h. The reaction mixture was then cooled and concentrated in vacuo. The crude residue was taken up in DMSO (1.5 mL) and then was purified directly by mass-directed preparative reverse phase HPLC (elution with 5-95% ACN/H2O containing 0.25% formic acid). The desired fractions were combined and partially concentrated in vacuo. To this was added 3N HCl (˜10 mL) and the volatiles were removed in vacuo. This treatment with 3N HCl was repeated to ensure formation of the hydrochloride salt. The mixture was then concentrated in vacuo and the residue was taken up in H2O and a small amount of ACN was added to provide a clear solution which was lyophilized to provide 26 mg (38%) of Example 3 as a yellow solid.1H NMR (400 MHz, CD3OD): δ 7.53 (d, 1H), 7.46 (d, 1H), 7.32-7.19 (m, 6H), 5.07 (dd, 1H), 4.97 (m partially obscured by H2O peak, 1H), 4.38 (ABq, 2H), 4.12 (m, 1H), 4.03-3.88 (m, 3H), 3.85-3.79 (m, 2H), 3.65 (m, 1H), 3.34 (m partially obscured by solvent peak, 1H), 3.20 (m, 1H), 2.77 (m, 1H), 2.60 (m, 1H), 2.36-2.25 (m, 2H), 2.17 (m, 1H), 2.05 (m, 1H), 1.87 (m, 1H) ppm; LCMS (Method A): tR=0.97 min, m/z 519.4/521.4 (M+H)+.

Following the methods described above for Examples 1-3, and substituting the corresponding intermediates, the following examples were prepared as indicated in Table 3. Unless indicated otherwise, the Examples in Table 3 were prepared from the major diastereomer obtained in step 4 (intramolecular reductive amination) of the synthesis.

TABLE 3IntermediatesLCMStR(M + H)+ExampleStructureand reagentsMethod(min)observed4I.7 and II.1A1.08595.5/597.45I.8 and II.1A1.00503.56I.9 and II.1A1.07545.57I.6 and II.1A1.12559.58I.5 and II.1A0.98477.59I.4 and II.1A1.00491.510I.2 and II.2A1.14531.5/533.511I.2 and II.3A1.27579.5/581.4/  583.412I.10 and II.1A1.07483.613I.2 and 3-buten-2-oneA0.96455.4/457.414AI.11 and II.1A1.08561.4/563.414BI.11 and II.1A0.94561.3/563.315I.12 and II.1A1.02547.3/549.316I.13 and II.1A0.99560.4/562.317I.11 and  phenoxymethyl vinyl  ketoneA0.96563.5/565.518I.2 and  (phenylsulfanyl)methyl  vinyl ketoneA1.15563.5/565.519I.2 and II.4A1.11547.4/549.4

Compound 22-3 was prepared from Compound 22-1 (92 mg, 0.21 mmol) using the same general procedures described for the preparation of Compound 1-3 in steps 2-4 in Example 1. After work up, the crude product was purified by FCC (SiO2, elution with 0-70% EtOAc/hexanes). Partial separation of the two diastereomers was achieved providing two fractions; one containing 29 mg of mainly the first eluting, major diastereomer (Compound 22-3A) and the other containing 17 mg of mainly the second eluting, minor diastereomer (Compound 22-3B). A small amount of the other diastereomer was present in each of the two fractions. The fraction containing mainly the minor diastereomer 22-3B was carried into the next step. Data for Compound 22-3A (major diastereomer): LCMS (Method C): tR=0.92 min, m/z 631.4/633.3 (M+H)+. Data for Compound 22-3B (minor diastereomer): LCMS (Method C): tR=0.99 min, m/z 631.4/633.3 (M+H)+.

Compound 22-3B (16 mg, 0.025 mmol) was taken up in 3N HCl in MeOH (1 mL) and the resultant solution was stirred at 50° C. in a tightly capped reaction vial for 90 min. The reaction mixture was then cooled, concentrated in vacuo and the crude residue was purified directly by preparative reverse phase MPLC (C18 column, elution with 5-95% ACN/H2O). The desired fractions were combined and partially concentrated in vacuo. To this was added 3N HCl (˜10 mL) and the volatiles were removed in vacuo. This treatment with 3N HCl was repeated to ensure formation of the hydrochloride salt. The mixture was then concentrated in vacuo and the residue was taken up in H2O and a small amount of ACN was added to provide a clear solution which was lyophilized to provide 11 mg (79%) of Example 22 as a white solid.1H NMR (400 MHz, CD3OD): δ 7.47 (d, 1H), 7.43 (d, 1H), 7.32-7.27 (m, 4H), 7.23-7.20 (m, 2H), 5.17 (dd, 1H), 4.55 (m, 1H), 4.33 (ABq, 2H), 4.10-3.42 (m, 6H), 2.90-2.76 (m, 3H), 2.45 (m, 1H), 2.15 (m, 1H), 2.00 (m, 1H), 1.81 (m, 1H), 1.66 (m, 1H), 1.45 (m, 1H), 1.01 (d, 3H), 0.94 (d, 3H) ppm; LCMS (Method A): tR=1.13 min, m/z 531.5/533.5 (M+H)+.

Additional examples as set forth in Table 4 below were prepared in accordance with the present invention:

Biology

It is desirable to find compounds with advantageous and improved characteristics compared with known TREX1 inhibitors, in one or more of the following categories that are given as examples, and are not intended to be limiting: (a) pharmacokinetic properties, including oral bioavailability, half-life, and clearance; (b) pharmaceutical properties; (c) dosage requirements; (d) factors that increase the concentration of active drug at the receptor; (e) factors that decrease the liability for clinical drug-drug interactions; (f) factors that decrease the potential for adverse side-effects, including selectivity versus other biological targets; and (g) factors that improve manufacturing costs or feasibility.

The term “compound”, as used herein, means a chemical, be it naturally-occurring or artificially-derived. Compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.

As used herein, the term “patient” encompasses all mammalian species.

As used herein, the term “subject” refers to any human or nonhuman organism that could potentially benefit from treatment with a TREX1 inhibitor. Exemplary subjects include human beings of any age with risk factors for a disorder, disease, syndrome, or condition affected by the inhibition of TREX1, or patients that have already experienced one episode of a disorder, disease, syndrome, or condition affected by the inhibition of TREX1.

As used herein, “treating” or “treatment” cover the treatment of a disease-state in a mammal, particularly in a human, and include: (a) inhibiting the disease-state, i.e., arresting its development; and/or (b) relieving the disease-state, i.e., causing regression of the disease state.

As used herein, “prophylaxis” or “prevention” covers the preventive treatment of a subclinical disease-state in a mammal, particularly in a human, aimed at reducing the probability of the occurrence of a clinical disease-state. Patients are selected for preventative therapy based on factors that are known to increase risk of suffering a clinical disease state compared to the general population. “Prophylaxis” therapies can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in a subject that has not yet presented with a clinical disease state, whereas secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.

As used herein, “risk reduction” covers therapies that lower the incidence of development of a clinical disease state. As such, primary and secondary prevention therapies are examples of risk reduction.

“Therapeutically effective amount” is intended to include an amount of a compound of the present invention that is effective when administered alone or in combination to modulate TREX1 and/or to prevent or treat the disorders listed herein. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the preventive or therapeutic effect, whether administered in combination, serially, or simultaneously.

As TREX1 inhibitors, it is believed that the compounds of Formula I, and the examples are useful in methods for treating or preventing cancer, disease, a syndrome, a condition or a disorder in a subject, including an animal, a mammal and a human in which the cancer, disease, the syndrome, the condition or the disorder is affected by the modulation, including inhibition, of the TREX1 protein. Such methods comprise, consist of and/or consist essentially of administering to a subject, including an animal, a mammal, and a human, in need of such treatment or prevention, a therapeutically effective amount of a compound, salt or solvate of Formula I.

In another embodiment, the present invention is directed to a compound of Formula I, or a stereoisomer, a tautomer, a salt, a solvate or a prodrug form thereof, for use in the treatment of a disorder affected by the inhibition of TREX1 selected from the group consisting of melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, bladder cancer, fibrosarcoma, and HIV.

In another embodiment, the present invention is directed to a compound of Formula I, or a stereoisomer, a tautomer, a salt, a solvate or a prodrug form thereof, for use in the treatment of a disorder affected by the inhibition of TREX1 wherein the disorder is bladder cancer.

The term “pharmaceutical composition,” as used herein, means any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Gennaro, A. R., ed.,Remington: The Science and Practice of Pharmacy,20th Edition, Mack Publishing Co., Easton, Pa. (2000).

The invention includes administering to a subject a pharmaceutical composition that includes a compound that modulates TREX1 (referred to herein as a “TREX1 inhibitor” or “therapeutic compound”).

The compounds of this disclosure can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. They may also be administered in intravenous (bolus or infusion), intraperitoneal, intravesical, subcutaneous, or intramuscular form, and all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. They can be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The preferred dose of the TREX1 inhibitor is a biologically active dose. A biologically active dose is a dose that will modulate the TREX1 protein and have an appropriate effect. Desirably, the dose includes a dose range from about 0.0005 mg to about 3000 mg, or any particular amount or range therein, in particular from about 0.0005 mg to about 1000 mg, or any particular amount or range therein, or, more particularly, from about 0.0005 mg to about 250 mg, or any particular amount or range therein, of active ingredient in a regimen of about 1 to about 4 times per day for an average (70 kg) human; although, it is apparent to one skilled in the art that the therapeutically effective amount for a compound of Formula I will vary as will the diseases, syndromes, conditions, and disorders being treated.

Compounds of this invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using transdermal skin patches. When administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

Compounds of this invention can be administered in intravesical form via a solution that is run through a tube (instilled through a catheter) into the particular organ, for example, the bladder, to treat the cancer.

The compounds are typically administered in a mixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as pharmaceutical carriers) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

Dosage forms (pharmaceutical compositions) suitable for administration will ordinarily contain the active ingredient in an amount of about 0.5-95% by weight based on the total weight of the composition.

Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

Representative useful pharmaceutical dosage-forms for administration of the compounds of this invention can be illustrated as follows:

Capsules

A large number of unit capsules can be prepared by filling standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil may be prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient. The capsules should be washed and dried.

Tablets

Tablets may be prepared by conventional procedures so that the dosage unit is 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

Dispersion

A spray dried dispersion can be prepared for oral administration by methods know to one skilled in the art.

A parenteral composition suitable for administration by injection may be prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution should be made isotonic with sodium chloride and sterilized.

Suspension

An aqueous suspension can be prepared for oral administration so that each 5 mL contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 mL of vanillin.

Where two or more of the foregoing second therapeutic agents are administered with the compound of Formula I, preferably, a compound selected from one of the examples, generally the amount of each component in a typical daily dosage and typical dosage form may be reduced relative to the usual dosage of the agent when administered alone, in view of the additive or synergistic effect of the therapeutic agents when administered in combination.

Additionally, certain compounds disclosed herein may be useful as metabolites of other compounds. Therefore, in one embodiment, compounds may be useful either as a substantially pure compound, which may also then be incorporated into a pharmaceutical composition, or may be useful as metabolite which is generated after administration of the prodrug of that compound. In one embodiment, a compound may be useful as a metabolite by being useful for treating disorders as described herein.

The disclosed compounds of Formula I may be useful in combination with one or more additional therapeutic agents. The additional therapeutic agent may be, e.g., radiation therapy, a chemotherapeutic, a biotherapeutic agent (including but not limited to antibodies to VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, and ICOS), an immunogenic agent (for example, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor derived antigen or nucleic acids, immune stimulating cytokines (for example, IL-2, IFNa2, GM-CSF), and cells transfected with genes encoding immune stimulating cytokines such as but not limited to GM-CSF).

The additional therapeutic agent(s) may be administered in a single dosage form with at least one compound of the present invention, or the additional therapeutic agent(s) may be administered in separate dosage form(s) from the dosage form containing the compound of the present invention.

The compound of the present invention disclosed herein may be used in combination with one or more other therapeutic agents, including but not limited to, other anti-cancer agents that are used in the prevention, treatment, control, amelioration, or reduction of risk of a particular disease or condition. In one embodiment, a compound disclosed herein is combined with one or more other anti-cancer agents for use in the prevention, treatment, control amelioration, or reduction of risk of a particular disease or condition for which the compounds disclosed herein are useful. Such other therapeutic agents may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure.

The additional therapeutic agent(s) may be one or more agents selected from the group consisting of radiation therapy, a STING agonist, anti-viral compounds, antigens, adjuvants, anti-cancer agents, another TREX1 inhibitor, CTLA-4, LAG-3, PD-1 pathway antagonists, PD-L1 antibodies, lipids, liposomes, peptides, cytotoxic agents, chemotherapeutic agents, immunomodulatory cell lines, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, topoisomerase II inhibitors, smoothen inhibitors, alkylating agents, anti-tumor antibiotics, anti-metabolites, retinoids, and immunomodulatory agents including but not limited to anti-cancer vaccines. It will be understood the descriptions of the above additional therapeutic agents may be overlapping. It will also be understood that the treatment combinations are subject to optimization, and it is understood that the best combination to use of the TREX1 inhibitor, and one or more additional therapeutic agents will be determined based on the individual patient needs.

When the compound disclosed herein is used contemporaneously with one or more other therapeutic agents, the compound may be administered either simultaneously with, or before or after, one or more other therapeutic agent(s).

The weight ratio of the compound of the present invention may be varied and will depend upon the therapeutically effective dose of each agent. Generally, a therapeutically effective dose of each will be used. Combinations including at least one compound of the present invention, and other therapeutic agents will generally include a therapeutically effective dose of each active agent. In such combinations, the compound of the present invention disclosed herein and other therapeutic agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent with, or subsequent to the administration of other agent(s).

In one embodiment, this disclosure provides at least one compound of the present invention, and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disorder affected by the inhibition of TREX1, such as cancer.

The disclosure also provides the use of a compound of Formula I for treating a disorder affected by inhibition of TREX1, where the patient has previously (e.g., within 24 hours) been treated with another therapeutic agent.

Anti-viral compounds that may be used in combination with the compounds disclosed herein include hepatitis B virus (HBV) inhibitors, hepatitis C virus (HCV) protease inhibitors, HCV polymerase inhibitors, HCV NS4A inhibitors, HCV NSSA inhibitors, HCV NSSb inhibitors, and human immunodeficiency virus (HIV) inhibitors.

Antigens and adjuvants that may be used in combination with the compounds disclosed herein include B7 costimulatory molecule, interleukin-2, interferon-y, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. Adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to trigger, enhance, or prolong an immune response. Additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, like CpG, a toll-like receptor (TLR) 9 agonist as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoprotein, lipopolysaccharide (LPS), monophosphoryllipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition retinoic acid-inducible gene I (RIG-I) agonists such as poly I:C, used separately or in combination are also potential adjuvants.

Examples of cytotoxic agents that may be used in combination with the compounds disclosed herein include, but are not limited to, arsenic trioxide, asparaginase, andErwiniaL-asparaginase.

Examples of vascular endothelial growth factor (VEGF) receptor inhibitors include, but are not limited to, bevacizumab, Brivanib Alaninate, motesanib, pasireotide, and sorafenib.

Examples of topoisomerase II inhibitors, include but are not limited to, etoposide and teniposide.

Examples of retinoids include, but are not limited to, alitretinoin, tretinoin, isotretinoin and bexarotene.

Examples of PD-1 antagonists include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, and AMP-224.

Examples of PD-L1 antibodies include, but are not limited to, Atezolizumab, Avelumab, and Durvalumab.

An example of a CLT-4 antagonist is ipilimumab.

Examples of STING agonists include, but are not limited to, those disclosed in International Published Patent Application Nos. WO 2017/027645 A1, WO 2017/027646 A1 and WO 2018/118664 A1.

The activity of the TREX1 inhibitors of the present invention can be measured in a variety of in vitro assays. Exemplary assays are shown in the Examples below.

The TREX1 Exonuclease assay is an exemplary in vitro assay for measuring the activity of the TREX1 inhibitors of the present invention. In this assay, TREX1 exonuclease activity was evaluated by measuring the increase in fluorescence resulting from TREX1-catalyzed removal of a quencher from the 3′ end of a FAM-labeled DNA oligonucleotide (5′FAM-CCA CGA GAG CGT-BHQ1-3′). The assay was conducted using Wild Type (WT) human or murine TREX1 enzyme constructs corresponding to amino acids 55-297 of UniProt ID Q9NSU2-1 (SEQ ID NO: 1), or amino acids 1-242 of UniProt ID Q91XB0-1 (SEQ ID NO: 2), respectively. See, e.g., Example A.

Example B is a protein thermal shift assay used to assess the binding of TREX1 inhibitors to TREX1 protein. In this assay, TREX1 protein unfolding with increasing temperature was measured in the presence and absence of TREX1 inhibitors by following the increase in fluorescence of a thiol-reactive dye (e.g BODIPY FL-cystine) as it reacts with exposed TREX1 cysteine residues. The assay was conducted using human (SEQ ID NO: 1), or murine (SEQ ID NO: 2) TREX1 protein constructs.

Example C is a cell-based assay for TREX1 inhibitors using THP1 Dual™ cells (Invivogen), a human monocyte cell line that has stable integration of an Interferon Stimulated Response Element (ISRE) Lucia reporter gene. The Lucia gene encodes a secreted luciferase reporter protein, under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements. Activation of the cGAS-STING pathway in THP1 Dual™ cells leads to enhanced luciferase secretion and increased luminescence.

Example D is a cell based assay that measures the increase in the expression of interferon stimulated genes (ISG) by RT-qPCR that are induced by TREX1 inhibitors when THP1 Dual™ cells are transfected with double-stranded DNA. The double stranded DNA used to transfect cells was VACV-70 (Invivogen), a 70 base pair oligonucleotide containing viral DNA motifs (Unterholzner L. et al., 2010. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 11(11):997-1004). Activation of cGAS-STING pathway in THP1 Dual™ cells increases the expression of ISGs.

Assays

While it is apparent that the embodiments of the application herein disclosed are well suited to fulfill the objectives stated above, it will be appreciated that numerous modifications and other embodiments may be implemented by those skilled in the art, and it is intended that the appended claims cover all such modifications and embodiments that fall within the true spirit and scope of the present application.

Example A

For human TREX1, the nucleotide sequence of the gene construct was codon optimized for expression in the bacterial host. The sequence was incorporated into the pMAL-c5e vector (NEB) downstream of a solubility-promoting MBP (maltose binding protein) fusion partner. The fusion protein product was expressed inE. colistrain BL21 (DE3) (Millipore) and purified in a series of chromatographic steps. Initial steps were conducted using a dextrin sepharose affinity column followed by a Q-sepharose ion exchange column (both GE Healthcare). After the second column the MBP partner was removed by incubation with enterokinase (NEB). Further purification was carried out with the second application of a Q-sepharose column followed by a Superdex 75 (GE Healthcare) size exclusion column. Finally, a heparin sepharose column (GE Healthcare) was applied to remove contaminating nucleotides. Similar methods were used in the preparation of the murine TREX1 enzyme and are described in Example B set forth below.

To evaluate the effect of compounds on TREX1 activity, test compounds were serially diluted (11-point, 3-fold) from 10 mM stock solutions and delivered to 384-well low-volume assay plates in 80 nL DMSO using an acoustic dispenser. Next, 4 μL of human TREX1 (0.5 nM), or murine TREX1 (1 nM), diluted in assay buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100 μg/mL BSA, 0.002% Triton X-100, 2 mM DTT), was added to the assay plate. After incubating for 30 minutes, 4 μL of labeled DNA oligonucleotide (500 nM) in assay buffer was added to initiate TREX1 exonuclease activity. The reaction was allowed to proceed for 45 minutes at room temperature prior to the addition of 4 μL of 150 mM EDTA to halt TREX1 activity. Assay plates were equilibrated for an additional 30 minutes and read on an EnVision Plate Reader (Perkin Elmer) to measure fluorescence emission at 535 nm following excitation at 485 nm. Fluorescence was plotted as a function of log molar compound concentration and fit to four-parameter dose-response equation to determine compound IC50.

Compounds of the present invention were tested in the TREX1 Exonuclease assay described immediately above and the results shown in Table 5 below were obtained.

Example B

TREX1 Thermal Shift Binding Assay

The thermal shift assays used recombinant protein corresponding to human (SEQ ID NO: 1) and murine (SEQ ID NO: 2) TREX1. The protein encoding plasmid contained a N-terminal MBP (maltose binding protein) tag followed by the TREX1 coding sequence within the pMAL-c5E vector. The plasmid was transfected into a BL21 (DE3)E. coliexpression strain. Protein was purified from the bacterial lysate using an Amylose resin column. The MBP tag was cleaved from the purified protein using recombinant enterokinase (EMD Millipore #69066-3). The cleaved TREX1 protein was further purified on a Q-Sepharose column followed by a heparin column.

Binding of compounds to both human and murine TREX1 were measured by protein thermal shift assays. (Huynh K, Partch C L. Current Protocols in Protein Science: Analysis of protein stability and ligand interactions by thermal shift assay.Current protocols in protein science/editorial board, John E Coligan. [et al]. 2015; 79:28.9.1-28.9.14. doi:10.1002/0471140864.ps2809s79.). Thermal shift assays were conducted in sealed 96-well PCR plates containing 5 μM TREX1 protein, 100 μM compound, and 2 μM BODIPY FL-cystine (Sigma) in either 20 μL assay buffer (20 mM Tris, 7.5, 5 mM MgCl2, 0.002% Triton X-100) or 20 μL phosphate buffered saline (PBS). Using a RT-qPCR machine (Mx3005P, Stratagene) changes in fluorescence (excitation at 492 nm and emission at 516 nm) were monitored as temperature was increased from 25° C. to 96° C. at a rate of 1° C. every 2 minutes. Tmvalues were calculated from the first derivative plot of fluorescence intensity versus temperature. The results shown in Table 6 indicate the examples bind to human TREX1 and murine TREX1. ND indicates assay was not done.

Example C

A cell-based assay was established using THP1 Dual™ cells (Invivogen), a human monocyte cell line that has stable integration of an Interferon Stimulated Response Element (ISRE) Lucia reporter gene. The Lucia gene encodes a secreted luciferase reporter protein, under the control of an ISG54 minimal promoter in conjunction with five IFN-stimulated response elements. Activation of the cGAS-STING pathway in these cells leads to enhanced luciferase secretion. The assay was conducted in standard 384-well white tissue culture plates. Cells (25,000 in 25 μL RPMI 1640 containing 10% Heat-Inactivated FBS, 1× Glutagro, 10 mM HEPES, 1 mM sodium pyruvate, 1× Pen/Strep, 100 μg/mL Normocin, 100 μg/mL Zeocin, 10 μg/mL Blasticidin) were added to the plate followed by 25 μL of compound in the same media+0.2% DMSO. The assay was incubated for 48 hours at 37° C. in 5% CO2. An aliquot of 5 μl was removed to combine with 12.5 μL of the luciferase detection reagent, QUANTI-Luc™ Gold (Invivogen) in a white 384-well plate. The luminescent signal was then read using an EnVision plate reader (Perkin Elmer). The fold activation was calculated by dividing the signal in wells containing test compound by the signal in the control wells without compound (see Table 7).

Example D

The RT-qPCR assay was used to assess the ability of TREX1 inhibitors to enhance the expression of interferon stimulated genes (ISGs) in cells transfected with double-stranded DNA. The double stranded DNA used to transfect cells was VACV-70 (Invivogen), a 70 base pair oligonucleotide containing viral DNA motifs (Unterholzner L. et al., 2010. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 11(11):997-1004). Human THP1 Dual™ cells (Invivogen), were treated with test compound in the presence or absence of 1,200 ng/mL VACV-70, or with VACV-70 alone, for 24 hours. Cells were harvested by centrifugation and washed with phosphate buffer saline. Total RNA was isolated using Qiagen RNeasy mini kit. Genomic DNA was degraded using Thermo Scientific DNase 1 kit. Total RNA was quantified using a Nanodrop spectrophotometer. Using equal amounts of RNA, cDNA was synthesized using Invitrogen Superstrand III First Strand synthesis kit. Target and reference gene expression levels were determined by RT-qPCR using TaqMan gene expression assays and a Stratagene Mx3005P QPCR system (all reagents were from TaqMan, Applied Biosystems). Data were analyzed by the Comparative CTmethod. GAPDH was used for normalization. Target genes that were analyzed included interferon stimulated genes (INF-β, CXCL10, IFIT1, IFIT2, IFIT3, IFI44, and IFI44L). The data are shown in Table 8.

While it is apparent that the embodiments of the application herein disclosed are well suited to fulfill the objectives stated above, it will be appreciated that numerous modifications and other embodiments may be implemented by those skilled in the art, and it is intended that the appended claims cover all such modifications and embodiments that fall within the true spirit and scope of the present application.