Small molecule inhibitors of DNA pkcs

Abstract

The present disclosure is directed to DNA-PKcs inhibitors designed to enhance bioavailability. The disclosed inhibitors may inhibit DNA-PKcs kinase activity resulting in increased tumor cell sensitivity to DNA-damage inducing chemotherapy and inhibition of human T cell function. The present disclosure is also directed to a method of making and a method of using the disclosed DNA-PKcs inhibitors.

Description

PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) SMALL MOLECULE INHIBITORS OF DNA PKCS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 672,891, filed July 18, 2024, entitled “SMALL MOLECULE INHIBITORS OF DNA PKCS”, the disclosure of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under P20GM109005 awarded by the National Institutes of General Medical Sciences. The government has certain rights in the invention. BACKGROUND

[0003] DNA PKcs is an important pro-inflammatory kinase, and activation of this kinase can lead to organ transplant rejection. Because of this, drug discovery programs have focused on identifying DNA PKcs inhibitors that can be used as anti-rejection agents. Current agents, however, have limited efficacy because of poor pharmacokinetic and / or pharmacodynamic profiles. SUMMARY OF INVENTION

[0004] The present disclosure is directed to a compound, or a pharmaceutically acceptable salt thereof, according to Formula (I): 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) wherein R1, R2, and R4are hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; and R3 is an azole or a substituted azole.

[0005] In some embodiments, the azole or substituted azole is pyrazole or substituted pyrazole, and with Formula (II):Formula (II); wherein R1, R2, and R4are hydrogen; and R5is hydrogen, C1-C6alkyl, or substituted C1-C6alkyl.

[0006] In some embodiments, R5 is methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0007] In some embodiments, R5is substituted C1-C6alkyl.

[0008] In some embodiments, the substituted C1-C6alkyl is substituted with one or more halogens, amine, substituted amine, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, or substituted C3-C6 heterocycloalkyl.

[0009] In some embodiments, the substituted amine is substituted with one or more C1-C6alkyl. The substituted C1-C6 alkyl may be substituted with one or more halogens, amine, substituted amine, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, or substituted C3-C6heterocycloalkyl. 2 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0010] In some embodiments, R5 is substituted ethyl, and with Formula (III):ormu a ; wherein R6 is amine, substituted amine, C3-C6 heterocycloalkyl, or substituted C3-C6 heterocycloalkyl.

[0011] In some embodiments, R6is substituted amine.

[0012] In some embodiments, the substituted amine is substituted with one or more C1-C6 alkyl.

[0013] In some embodiments, R6is substituted C3-C6heterocycloalkyl.

[0014] In some embodiments, the substituted C3-C6heterocycloalkyl is substituted piperidine.

[0015] In some embodiments, the substituted piperidine is substituted with one or more halogens.

[0016] In some embodiments, the halogen is fluorine, chlorine, or bromine.

[0017] In some embodiments, the C3-C6 heterocycloalkyl is aziridine, azetidine, pyrrolidine, piperidine, piperazine, substituted aziridine, substituted azetidine, substituted pyrrolidine, substituted piperidine, or substituted piperazine.

[0018] In some embodiments, the C3-C6 heterocycloalkyl is substituted piperazine.

[0019] In some embodiments, the substituted piperazine is substituted with a C1-C6 alkyl or one or more halogens.

[0020] In some embodiments, the C3-C6 heterocycloalkyl is substituted piperidine.

[0021] In some embodiments, the substituted piperidine is substituted with one or more halogens.

[0022] In some embodiments, the halogen is fluorine, chlorine, or bromine. 3 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0023] In some embodiments, the compound is selected from the group consisting of: 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 6; 5 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 12; and 7 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)p .

[0024] In some embodiments, the compound has a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.2.

[0025] In some embodiments, the compound has a solubility limit of greater than 14 μg / mL in phosphate buffer saline at pH 7.2.

[0026] The present disclosure is directed to a composition comprising a DNA-PKcs inhibitor with a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.2.

[0027] The present disclosure is also directed to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I):Formula (I); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3is an azole or a substituted azole. 8 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0028] The present disclosure is directed to a method of inhibiting DNA-PKcs kinase activity in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I):Formula (I); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3 is an azole or a substituted azole.

[0029] The present disclosure is directed to a method of treating a transplanted solid organ for anti-rejection therapy in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I): 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) Formula (I); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3 is an azole or a substituted azole.

[0030] In some embodiments, the anti-rejection therapy is T-cell mediated. BRIEF DESCRIPTION OF FIGURES

[0031] FIG. 1A depicts morpholine segment of NU7441 plays a crucial role in binding to the DNA-PKcs hinge, and any modification at this region will result in a loss of binding affinity.

[0032] FIG. 1B depicts morpholine segment remains unchanged, while modifications were made to the solvent-accessible region in DA-138 (Compound 1) and DA-143 (Compound 10) in accordance with some embodiments. The pyrazole on DA-138 (Compound 1) may serve as a tetherable site to introduce an aliphatic basic amine, enhancing solubility.

[0033] FIGS. 1C and 1D show predicted orientation of the pyrazole in both DA-138 (Compound 1) and DA-143 (Compound 10) is towards the solvent in accordance with some embodiments. Additionally, the pyrrolidine on DA-143 (Compound 10), being highly ionizable, may have the potential to improve water solubility.

[0034] FIG. 2A shows DNA-PK inhibitors NU7441, DA-138 (Compound 1), DA-143 (Compound 10), and DA-147 (Compound 13) in accordance with some embodiments.

[0035] FIG. 2B shows inhibitor activity with an in vitro assay measuring ATP to ADP conversion by DNA-PKcs enzyme in the presence of a polypeptide substrate in accordance with some embodiments. * indicates p < 0.001 by Student’s T test. *** indicates p > 0.1 by T test between each sample pairing.

[0036] FIG.2C shows solubility of NU7441 and DA-143 (Compound 10) in phosphate buffered saline (PBS) at pH 7.2 determined via turbidimetric solubility measurement in accordance with some embodiments.

[0037] FIG.3A depicts dot plots of flow cytometry plots with Annexin V staining on the X axis and 7-AAD staining on the Y axis in accordance with some embodiments. MC38 mouse colon 10 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) cancer cells were pre-treated with inhibitors (5 µM) then exposed to a low dose (25 nM) of doxorubicin for 48 hours.

[0038] FIG. 3B shows the quantification of the flow cytometry plots in FIG. 3A. Error bars = s.d. of the mean of three replicates. * p < 0.001 by Student’s T test.

[0039] FIG. 3C depicts Western Blot of MC38 cells treated with the indicated combination of doxorubicin and inhibitor for 24 hours in accordance with some embodiments. The indicated proteins were detected from whole cell lysates by Western blotting. Blot has been cropped to show only relevant proteins.

[0040] FIG. 4A depicts Western Blot for DNA-PKcs autophosphorylation at serine 2056 and Kap1 phosphorylation at serine 824 of Jurkat T cells pre-treated with DNA-PKcs inhibitors and stimulated for 10 minutes before harvesting in accordance with some embodiments. Blots have been cropped to show only relevant proteins. Blots have been cropped to show only relevant proteins.

[0041] FIG.4B depicts Western Blot for AKT phosphorylation at serine 473 of Jurkat cells pre- treated with inhibitors and stimulated for 2 hours before harvesting in accordance with some embodiments. Blots have been cropped to show only relevant proteins.

[0042] FIG. 5A depicts flow cytometry gating on CD3+ cells data showing proliferation of isolated human PBMCs stained with CellTrace Violet, then treated with 5 µM inhibitor or vehicle and stimulated with anti-CD3 / anti-CD28 and cultured for 5 days in accordance with some embodiments.

[0043] FIG. 5B depicts flow cytometry data showing DNA-PKcs inhibitors and cytokine expression in isolated mouse CD8+ cells stained with CellTrace Violet, then treated with 5 µM inhibitor or vehicle and stimulated with anti-CD3 and cultured for 3 days in accordance with some embodiments.

[0044] FIG.5C shows bar plots showing IL2 expression in human PBMCs after 48 hours with DNA-PKcs inhibitors (10 µM) or vehicle control measured by ELISA from cell culture media in accordance with some embodiments. * indicates p < 0.05 by Student’s T test (1-tailed). ** indicates p < 0.5 by T test (2 tailed). 11 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0045] FIG.5D shows bar plots showing IL2 expression in mouse CD4+ T cells after 24 hours with DNA-PKcs inhibitors (10 µM) or vehicle control measured by ELISA from cell culture media. * indicates p < 0.05 by Student’s T test (1-tailed). ** indicates p < 0.5 by T test (2 tailed).

[0046] FIG. 6A is a UHPLC chromatograph of 1-(3-bromo-2-hydroxyphenyl)-3- (dimethylamino)prop-2-en-1-one, (3) in accordance with some embodiments.

[0047] FIG.6B is a UHPLC chromatograph of 8-bromo-4H-chromen-4-one, (4) in accordance with some embodiments.

[0048] FIG. 6C is a UHPLC chromatograph of 8-bromo-2-(1H-1,2,4-triazol-1-yl)-4H- chromen-4-one (6) in accordance with some embodiments.

[0049] FIG.6D is a UHPLC chromatograph of 8-bromo-2-morpholino-4H-chromen-4-one, (8) in accordance with some embodiments.

[0050] FIG. 6E is a UHPLC chromatograph of 6-bromo-2-iododibenzo[b,d]thiophene, (12) in accordance with some embodiments.

[0051] FIG. 6F is a UHPLC chromatograph of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1- methyl-1H-pyrazole (14a) in accordance with some embodiments.

[0052] FIG. 6G is a UHPLC chromatograph of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-(2- (pyrrolidin-1-yl)ethyl)-1H-pyrazole, (14b) in accordance with some embodiments.

[0053] FIG. 6H is a UHPLC chromatograph of 1-(6-bromodibenzo[b,d]thiophen-2-yl)-4- methylpiperazine, (14c) in accordance with some embodiments.

[0054] FIG. 6I is a UHPLC chromatograph of 8-(8-(1-methyl-1H-pyrazol-4- yl)dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one, DA-138, (Compound 1) in accordance with some embodiments.

[0055] FIG. 6J is a UHPLC chromatograph of 8-(8-(4-methylpiperazin-1- yl)dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one, DA-143, (Compound 10) in accordance with some embodiments.

[0056] FIG. 6K is a UHPLC chromatograph of 2-morpholino-8-(8-(1-(2-(pyrrolidin-1- yl)ethyl)-1H-pyrazol-4-yl)dibenzo[b,d]thiophen-4-yl)-4H-chromen-4-one, DA-147, (Compound 13) in accordance with some embodiments. 12 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0057] FIG. 7A is a1H NMR spectra of (3-bromo-2-hydroxyphenyl)-3-(dimethylamino)prop- 2-en-1-one, (3) in accordance with some embodiments.

[0058] FIG.7B is a13C NMR spectra of (3-bromo-2-hydroxyphenyl)-3-(dimethylamino)prop- 2-en-1-one, (3) in accordance with some embodiments.

[0059] FIG. 7C is a1H NMR spectra of 8-bromo-4H-chromen-4-one, (4) in accordance with some embodiments.

[0060] FIG. 7D is a13C NMR spectra of 8-bromo-4H-chromen-4-one, (4) in accordance with some embodiments.

[0061] FIG. 7E is a1H NMR spectra of 8-bromo-2-(1H-1,2,4-triazol-1-yl)-4H-chromen-4-one (6) in accordance with some embodiments.

[0062] FIG.7F is a13C NMR spectra of 8-bromo-2-(1H-1,2,4-triazol-1-yl)-4H-chromen-4-one (6) in accordance with some embodiments.

[0063] FIG. 7G is a1H NMR spectra of 8-bromo-2-morpholino-4H-chromen-4-one, (8) in accordance with some embodiments.

[0064] FIG. 7H is a13C NMR spectra of 8-bromo-2-morpholino-4H-chromen-4-one, (8) in accordance with some embodiments.

[0065] FIG. 7I is a1H NMR spectra of 6-bromo-2-iododibenzo[b,d]thiophene, (12) in accordance with some embodiments.

[0066] FIG. 7J is a13C NMR spectra of 6-bromo-2-iododibenzo[b,d]thiophene, (12) in accordance with some embodiments.

[0067] FIG. 7K is a1H NMR spectra of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-(2- (pyrrolidin-1-yl)ethyl)-1H-pyrazole, (14b) in accordance with some embodiments.

[0068] FIG. 7L is a13C NMR spectra of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-(2- (pyrrolidin-1-yl)ethyl)-1H-pyrazole, (14b) in accordance with some embodiments.

[0069] FIG.7M is a1H NMR spectra of 8-(8-(4-methylpiperazin-1-yl)dibenzo[b,d]thiophen-4- yl)-2-morpholino-4H-chromen-4-one, DA-143, (Compound 10) in accordance with some embodiments. 13 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0070] FIG. 7N is a13C NMR spectra of 8-(8-(4-methylpiperazin-1-yl)dibenzo[b,d]thiophen- 4-yl)-2-morpholino-4H-chromen-4-one, DA-143, (Compound 10) in accordance with some embodiments.

[0071] FIG. 7O is a1H NMR spectra of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-methyl-1H- pyrazole (14a) in accordance with some embodiments.

[0072] FIG. 7P is a13C NMR spectra of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-methyl-1H- pyrazole (14a) in accordance with some embodiments.

[0073] FIG.7Q is a1H NMR spectra of 8-(8-(1-methyl-1H-pyrazol-4-yl)dibenzo[b,d]thiophen- 4-yl)-2-morpholino-4H-chromen-4-one, DA-138, (Compound 1) in accordance with some embodiments.

[0074] FIG. 7R is a13C NMR spectra of 8-(8-(1-methyl-1H-pyrazol-4- yl)dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one, DA-138, (Compound 1) in accordance with some embodiments.

[0075] FIG. 7S is a1H NMR spectra of 1-(6-bromodibenzo[b,d]thiophen-2-yl)-4- methylpiperazine, (14c) incordance with some embodiments.

[0076] FIG. 7T is a13C NMR spectra of 1-(6-bromodibenzo[b,d]thiophen-2-yl)-4- methylpiperazine, (14c) in accordance with some embodiments.

[0077] FIG. 7U is a1H NMR spectra of 2-morpholino-8-(8-(1-(2-(pyrrolidin-1-yl)ethyl)-1H- pyrazol-4-yl)dibenzo[d]thiophen-4-yl)-4H-chromen-4-one, DA-147, (Compound 13) in accordance with some embodiments.

[0078] FIG. 7V is a13C NMR spectra of 2-morpholino-8-(8-(1-(2-(pyrrolidin-1-yl)ethyl)-1H- pyrazol-4-yl)dibenzo[b,d]thiophen-4-yl)-4H-chromen-4-one, DA-147, (Compound 13) in accordance with some embodiments. DETAILED DESCRIPTION

[0079] The present disclosure is directed to a compound. The compound may be a DNA PKcs inhibitor. The disclosed DNA PKcs inhibitors may improve upon clinical shortcomings with current agents. Specifically, the disclosed DNA PKcs inhibitors may have improved solubility profiles as well as higher potency when blocking pro-inflammatory signaling. The disclosed DNA PKcs inhibitors may be more effective at reducing T cell proliferation and cytokine production 14 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) at the same concentration of M3814 and NU7741, two clinical DNA PKcs inhibitors. The disclosed agents may be at least twice as soluble as NU7741. NU7741

[0080] In some aspects, the present disclosure may improve upon clinical limitation of DNA PKcs inhibitors. In other aspects, the present disclosure is directed to DNA PKcs inhibitor scaffolds that may maximize solubility of the agent as well as binding energies to the kinase. The DNA PKcs inhibitor NU7441 has poor solubility. By analyzing a cocrystal structure of NU7441 with DNA PKcs, a solvent accessible region may be identified.

[0081] In some aspects, a synthetic protocol may be designed to access the solvent accessible region to generate the disclosed scaffold, which improves solubility and binding energies compared to NU7441. In in vitro testing, the agent potently may reduce T cell proliferation and cytokine production, which may be the main drivers of acute transplant rejection. At the same concentration, the disclosed scaffold may have a more pronounced reduction in T cell proliferation and cytokine production compared to M3814 and NU7741, two clinical DNA PKcs inhibitors. This suggests that the disclosed scaffold may be more effective as an anti-rejection therapy. 15 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) (I) Compositions

[0082] One aspect of the present disclosure encompasses a compound, or a pharmaceutically acceptable salt thereof, according to Formula (I):Formula (I).

[0083] In at least one example, R1may be hydrogen. In at least one example, R1may be C1-C6alkyl. In at least one example, R1 may be substituted C1-C6 alkyl.

[0084] In at least one example, R2 may be hydrogen. In at least one example, R2 may be C1-C6 alkyl. In at least one example, R2may be substituted C1-C6alkyl.

[0085] In at least one example, R4 may be hydrogen. In at least one example, R4 may be C1-C6 alkyl. In at least one example, R4 may be substituted C1-C6 alkyl. In at least one example, R3 may be an azole or a substituted azole.

[0086] In some embodiments of Formula (I), the azole or substituted azole may be pyrazole or substituted pyrazole, and with Formula (II): 16 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Formu a (II).

[0087] In at least one example, R1may be hydrogen. In at least one example, R2, may be. In at least one example, R4 may be hydrogen.

[0088] In at least one example, R5 may be hydrogen. In at least one example, R5 may be C1-C6 alkyl. In at least one example, R5may be substituted C1-C6alkyl.

[0089] In some embodiment R5 may be C1-C6 alkyl. The C1-C6 alkyl may be methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0090] In some embodiments, R5may be substituted C1-C6alkyl. The substituted C1-C6alkyl may be substituted with one or more halogens, amine, substituted amine, C3-C6cycloalkyl, substituted C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, or substituted C3-C6 heterocycloalkyl. The substituted amine may be substituted with one or more C1-C6alkyl. For example, the substiuted amine may be substituted with one or more methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0091] In some embodiments of Formula (II), R5 may be substituted ethyl, and with Formula (III): 17 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)o u a .

[0092] In at least one example, R6may be amine. In at least one example, R6may be substituted amine. In at least one example, R6 may be C3-C6 heterocycloalkyl. In at least one example, R6 may be substituted C3-C6 heterocycloalkyl.

[0093] In some embodiments, R6may be substituted amine. The substituted amine may be substituted with one or more C1-C6 alkyl. For example, the substiuted amine may be substituted with one or more methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0094] In some embodiments, R6may be substituted C3-C6heterocycloalkyl. The substituted C3-C6heterocycloalkyl may be substituted piperidine.

[0095] In some embodiments, the substituted piperidine may be substituted with one or more halogens.

[0096] In some embodiments, the halogen may be fluorine, chlorine, or bromine.

[0097] In some embodiments, the C3-C6 heterocycloalkyl may be aziridine, azetidine, pyrrolidine, piperidine, piperazine, substituted aziridine, substituted azetidine, substituted pyrrolidine, substituted piperidine, or substituted piperazine.

[0098] In some embodiments, the C3-C6 heterocycloalkyl may be substituted piperazine.

[0099] In some embodiments, the substituted piperazine may be substituted with a C1-C6 alkyl or one or more halogens.

[0100] In some embodiments, the C3-C6 heterocycloalkyl may be substituted piperidine.

[0101] In some embodiments, the substituted piperidine may be substituted with one or more halogens.

[0102] In some embodiments, the halogen may be fluorine, chlorine, or bromine. 18 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0103] In exemplary embodiments, a compound of the disclosure comprises Formula (I) or Formula (II) as shown below:Compound 3; 19 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 6; 20 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 12; and 22 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)p .

[0104] In some embodiments, the compound may have a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.2. For example, the solubility limit may be about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, or about 50 μM in phosphate buffer saline at pH 7.2.

[0105] In some embodiments, the compound has a solubility limit of greater than 14 μg / mL in phosphate buffer saline at pH 7.2. For example, the solubility limit may be about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL¸ about 18 μg / mL, about 19 μg / mL, about 20 μg / mL¸ about 21 μg / mL, about 22 μg / mL, about 23 μg / mL, about 24 μg / mL, about 25 μg / mL, about 26 μg / mL, about 27 μg / mL¸ about 28 μg / mL, about 29 μg / mL, about 20 μg / mL¸ about 31 μg / mL, about 32 μg / mL, about 33 μg / mL, about 34 μg / mL, about 35 μg / mL, about 36 μg / mL, about 37 μg / mL¸ about 38 μg / mL, about 39 μg / mL, about 40 μg / mL¸ about 41 μg / mL, about 42 μg / mL, about 43 μg / mL, about 44 μg / mL, about 45 μg / mL, , about 46 μg / mL, about 47 μg / mL¸ about 48 μg / mL, about 49 μg / mL, or about 50 μg / mL in phosphate buffer saline at pH 7.2.

[0106] The present disclosure is directed to a composition comprising a DNA-PKcs inhibitor with a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.2. For example, the solubility limit may be about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about 31 μM, about 32 μM, about 33 μM, about 34 μM, about 35 μM, about 36 μM, about 37 μM, about 38 μM, about 39 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM, about 44 23 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about 49 μM, or about 50 μM in phosphate buffer saline at pH 7.2. (II) Pharmaceutical Compositions

[0107] The present disclosure also provides pharmaceutical compositions. The pharmaceutical composition comprises a compound of Formula (I), as an active ingredient, and at least one pharmaceutically acceptable excipient.

[0108] The pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, taste-masking agent, a flavoring agent, or a coloring agent. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science. (i) Diluent

[0109] In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate. (ii) Binder

[0110] In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof. (iii) Filler 24 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0111] In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non- limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol. (iv) Buffering Agent

[0112] In still another embodiment, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline). (v) pH Modifier

[0113] In various embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid. (vi) Disintegrant

[0114] In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid. (vii) Dispersant

[0115] In yet another embodiment, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose. (viii) Excipient 25 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0116] In another alternate embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol. (ix) Lubricant

[0117] In a further embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate, or stearic acid. (x) Taste-Masking Agent

[0118] In yet another embodiment, the excipient may be a taste-masking agent. Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers; cellulose acetate phthalate; and combinations thereof (xi) Flavoring Agent

[0119] In an alternate embodiment, the excipient may be a flavoring agent. Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof. (xii) Coloring Agent

[0120] In a further embodiment, the excipient may be a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).

[0121] The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition. (III) Administration 26 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0122] In certain aspects, a therapeutically effective amount of a composition of the invention may be administered to a subject. Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system. Peripheral administration includes but is not limited to oral, inhalation, intravenous, intraperitoneal, intra-articular, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including directly into the central nervous system (CNS) includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation. The route of administration may be dictated by the disease or condition to be treated. For example, if the disease or condition is COPD or IPF, the composition may be administered via inhalation. Alternatively, is the disease or condition is osteoarthritis, the composition may be administered via intra-articular invention. It is within the skill of one in the art, to determine the route of administration based on the disease or condition to be treated. In a specific embodiment, a composition of the invention is administered orally.

[0123] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents, and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners.

[0124] For therapeutic applications, a therapeutically effective amount of a composition of the invention is administered to a subject. A “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response (e.g., cell death of senescent cells, an anti-aging response, an improvement in symptoms associated with a degenerative disease, or an improvement in symptoms associated with a function-decreasing disorder). Actual dosage levels of active ingredients in a therapeutic composition of the invention can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, age, the age-related disease or 27 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) condition, the degenerative disease, the function-decreasing disorder, the symptoms, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.

[0125] The frequency of dosing may be daily or once, twice, three times, or more per week or per month, as needed as to effectively treat the symptoms. The timing of administration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic. Duration of treatment could range from a single dose administered on a one- time basis to a life-long course of therapeutic treatments.

[0126] Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.

[0127] A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In still another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human.

[0128] The human subject may be of any age. However, since senescent cells are normally associated with aging, a human subject may be an older human subject. In some embodiments, the human subject may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 years of age or older. In some preferred embodiments, the human subject is 30 years of age or older. In other preferred embodiments, the human subject is 40 years of age or older. In other preferred embodiments, the human subject is 45 years of age or older. In yet other preferred embodiments, the human subject is 50 years of age or older. In still other preferred embodiments, the human 28 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) subject is 55 years of age or older. In other preferred embodiments, the human subject is 60 years of age or older. In yet other preferred embodiments, the human subject is 65 years of age or older. In still other preferred embodiments, the human subject is 70 years of age or older. In other preferred embodiments, the human subject is 75 years of age or older. In still other preferred embodiments, the human subject is 80 years of age or older. In yet other preferred embodiments, the human subject is 85 years of age or older. In still other preferred embodiments, the human subject is 90 years of age or older. (IV) Method of use

[0129] The present disclosure is directed to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I):Formula (I).

[0130] In at least one example, R1may be hydrogen.

[0131] In at least one example, R2may be hydrogen.

[0132] In at least one example, R4 may be hydrogen. In at least one example, R4 may be C1-C6 alkyl. In at least one example, R4 may be substituted C1-C6 alkyl.

[0133] In at least one example, R3may be an azole or a substituted azole. 29 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0134] The present disclosure is directed to a method of inhibiting DNA-PKcs kinase activity in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I):Formula (I).

[0135] In at least one example, R1may be hydrogen. In at least one example, R1may be C1-C6alkyl. In at least one example, R1 may be substitued C1-C6 alkyl.

[0136] In at least one example, R2 may be hydrogen. In at least one example, R2 may be C1-C6 alkyl. In at least one example, R2may be substitued C1-C6alkyl.

[0137] In at least one example, R4 may be hydrogen. In at least one example, R4 may be C1-C6 alkyl. In at least one example, R4 may be substitued C1-C6 alkyl.

[0138] In at least one example, R3may be an azole or a substituted azole.

[0139] The present disclosure is directed to a method of treating a transplanted solid organ for anti-rejection therapy in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I): 30 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)ormu a ( ).

[0140] In at least one example, R1may be hydrogen. In at least one example, R1may be C1-C6alkyl. In at least one example, R1 may be substitued C1-C6 alkyl.

[0141] In at least one example, R2 may be hydrogen. In at least one example, R2 may be C1-C6 alkyl. In at least one example, R2may be substitued C1-C6alkyl.

[0142] In at least one example, R4 may be hydrogen. In at least one example, R4 may be C1-C6 alkyl. In at least one example, R4 may be substitued C1-C6 alkyl.

[0143] In at least one example, R3may be an azole or a substituted azole.

[0144] In some embodiments, the anti-rejection therapy is T-cell mediated. (V) Definitions

[0145] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0146] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75thEd. 1994. Additionally, general principles of organic chemistry are described in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic 31 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) Chemistry,” 5thEd., Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0147] The term “alkyl” as used herein alone or as part of a group refers to saturated monovalent hydrocarbon radicals having straight or branched hydrocarbon chains or, in the event that at least 3 carbon atoms are present, cyclic hydrocarbons or combinations thereof and contains 1 to 20 carbon atoms (C1-20alkyl), suitably 1 to 10 carbon atoms (C1-10alkyl), preferably 1 to 8 carbon atoms (C1-8 alkyl), more preferably 1 to 6 carbon atoms (C1-6 alkyl), and even more preferably 1 to 4 carbon atoms (C1-4alkyl). Examples of alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0148] The term “aryl” as used herein, alone or as part of a group, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, and includes monocyclic and polycyclic radicals, such as phenyl, biphenyl, naphthyl, and the like.

[0149] The term “cycloalkyl” as used herein, alone or in combination, means a saturated or partially saturated monocyclic, bicyclic or tricyclic alkyl radical wherein each cyclic moiety contains from about 3 to about 8 carbon atoms, more preferably from about 3 to about 6 carbon atoms. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0150] The term “C3-C6cycloalkyl” means a saturated cyclic alkyl radical wherein each cyclic moiety contains from about 3 to about 6 carbon atoms.

[0151] The term “azole” as used herein refers to five-membered heterocyclic compounds containing a nitrogen atom and at least one other non-carbon atom (i.e. nitrogen, sulfur, or oxygen) as part of the ring. Examples include but are not limited to imidazole, pyrazole, 1,2,3- triazole, tetrazole, pentazole, oxazole, isoxazole, 1,2,3-oxadiazole, oxadiazole, furazan, 1,3,4- oxadiazole, thiazole, isothiazole, thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, and 1,3,4- thiadiazole, and the like.

[0152] The term “pyrazole” as used herein refers to a 5-membered ring of three carbon atoms and two adjacent nitrogen atoms, which are in ortho-substitution.

[0153] The term “aziridine” as used herein refers to a heterocyclic cyclopropane ring containing one nitrogen atom. 32 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0154] The term “azetidine” as used herein refers to a saturated heterocyclic organic compound containing three carbon atoms and one nitrogen atom.

[0155] The term “pyrrolidine” as used herein refers to a cyclic amine whose five-membered ring contains four carbon atoms and one nitrogen atom.

[0156] The term “piperidine” as used herein refers to heterocyclic amine consisting of a six- membered ring containing five methylene bridges (–CH2–) and one amine bridge (–NH–).

[0157] The term “piperazine” as used herein refers to a compound that consists of a six- membered ring containing two opposing nitrogen atoms.

[0158] The term “substituted” as used herein means that one or more of the hydrogen atoms bonded to carbon atoms in the chain or ring have been replaced with other substituents. Suitable substituents include monovalent hydrocarbon groups including alkyl groups such as methyl groups and monovalent heterogeneous groups including alkoxy groups such as methoxy groups.

[0159] The term “unsubstituted” as used herein means that the carbon chain or ring contains no other substituents other than carbon and hydrogen.

[0160] The term “branched” as used herein means that the carbon chain is not simply a linear chain. “Unbranched” means that the carbon chain is a linear carbon chain.

[0161] The term “saturated” as used herein means that the carbon chain or ring does not contain any double or triple bonds. “Unsaturated” means that the carbon chain or ring contains at least one double bond. An unsaturated carbon chain or ring may include more than one double bond.

[0162] The term “hydrocarbon group” means a chain of 1 to 25 carbon atoms, suitably 1 to 12 carbon atoms, more suitably 1 to 10 carbon atoms, and most suitably 1 to 8 carbon atoms. Hydrocarbon groups may have a linear or branched chain structure. Suitably the hydrocarbon groups have one branch.

[0163] The term “carbocyclic group” means a saturated or unsaturated hydrocarbon ring. Carbocyclic groups are not aromatic. Carbocyclic groups are monocyclic or polycyclic. Polycyclic carbocyclic groups can be fused, spiro, or bridged ring systems. Monocyclic carbocyclic groups contain 4 to 10 carbon atoms, suitably 4 to 7 carbon atoms, and more suitably 5 to 6 carbon atoms in the ring. Bicyclic carbocyclic groups contain 8 to 12 carbon atoms, preferably 9 to 10 carbon atoms in the rings. 33 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0164] The term “heteroatom” means an atom other than carbon e.g., in the ring of a heterocyclic group or the chain of a heterogeneous group. Preferably, heteroatoms are selected from the group consisting of sulfur, phosphorous, nitrogen and oxygen atoms. Groups containing more than one heteroatom may contain different heteroatoms.

[0165] The term “heterocyclic group” means a saturated or unsaturated ring structure containing carbon atoms and 1 or more heteroatoms in the ring. Heterocyclic groups are not aromatic. Heterocyclic groups are monocyclic or polycyclic. Polycyclic heteroaromatic groups can be fused, spiro, or bridged ring systems. Monocyclic heterocyclic groups contain 4 to 10 member atoms (i.e., including both carbon atoms and at least 1 heteroatom), suitably 4 to 7, and more suitably 5 to 6 in the ring. Bicyclic heterocyclic groups contain 8 to 18 member atoms, suitably 9 or 10 in the rings.

[0166] The term “C3-C6heterocycloalkyl” means a stable 3-6 membered cyclic group having carbon atoms and 1 to 3 heteroatoms independently selected from S, N or O, wherein when two O atoms or one O atom and one S atom are present, the two O atoms or one O atom and one S atom are not bonded to each other, respectively

[0167] As various changes could be made in the above-described materials and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. EXAMPLES Abstract

[0168] DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a vital role in DNA damage repair and lymphocyte function, presenting a significant target for cancer and immune disease therapies. Current DNA-PKcs inhibitors are undergoing Phase I / II trials as adjuncts to radiotherapy and chemotherapy for cancer. Nevertheless, clinical utility is limited by suboptimal bioavailability. This study introduces DNA-PKcs inhibitors designed to enhance bioavailability. DA-143 (Compound 9) surpasses NU7441 in aqueous solubility as well as additional available inhibitors. Consistent with current inhibitors, DA-143 (Compound 9) inhibits DNA-PKcs kinase activity resulting in increased tumor cell sensitivity to DNA-damage inducing chemotherapy and 34 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) inhibition of human T cell function. DA-143 (Compound 9)'s improved solubility is critical for enhanced efficacy at reduced doses and facilitates more effective evaluation of DNA-PKcs inhibition in both preclinical and clinical development. Introduction

[0169] The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is crucial for genomic stability, orchestrating the repair of DNA double-strand breaks (DSBs) primarily through the non-homologous end-joining (NHEJ) pathway. Unlike homologous recombination (HR) which predominates during the S and G2 phases, NHEJ, facilitated by DNA-PKcs, repairs DSBs throughout the cell cycle. DNA-PKcs partners with the Ku70 / Ku80 heterodimer, forming the active DNA-PK holoenzyme at DSB sites. This complex initiates a series of reactions that, while sometimes error-prone, are essential for the reconnection of DNA ends, leveraging the exonuclease Artemis among other factors for end processing. As a kinase, DNA-PKcs phosphorylates several crucial proteins involved in DNA repair, cell cycle progression, and apoptosis, modulating the cell's response to DNA damage. Its key role in DNA repair pathways makes DNA-PKcs a compelling target for therapeutic intervention, and it is currently being investigated for its synergy with anticancer agents like doxorubicin. Inhibitors of DNA-PKcs enhance the efficacy of radiation and chemotherapy by targeting its central function in the NHEJ repair mechanism. Beyond DNA repair, DNA-PKcs is essential to the development of the adaptive immune system, particularly in the recombination processes of antibody and receptor genes, further underscoring its therapeutic potential in immune system-related pathologies. For example, mice lacking functional DNA-PKcs exhibit a SCID (severe combined immunodeficiency) phenotype. Recent studies have identified DNA-PKcs as a crucial element in T cell signaling and the cytoplasmic detection of foreign DNA. The role of DNA-PKcs in T cell activation is currently under investigation. Beyond its implications in cancer therapy, its utility in modulating the immune response is being investigated, especially in mature T cells. DNA- PKcs is vital for mature T cell activation; inhibitors of DNA-PKcs impede proper T cell response post-stimulation, highlighting its essential role in this process. Given the advancement of two DNA-PKcs inhibitors to phase I trials for safety assessment, there is promising potential for these inhibitors in treating immunological conditions. Particularly in transplantation, where therapeutic innovation has been limited, DNA-PKcs presents a new target for preventing tissue rejection but 35 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) more preclinical testing is required. There exists a significant problem with currently available inhibitors that greatly limits effective preclinical and clinical evaluation.

[0170] Belonging to the PI3K kinase family, DNA-PKcs is inadvertently affected by traditional PI3K inhibitors like Wortmannin and LY294002. However, specific inhibitors such as NU7026 were among the first developed to target DNA-PKcs, enhancing cellular sensitivity to anticancer agents. Among the notable inhibitors are ATP-competitive agents like NU7441, AZD7648, and M3814. NU7441, in particular, is a potent DNA-PKcs inhibitor with an IC50 of approximately 14 nM, demonstrating specificity and potential in augmenting anti-cancer therapeutic strategies. However, NU7441 as well as other DNA-PKcs inhibitors face significant challenges with water solubility, complicating their preparation for in vivo studies, especially in the larger animal models required for clinical assessments. To overcome this limitation, a new inhibitor, DA-143 (Compound 9), has been synthesized and assessed, which maintains structural similarity to NU7441 but unexpectedly exhibits improved solubility. An in-depth characterization of DA-143 (Compound 9) in vitro is provided, demonstrating its efficacy in cells undergoing DNA damage and its impact on activated T cells. Methods Chemicals synthesis of compounds

[0171] General Experimental Methods:

[0172] General Methods:1H NMR and13C NMR spectra were recorded on an Agilent 400 NMR spectrometer or NMR AscendTM500, Bruker. Deuterated chloroform and deuterated dimethyl sulfoxide were used as solvents, unless stated otherwise. The spectra were calibrated against the residual solvent peak or TMS. Chemical shifts (δ) and coupling constants (J) are given in ppm (parts per million) and Hz (Hertz). The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet.1H NMR and13C NMR spectra of the intermediates and final compounds are provided in FIGS.7A-7V. Purity of final compounds was assessed using a Thermo Vanquish UHPLC with ISQ EM mass spectrometer at a wavelength of 254 nm and confirmed >95%. All commercially available compounds were used without purification. UHPLC chromatograms of the intermediates and final compounds are provided in FIGS.6A-6K. Purity analysis details are listed in Table 1 below, unless stated otherwise: 36 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0173] Table 1 Flow rate 0.5 mL / min [0 e, (3):

[0175] 1.0 eqo - - o o- - y o yp e y e a - -o e a . eq of dimethyl formamide acetal (2) was dissolved in DMF and heated to 75 °C for an hour. The reaction mixture was then poured into ice-water. The precipitate was filtered and washed with water and hexanes. The precipitate was then used for next step. Yield = 100%. [M+H]+= 270.0

[0176] 1H NMR (500 MHz, CDCl3) δ 15.00 (s, 1H), 7.92 (dt, J = 11.8, 1.9 Hz, 1H), 7.65 (dd, J = 8.1, 2.3 Hz, 1H), 7.61 (dd, J = 7.8, 2.3 Hz, 1H), 6.71 (t, J = 3.9 Hz, 1H), 5.75 (d, J = 12.0 Hz, 1H), 3.21 (s, 3H), 2.99 (s, 3H).

[0177] 13C NMR (126 MHz, CDCl3) δ 190.44, 159.66, 155.43, 136.88, 127.40, 121.30, 118.55, 111.85, 89.57, 45.57, 37.51.

[0178] Synthesis of 8-bromo-4H-chromen-4-one, (4): 37 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0179] 1 mmol of 1-(3-bop-2-en-1-one (3) was dissolved in 8 mL of DCM, followed by addition of 0.8 mL of HCl, and refluxed for an hour. The solvent was then evaporated, and the resulting product was used for another step without any purification. Yield = 90%.

[0180] 1H NMR (500 MHz, CDCl3) δ 8.16 (dd, J = 8.0, 1.9 Hz, 1H), 7.95 (d, J = 5.8 Hz, 1H), 7.91 (dd, J = 8.0, 2.1 Hz, 1H), 7.29 (t, J = 8.1, 1H), 6.39 (d, J = 6.2 Hz, 1H).

[0181] 13C NMR (126 MHz, CDCl3) δ 176.90, 155.37, 153.11, 137.33, 126.17, 125.88, 125.23, 113.15, 111.79.

[0182] Synthesis of 8-bromo-2-(1H-1,2,4-triazol-1-yl)-4H-chromen-4-one, (6):

[0183] 1.0 eq of 8-romo- -c romen- -one ( ), . eq o tr azo e ( ), . eq of iodine, and 5 eq of K2CO3 was added to a vial followed by the addition of DMF. The crude mixture was heated for 80 °C for 17h. The crude mixture was cooled down to allow the precipitate to form. The precipitate was filtered, washed with saturated sodium thiosulfate solution and then with hexanes. Yield = 70%, [M+H]+= 291.9.

[0184] 1H NMR (500 MHz, CDCl3) δ 9.00 (s, 1H), 8.21 (s, 2H), 7.97 (d, J = 1.7 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 1.9 Hz, 1H).

[0185] 13C NMR (126 MHz, DMSO) δ 177.01, 154.63, 153.58, 151.01, 144.55, 138.35, 127.78, 125.23, 125.09, 111.42, 97.98.

[0186] Synthesis of 8-bromo-2-morpholino-4H-chromen-4-one, (8): 38 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0187] 1.0 eq2.0 eq of morpholine (7) and 2.0 eq of K2CO3were added to a vial followed by addition of DMF. The crude mixture was heated at 80 °C overnight. Following the completion of reaction, water and ethyl acetate was added and extracted into ethyl acetate. The organic layer was washed with brine, evaporated, and purified via flash chromatography. Yield = 80%, [M+H]+= 310.0.

[0188] 1H NMR (500 MHz, CDCl3) δ 8.10 (dd, J = 7.9, 1.6 Hz, 1H), 7.77 (dd, J = 7.8, 1.5 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 5.50 (s, 1H), 3.90 – 3.79 (m, 4H), 3.61 – 3.56 (m, 4H).

[0189] 13C NMR (126 MHz, CDCl3) δ 176.27, 162.26, 150.24, 135.61, 125.50, 124.95, 124.49, 110.17, 86.87, 65.97, 44.81.

[0190] Synthesis of 2-morpholino-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H- chromen-4-one, (10):

[0191] 1.0 eq of 8-bromo-2-morpholino-4H-chromen-4-one (8), 1.3 eq of 2 bis(pinacolato)diboron (9), and 3 eq of KOAc were added to a vial. Dioxane was added, and the vial was flushed with nitrogen for 10 minutes followed by addition of 0.05 eq of Pd(dppf)Cl2. The reaction mixture was heated at 90 °C for overnight and extracted into ethyl acetate and washed with brine. The organic layer was evaporated, and the compound was used for the next step without any purification. Yield: 100%. [M+H]+: 358.1

[0192] Synthesis of 6-bromo-2-iododibenzo[b,d]thiophene, (12): 39 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0193] 1 eq of 4-bromodibenzo[b,d]thiophene (11),. ne and 0.5 eq of iodobenzene diacetate was dissolved in 1:1 mixture of acetic acid and acetic anhydride. 1-2 drops of H2SO4 were added and the crude mixture was stirred at RT overnight. Following the completion of reaction, water and ethyl acetate were added to the crude mixture and extracted. The organic layer was washed with saturated sodium thiosulfate and brine. The organic layer was evaporated followed by purification to afford 6-bromo-2-iododibenzo[b,d]thiophene. Yield = 45%.

[0194] 1H NMR (400 MHz, dmso) δ 8.81 (s, 1H), 8.48 (dd, J = 8.0, 1.0 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.86 (dd, J = 8.4, 1.6 Hz, 1H), 7.79 (dd, J = 7.8, 0.9 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H).

[0195] 13C NMR (126 MHz, DMSO) δ 140.77, 138.25, 137.86, 136.35, 135.75, 131.90, 130.61, 127.32, 125.74, 122.25, 115.80, 91.22.

[0196] Synthesis of intermediates 14a, 14b, and 14c:

[0197] 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-methyl-1H-pyrazole, 14a:

[0198] 1.0 eq of 6-bromo-2-iododibenzo[b,d]thiophene (12), 1.3 eq of 1-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (13), and 3 eq of Na2CO3 were added to a vial. 5:1 DMF:H2O was added, and the vial was flushed with nitrogen for 10 minutes followed by addition of 0.05 eq of Pd(dppf)Cl2. The reaction mixture was heated at 80 °C overnight and 40 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) extracted into ethyl acetate and washed with brine. The organic layer was then evaporated, and the crude mixture was purified via flash chromatography. Yield: 32%. [M+H]+: 342.98

[0199] 1H NMR (500 MHz, CDCl3) δ 8.13 (d, J = 1.7 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.86 (s, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.63 – 7.53 (m, 2H), 7.33 (t, J = 7.8 Hz, 1H), 3.97 (s, 3H).

[0200] 13C NMR (126 MHz, CDCl3) δ 141.80, 136.97, 136.85, 136.69, 136.57, 129.64, 129.51, 127.07, 125.76, 125.30, 123.16, 123.00, 120.29, 118.71, 116.42, 39.20.

[0201] 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazole, 14b:

[0202]

[0203] 1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 1.7 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.85 (s, 1H), 7.75 (d, J = 7.9 Hz, 2H), 7.57 – 7.49 (m, 2H), 7.27 (t, J = 7.9 Hz, 1H), 4.31 (t, J = 7.0 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.61 – 2.51 (m, 4H), 1.83 – 1.73 (m, 4H).

[0204] 13C NMR (126 MHz, CDCl3) δ 141.73, 136.85, 136.83, 136.76, 136.71, 136.66, 136.58, 136.48, 129.67, 129.43, 126.59, 125.69, 125.25, 123.05, 122.65, 120.27, 118.61, 116.36, 55.98, 55.94, 54.35.

[0205] 1-(6-bromodibenzo[b,d]thiophen-2-yl)-4-methylpiperazine, 14c: 104772379PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0206] 1.0 eq of 6-bromo-2-iododibenzo[b,d]thiophene (12), 1.3 eq of 1-methylpiperazine (16), and 3 eq of t-BuOK were added to a vial. Toluene was added, and the vial was flushed with nitrogen for 10 minutes followed by addition of 0.05 eq of Pd(dppf)Cl2 and BINAP. The reaction mixture was heated at 80 °C overnight and extracted into ethyl acetate and washed with brine. The organic layer was then evaporated, and the crude mixture was purified via flash chromatography. Yield: 40%. [M+H]+: 361.03

[0207] 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.60 – 7.55 (m, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.18 (dd, J = 8.8, 2.4 Hz, 1H), 3.34 – 3.28 (m, 4H), 2.68 – 2.62 (m, 4H), 2.40 (s, 3H).

[0208] 13C NMR (126 MHz, CDCl3) δ 149.50, 142.24, 136.95, 136.91, 130.45, 129.19, 125.41, 123.14, 120.17, 118.45, 116.42, 108.64, 55.19, 49.99, 46.20.

[0209] Synthesis of final compounds DA-138 (Compound 1), DA-143 (Compound 10), and DA-147 (Compound 13):

[0210] 8-(8-(1-methyl-1H-pyrazol-4-yl)dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H- chromen-4-one, DA-138 (Compound 1)

[0211] 1.0 eq of 4-(6-bromodibenzo[b,d]thiophen-2-yl)-1-methyl-1H-pyrazole (14c), 1.3 eq of 2-morpholino-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one (10), and 3 eq of K2CO3 were added to a vial. Dioxane was added, and the vial was flushed with nitrogen for 10 minutes followed by addition of 0.05 eq of tetrakis. The reaction mixture was heated at 90 °C for 48 hours and extracted into ethyl acetate and washed with brine. The organic layer was then evaporated, and the crude mixture was purified via flash chromatography. Yield: 40%. [M+H]+: 494.2. 42 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0212] 1H NMR (500 MHz, CDCl3) δ 8.33 – 8.23 (m, 3H), 7.90 (s, 1H), 7.81 – 7.74 (m, 3H), 7.63 – 7.56 (m, 2H), 7.49 (t, J = 7.6 Hz, 2H), 5.51 (s, 1H), 4.01 (s, 3H), 3.54 – 3.48 (m, 4H), 3.09 (t, J = 4.9 Hz, 4H).

[0213] 13C NMR (126 MHz, CDCl3) δ 177.00, 162.09, 150.56, 140.38, 137.07, 136.83, 136.06, 135.88, 133.26, 131.40, 129.56, 128.47, 128.16, 127.10, 125.98, 125.22, 124.84, 124.68, 123.56, 123.03, 123.01, 121.37, 118.39, 86.87, 65.75, 44.49, 39.22.

[0214] 8-(8-(4-methylpiperazin-1-yl)dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H- chromen-4-one, DA-143 (Compound 10):

[0215] Prepared following procedure same as Compound 1 (DA-138).Yield: 32%, [M+H]+= 577.2

[0216] 1H NMR (500 MHz, CDCl3) δ 8.34 – 8.25 (m, 3H), 7.93 (s, 1H), 7.87 (s, 1H), 7.83 – 7.77 (m, 2H), 7.65 – 7.58 (m, 2H), 7.52 (ddd, J = 7.6, 4.5, 3.4 Hz, 2H), 5.53 (s, 1H), 4.38 (t, J = 6.9 Hz, 2H), 3.56 – 3.50 (m, 4H), 3.11 (t, J = 4.9 Hz, 4H), 3.06 (t, J = 6.9 Hz, 2H), 2.62 (m, 4H), 1.84 (p, J = 3.1 Hz, 4H).

[0217] 13C NMR (126 MHz, CDCl3) δ 177.00, 162.08, 150.56, 140.38, 137.05, 136.87, 136.05, 135.88, 133.25, 131.40, 129.58, 128.48, 128.15, 126.77, 125.98, 125.23, 124.83, 124.68, 123.56, 122.99, 122.76, 121.39, 118.41, 86.87, 65.75, 55.88, 54.35, 51.62, 44.49, 23.58.

[0218] 2-morpholino-8-(8-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazol-4- yl)dibenzo[b,d]thiophen-4-yl)-4H-chromen-4-one, DA-147 (Compound 13): 43 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0219] ++H] = 512.19

[0220] 1H NMR (500 MHz, CDCl3) δ 8.29 (dd, J = 7.9, 1.7 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.78 (dd, J = 7.4, 1.7 Hz, 1H), 7.72 – 7.64 (m, 2H), 7.56 (t, J = 7.6 Hz, 1H), 7.52 – 7.45 (m, 2H), 7.20 (dd, J = 8.8, 2.4 Hz, 1H), 5.51 (s, 1H), 3.52 (t, J = 4.9 Hz, 4H), 3.36 (t, J = 4.9 Hz, 4H), 3.10 (t, J = 4.9 Hz, 4H), 2.71 (t, J = 4.9 Hz, 4H), 2.44 (s, 3H).

[0221] 13C NMR (126 MHz, CDCl3) δ 177.06, 162.09, 150.58, 149.42, 140.89, 136.38, 136.11, 133.27, 131.38, 130.69, 128.62, 127.85, 125.89, 124.80, 124.33, 123.53, 123.01, 121.20, 118.41, 108.45, 86.86, 65.78, 55.17, 50.02, 46.11, 44.51. Solubility measurement

[0222] DA-143 (Compound 10) and NU7441 were tested for aqueous solubility in phosphate buffered saline, pH7.2 (PBS). 10 mM DMSO stock solutions of the compounds were used to prepare a 2-fold serial dilution of each in PBS at a concentration range 100 μM – 0.1 μM. The absorbance was then measured at 600 nM to determine the solubility limit. DMSO percentage did not exceed 1% in the serial dilutions. Cell lines and culture

[0223] Jurkat E6.1 human T cell leukemia and MC38 murine colon cancer cell lines were obtained from ATCC (American Type Culture Collection). Mice used in the study were housed in the Arkansas Children’s Research Institute Animal Facility. All animal studies were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences / Arkansas Children’s Research Institute. Study is reported in accordance with The ARRIVE (Animal Research: 44 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) Reporting of in vivo Experiments) guidelines. Mouse T cells were isolated from the spleens of C56BL / 6 mice using StemCell EasySep Mouse CD8+ T cell isolation kit #19853A and stimulated using 5 µg / ml plate-bound anti-CD3 (Biolegend #100340) and 5 µg / ml of anti-CD28 (Biolegend #102116). Human PBMCs were received frozen from a healthy donors (StemCell #70025) and stimulated with plate-bound anti-CD3 (Biolegend #317347) and anti-CD28 (Biolegend #302934) both at 5 µg / ml. T cells and PBMCs were cultured in RPMI with 10% FBS and 1X Penstrep (Corning #30-002-CL). MC38 cells were cultured in DMEM with 10% FBS and Penstrep. Inhibitors

[0224] NU7441 and DA-143 (Compound 10) were dissolved and stored frozen in DMSO at 5 mM, -80oC. DA-143 (Compound 10) was synthesized in-house as described. NU7441 was purchased from Selleckchem. Assays

[0225] In vitro DNA-PK kinase assay. The DNA-PK Kinase Enzyme System (Cat #V4106) and ADP-Glo Assay (Cat #V6930) from Promega were used to perform this assay. Inhibitors were used at 100 nM concentration and equivalent amounts of DMSO were added to samples without inhibitor.

[0226] Western blotting. Samples for Western blots of MC38 cells were pre-treated for 1 hour with inhibitor, then treated with Doxorubicin for the indicated time. Samples were lysed in Cell Extraction Buffer (Thermo FNN0011) with protease inhibitors (Thermo Scientific #1860932) and phosphatase inhibitors (Thermo Scientific #78428) followed by sonication in a QSonica Q800R3 with the settings 30% amplitude, 30s on / off, and 15 min sonication time. Jurkat Western blot samples for FIG. 4A were pre-treated with inhibitors, stimulated with 5 µg / ml anti-human CD3 and anti-human CD28 for 15 minutes, and lysed in 0.1% NP40 in water with protease and phosphatase inhibitors. Jurkat Western blot samples for FIG.4B were pre-treated with inhibitors, stimulated with 1 µg / ml PHA (phytohaemagglutinin) and 50 ng / ml PMA (phorbol 12-myristate 13-acetate), and lysed similarly to the MC38 cells. Lysed samples were pelleted at 10,000 xg for 10 minutes, and supernatants were normalized by protein concentration determined using the bicinchoninic assay (BCA) (Thermo Scientific #23225). Samples were heated in LDS (lithium dodecyl sulfate) loading buffer (Thermo Scientific #B0007) then loaded into 4–12% bis-tris gels 45 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) (Thermo Scientific #NW04122BOX). Transfer to a PVDF membrane was completed using a Pierce Power Blotter system run at 25 V for 10 minutes.

[0227] Primary antibodies. Anti-DNA-PKcs (Cell Signaling #38168), Anti-phospho-DNA- PKcs ser2056 (Invitrogen #PA5-78130), Anti-AKT (Cell Signaling #2938S), Anti-phospho-AKT ser473 (Cell Signaling #4058S), Anti-Kap1 (abcam #ab10483), Anti-phospho-Kap1 ser824 (Abcam #ab70369), Anti-Beta-actin (Thermo Scientific #MA1-140) Anti-GAPDH (Thermo Scientific #MA5-15738), anti-H2A (Cell signaling #12349S) and anti-pH2AX (Cell signaling #25775). Secondary antibodies are as follows: Thermo Scientific goat anti-mouse IgG (H+L) Alexa Fluor Plus 647 (#A32728) and GE Healthcare donkey anti-rabbit HRP (#NA934V). Imaging was done with a GE ImageQuant LAS4000.

[0228] Apoptosis assay. Trypsinized MC38 cells were plated at 10,000 cells / well in a 96 well plate. The next day, 1000X stocks of each inhibitor was made in DMSO such that each well would receive the same amount of DMSO. Aliquots of each drug at assay concentration were then made in culture media. The media from the plated cells was removed by vacuum and the indicated inhibitor concentration in 100 µl of media was added to each well. Cells were pre- treated this way for 1 hour before adding another 100 µl of media containing inhibitor and a 2X concentration of doxorubicin (from a 1 mM frozen stock). Cells grew at 37C with 5% CO2 for 48 hours. Cells were harvested by trypsinization and transferred to a 96 well V-bottom plate, and an eBiosciences Annexin V efluor 450 Apoptosis detection kit (#88-8006-74) was used to stain the cells. Flow cytometry was performed on an Attune NxT flow cytometer. The gating strategy used for flow cytometry analysis is as follows: Doublet discrimination was used based on forward scatter area and height profiles to gate on single cells.

[0229] Proliferation assay. After isolation or thawing, T cells were counted by flow cytometry to measure total CD8+ (mouse) or CD3+ (human) cells within the total population. The cells were stained with CellTrace Violet (Thermo Scientific #C34557) according to the kit. Cells were then plated at 1 million cells / ml and stimulated with anti-CD3 and anti-CD28 antibodies after being pre-treated with inhibitor. They were allowed to grow in culture for the indicated times at 37°C / 5% CO2. Analysis was done by flow cytometry on an Attune NxT flow cytometer. Results Design of new DNA-PKcs inhibitors 46 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0230] Due to poor aqueous solubility of the DNA-PKcs inhibitor NU7441, a new DNA-PKcs inhibitor featuring enhanced water solubility was developed. The intention behind developing such an inhibitor with improved solubility characteristics is to streamline drug administration through various routes, including intravenous, while also enhancing potency by reducing desolvation penalties. This advancement may enhance the feasibility of studying DNA-PKcs across diverse animal models and may also simplify the adaptation of DNA-PKcs inhibitors in a clinical setting.

[0231] A co-crystal structure of NU7441 bound to DNA-PKcs was employed to identify critical binding regions and areas suitable for modification to enhance aqueous solubility. Analysis of the co-crystal structure revealed that the dibenzothiophene segment of NU7441 occupies a solvent-exposed pocket that is non-essential for binding, suggesting this region could be altered to enhance solubility (FIG.1A). The morpholine segment in NU7441 establishes a crucial hydrogen bond with the DNA-PKcs hinge, and any modifications at this site would jeopardize binding interactions. Consequently, water-soluble groups were incorporated into the dibenzothiophene motif by introducing a pyrazole (DA-138 (Compound 1)) and subsequently linking the pyrazole to pyrrolidine (DA-143 (Compound 10)) (FIGs.1B- D). Pyrrolidine, characterized by an ionizable functional group with a pKa range of 9-11, is readily ionizable at physiological pH, thereby offering a substantial potential to enhance aqueous solubility. Additionally, another derivative of NU7441 was explored, incorporating an N-methylpiperazine at the solvent-exposed region (DA-147 (Compound 13)). However, this particular derivative was not given priority due to predictions indicating a suboptimal pharmacokinetic profile, attributed to the rapid N-dealkylation of the methyl group.

[0232] The DNA-PKcs inhibitors were synthesized as described above. Biochemical Evaluation of New DNA-PKcs Inhibitors

[0233] From the synthesis outlined, three candidate DNA-PKcs inhibitors were generated: DA-138 (Compound 1), DA-143 (Compound 10), and DA-147 (Compound 13) (FIG.2A). To assess their capability to inhibit DNA-PKcs kinase activity, a comparative analysis with known DNA-PKcs inhibitors NU7441 and M3814 was completed. An in vitro phosphorylation assay (FIG.2B) was employed, which indirectly measured kinase activity by monitoring ATP to ADP conversion. Treatment with NU7441, M3814, DA-143 (Compound 10), and DA-147 47 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) (Compound 13) led to an almost complete loss of detectable kinase activity compared to the vehicle-treated sample. NU7441, DA-147 (Compound 13), and DA-143 (Compound 10) demonstrated statistically comparable inhibitory effects in relation to each other. DA-138 (Compound 1) exhibited inhibition of DNA-PKcs, but to a much lesser amount. Solubility Studies of DA-143 (Compound 9)

[0234] Having confirmed that the new inhibitors exhibited a comparable impact on DNA- PKcs kinase activity to inhibitors in clinical studies, the next objective was to assess their solubility in comparison to NU7441. Despite both DA-143 (Compound 10) and DA-147 (Compound 13) exhibiting similar activity, DA-143 (Compound 10) was advanced with due to considerations of metabolic stability. The N-methyl group in the solvent front region of DA- 147 (Compound 13) is susceptible to N-dealkylation through CYP enzymes. In contrast, the presence of steric bulk around the pyrrolidine nitrogen in DA-143 (Compound 10) is expected to impede N-dealkylation, making it a more favorable choice from a metabolic stability standpoint.

[0235] A comparative solubility analysis between the DNA-PKcs inhibitor NU7441 and DA- 143 (Compound 10) was conducted. The key distinction between the two inhibitors is the 1-(2- (pyrrolidine-1-yl)ethyl)-1H-pyrazole motif at the solvent front region of DA-143 (Compound 10), which is hypothesized to enhance solubility due to the ionizable nature of the pyrrolidine ring at physiological pH. Stock solutions of both inhibitors in DMSO and N-methyl pyrrolidine (NMP) were prepared. The maximum concentration of NU7441 achievable without precipitation in dimethyl sulfoxide (DMSO) and N-methyl-2-pyrrolidone (NMP) was 16.5 mg / mL (40 mM) and 42 mg / mL (100 mM), respectively. Beyond these concentrations, NU7441 was insoluble despite sonication or heating. In contrast, DA-143 (Compound 10) exhibited solubility at 200 mg / mL (~350 mM) in both solvents. This indicated that DA-143 (Compound 10) possesses approximately 9 times better solubility in DMSO and 4 times better solubility in NMP compared to NU7441.

[0236] After determining that DA-143 (Compound 10) exhibited improved solubility in both DMSO and NMP, the solubility of both compounds in an intravenous formulation containing PEG / NMP / EtOH / H2O at 50 / 12 / 10 / 28 v / v that has been successfully utilized in large animals was investigated. Stock solutions of both inhibitors in NMP were prepared and then added the 48 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) stock into PEG / EtOH / H2O to achieve a final concentration of 10 mg / mL. It is noteworthy that NU7441 exhibited immediate precipitation upon the addition of the NMP stock into the formulation, while DA-143 (Compound 10) remained soluble within the formulation. This showcases the superior solubility of DA-143 (Compound 10) compared to NU7441 and raises concerns about the potential formation of hazardous embolisms when NU7441 is administered intravenously. Emphasizing the importance of considering solubility characteristics, especially for intravenous use, this observation underscores the enhanced solubility of DA-143 (Compound 10) in the tested formulation suggesting its suitability for intravenous use, which will maximize applications in both pre-clinical and clinical settings.

[0237] Subsequently, the aqueous solubility of both DA-143 (Compound 10) and NU7441 was determined. Serially diluted stock solutions of both inhibitors were prepared and added to phosphate-buffered saline (PBS, pH = 7.2) at concentrations ranging from 0.1 μM to 10 μM. The results indicate that DA-143 (Compound 10) exhibits increased aqueous solubility (approximately 5 times) compared to NU7441 (FIG.2c and Table 2). Thus, the introduction of an ionizable group (i.e., 1-(2-(pyrrolidine-1-yl)ethyl)-1H-pyrazole) proved effective in enhancing the solubility of DA-143 (Compound 10) in comparison to NU7441. Table 2. Compound MW Lo Solubilit limit Solubilit limit- ( ompoun ) sens t zes cancer ce s to oxoru c n

[0238] DNA-PKcs inhibitors increase the sensitivity of cancer cells to DNA-damaging chemotherapies by preventing the ability of cancer cells to rapidly repair DNA damage. Therefore, to confirm that DA-143 (Compound 10) maintained the same affects as its parent 49 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) compound, the ability of DA-143 (Compound 10) to increase MC38 cancer cell chemosensitivity to doxorubicin was tested by measuring the level of apoptosis following treatment with the DNA DSB-inducing chemotherapy. As a popular anti-cancer therapy, doxorubicin administration in combination with DNA-PKcs inhibitors has been explored as a combination therapy to enhance cancer treatment regimens. As expected, MC38 cancer cells were largely resistant to doxorubicin treatment as evident by a low level of annexin V + cells following 48 hours of treatment. When MC38 cells were treated with either inhibitor alone at 5 µM concentration, very little differences in annexin V staining were observed compared to vehicle treated controls (No Treatment, 0 µM inhibitor) suggesting that the inhibitors alone did not induce apoptosis at 48 hours. However, combining doxorubicin treatment (25 nM) with NU7441 or DA-143 (Compound 10) (5 µM) resulted in a 2.5-fold increase in annexin V staining (>50% of cells), suggesting an increased vulnerability to DNA damage as would be expected in the absence DNA-PKcs activity (FIG.3A). Consistent with these findings, DA-143 (Compound 10) elicited an increase in phosphorylation of s139 of the histone variant H2AX after 24 hours of doxorubicin exposure, suggesting that the inhibitor efficiently blocks DNA- PKcs activity and therefore the ability to repair double-strand breaks (FIG.3B). This phosphorylation event serves as a marker for DNA damage induction and subsequent repair. These results provide evidence that DA-143 (Compound 10) is an efficient inhibitor of DNA- PKcs and its activity in DNA damage repair. DA-143 (Compound 10) blocks phosphorylation of DNA-PKcs substrates

[0239] The most direct method of determining if the new inhibitor is effective in a cellular context is to compare phosphorylation levels of known DNA-PKcs substrates. Activation of DNA-PKcs occurs through autophosphorylation at serine 2056. To validate that DA-143 (Compound 10) blocks the activation and kinase activity of DNA-PKcs in cells, phosphorylation of s2056 in Jurkat T cells following activation through the T cell receptor was analyzed. A stark increase in s2056 phosphorylation after about 15 minutes of activation was observed. However, cells treated with either inhibitor almost completely inhibited phosphorylation of serine 2056 indicating it is capable of inhibiting the activation and kinase activity of DNA-PKcs (FIG.4A). Phosphorylation of Kap1 / Trim28 was also observed. Kap1 phosphorylation at serine 824 is reported to be dependent on DNA-PKcs. Kap1 is a transcriptional mediator known to be involved in DNA damage responses as well as control the 50 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) expression of genes involved in T cell receptor signaling. A significant drop in the level of phosphorylation at serine 824 with NU7441 and DA-143 (Compound 10) treatment following T cell activation was observed. A third phosphorylation event observed was that of serine 473 on the kinase AKT. This is thought to be a target of multiple kinases, including DNA-PKcs. Treatment with DA-143 (Compound 10) also caused a drop in levels of AKT phosphorylation after stimulation in Jurkat cells (FIG.4B). These observations both provide evidence of DA- 143 (Compound 10) inhibitor activity toward DNA-PKcs as well as contribute to the unfolding narrative that DNA-PKcs has a role to play in early T cell activation. DA-143 (Compound 9) alters T cell function

[0240] DNA-PKcs is critical for the function of primary T cells. The DNA-PKcs inhibitors NU7441, M3814 and AZD7648 inhibited the proliferation and activity of both CD4+and CD8+T cells. Here, it was determined if DA-143 (Compound 10) had similar effects on primary T cell function. Both human PBMCs and isolated mouse CD8+T cells were activated with anti- CD3 / anti-CD28 antibodies and tracked proliferation using CellTrace Violet dye. While activated vehicle-treated (DMSO) T cells proliferated aptly, cells treated with NU7441 or DA- 143 (Compound 10) had a significant disruption in proliferation. At a concentration of 5 µM DA-143 (Compound 10), T cells were unable to divide for more than 1 generation (both human and mouse). The same concentration of NU7441 also inhibited proliferation, but to a lesser extent than DA-143 (Compound 10) (FIGS.5A-B).

[0241] The cytokine Interleukin-2 (IL2) produced by T cells is vital for the expansion and activity of numerous immune cell populations. The previous studies indicate that DNA-PKcs is critical for the proper expression of IL2 following T cell activation as inhibitors NU7441, M3814 and AZD7648 significantly reduced IL2 production in T cells. DA-143 (Compound 10) had similar effects reducing IL2 production in primary human T cells as well as mouse CD4+T cells following activation with anti-CD3 / anti-CD28 antibodies. As observed with proliferation, DA-143 (Compound 10) at lower concentrations had a stronger inhibitory effect on IL2 production than NU7441. These results further confirm the necessary role of DNA-PKcs in T cell activity and highlight the potential use of this new inhibitor in the treatment of T cell mediated diseases and disorders. (FIGS.5C-D). Discussion 51 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)

[0242] As the role of DNA-PKcs extends beyond DNA repair, encompassing functions in specific cell types like neurons and immune cells, the application of DNA-PKcs inhibitors for a broad spectrum of diseases is garnering significant interest. The exploration of these inhibitors' clinical utility is often hampered by their poor aqueous solubility. The DNA-PKcs inhibitor DA- 143 (Compound 10) was developed to enhance water solubility. Beginning with the structure of NU7441, an ionizable group to create a new compound that retains the inhibitory capabilities of NU7441 yet demonstrates a five-fold increase in aqueous solubility was incorporated. This property is crucial for intravenous administration, affecting the maximum concentration at which a drug can be administered without the risk of precipitation in aqueous, minimally organic solvent-containing therapeutic formulations.

[0243] In the comparative studies, DA-143 (Compound 10) matched NU7441 in inhibitory efficacy in kinase assays and exhibited significant effectiveness in cell culture models. The impact of DA-143 (Compound 10) on the DNA repair function of DNA-PKcs was assessed by examining apoptosis and DNA damage markers post-doxorubicin treatment. The results showed that DA-143 (Compound 10) enhances the sensitivity of cancer cells to doxorubicin, indicated by increased apoptosis rates, likely due to persistent DNA damage as suggested by elevated H2AX phosphorylation levels. Moreover, DA-143 (Compound 10) also demonstrated an ability to inhibit T cell activity, reducing proliferation and IL2 cytokine production. These findings position DA-143 (Compound 10) as a promising candidate for further preclinical and clinical evaluations, offering insights into DNA-PKcs inhibitors' roles not only in cancer therapy but also in managing T cell-mediated conditions, including autoimmune disorders and transplant rejection. Inhibitors

[0244] NU7441 and DA-143 (Compound 10) were dissolved and stored frozen in DMSO at 5 mM, -80oC. DA-143 (Compound 10) was synthesized in-house as described. NU7441 was purchased from Selleckchem. Computational Modeling

[0245] Computational modeling studies were performed to determine the binding pose of compounds in the DNA-PKcs kinase. The co-crystal structure between NU7441 and DNA-PKcs was utilized for modeling studies (PDB ID: 7OTM). NU7441 was removed from the active site, 52 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) and DA-138 (Compound 1) or DA-143 (Compound 10) was then docked into the kinase. Briefly, all hydrogens in the protein were added as polar only and the ATP binding site was selected. The compound was then docked using AutoDock Vina. The modeling results were visualized in Discovery Studio 2021 Client. 53 104772379.1

Claims

PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) CLAIMS What is claimed is:

1. A compound, or a pharmaceutically acceptable salt thereof, according to Formula (I):Formula (I); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3 is an azole or a substituted azole.

2. The compound of claim 1, wherein the azole or substituted azole is pyrazole or substituted pyrazole, and with Formula (II): 54 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Formu a (II); wherein R1, R2, and R4 are hydrogen; and R5is hydrogen, C1-C6alkyl or substituted C1-C6alkyl.

3. The compound of claim 2, wherein R5 is methyl, ethyl, propyl, butyl, pentyl, or hexyl.

4. The compound of claim 2, wherein R5 is substituted C1-C6 alkyl.

5. The compound of claim 4, wherein the substituted C1-C6alkyl is substituted with one or more halogens, amine, substituted amine, C3-C6cycloalkyl, substituted C3-C6cycloalkyl, C3-C6heterocycloalkyl, or substituted C3-C6 heterocycloalkyl.

6. The compound of claim 5, wherein the substituted amine is substituted with one or more C1- C6alkyl.

7. The compound of claim 4, wherein R5 is substituted ethyl, and with Formula (III): 55 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)o u a ; wherein R6 is amine, substituted amine, C3-C6 heterocycloalkyl, or substituted C3-C6 heterocycloalkyl.

8. The compound of claim 7, wherein R6is substituted amine.

9. The compound of claim 8, wherein the substituted amine is substituted with one or more C1- C6 alkyl.

10. The compound of claim 7, wherein R6is substituted C3-C6heterocycloalkyl.

11. The compound of claim 10, wherein the substituted C3-C6heterocycloalkyl is substituted piperidine.

12. The compound of claim 11, wherein the substituted piperidine is substituted with one or more halogens.

13. The compound of claim 12, wherein the halogen is fluorine, chlorine, or bromine.

14. The compound of claim 5, wherein the C3-C6 heterocycloalkyl is aziridine, azetidine, pyrrolidine, piperidine, piperazine, substituted aziridine, substituted azetidine, substituted pyrrolidine, substituted piperidine, or substituted piperazine.

15. The compound of claim 14, wherein the C3-C6 heterocycloalkyl is substituted piperazine.

16. The compound of claim 15, wherein the piperazine is substituted with a C1-C6alkyl or one or more halogens. 56 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) 17. The compound of claim 14, wherein the C3-C6 heterocycloalkyl is substituted piperidine.

18. The compound of claim 17, wherein the substituted piperidine is substituted with one or more halogens.

19. The compound of claim 18, wherein the halogen is fluorine, chlorine, or bromine.

20. The compound of claim 1, wherein the compound is selected from the group consisting of:Compound 2; 57 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 5; 58 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 8; 59 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 11; 60 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)Compound 13 (DA-147).

21. The compound of claim 1, wherein the compound has a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.

2.

22. The compound of claim 1, wherein the compound has a solubility limit of greater than 14 μg / mL in phosphate buffer saline at pH 7.

2.

23. A composition comprising a DNA-PKcs inhibitor with a solubility limit of greater than 20 μM in phosphate buffer saline at pH 7.

2.

24. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I): 61 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009)ormu a ( ); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3is an azole or a substituted azole.

25. A method of inhibiting DNA-PKcs kinase activity in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I): 104772379.1PATENT - PCT Atty Docket No: 052592-843981 (BV 2024-009) wherein R1, R2, and R4are hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; and R3 is an azole or a substituted azole.

26. A method of treating a transplanted solid organ for anti-rejection therapy in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a compound of Formula (I):Formula (I); wherein R1, R2, and R4 are hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl; and R3is an azole or a substituted azole.

27. The method of claim 26, wherein the anti-rejection therapy is T-cell mediated. 63 104772379.1

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