WO2023192864A2

WO2023192864A2 - Covalent parp16 inhibitors

Info

Publication number: WO2023192864A2
Application number: PCT/US2023/065045
Authority: WO
Inventors: Michael Cohen; Daniel BEJAN; Sunil SUNDALAM
Original assignee: Oregon Health and Science University
Current assignee: Oregon Health and Science University
Priority date: 2022-03-28
Filing date: 2023-03-28
Publication date: 2023-10-05
Anticipated expiration: 2024-09-28
Also published as: WO2023192864A3

Abstract

This invention provides a PARP16 inhibiting compound of Formula (I): (I) wherein: R₁ is selected from the group of H, halo, -OH, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-alkyl,-O-alkenyl,-O-alkynyl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, -O-heteroaryl, - SO₂-cycloalkyl, -NH₂, -NH(alkyl), -N(alkyl)₂, alkynyl-NH₂, alkynyl-NH(alkyl), and alkynyl-N(alkyl)₂; R₂ and R₃ are each selected from the group of H, F, Cl, Br, I, CH₃, CN, -CH₂CN, CF₃, and CH₂-CF₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co-crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof.

Description

COVALENT PARP16 INHIBITORS FIELD OF THE INVENTION The present invention concerns phthalazinone compounds useful as inhibitors of PARP16 and methods of their use in the treatment of cancers and other diseases and disorders. BACKGROUND OF THE INVENTION Phthalazinone compounds have been studied for use as PARP inhibitors, including olaparib, marketed as LYNPARZA® BY AstraZeneca and Merck, and those described in U.S. Pat. No. 6,903,098 (Lubisch et al.), U.S. Patent No. 7,196,085 (Martin et al.), U.S. Patent No. 7,407,957 (Javaid et al.), U.S. Pat. No. 7,803,795 (Mevellec et al.), U.S. Pat. 7,981,889 (Barr Martin et al.), U.S. Pat. No. 8,129,380 (Menear et al.), U.S. Pat. 8,999,985 (Gao et al.), U.S. Patent No. 9,273,052 (Tang et al.), U.S. Pat. No. 9,682,973 (Kang et al.), and U.S. Patent Publication No. 2011/0015393 (Hawkins). There remains a need for new inhibitors of PARP, including inhibitors of PARP 16. SUMMARY OF THE INVENTION PARP16 is an endoplasmic reticulum‐resident (ER‐resident), mono‐ADP‐ribosyl transferase that has been gaining attention as a novel therapeutic target. Recent studies have revealed PARP16‐ dependent vulnerabilities, such as regulation of protein synthesis, can be exploited to treat cancer. Additionally, PARP16 has been identified as an off‐target of talazoparib—an approved PARP1 inhibitor— in small cell lung cancer, suggesting a potential pharmacology‐based mechanism of action for talazoparib. These studies highlight the therapeutic potential for PARP16 inhibition. However, there is a lack of selective chemical tools available to validate the catalytic‐dependent roles of PARP16 obtained by genetic methods (i.e., RNA interference or CRISPR). Provided herein are first‐in‐class covalent PARP inhibitors, targeting PARP16, that elucidate the catalytic function of PARP16 in normal physiology and diseased states. A first embodiment provides a compound of Formula (I):

wherein: R₁ is selected from the group of H, halo, ‐OH, C₁‐₆alkyl, C_2‐6alkenyl, C_2‐6 alkynyl, C_3‐6 cycloalkyl, C_3‐ ₆ heterocyclyl, C_5‐6 aryl, C_5‐6 heteroaryl, ‐O‐C_1‐6 alkyl,‐O‐C_2‐6alkenyl,‐O‐C_2‐6alkynyl, ‐O‐C_3‐6cycloalkyl, ‐O‐ C_5‐6 heterocyclyl, ‐O‐ C_5‐6 aryl, ‐O‐ C_5‐6 heteroaryl, ‐SO₂‐C_3‐6cycloalkyl, ‐NH₂, ‐NH(C_1‐3alkyl), ‐N(C_1‐3alkyl)₂, C_2‐6alkynyl‐NH₂, C_2‐6alkynyl‐NH(C_1‐3alkyl), and C_2‐6alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, Cl, Br, I, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐CF₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. BRIEF DESCRIPTIONS OF THE MANY VIEWS OF THE DRAWINGS FIGURE 1A represents the crystal structure of the active site of PARP16 (PDB: 4F0D) overlaid with PARP1 (cyan, PDB: 5DS3) bound to olaparib. FIGURE 1B provides a PARP family sequence alignment generated with T‐Coffee multiple sequence alignment algorithm. The non‐conserved D‐loop cysteine (C169) of PARP16 is highlighted. FIGURE 1C presents the structure of HJ‐52 and DB008, with the acrylamide warhead shown lighter gray in lighter gray at the lower right, and the dual selectivity/clickable alkyne handle at the left side of the structure. FIGURE 1D graphs a biochemical activity assay to assess potency of olaparib, HJ‐52, and DB008 against PARP16; n ≥ 3 biological replicates. FIGURE 1E graphs a biochemical activity assay to assess potency of olaparib, HJ‐52, and DB008 against PARP16PARP1; n ≥ 3 biological replicates. FIGURE 2A charts DB008 potency against the PARP family determined using a previously described trans‐modification biochemical plate assay¹, unless otherwise indicated: *Auto‐modification assay format using native NAD⁺ (100 µM for PARP3, PARP4, and PARP7; 400 µM for PARP16). N.D. = not determined. FIGURE 2B represents cellular inhibition of PARP1 determined by treating HEK293T cells with a dose response of PARP inhibitors (30 min) followed by PARG inhibitor (15 min) to amplify the PARylation signal. Western blotting for PARylation was done using a Mono/Poly ADPr antibody from Cell Signaling Technology. FIGURE 2C graphs quantification of inhibition from Figure 2 B; n = 2 biological replicates. FIGURE 2D represents a cellular PARP1/2 inhibition assay performed as in Figure 2B, including a washout condition before PARG inhibitor treatment; n = 2 biological replicates. FIGURE 3A presents a model of DB008 covalently bound to C169 of PARP16 generated using Nir London method. FIGURE 3B represents HEK293T cells transfected with Myc2x‐tagged PARP16 WT or the C169S mutant, treated with a DB008 dose response for 2 hours, followed by lysis and clicking to TAMRA‐azide for in‐gel fluorescence detection of PARP16 labeling. FIGURE 3C graphs quantification of TAMRA signal from Fig. 3B; n = 3 biological replicates. FIGURE 3D represents HEK293T cells transfected with Myc2x‐tagged PARP16 WT, treated with a 300 nM DB008 on a time course, followed by lysis and clicking to TAMRA‐azide for in‐gel fluorescence detection of PARP16 labeling. FIGURE 3E graphs quantification of TAMRA signal from Fig. 3D; n = 3 biological replicates. FIGURE 3F represents HAP1 WT and HAP1 PARP16 KO cells treated with a DB008 dose response for 2 hours, followed by lysis and clicking to TAMRA‐azide for in‐gel fluorescence detection of PARP16 labeling. FIGURE 3G graphs quantification of TAMRA signal from Fig. 3F; n = 2 biological replicates. FIGURE 4A presents chemical structures of talazoparib and epigallocatechin gallate (EGCG). FIGURE 4B graphs an in vitro competition assay wherein talazoparib and EGCG were incubated with purified PARP16 (40 min) followed by treatment with DB008 (20 min) and clicking to TAMRA‐azide for in gel‐fluorescence detection of PARP16 labeling; n = 2 biological replicates. FIGURE 4C represents a cellular competition assay wherein Myc2x‐PARP16 expressing HEK293T cells were dosed with talazoparib and EGCG for 1 hour, then treated with DB008 (0.3 µM) for 30 min, followed by lysis and clicking to TAMRA‐azide for in gel‐fluorescence detection of PARP16 labeling. FIGURE 4D graphs quantification of TAMRA signal from Fig. 4C; n = 2 biological replicates. DETAILED DESCRIPTION OF THE INVENTION A second embodiment provides a compound of Formula (I), as defined above, with the proviso that when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A third embodiment provides a compound of Formula (I), above, wherein: R₁ is selected from the group of H, halo, ‐OH, C₁‐₄alkyl, C_2‐4alkenyl, C_2‐4 alkynyl, C_3‐6 cycloalkyl, C_3‐ ₆ heterocyclyl, C_5‐6 aryl, C_5‐6 heteroaryl, ‐O‐C_1‐4 alkyl,‐O‐C_2‐4alkenyl,‐O‐C_2‐4alkynyl, ‐O‐cycloalkyl, ‐O‐ C_5‐6 heterocyclyl, ‐O‐C_5‐6 aryl, ‐O‐ C_5‐6 heteroaryl, ‐SO₂‐C_3‐6cycloalkyl, ‐NH₂, ‐NH(C_1‐3alkyl), ‐N(C_1‐3alkyl)₂, C_2‐6 alkynyl‐NH₂, C_2‐4alkynyl‐NH(C_1‐3alkyl), and C_2‐4alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, Cl, Br, I, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐CF₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A fourth embodiment provides a compound of Formula (I), as defined for the third embodiment, above, with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A fifth embodiment provides a compound of Formula (I), above, wherein: R₁ is selected from the group of H, C₁‐₄alkyl, C_2‐3alkenyl, C_2‐3 alkynyl, C_2‐6alkynyl‐NH₂, C_2‐4alkynyl‐ NH(C_1‐3alkyl), and C_2‐4alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, Cl, Br, I, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐CF₃; with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A sixth embodiment provides a compound of Formula (I), above, wherein: R₁ is selected from the group of H, C₁‐₃alkyl, C_2‐3alkenyl, C_2‐3 alkynyl, C_2‐3alkynyl‐NH₂, C_2‐3alkynyl‐ NH(C_1‐3alkyl), and C_2‐3alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐ CF₃; with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; R₄ is selected from the group of H, F, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A seventh embodiment provides a compound of Formula (II):

wherein: R₂ and R₃ are each independently selected from the group of H, F, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐ CF₃; with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; R₄ is selected from the group of H, F, CH₃, and CF₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. An eighth embodiment provides a compound of Formula (II), above: wherein: R₂ and R₃ are each independently selected from the group of H, F, and CH₃; R₄ is selected from the group of H, F, CH₃, and CF₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A ninth embodiment provides a compound of Formula (II), above: wherein: R₂ and R₃ are each independently selected from the group of H and CH₃; R₄ is selected from the group of H, F, and CH₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A tenth embodiment provides a compound of Formula (II), above: wherein: R₂ is H; R₃ is selected from the group of H and CH₃; R₄ is selected from the group of H, F, and CH₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. An eleventh embodiment provides a compound of Formula (III):

wherein: R₄ is selected from the group of H, F, and CH₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A twelfth embodiment provides a compound of Formula (III), above: wherein: R₄ is selected from the group of H, and CH₃; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. An thirteenth embodiment provides a compound of Formula (III), above: wherein: R₄ is H; R₅ and R₇ are each independently selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. An fourteenth embodiment provides a compound of Formula (III), above: wherein: R₄ is CH₃; and R₅ and R₆ are each independently selected from the group of H and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. Within each of the embodiments one through fourteen, there is another embodiment in which both R₂ and R₃ are methyl. Within each of embodiments one through fourteen, there is also another embodiment in which both R₂ is H and R₃ is methyl. Within each of embodiments one through fourteen, there is a further embodiment in which both R₂ and R₃ are H. Another embodiment provides a method of enhancing endoplasmic reticulum (ER) stress‐ induced cell apoptosis of cancer cells in a subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. ER stress‐related conditions that may be treated using a pharmaceutically effective amount of a compound of Formula (I), as defined herein, include those in cancers, protein folding/misfolding disease, diabetes mellitus, Wolcott‐Rallison syndrome, ischemia/reperfusion injury, stroke, neurodegeneration, atherosclerosis, neoplasia, hypoxia, or hypoglycemia. Cancers of this ER stress‐related group include colon adenocarcinoma, esophagus adenocarcinoma, liver hepatocellular carcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet cell tumor, rectum adenocarcinoma, gastrointestinal stromal tumor, stomach adenocarci noma, adrenal cortical carcinoma, renal cancer, thyroid cancer, melanoma, testicular cancer, follicular carcinoma, papillary carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, intraductal carcinoma, mucinous carcinoma, phyllodes tumor, Ewing’s sarcoma, ovarian adenocarcinoma, endometrium adenocarcinoma, granulose cell tumor, mucinous cystadenocarcinoma, cervix adenocarcinoma, vulva squamous cell carcinoma, basal cell carcinoma, prostate adenocarcinoma, giant cell tumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma, kidney carcinoma, urinary bladder carcinoma, Wilm’s tumor, and lymphoma. ER stress‐related protein folding/misfolding diseases include Huntington’s disease, spinobulbar muscular atrophy (Kennedy disease), Machado‐Joseph disease, dentatorubralpallidoluysian atrophy (Haw River Syndrome), spinocerebellar ataxia, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Creutzfeldt‐Jakob disease, bovine spongiform encephalopathy (BSE), and light chain amyloidosis (AL). Provided herein are individual methods for the treatment of each of the ER stress‐related conditions referenced herein, each method comprising administering to a subject in need of such treatment a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. It is understood that such methods also exist for treating the subject in need thereof by administering to the subject a pharmaceutically effective amount of a compound of Formula (II) and/or a compound of Formula (III), as defined herein. Another embodiment provides a method of treating ovarian cancer in a subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. A further embodiment provides a method of small cell lung cancer in a subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. Another embodiment provides a method of treating a carcinoid tumor in a subject, the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. Definitions The term "alkyl" refers herein to a straight or branched hydrocarbon. For non‐limiting examples, an alkyl group may have from 1 to 6 carbon atoms (i.e., C₁‐C₆ alkyl or C_1‐6alkyl), 1 to 4 carbon atoms (i.e., C₁‐C₄ alkyl or C_1‐4alkyl), or 1 to 3 carbon atoms (i.e., C₁‐C₃ alkyl or C_1‐3alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, ‐‐CH₃), ethyl (Et, ‐CH₂CH₃), 1‐propyl (n‐Pr, n‐propyl, ‐ CH₂CH₂CH₃), 2‐propyl (i‐Pr, i‐propyl, ‐CH(CH₃)₂), 1‐butyl (n‐Bu, n‐butyl, ‐CH₂CH₂CH₂CH₃), 2‐methyl‐1‐ propyl (i‐Bu, i‐butyl, ‐‐CH₂CH(CH₃)₂), 2‐butyl (s‐Bu, s‐butyl, ‐CH(CH₃)CH₂CH₃), 2‐methyl‐2‐propyl (t‐Bu, t‐ butyl, ‐C(CH₃)₃), 1‐pentyl (n‐pentyl, ‐CH₂CH₂CH₂CH₂CH₃), 2‐pentyl (‐CH(CH₃)CH₂CH₂CH₃), 3‐pentyl (‐ CH(CH₂CH₃)₂), 2‐methyl‐2‐butyl (‐C(CH₃)₂CH₂CH₃), 3‐methyl‐2‐butyl (‐CH(CH₃)CH(CH₃)₂), 3‐methyl‐1‐butyl (‐CH₂CH₂CH(CH₃)₂), 2‐methyl‐1‐butyl (‐CH₂CH(CH₃)CH₂CH₃), 1‐hexyl (‐CH₂CH₂CH2CH₂CH₂CH₃), 2‐hexyl (‐ CH(CH₃)CH₂CH₂CH₂CH₃), 3‐hexyl (‐CH(CH₂CH₃)(CH₂CH₂CH₃)), 2‐methyl‐2‐pentyl (‐C(CH₃)₂CH₂CH₂CH₃), 3‐ methyl‐2‐pentyl (‐CH(CH₃)CH(CH₃)CH₂CH₃), 4‐methyl‐2‐pentyl (‐CH(CH₃)CH₂CH(CH₃)₂), 3‐methyl‐3‐pentyl (‐C(CH₃)(CH₂CH₃)₂), 2‐methyl‐3‐pentyl (‐CH(CH₂CH₃)CH(CH₃)₂), and 2,3‐dimethyl‐2‐butyl (‐ C(CH₃)₂CH(CH₃)₂). Groups such as ‐O‐C_1‐6 alkyl, ‐O‐C_1‐4 alkyl, and ‐O‐C_1‐3 alkyl are understood to be straight or branched alkoxy groups of the number of carbon atoms indicated. The term "alkenyl" refers to a straight or branched hydrocarbon with at least one site of unsaturation, i.e. a carbon‐carbon, sp² double bond. As seen for alkyl groups, for example, an alkenyl group can have 2 to 6 carbon atoms (i.e., C₂‐C₆ or C_2‐6alkenyl), 2 to 4 carbon atoms (i.e., C₂‐C₄ or C_2‐4 alkenyl), or 2 to 3 carbon atoms (i.e., C₂‐C₃ or C_2‐3alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (‐CH=CH₂) and allyl (‐CH₂CH=CH₂) groups. The term "alkynyl" refers to a straight or branched hydrocarbon with at least one site of unsaturation, i.e. a carbon‐carbon, sp triple bond. For example, an alkynyl group can have 2 to 6 carbon atoms (i.e., C₂‐C₆ or C_2‐6alkynyl). Examples of suitable alkynyl groups include, but are not limited to, acetylenic (‐CΞCH), propargyl (‐CH₂CΞCH), and the like. Reference to groups such as C_2‐6alkynyl‐NH₂ indicates an alkynylene chain terminating in another listed moiety, an amino group in this particular example. The term "cycloalkyl" refers to a saturated ring having 3 to 6 carbon atoms as a monocycle, including cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. The terms “halo” and “halogen” refer to an element or substituent selected from the group of F, Cl, Br, and I. A “heterocyclyl,” “heterocycle,” or “heterocyclic” group herein refers to a chemical ring containing carbon atoms and at least one ring heteroatom selected from O, S, and N. Examples of 5‐ membered and 6‐membered heterocycles include, by way of example and not limitation, pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, 4‐ piperidinyl, pyrrolidinyl, 2‐pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, triazinyl, 6H‐1,2,5‐thiadiazinyl, 2H,6H‐1,5,2‐dithiazinyl, thienyl, thianthrenyl, pyranyl, 2H‐pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, morpholinyl, and oxazolidinyl. The terms "heteroaryl" and “heteroaromatic” refers to an aromatic heterocyclyl having at least one heteroatom in the ring. Non‐limiting examples of suitable heteroatoms which can be included in the aromatic ring include oxygen, sulfur, and nitrogen. Non‐limiting examples of 5‐ and 6‐membered heteroaryl rings include pyridinyl, pyrrolyl, oxazolyl, furanyl, thienyl (thiophenyl), imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl etc. The terms "therapeutically effective amount" and "pharmaceutically effective amount" may be used interchangeably and refer to an amount that is sufficient to effect treatment, as defined below, when administered to a subject (e.g., a mammal, such as a human) in need of such treatment. The therapeutically or pharmaceutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For example, a "therapeutically effective amount" or a "pharmaceutically effective amount" of a compound of Formula I, or a pharmaceutically acceptable salt or co‐crystal thereof, is an amount sufficient to modulate PARP16 expression or activity, and thereby treat a subject (e.g., a human) suffering an indication, or to ameliorate or alleviate the existing symptoms of the indication. For example, a therapeutically or pharmaceutically effective amount may be an amount sufficient to decrease a symptom of a disease or condition responsive to inhibition of PARP16 activity. In some embodiments, the compound of Formula (I) may be administered to a subject in need thereof at a dose of from about 0.1 mg to about 1000 mg per day in a single dose or in divided doses. In some embodiments, each pharmaceutically effective dosage unit contains from 0.1 mg to 1 g, 0.1 mg to 700 mg, or 0.1 mg to 100 mg of a compound of Formula I, or a pharmaceutically acceptable salt or co‐crystal thereof. In some embodiments, a therapeutically effective amount or a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, comprises from about 0.1 mg to about 500 mg per dose, given once or twice daily. In some embodiments, the individual pharmaceutically effective dose is selected from the group of 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, and 500 mg per dose. The term "pharmaceutically acceptable salt" include, for example, salts with inorganic acids and salts with an organic acid. Examples of salts may include hydrochloride, phosphate, diphosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate (mesylate), benzenesuflonate (besylate), p‐toluenesulfonate (tosylate), 2‐ hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate (such as acetate, HOOC‐(CH₂)_n‐ COOH where n is an integer from 0‐4). In addition, if the compounds described herein are obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare nontoxic pharmaceutically acceptable addition salts. A pharmaceutical composition refers to a composition containing a pharmaceutically effective amount of one or more of the compounds described herein, or a pharmaceutically acceptable salt thereof, formulated with a pharmaceutically acceptable carrier, which can also include other additives, as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21^st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005) and in The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013. As used herein, "pharmaceutically acceptable excipient" is a pharmaceutically acceptable vehicle that includes, without limitation, any and all carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The term "carrier" refers to an excipient or vehicle that includes without limitation diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, and the like with which the compound is administered. Carriers are generally described herein and also in "Remington's Pharmaceutical Sciences" by E. W. Martin. Examples of carriers include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, carboxymethylcellulose sodium, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxyoctacosanyl hydroxystearate, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 188, poloxamer 237, poloxamer 407, povidone, silicon dioxide, colloidal silicon dioxide, silicone, silicone adhesive 4102, and silicone emulsion. It should be understood, however, that the carriers selected for the pharmaceutical compositions, and the amounts of such carriers in the composition, may vary depending on the method of formulation (e.g., dry granulation formulation, solid dispersion formulation). The term "subject" refers to an animal, such as a mammal, that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in both human therapy and veterinary applications. In some embodiments, the subject is a mammal; in some embodiments the subject is human; and in some embodiments the subject is chosen from cats and dogs. "Subject in need thereof" or "human in need thereof" refers to a subject, such as a human, who may have or is suspected to have diseases or conditions that would benefit from certain treatment; for example treatment with a compound of Formula I, or a pharmaceutically acceptable salt or co‐crystal thereof, as described herein. This includes a subject who may be determined to be at risk of or susceptible to such diseases or conditions, such that treatment would prevent the disease or condition from developing. All ranges disclosed and/or claimed herein are inclusive of the recited endpoint and independently combinable. For example, the ranges of "from 2 to 6" and “2‐6” are inclusive of the endpoints, 2 and 6, and all the intermediate values between in context of the units considered. For instance, reference to “Claims 2‐6” or “C₂‐C₆ alkyl” includes units 2, 3, 4, 5, and 6, as claims and atoms are numbered in sequential numbers without fractions or decimal points, unless described in the context of an average number. The context of “pH of from 5‐9” or “a temperature of from 5˚C to 9˚C”, on the other hand, includes whole numbers 5, 6, 7, 8, and 9, as well as all fractional or decimal units in between, such as 6.5 and 8.24. Compounds of Formula (I) may be prepared by methods known in the art, including the general scheme below representing synthesis of Compound DB008.

General 1H NMR spectra were recorded on a Bruker DPX spectrometer at 400 MHz. Chemical shifts are reported as parts per million (ppm) downfield from an internal tetramethylsilane standard or solvent references. For air and water sensitive reactions, glassware was oven dried prior to use and reactions were performed under argon. Tetrahydrofuran (THF), N,N‐dimethylformamide (DMF), and dichloromethane (CH₂Cl₂) were dried using a solvent purification system manufactured by Glass Contour, Inc. (Laguna Beach, CA). All other solvents were of ACS chemical grade (Fisher Scientific) and used without further purification unless otherwise stated. Commercially available starting reagents were used without further purification. Flash column chromatography was conducted using self‐packed columns containing 200‐ 400 mesh silica gel (SiliCycle) on a Teledyne ISCO Combiflash Rf 150. Microwave reactions were performed using a Biotage Initiator+ SP Wave Microwave Reactor. For NMR data, the abbreviation brs is used to define a broad singlet. 2‐fluoro‐5‐((4‐oxo‐7‐((triisopropylsilyl)ethynyl)‐3,4‐dihydrophthalazin‐1‐yl)methyl)benzoic (2). Compound 1, 5‐((7‐bromo‐4‐oxo‐3,4‐dihydrophthalazin‐1‐yl)methyl)‐2‐fluorobenzoic acid, was prepared following previously reported methods¹.To a dry 10 mL microwave vial, compound 1 (0.5 g, 1.33 mmol) was added and dissolved in anhydrous DMF (1.6 mL), followed by addition of PdCl₂(PPh₃) (51.2 mg, 0.073 mmol), CuI (13.9 mg, 0.073 mmol), triphenylphosphine (69.5 mg, 0.265 mmol), and triethylamine (2.8 mL, 20.0 mmol). After 10 min stirring at room temperature, (triisopropylsilyl)acetylene (0.44 mL, 1.99 mmol) was added dropwise and the microwave vial was sealed, purged with argon, and reacted in a microwave reactor for 25 min at 120°C. The reaction mixture was diluted with ethyl acetate (10 mL) and washed once with 1M HCl (10 mL). The aqueous layer was extracted with ethyl acetate (3 x 10 mL), and the combined layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The resulting crude residue was purified on a Teledyne ISCO CombiFlash (0‐10% MeOH‐containing 2% acetic acid, in CH₂Cl₂). The product was isolated as a white solid (0.482 g, 76% yield). ¹H NMR (400 MHz, DMSO) δ 13.24 (brs, 1H), 12.68 (s, 1H), 8.23 (d, J = 7.0 Hz, 1H), 7.96 (s, 1H), 7.83 (s, 2H), 7.54 (s, 1H), 7.30 – 7.15 (m, 1H), 4.40 (s, 2H), 1.17 – 1.04 (m, 21H). tert‐butyl 4‐(2‐fluoro‐5‐((4‐oxo‐7‐((triisopropylsilyl)ethynyl)‐3,4‐dihydrophthalazin‐1‐ yl)methyl)benzoyl)piperazine‐1‐carboxylate (3). To a dry 4 mL dram vial, compound 2 (80 mg, 0.167 mmol) and tert‐butyl piperazine‐1‐ carboxylate (47 mg, 0.251 mmol) were combined, dissolved in dry DMF (0.33 mL), and cooled to 0°C. DIPEA (0.087 mL, 0.5 mmol) was added dropwise, followed by stirring for 5 min, then propylphosphonic anhydride solution (0.21 mL, 0.33 mmol) was added dropwise .The reaction mixture was stirred for 1.5 hr at room temperature, diluted with ethyl acetate (1 mL) and quenched with sat. sodium bicarbonate (1 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 x 1 mL). The combined organic layers were washed once with 1M HCl (3 mL), dried over sodium sulfate, filtered, co‐evaporated with heptane (3 x 10 mL) and concentrated in vacuo to yield an off‐white, semi‐ transparent solid (101.6 mg crude, 94% yield) that was used in the TIPS deprotection step without further purification.¹H NMR (400 MHz, CDCl₃) δ 9.91 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.31 – 7.20 (m, 2H), 6.99 (t, J = 8.8 Hz, 1H), 4.19 (s, 2H), 3.67 (brs, 2H), 3.54 – 3.35 (m, 3H), 3.29 (t, J = 5.1 Hz, 2H), 3.18 (brs, 2H), 1.39 (s, 9H), 1.15 – 1.00 (m, 21H). tert‐butyl 4‐(5‐((7‐ethynyl‐4‐oxo‐3,4‐dihydrophthalazin‐1‐yl)methyl)‐2‐fluorobenzoyl)piperazine‐1‐ carboxylate (4). Compound 3 (67.8 mg, 0.10 mmol) was dissolved in anhydrous THF (1 mL) in a dry 4 mL dram vial and cooled to 0ºC. A 1M solution of TBAF in THF (0.12 mL, 0.12 mmol) was added dropwise and the solution was stirred for 15 min at 0ºC, then 45 min at room temperature. The reaction mixture was diluted with ethyl acetate (1 mL) and washed once with brine (1 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 x 1 mL). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo, and the resulting residue was purified on a Teledyne ISCO CombiFlash (0‐75% ethyl acetate in hexanes). The produce was isolated as a white solid (36.0 mg, 70% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.86 (s, 1H), 8.34 (dd, J = 8.0, 0.9 Hz, 1H), 7.78 – 7.69 (m, 2H), 7.27 – 7.21 (m, 2H), 6.99 (t, J = 8.7 Hz, 1H), 4.18 (s, 2H), 3.68 (brs, 2H), 3.43 (s, 2H), 3.32 (t, J = 5.1 Hz, 2H), 3.26 (s, 1H), 3.21 (brs, 2H), 1.40 (s, 9H). 4‐(3‐(4‐acryloylpiperazine‐1‐carbonyl)‐4‐fluorobenzyl)‐6‐ethynylphthalazin‐1(2H)‐one (5, DB008). Compound 4 (34.5 mg, 0.07 mmol) was dissolved in anhydrous CH₂Cl₂ (1 mL) and cooled to 0ºC. TFA (0.21 mL, 2.8 mmol) was added dropwise, and the reaction was stirred for 1 hr at room temperature. Next, CH₂Cl₂ (0.5 mL) was added and the reaction mixture was cooled to ‐10ºC, followed by the dropwise addition of DIPEA (0.61 mL, 3.5 mmol). The reaction mixture was stirred for 10 minutes, then acryloyl chloride (6.8 µL, 0.084 mmol) was added dropwise and the solution was stirred for 15 min at ‐10ºC. The reaction mixture was quenched with water (2 mL) and the organic layer was separated, washed with brine (1 x 2 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was co‐evaporated with toluene (4 x 5 mL), then purified on a Teledyne ISCO CombiFlash (0‐5% MeOH in CH₂Cl₂). The product was isolated as a white solid (19.5 mg, 62.5% over two steps). ¹H NMR (400 MHz, CDCl₃) δ 11.26 (s, 1H), 8.35 (d, J = 8.5 Hz, 1H), 7.77 – 7.69 (m, 2H), 7.34 – 7.20 (m, 2H), 6.99 (t, J = 8.7 Hz, 1H), 6.49 (brs, 1H), 6.31 – 6.19 (m, 1H), 5.68 (s, 1H), 4.21 (s, 2H), 3.87 – 3.15 (m, 9H). Reference: Phthalazinone derivatives and their pharmaceutical compositions as PARP inhibitors useful for the treatment of diseases and preparation thereof. WO 2009093032 A1, Jul 30, 2009. Other protected piperazine carboxylic acids may be utilized to prepare compounds of Formula (I), including tert‐butyl (S)‐3‐methylpiperazine‐1‐carboxylate (CAS Reg. No. 147081‐29‐6), tert‐butyl (3S,5R)‐3,5‐dimethylpiperazine‐1‐carboxylate (CAS Registry No. 129779‐30‐2), tert‐butyl (S)‐2‐ methylpiperazine‐1‐carboxylate (CAS Reg. No. 169447‐70‐5), and tert‐butyl (R)‐2‐methylpiperazine‐1‐ carboxylate (CAS Reg. No. 170033‐47‐3). ADP‐ribosylation is a critical post‐translational modification carried out by a family of enzymes known as PARPs. Upon binding nicotinamide adenine dinucleotide (NAD⁺), PARPs cleave the nicotinamide group and transfer the resulting ADP‐ribose (ADPr) to generate ADP‐ribosylated targets— primarily on proteins but also on nucleic acids¹. The PARP family is divided based on the ability to transfer ADPr in the form of polymers (poly‐ADP‐ribosylation or PARylation) or monomers (mono‐ADP‐ ribosylation or MARylation). PARP1, PARP2, and the tankyrases—TNKS1 (PARP5a) and TNKS2 (PARP5b)— catalyze PARylation, while the remaining members (PARP 3, 4, 6‐12, 14‐16) catalyze MARylation, with the exception of PARP13 being inactive². The role of PARylation has been extensively studied, particularly in cancer, resulting in four clinically approved PARP1/2 inhibitors developed to date (olaparib, rucaparib, niraparib, talazoparib)³. In contrast, the physiological roles of MARylation are far less understood, in large part due to the lack of chemical tools currently available. However, there have been recent advances in inhibitor development focusing on MARylating PARPs as these PARPs are being implicated in diseases such as cancer and inflammation. For example, a PARP7 inhibitor (RBN‐2397)⁴ and PARP14 inhibitor (RBN‐3143)⁵ have recently entered clinical trials for the treatment of lung cancer (NCT05127590) and atopic dermatitis (NCT05215808), respectively. PARP16 is an example of another MARylating PARP that has been gaining attention as a novel therapeutic target. PARP16 contains a C‐terminal transmembrane (TM) domain that localizes to the endoplasmic reticulum (ER) membrane, with the N‐terminal catalytic domain facing the cytoplasm. Upon ER stress, PARP16 has been shown to MARylate two sensors of the unfolded protein response (UPR), PKR‐like ER kinase (PERK) and inositol‐requiring enzyme 1α (IRE1α), leading to a suppression of protein synthesis⁶. The role of PARP16 in UPR has motivated additional studies to explore opportunities for therapeutic intervention, since the UPR pathway is implicated in a variety of diseases including diabetes, neurodegeneration, and cancer⁷. Two recent studies have revealed PARP16‐dependent vulnerabilities that can be exploited to treat cancer. First, Palve et al. discovered PARP16 as a novel off‐ target to the clinically approved PARP1/2 inhibitor, talazoparib, in small cell lung cancer (SCLC)⁸. Talazoparib displayed a half maximal inhibitory concentration (IC₅₀) between 160–289 nM against recombinant PARP16 in a biochemical assay. They also find that transient knockdown of PARP16 reduces SCLC cell viability, and that PARP16 knockdown in combination with olaparib (PARP1/2 inhibitor) treatment, results in a greater decrease in viability. In the second study, Challa et al. identify PARP16 as an important driver of ovarian cancer. They show that MARylation of ribosomes by PARP16 attenuates protein synthesis, enabling maintenance of protein homeostasis for cancer cell survival. When PARP16 is silenced or knocked‐out, protein synthesis increases uncontrollably, forcing the cancer cell to enter a state of proteotoxic stress, which ultimately leads to cell death⁹. These studies highlight PARP16 as an actionable drug target in cancer. However, since genetic methods were used, there is a need for development of a potent and selective PARP16 inhibitors to validate whether catalytic inhibition of PARP16 phenocopies PARP16 knockdown results. The reported PARP16 inhibitors to date are not potent, displaying IC₅₀ values in the high micromolar range, and/or lacking PARP family‐wide selectivity evaluation^10,11. Here, we describe the rationale‐based design of the first potent, selective, and cell‐active covalent PARP inhibitor targeting PARP16, and evaluate target engagement using click chemistry. Results and Discussion Developing selective inhibitors for PARPs is challenging due to the highly conserved NAD‐binding pocket shared between PARP family members. To achieve inhibitor selectivity, we employed a structural bioinformatics‐based approach that uses two selectivity filters (Fig. 1A). The first selectivity filter exploits a hydrophobic sub‐pocket present in PARPs containing the HYΦ active site motif (where ^ is a hydrophobic amino acid) but lacking in HYE PARPs. Scaffolds with strategically placed hydrophobic groups are predicted to interact favorably with HYΦ PARPs, but clash with the glutamate of HYE PARPs, resulting in selective inhibition of MARylating PARPs (except for PARP3 and PARP4) over PARylating PARPs. Indeed, we have successfully applied this general strategy to develop selective PARP10 and PARP11 inhibitors^12,13. To generate a specific inhibitor for PARP16, we found a second selectivity filter in the flexible region adjacent to the active site known as the D‐loop. Family‐wide sequence alignments revealed a non‐conserved cysteine (C169) present only in the D‐loop of PARP16 (Fig. 2B). We started with olaparib, an approved PARP1 inhibitor, because it displays moderate potency against PARP16 (IC₅₀ = 2‐3 µM)^8,14 and crystal structures of PARP1‐bound olaparib overlaid with PARP16 show the cyclopropyl amide in close proximity (3.7 Å) with C169 of PARP16 (Fig. 1A). As a proof‐of‐concept, we synthesized HJ‐52 (Fig. 2C), an olaparib analog in which the cyclopropyl amide is replaced with an acrylamide warhead to promote covalent bond formation with C169. Gratifyingly, we identified a covalent adduct between purified PARP16 and HJ‐52 by mass spectrometry. HJ‐52 inhibited PARP16 about 2.5‐times more than olaparib in a biochemical assay (Fig. 1D), however, as expected, HJ‐52 is not selective and inhibits PARP1 with a low nanomolar IC₅₀ similarly as olaparib (Fig. 1E). To improve HJ‐52 selectivity, we applied the first selectivity filter and decided to install an ethynyl group on the C6 position of the phthalazinone scaffold. This terminal alkyne serves two functions: i) promotes selective binding to HYΦ PARPs over HYE PARPs and ii) provides a clickable handle for assessment of target engagement. The final compound, DB008 (Fig. 1C), displayed a 4.3‐fold improvement in potency against PARP16 (IC₅₀ = 275 nM)compared to HJ‐52 (Fig. 1D). Importantly, PARP1 inhibition by DB008 decreased 487‐fold relative to HJ‐52, with an IC₅₀ of 0.925 µM (Fig. 1E). We next sought out to assess the selectivity of DB008 across the PARP family using our PASTA (PARP Activity Screening and Inhibitor Testing Assay) biochemical plate assay (Fig. 2A)¹⁵. We were encouraged to see that DB008 displayed an excellent selectivity profile among the MARylating PARPs and the majority of the PARylating PARPs. One of the prominent off‐targets of DB008 is PARP2 (IC₅₀ = 139 nM), which is not uncommon since all of the approved PARP1 inhibitors (olaparib, rucaparib, niraparib, talazoparib) and the recent clinical PARP7 candidate (RBN‐2397) potently inhibit PARP2 as well^4,16. Nevertheless, we wanted to determine if the inhibition of PARP1/2 by DB008 was due to reversible or irreversible binding. We first confirmed inhibition of PARylation in a cellular context by treating HEK293T cells with DB008, HJ‐52, and olaparib as a positive control, followed by treatment with a PARG inhibitor¹⁷ to boost the PARylation signal for detection by western blot (Fig. 2B). Our cellular PARylation assay recapitulated the results from our in vitro PASTA assay, with olaparib and HJ‐52 displaying low nanomolar potency against PARP1, and DB008 inhibiting PARP1 with an IC₅₀ ~ 1 µM (Fig. 2C). We did not detect auto‐PARylation of PARP2 (molecular weight = 66 kDa), which is likely due to the relatively low expression levels or PARP2 (170 nM) compared to PARP1 (4900 nM) in HEK293T cells¹⁸. To determine whether DB008 inhibits PARylation in a reversible or irreversible manner, we performed a washout experiment in which HEK293T cells were treated with DB008, followed by a series of washouts (replacing media with fresh media lacking DB008). We observed a complete rescue of PARylation after the washout conditions, suggesting that DB008 is not inhibiting PARP1/2 in a covalent manner (Fig. 2D). Having characterized off‐target engagement in the reversible binding mode, we next wanted to profile the selectivity and potency of DB008 in the covalent binding mode. We generated a model to show the predicted covalent binding of DB008 to C169 of PARP16 (Fig. 3A). To confirm covalent binding of PARP16, we transiently expressed Myc2x‐tagged PARP16 in HEK293T cells, then treated cells with a dose response of DB008 for 2 hours. After lysing cells, we performed an azide‐alkyne copper‐catalyzed cycloaddition (CuAAC) reaction to conjugate a TAMRA‐azide to the alkyne of DB008 for monitoring target engagement by in‐gel fluorescence. Excitingly, we observed highly selective labeling of Myc2x‐ PARP16, with saturation occurring at ~300 nM DB008 (Fig. 3B) and an apparent K_d of 110.8 nM (Fig. 3C). We also expressed a Myc2x‐PARP16 C169S mutant and observed a near complete reduction in labeling by DB008, suggesting that DB008 is indeed binding to PARP16 in a covalent manner dependent on C169 (Fig. 3B). We also performed a time course using the 300 nM saturating dose of DB008 (Fig. 3D) and determined that saturation occurs at approximately 120 minutes (Fig. 3E). Finally, we wanted to examine target engagement of DB008 in a non‐overexpressed system. To profile endogenous labeling of PARP16, we treated HAP1 wild‐type (WT) and HAP1 PARP16 knock‐out (KO) cells with a dose response of DB008 and observed selective labeling of a PARP16 (~37 kDa), with saturation occurring at 300 nM DB008 (Fig. 3F), and an apparent K_d of 60.9 nM (Fig. 3G). Importantly, no labeling of PARP16 was detected in the PARP16 KO cells, validating PARP16 target engagement by DB008. These data support DB008 as a membrane permeable, potent, and selective covalent binder of PARP16. It is worth noting that despite decent expression and labeling of PARP16, we have not been able to detect PARP16‐dependent auto‐MARylation or trans‐MARylation activity in cells (data not shown). We do see auto‐MARylation in vitro using a modified version of our PASTA assay (illustrated in Fig. 1D), however, only at high concentrations of NAD⁺ (400 µM) and PARP16 (1 µM added to the plate), which are conditions that force self‐modification similar to assay developed by Wigle et al.¹⁴. We are therefore unable to determine a cellular IC₅₀ value for DB008 against PARP16. However, the IC₅₀ is not the best measure of potency for irreversible inhibitors because it is time‐dependent—longer pre‐incubation times shift the IC₅₀ to lower values. Instead, the more appropriate parameter for measuring potency of covalent inhibitors is K_inact/K_I, a second‐order rate constant that describes the efficiency of covalent bond formation¹⁹. To calculate K_inact/K_I, we conducted time‐dependent labeling of Myc2x‐PARP16 expressed in HEK293T cells, with increasing concentrations of DB008, and determined the K_inact/K_I of DB008 to be 5.95 x10³ M^‐1s^‐1. Importantly, the relationship between inhibitor concentration and K_obs resulted in a saturation binding curve, indicative of a two‐step, specific binding model wherein the inhibitor binds the protein first, forming a reversible protein‐inhibitor complex, followed by covalent bond formation between the nucleophilic residue and electrophile. The ability to label and visualize cellular PARP16, enables use of DB008 as a probe to validate previously reported PARP16 inhibitors in a competition‐based assay, since PARP16 cellular activity has been difficult to detect. We chose to focus on talazoparib and epigallocatechin‐3‐gallate (EGCG) (Fig. 4A), two recently reported PARP16 inhibitors that are commercially available and have been shown to induce cancer cell deathand/or mitigate the unfolded protein response in the context of vascular stress^8,10,20,21. Talazoparib, a potent PARP1 inhibitor, was found to bind PARP16 in SCLC using inhibitor‐ bead conjugates in a chemical proteomics‐based approach.⁸ Using our modified PASTA assay, we determined talazoparib inhibition of PARP16 catalytic activity with an IC₅₀ in the 100‐300 nM range⁸, validating that binding is occurring in the active site. EGCG, a major catechin found in tea, was identified as a binder of purified GST‐PARP16 from a high‐throughput optical‐based microarray screen. An in vitro ADP‐ribosylation assay using biotinylated‐NAD⁺ was used to calculate an IC₅₀ of 14.52 µM for EGCG against GST‐PARP16¹⁰. EGCG, used at 100 µM, was shown to suppress PERK‐mediated UPR in response to ER‐stress in QGY‐7703 and HeLa cells. EGCG was also used at 30‐100 µM to reduce PARP16‐ dependent ER stress in models of vascular aging²⁰ and neointimal hyperplasia²¹. Given the prevalent use of EGCG in PARP16 studies, we wanted to evaluate whether EGCG binds the active site of PARP16. We first performed an in vitro competition assay wherein EGCG and talazoparib (positive control) were pre‐incubated with recombinant PARP16, followed by competition with DB008, clicking to TAMRA‐azide, and visualization of PARP16 binding by in gel‐fluorescence (Fig. 4B). While 1 µM talazoparib competed DB008 labeling effectively (~70%), 100 µM EGCG only partially competed (~30%) labeling. We then wanted to evaluate how effectively talazoparib and EGCG compete labeling in HEK293T cells expressing Myc2x‐PARP16 (Fig. 4C). While talazoparib bound PARP16 with an IC₅₀ of 949 nM, we observed no competition of labeling with EGCG up to 100 µM, suggesting that EGCG does not bind the active site of PARP16 in cells (Fig. 4D). Therefore, we caution the use of EGCG as a probe to study PARP16 biology. The effects on the UPR previously observed with EGCG may be due to off‐target binding, especially at 100 µM, or binding of PARP16 at an allosteric site. Lastly, we wanted to validate whether PARP16 KO or inhibition of PARP16 by DB008 prevents UPR signaling through PERK in response to ER‐stress, as observed in previous studies^6,10,20,21. Upon treating HAP1 WT and PARP16 KO cells with DB008 and tunicamycin to induce ER‐stress, we observed no change in levels of ATF4 or phosphorylated eiF2α. Palve et al. also examined the role of PARP16 in regulating IRE1α‐mediated UPR signaling in SCLC and Ewings Sarcoma under tunicamycin‐induced ER stress. They also observed no effect on XBP1 splicing upon siRNA knockdown of PARP16⁸. Together these data suggest that PARP16 may not be involved in the UPR as previously described, or perhaps the role of PARP16 in UPR is tissue‐ specific. (1) Cohen et al., Insights into the Biogenesis, Function, and Regulation of ADP‐Ribosylation. Nat Chem Biol 2018, 14 (3), 236–243. https://doi.org/10.1038/nchembio.2568. (2) Sanderson et al., Mechanisms Governing PARP Expression, Localization, and Activity in Cells. Crit Rev Biochem Mol 2020, 55 (6), 1–14.

(3) Cohen et al., Insights into the Biogenesis, Function, and Regulation of ADP‐Ribosylation. Nat Chem Biol 2018, 14 (3), 236–243. https://doi.org/10.1038/nchembio.2568. (4) Sanderson et al.,Mechanisms Governing PARP Expression, Localization, and Activity in Cells. Crit Rev Biochem Mol 2020, 55 (6), 1–14. https://doi.org/10.1080/10409238.2020.1818686. (5) Mateo et al., A Decade of Clinical Development of PARP Inhibitors in Perspective. Ann Oncol 2019, 30 (9), 1437–1447. https://doi.org/10.1093/annonc/mdz192. (6) Gozgit et al., PARP7 Negatively Regulates the Type I Interferon Response in Cancer Cells and Its Inhibition Triggers Antitumor Immunity. Cancer Cell 2021, 39 (9), 1214‐1226.e10. https://doi.org/10.1016/j.ccell.2021.06.018. (7) Schenkel et al., A Potent and Selective PARP14 Inhibitor Decreases Protumor Macrophage Gene Expression and Elicits Inflammatory Responses in Tumor Explants. Cell Chem Biol 2021, 28 (8), 1158‐ 1168.e13. https://doi.org/10.1016/j.chembiol.2021.02.010. (8) Jwa et al., PARP16 Is a Tail‐Anchored Endoplasmic Reticulum Protein Required for the PERK‐ and IRE1α‐Mediated Unfolded Protein Response. Nat Cell Biol 2012, 14 (11), 1223–1230. https://doi.org/10.1038/ncb2593. (9) Hetz et al., Targeting the Unfolded Protein Response in Disease. Nat Rev Drug Discov 2013, 12 (9), 703–719. https://doi.org/10.1038/nrd3976. (10) Palve et al., The Non‐Canonical Target PARP16 Contributes to Polypharmacology of the PARP Inhibitor Talazoparib and Its Synergy with WEE1 Inhibitors. Cell Chem Biol 2021. https://doi.org/10.1016/j.chembiol.2021.07.008. (11) Challa et al., Ribosome ADP‐Ribosylation Inhibits Translation and Maintains Proteostasis in Cancers. Cell 2021. https://doi.org/10.1016/j.cell.2021.07.005. (12) Wang et al., Epigallocatechin‐3‐Gallate Enhances ER Stress‐Induced Cancer Cell Apoptosis by Directly Targeting PARP16 Activity. Cell Death Discov 2017, 3 (1), 17034. https://doi.org/10.1038/cddiscovery.2017.34. (13) Centko et al., Combination of Selective PARP3 and PARP16 Inhibitory Analogues of Latonduine A Corrects F508del‐CFTR Trafficking. Acs Omega 2020, 5 (40), 25593–25604. https://doi.org/10.1021/acsomega.0c02467. (14) Morgan et al., Rational Design of Cell‐Active Inhibitors of PARP10. Acs Med Chem Lett 2018, 10 (1), 74–79. https://doi.org/10.1021/acsmedchemlett.8b00429. (15) Kirby et al., A Potent and Selective PARP11 Inhibitor Suggests Coupling between Cellular Localization and Catalytic Activity. Cell Chem Biol 2018, 25 (12), 1547‐1553.e12. https://doi.org/10.1016/j.chembiol.2018.09.011. (16) Wigle et al., Forced Self‐Modification Assays as a Strategy to Screen MonoPARP Enzymes. Slas Discov 2020, 25 (3), 241–252. https://doi.org/10.1177/2472555219883623. (17) Kirby et al., PASTA: PARP Activity Screening and Inhibitor Testing Assay. Star Protoc 2021, 2 (1), 100344. https://doi.org/10.1016/j.xpro.2021.100344. (18) Thorsell et al., Structural Basis for Potency and Promiscuity in Poly(ADP‐Ribose) Polymerase (PARP) and Tankyrase Inhibitors. J Med Chem 2016, 60 (4), 1262–1271. https://doi.org/10.1021/acs.jmedchem.6b00990. (19) James et al., First‐in‐Class Chemical Probes against Poly(ADP‐Ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib. Acs Chem Biol 2016, 11 (11), 3179–3190. https://doi.org/10.1021/acschembio.6b00609. (20) Cho et al., OpenCell: Endogenous Tagging for the Cartography of Human Cellular Organization. Science 2022, 375 (6585), eabi6983. https://doi.org/10.1126/science.abi6983. (21) Strelow, A Perspective on the Kinetics of Covalent and Irreversible Inhibition. J Biomol Screen 2016, 22 (1), 3–20. https://doi.org/10.1177/1087057116671509. (22) Yang et al., Smyd3‐PARP16 Axis Accelerates Unfolded Protein Response and Vascular Aging. Aging. (23) Long et al., SMYD3–PARP16 Axis Accelerates Unfolded Protein Response and Mediates Neointima Formation. Acta Pharm Sinica B 2021, 11 (5), 1261–1273. https://doi.org/10.1016/j.apsb.2020.12.010.

Claims

What is claimed: 1. A compound of Formula (I): wherein:

R₁ is selected from the group of H, halo, ‐OH, C₁‐₆alkyl, C_2‐6alkenyl, C_2‐6 alkynyl, C_3‐6 cycloalkyl, C_3‐ ₆ heterocyclyl, C_5‐6 aryl, C_5‐6 heteroaryl, ‐O‐C_1‐6 alkyl,‐O‐C_2‐6alkenyl,‐O‐C_2‐6alkynyl, ‐O‐C_3‐6cycloalkyl, ‐O‐ C_5‐6 heterocyclyl, ‐O‐ C_5‐6 aryl, ‐O‐ C_5‐6 heteroaryl, ‐SO₂‐C_3‐6cycloalkyl, ‐NH₂, ‐NH(C_1‐3alkyl), ‐N(C_1‐3alkyl)₂, C_2‐6alkynyl‐NH₂, C_2‐6alkynyl‐NH(C_1‐3alkyl), and C_2‐6alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, Cl, Br, I, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐CF₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 2. The compound of Claim 1, with the proviso that when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 3. The compound of Claim 1, wherein: R₁ is selected from the group of H, halo, ‐OH, C₁‐₄alkyl, C_2‐4alkenyl, C_2‐4 alkynyl, C_3‐6 cycloalkyl, C_3‐ ₆ heterocyclyl, C_5‐6 aryl, C_5‐6 heteroaryl, ‐O‐C_1‐4 alkyl,‐O‐C_2‐4alkenyl,‐O‐C_2‐4alkynyl, ‐O‐cycloalkyl, ‐O‐ C_5‐6 heterocyclyl, ‐O‐C_5‐6 aryl, ‐O‐ C_5‐6 heteroaryl, ‐SO₂‐C_3‐6cycloalkyl, ‐NH₂, ‐NH(C_1‐3alkyl), ‐N(C_1‐3alkyl)₂, C_2‐6 alkynyl‐NH₂, C_2‐4alkynyl‐NH(C_1‐3alkyl), and C_2‐4alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, Cl, Br, I, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐CF₃; R₄ is selected from the group of H, F, Cl, Br, I, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 4. The compound of Claim 1, wherein: R₁ is selected from the group of H, C₁‐₃alkyl, C_2‐3alkenyl, C_2‐3 alkynyl, C_2‐3alkynyl‐NH₂, C_2‐3alkynyl‐ NH(C_1‐3alkyl), and C_2‐3alkynyl‐N(C_1‐3alkyl)₂; R₂ and R₃ are each independently selected from the group of H, F, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐ CF₃; with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; R₄ is selected from the group of H, F, CH₃, and CF₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 5. The compound of Claim 1, wherein R₁ is selected from the group of C_2‐3alkenyl and C_2‐3 alkynyl; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 6. A compound of Formula (II):

wherein:

R₂ and R₃ are each independently selected from the group of H, F, CH₃, CN, ‐CH₂CN, CF₃, and CH₂‐ CF₃; with the proviso that, when R₂ is CN, ‐CH₂CN, CF₃, or CH₂‐CF₃, R₃ is selected from the group of H, F, Cl, Br, I, and CH₃; R₄ is selected from the group of H, F, CH₃, and CF₃; R₆ is selected from the group of H, F, and CH₃; and R₅ and R₇ are each independently selected from the group of H and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 7. The compound of Claim 6, wherein: R₂ is H; R₃ is selected from the group of H and CH₃; R₄ is selected from the group of H, F, and CH₃; R₆ is selected from the group of H, F, and CH₃; and R₅ and R₇ are each independently selected from the group of H and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 8. A compound of Formula (III):

wherein: R₄ is selected from the group of H, F, and CH₃; R₆ is selected from the group of H, F, and CH₃; and R₅ and R₇ are each independently selected from the group of H and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 9. The compound of Claim 8, wherein: R₄ is selected from the group of H, and CH₃; R₅ is selected from the group of H and CH₃; and R₆ is selected from the group of H, F, and CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 10. The compound of Claim 1, wherein R₂ is H and R₃ is CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, prodrug thereof. 11. The compound of Claim 1, wherein R₂ and R₃ are each CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 12. The compound of Claim 1, wherein R₂ and R₃ are bound to the piperazine ring as indicated in the structure below:

or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 13. The compound of Claim 1, wherein R₂ and R₃ are bound to the piperazine ring as indicated in the structure below:

or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 14. The compound of Claim 1, wherein R₂ and R₃ are bound to the piperazine ring as indicated in the structure below:

or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 15. The compound of Claim 1, wherein R₂ and R₃ are bound to the piperazine ring as indicated in the structure below:

or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 16. The compound of Claim 1, wherein R₄ is H; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 17. The compound of Claim 1, wherein R₄ is CH₃; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 18. The compound of Claim 1, which is: or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate,

isomer, tautomer, isotope, polymorph, prodrug thereof. 19. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of a compound of Claim 1; or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof. 20. A method of treating in a subject an ER stress‐related cancer selected from the group of colon adenocarcinoma, esophagus adenocarcinoma, liver hepatocellular carcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet cell tumor, rectum adenocarcinoma, gastrointestinal stromal tumor, stomach adenocarci noma, adrenal cortical carcinoma, renal cancer, thyroid cancer, melanoma, testicular cancer, follicular carcinoma, papillary carcinoma, breast cancer, ductal carcinoma, lobular carcinoma, intraductal carcinoma, mucinous carcinoma, phyllodes tumor, Ewing’s sarcoma, ovarian adenocarcinoma, endometrium adenocarcinoma, granulose cell tumor, mucinous cystadenocarcinoma, cervix adenocarcinoma, vulva squamous cell carcinoma, basal cell carcinoma, prostate adenocarcinoma, giant cell tumor of bone, bone osteosarcoma, larynx carcinoma, lung adenocarcinoma, kidney carcinoma, urinary bladder carcinoma, Wilm’s tumor, and lymphoma; the method comprising administering to the subject in need thereof a pharmaceutically effective amount of a compound of Claim 1, or a pharmaceutically acceptable salt, co‐crystal, solvate, hydrate, isomer, tautomer, isotope, polymorph, prodrug thereof.