Sirtuin-modulating compounds, including sirtuin-activating compounds, and applications thereof

Abstract

In one aspect, described herein are arylcarboximidamide compounds that comprise a carboximidamide molecular structure and modulate mammalian sirtuin enzymes (SIRT1-7), particularly compounds for modulating SIRT3. Using DNA-encoded library technology (ELT), novel small molecules that modulate sirtuin activity have been developed. Modulation of sirtuin activity includes sirtuin activation and / or sirtuin inhibition.

Description

[Technical Field]

[0001] Related application data This application claims priority pursuant to 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 353,248, filed June 17, 2022, which is incorporated herein by reference in its entirety.

[0002] Field The present application addresses compounds that exhibit sirtuin modulating functionality, particularly compounds for the modulation of SIRT3. [Background technology]

[0003] NAD + Sirtuins (silent signal regulators) enzymes, which catalyze NAD-dependent protein post-translational modifications, have emerged as critical regulators of many cellular pathways. In particular, these enzymes protect against age-related diseases and act as key mediators of longevity in evolutionarily distant organismal models. Sirtuins regulate NAD + is a NAD-dependent lysine deacetylase that requires the cofactor NAD to cleave the acyl group from the lysine side chain of its substrate protein. + requires.

[0004] A complete understanding of sirtuin chemistry is not only of fundamental importance, but also of considerable medical importance, as there is currently great interest in developing novel mechanism-based sirtuin modulators. The overall catalytic process has been suggested to proceed in two successive steps. The first step is the cleavage of NAD + This reaction involves cleavage of the nicotinamide moiety of NAD and nucleophilic attack of the acetyl-Lys side chain of the protein substrate, forming a positively charged O-alkylimidate intermediate. Nicotinamide-induced reversal of the intermediate (the so-called base exchange reaction) results in the formation of NAD + and acetyl-Lys protein reformation. The energetics of this reversible reaction depend on the strength of nicotinamide (NAM) inhibition of sirtuins and the NAD + Michaelis constant (K m, NAD+The second stage of sirtuin catalysis, which contains the rate-limiting step, involves four sequential steps that ultimately result in deacetylation of the Lys side chain of the protein substrate and the formation of an O-acetyl ADP-ribose by-product.

[0005] In recent years, there has been intense interest in activating the seven mammalian sirtuin enzymes (SIRT1-7) to combat aging. Compared to enzyme inhibitors, which constitute the majority of today's drugs, enzyme activators offer considerable advantages. However, enzyme catalysis has been optimized over billions of years of evolution and is therefore much more difficult to design. Previous studies on sirtuin activation have focused exclusively on allosteric activation of the SIRT1 enzyme and have focused on experimental screening. Indeed, small molecule allosteric activators of SIRT1 have been shown to induce lifespan extension in model organisms such as mice. Allosteric activation is one of four known ways in which small molecules can activate enzymes. These are characterized by the substrate dissociation constant (for sirtuins, the acetylated protein dissociation constant, K ). d,Ac-Pr It works by reducing

[0006] Nearly all known sirtuin activators allosterically target SIRT1 and do not bind to the activation site. However, allosteric activators only work with specific substrates of SIRT1. Other sirtuins, including SIRT2, SIRT3, and SIRT6, are now known to play important roles in regulating mammalian lifespan. Therefore, a general strategy for activating any mammalian sirtuin (including activating SIRT1 with other substrates) is highly important but poorly understood. Generally, substrate K d Allosteric activation to reduce ATP is not an option for enzyme activation, and mechanism-based activation is important.

[0007] A basis for the rational design of mechanism-based activators has been lacking. Several types of mechanism-based sirtuin inhibitors, including Ex-527, have been reported in the literature in recent years. However, mechanism-based activation has proven much more elusive due to the difficulty of screening for the balance of properties required for a modulator to bind to the active site and accelerate catalysis. While there are many ways to inhibit enzyme mechanisms, there are far fewer ways to activate them. Only about a dozen distinct types of small molecule enzyme activators are currently known, and across all enzyme families, only four modes of activation are known. None of these modes of activation exploit the unique catalytic mechanism of the target enzyme. Summary of the Invention

[0008] In one aspect, compounds that modulate sirtuin activity are described herein. Modulation of sirtuin activity includes sirtuin activation and / or sirtuin inhibition. In some embodiments, the sirtuin-modulating compound and / or salt thereof has Formula I: [ka] In the formula, Ar 1 is aryl or heteroaryl, and R1-R7 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, —C(O)R8, alkoxy, halo, nitrile (—NO2), and hydroxy; Ar 1 , alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, and hydroxy; R8 is alkyl, alkenyl, and NR9R 10 R9 and R 10are independently selected from the group consisting of hydrogen, alkyl, and cycloalkyl, and m and n are integers each having a value independently selected from 0 to 10. In some embodiments, the compound of formula I and / or its salt is: [ka] is.

[0009] In another embodiment, the sirtuin-modulating compound and / or salt thereof has Formula II: [ka] wherein Ar is aryl or heteroaryl; R1-R6 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, —C(O)R7, alkoxy, halo, nitrile (—NO2), and hydroxy; and Ar 1 , alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally (C1-C 10 )-Alkyl, (C1-C 10 )-alkenyl, alkoxy, halo, amine, alkoxy-amido, and hydroxy; R7 is selected from the group consisting of alkyl, alkenyl, and NR8R9; R8 and R9 are independently selected from the group consisting of hydrogen, alkyl, and cycloalkyl; and m and n are each integers having values independently selected from 0 to 10. In some embodiments, for example, the compound of Formula II and / or its salt is: [ka] is.

[0010] In another embodiment, the compound of formula II and / or its salt is: [ka] is.

[0011] In another embodiment, the sirtuin-modulating compound and / or salt thereof has Formula III: [ka] In the formula, Ar 1 is aryl or heteroaryl, R is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine, and —C(O)NR R ; 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and amine are optionally selected from the group consisting of (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, and hydroxy; R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and heteroaryl; R 2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, amine, alkylamine, and O; Y is selected from the group consisting of N and CH; and X is selected from the group consisting of NH, S, and O. In some embodiments, the compound of Formula III and / or its salt is: [ka] Contains JPEG2025525358000009.jpg104165.

[0012] In another embodiment, the sirtuin-modulating compound and / or salt thereof has Formula IV: [ka] In the formula, Ar 1 is aryl or heteroaryl, R is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine, and —C(O)NR R ; 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and amine are optionally selected from the group consisting of (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, and hydroxy; R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and heteroaryl; R 2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, amine, alkylamine, and O; Y is selected from the group consisting of N and CH; and X is selected from the group consisting of NH, S, and O. In some embodiments, the compound of Formula III and / or its salt is: [ka] Includes:

[0013] In a further embodiment, the sirtuin-modulating compound and / or salt thereof has Formula V: [ka] wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, and heterocycloalkyl; Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, and aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, and hydroxyl; X is selected from the group consisting of O, S, and N; and m and n are integers each having a value independently selected from 0 to 10. It is of the type.

[0014] In some embodiments, the compound of formula V and / or its salt is: [ka] JPEG2025525358000014.jpg63165.

[0015] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula VI: [ka] wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, and heterocycloalkyl; Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, and aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10)-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, and hydroxyl In some embodiments, the compound of formula VI and / or its salt is selected from one of the following:

[0016] In some embodiments, the compound of Formula VI and / or its salt is: [ka] is selected from one of the following:

[0017] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula VII: [ka] wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl; Ar1 and Ar2 are independently selected from aryl and heteroaryl, wherein aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of Formula VII and / or its salt is:

[0018] In some embodiments, the compound of Formula VII and / or its salt is: [ka] is.

[0019] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula VIII: [ka] wherein R1-R3 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, and heterocycloalkyl, and the alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of alkoxy, halo, amine, nitro, and hydroxyl. In some embodiments, the compound of Formula VIII and / or its salt is: [ka] is.

[0020] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula IX: [ka] wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, akynyl, acyl, heteroalkyl, cycloalkyl, heterocycloalkyl, amido, and keto, wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, amido, and keto are optionally substituted with one or more substituents selected from the group consisting of aryl, heteroaryl, alkoxy, halo, amine, nitro, and hydroxyl. In some embodiments, the compound of Formula IX and / or its salt is: [ka] is.

[0021] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula X: [ka] wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, and heterocycloalkyl; Ar 1 is selected from aryl and heteroaryl, and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, aryl, alkoxy, halo, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of formula X and / or its salt is: [ka] is.

[0022] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula XI: [ka] In the formula, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, and aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of formula XI and / or its salt is: [ka] is.

[0023] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula XII: [ka] In the formula, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, and aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of formula XII and / or its salt is: [ka] is.

[0024] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula XIII: [ka] In the formula, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, and aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of Formula XIII and / or its salt is: [ka] is.

[0025] In another embodiment, the sirtuin-modulating compound and / or salt thereof has the formula XIV: [ka] wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, heteroalkenyl, heteroalkynyl, and heterocycloalkyl; Ar is selected from aryl and heteroaryl, wherein aryl and heteroaryl are optionally selected from (C1-C 10 )-Alkyl, (C1-C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl In some embodiments, the compound of formula XIV is: [ka] is.

[0026] As further described herein, compounds of Formulas I-XIV and / or salts thereof, in some embodiments, can modulate SIRT3. Additionally, methods of treating a patient are also described herein. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound selected from Formulas III-XIV herein, wherein the compound elevates NAD+ in the patient. In some embodiments, administration of a compound described herein enhances cellular metabolism and / or energy production in the patient. Compounds of Formulas III-XIV, in some embodiments, can also restore the activity of the abundant mitochondrial sirtuins observed in younger individuals by increasing the sensitivity of sirtuins to NAD+, providing an effective treatment for age-related diseases, including mitochondrial diseases.

[0027] These and other embodiments are further described in the detailed description that follows. [Brief explanation of the drawings]

[0028] [Figure 1] Frequency of all compounds in the natural library. All derivatives of the 20 amino acids show a similar distribution with respect to their 194 carboxylic acid counterparts in the library. [Figure 2] SIRT3 selection in the presence of Carba-NAD and acetylated peptides. Derivatives of AC71 and AC92 show the highest enrichment. Among others, some derivatives of AC40 and AC56 also show significant enrichment. The Y-axis shows the calculated enrichment for each compound as the ratio of its frequency in the targeted selection to its frequency in the non-targeted selection. [Figure 3] SIRT3 selection in the presence of OAADPr and deacetylated peptides. Derivatives of AC71 and AC92 show the highest enrichment. Among others, some derivatives of AC40 and AC56 also show significant enrichment. The Y-axis shows the calculated enrichment for each compound as the ratio of its frequency in the targeted selection to its frequency in the non-targeted selection. [Figure 4] Effect of top-ranking hit compounds on Sirt3 deacetylation activity. Bar chart of % control with different compounds ([NAD+]=1 mM, [FdL2 peptide]=50 μM, [cpd]=1, 10, 50 μM or max, [E]0 / [NAD]0=0.000245, time point=30 min, n=2). [Figure 5] Sirt3 activation by top-ranking hit compounds. Bar graph of % control (HKL) by different compounds ([NAD+]=50 μM, [MnSOD K122]=600 μM, [cpd]=10 μM or max, [E]0 / [NAD]0=0.1223, time point=2 min, n=3). [Figure 6]DNA-tagged and C-terminally amidated forms of selected active and inactive ligands identified by affinity selection assays. (A) Structure of the DNA-tagged form of the active ligand, AA12-AC71; (B) Structure of the C-terminally amidated form of AA12-AC71; (C) Structure of the DNA-tagged form of another active ligand, AA20-AC92; (D) Structure of the C-terminally amidated form of AA20-AC92; (E) Structure of the DNA-tagged form of the inactive ligand, AA5-AC117; (F) Structure of the C-terminally amidated form of AA5-AC117. Compounds shown in B, D, and F were used for all ligand docking studies instead of those in A, C, and E. [Figure 7] Location of site 1 in 4BVG and top poses of docked hits. (A) The location of the first putative atypical site defined by pseudo-atoms in 4BVG is approximately antipodal to the known internal site. Cyan is used to highlight lining residues. (B) Close-up of the top docking pose of AA5 / 7 / 11 / 12-AC92 at the putative atypical site. (C) An overlay of the top docking pose of AA5 / 7 / 11 / 12-AC92 shows a high degree of overlap for the pharmacophore. (D) An overlay of the top docking pose of AA5 / 7 / 11 / 12-AC71 also shows a high degree of overlap for the pharmacophore at atypical site 1 in 4BVG. [Figure 8] Docking poses of hit compound 6875218 (Figure 13) in the SIRT3 open (4FVT) and closed (4BVG) loop conformations. The figure shows the ternary complex of SIRT3 bound to Ac-ACS peptide and Carba-NAD. The protein is shown in cyan, the peptide in green, NAD+ in blue, and the compound in pink. Protein residues that form interactions are shown as white sticks. Intermolecular hydrogen bonds are shown as yellow dotted lines with corresponding distance labels. [Figure 9] Modulatory effects of the top 70 selected compounds under steady-state conditions (N=3). [Figure 10]Modulatory effects of the top 20 selected compounds under non-steady-state conditions (N=3). [Figure 11] Common structural patterns among Sirt3 activator hit compounds. ChemBridge IDs (A) 6068318 (Figure 12), (B) 5749820, (C) 5761865, (D) 7965907 (Figure 31). All compounds presented here appear to share certain common structural features, specifically a scaffold consisting of a macrobicyclic aromatic ring linked to a six-membered aromatic by an amide group. [Figure 12] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 13] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 14] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 15] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 16] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 17] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 18] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 19] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 20] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 21] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 22] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 23] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 24] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 25] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 26] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 27] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 28] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 29] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 30] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 31] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 32] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 33] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 34] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 35] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 36] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 37] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 38] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 39] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 40] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 41] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 42]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 43] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 44] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 45] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 46] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 47] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 48] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 49] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 50] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 51] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 52] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 53] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 54] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 55] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 56] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 57] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 58] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 59]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 60] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 61] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 62] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 63] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 64] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 65] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 66] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 67] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 68] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 69] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 70] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 71] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 72] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 73] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 74] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 75] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 76]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 77] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 78] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 79] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 80] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 81] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 82] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 83] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 84] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 85] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 86] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 87] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 88] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 89] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 90] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 91] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 92] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 93]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 94] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 95] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 96] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 97] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 98] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 99] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 100] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 101] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 102] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 103] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 104] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 105] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 106] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 107] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 108] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 109] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 110]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 111] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 112] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 113] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 114] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 115] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 116] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 117] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 118] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 119] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 120] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 121] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 122] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 123] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 124] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 125] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 126] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 127]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 128] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 129] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 130] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 131] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 132] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 133] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 134] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 135] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 136] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 137] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 138] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 139] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 140] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 141] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 142] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 143] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 144]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 145] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 146] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 147] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 148] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 149] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 150] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 151] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 152] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 153] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 154] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 155] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 156] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 157] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 158] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 159] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 160] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 161]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 162] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 163] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 164] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 165] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 166] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 167] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 168] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 169] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 170] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 171] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 172] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 173] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 174] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 175] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 176] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 177] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 178]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 179] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 180] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 181] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 182] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 183] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 184] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 185] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 186] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 187] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 188] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 189] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 190] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 191] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 192] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 193] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 194] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 195]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 196] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 197] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 198] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 199] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 200] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 201] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 202] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 203] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 204] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 205] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 206] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 207] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 208] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 209] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 210] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 211] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 212]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 213] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 214] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 215] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 216] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 217] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 218] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 219] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 220] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 221] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 222] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 223] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 224] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 225] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 226] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 227] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 228] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 229]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 230] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 231] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 232] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 233] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 234] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 235] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 236] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 237] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 238] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 239] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 240] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 241] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 242] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 243] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 244] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 245] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 246]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 247] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 248] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 249] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 250] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 251] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 252] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 253] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 254] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 255] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 256] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 257] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 258] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 259] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 260] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 261] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 262] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 263]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 264] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 265] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 266] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 267] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 268] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 269] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 270] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 271] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 272] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 273] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 274] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 275] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 276] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 277] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 278] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 279] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 280]Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 281] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 282] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 283] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 284] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 285] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 286] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 287] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 288] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 289] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 290] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 291] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 292] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 293] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Fig. 294] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 295] Exemplary compounds of Formulas 3, 5-11, and 14 described herein. [Figure 296] FIG. 1 illustrates the compound screening process used in some embodiments of the present application. [Figure 297](A)-(D). Effect of VS hit compounds on SIRT3 deacetylase activity under steady-state conditions: dose-response curves and time series of enzyme activation. (A) Dose-response curves for three VS hit compounds in the presence of 1 mM NAD+ and 50 μM MnSOD peptide, [E]o / [NAD+]o = 0.00157, t = 30 min, N = 3. (B) Bar graph of % control of two VS hit compounds (20 μM 5329973 or 1 μM 5689785) on SIRT3 deacetylase activity under steady-state conditions, [NAD+] = 100 μM, [MnSOD K122] = 600 μM, [E]o / [NAD+]o = 0.00157, t = 10 min, N = 2. (*P < 0.001). Plot of product formation versus time in the presence and absence of (C) 20 μM 5329973 (N=2) for [NAD+]=100 and 500 μM, [MnSOD K122]=600 μM, [E]o / [NAD+]o=0.00157; (D) 1 μM 5689785 (N=2) for [NAD+]=100 and 500 μM, [MnSOD K122]=600 μM, [E]o / [NAD+]o=0.00157. [Figure 298] (A)-(E). Binding affinity measurements for complexes in the sirtuin reaction mechanism. (A, C, E) Pathway in the sirtuin reaction network; E, enzyme; Ac-Pr, acetylated peptide substrate; NAD, nicotinamide adenine dinucleotide; A, regulator (HKL or hit compound 5329973); NAM, nicotinamide adenine mononucleotide. (B) Carba-NAD binding in the ternary complex: Effect of the mechanism-based regulator HKL. Carba-NAD (c-NAD) binding to Sirt3. Ac-MnSOD complex in the presence and absence of 6.25 μM HKL measured using MST. (D) HKL binding: Effect of NAM. HKL binding to Sirt3. Ac-MnSOD complex in the presence and absence of NAM measured using MST. (F) Hit compound 5329973 binding to the apoenzyme Sirt3. [Figure 299](A)-(D). Steady-state kinetic characterization of the MnSOD substrate hSIRT3102-399 deacetylation by the non-steady-state activator HKL and the steady-state activator. Double-reciprocal plots of initial deacetylation rates measured in the presence and absence of HKL at (A) [NAM] = 0 μM and (B) [NAM] = 5 mM. Double-reciprocal plots of initial SIRT3 deacetylation rates measured in the presence and absence of (C) 20 μM 5329973 and (D) 1 μM 5689785. Initial rates were measured at a series of time points with different [NAD+] and at [MnSOD K122] = 600 μM (N = 2). DETAILED DESCRIPTION OF THE INVENTION

[0029] The embodiments described herein may be more readily understood by reference to the following detailed description and examples, as well as the preceding and subsequent descriptions thereof. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0030] definition The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain saturated hydrocarbon group, optionally substituted with one or more substituents. For example, alkyl refers to C1 to C6 30 or C1~C 18 may be.

[0031] The term "alkenyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0032] The term "aryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system, optionally substituted with one or more substituents. The term "aryl" includes fused and non-fused ring systems, which include alkylene and sulfonamide linkages between the rings.

[0033] The term "heteroaryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, such as nitrogen, oxygen, and / or sulfur. The term "heteroaryl" includes fused and non-fused ring systems, and non-fused ring systems include alkylene and sulfonamide linkages between the rings. For polycyclic ring systems, each ring need not be aromatic.

[0034] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic, monocyclic or polycyclic ring system, optionally substituted with one or more ring substituents.

[0035] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic, monocyclic or polycyclic ring system in which one or more of the atoms in the ring system, alone or in combination, is an element other than carbon, for example, nitrogen, oxygen, or sulfur, and the ring system is optionally substituted with one or more ring substituents.

[0036] The term "heteroalkyl," as used herein, alone or in combination, refers to an alkyl moiety as defined above having one or more carbon atoms in the chain, e.g., 1, 2 or 3 carbon atoms, substituted with one or more heteroatoms, which may be the same or different, and the point of attachment to the rest of the molecule is through a carbon atom of the heteroalkyl radical.

[0037] The term "alkoxy," as used herein, alone or in combination, refers to the moiety RO-, where R is alkyl or alkenyl as defined above.

[0038] The term "halo," as used herein, alone or in combination, refers to an element in Group VIIA (Halogen) of the Periodic Table. Depending on the chemical environment, a halo can be in a neutral or anionic state.

[0039] I. DNA-encoded library selection (Equations I and II) In an effort to develop novel small molecules that modulate sirtuin activity, we used DNA-encoded library technology (ELT). This technology provided a robust hit identification approach using large collections of diverse DNA-encoded small molecule libraries that were screened for affinity to desired protein targets. This technology provided an efficient way to screen a broad chemical space of structures. It is also an attractive strategy because it requires only small amounts of target protein to perform selection experiments and identifies ligands regardless of their functional activity.

[0040] Based on the design defined in Mannocci et al., a 3880-member DNA-encoded library consisting of 194 carboxylic acid and 20 amino acid couplings was generated and screened. Affinity-mediated selection against SIRT3 was performed using Carba-NAD (stable NAD). + Carba-NAD or OAADPr, along with acetylated or deacetylated peptide substrates, are required to display a functional SIRT3 conformation.

[0041] For each screening, a no-target screen was performed simultaneously, serving as a negative control. This allowed for the elimination of any compounds in the library that nonspecifically bound to the nickel beads. All screening experiments were performed at 4°C to preserve SIRT3 activity. During the selection process, special care was taken to wash the beads after incubation of the protein with the library to eliminate as many nonspecific binders as possible. Only one round of selection was performed, and the final eluate was amplified, clustered, and sequenced using an Illumina iseq100. Prior to selection, we sequenced the natural library to ensure that all compounds were present at roughly similar concentrations, thus ensuring that selection was not biased toward any compounds that may be present at higher concentrations than others. Figure 1 shows a 3D plot of the natural library, showing the DNA barcode reads corresponding to each compound. This is representative of the distribution of compounds in the library. We observed that all 194 derivatives of each of the 20 amino acids showed similar distributions in terms of frequency after next-generation sequencing. Only the AA5 derivative appeared to be slightly overrepresented in the natural library. The initial frequencies of the compounds helped us to normalize the final output and counteract any bias that may occur due to differences in the concentrations of individual compounds in the natural library.

[0042] Figures 2 and 3 show 3D plots of the enrichment of compounds above the calculated background (no-target selection) after normalizing their abundance with respect to the total number of DNA sequence reads obtained for each selection. The top 10 highly enriched samples were selected for characterization and are listed in Table 1. All of these compounds showed 12- to 16-fold enrichment over the no-target control selection.

[0043] Interestingly, for both selection conditions, the same carboxylic acid derivatives, AC71 and AC92, showed the highest enrichment in the presence of Carba-NAD and OAADPr. Among others, several derivatives of AC40 and AC56 showed significant enrichment, 4- to 8-fold, over negative selection, and these are worthy of further investigation. Compounds were not overrepresented in the natural library, further supporting the hypothesis that enrichment is observed solely due to binding to SIRT3. These compounds showed similar enrichment when run in duplicate for both selection conditions. This highlights the role of both AC71 and AC92 derivatives as putative binding partners of SIRT3, which was the rationale behind our selection of them for further analysis. We observed that nearly all compounds in the library did not exhibit any affinity for nickel beads, confirming the correct choice of immobilization matrix. All of the top 10 hits are derivatives of either AC71 or AC92, and four of their corresponding amino acids, AA5, AA8, AA11, and AA12, are common to both derivatives.

[0044] Therefore, it was decided to synthesize seven compounds: six (AA12AC92, AA12AC78, AA12AC71, AA8AC92, AA8AC71, AA8AC40) that were among the compounds showing optimal enrichment (top 10 hits), and one (AA12AC02Bis) that showed no enrichment and served as a negative control. These compounds were synthesized off-DNA (DNA strands substituted with butyl groups) to verify their binding affinity with Switchsense® and to test their modulating effect on SIRT3 activity. Table 1. Chemical structures of the top 10 hits from both SIRT3 selection experiments in the presence of Carba-NAD or OAADPr. These compounds were resynthesized without the oligonucleotide conjugate for further characterization. [Table 1]

[0045] SIRT3 regulatory effect of hit compounds Test compounds are expected to undergo three phases during the enzymatic reaction: pre-steady state, steady state, and post-steady state. The pre-steady state and post-steady state can also be considered as non-steady state (state II for activity testing), where the enzyme concentration versus NAD + The substrate concentration ratio is high. Depending on the initial ratio of enzyme to limiting substrate in the system (and the ratio of enzyme to test compound concentrations), the net effect of the test compound over the course of the reaction may be either inhibitory or activating; if this ratio is higher than a threshold, activation occurs.

[0046] The six top-ranked compounds from the DEL screening were tested for their SIRT3 modulatory effects using a fluorescence-based assay (Figure 4). Selected compounds exhibit different degrees of inhibitory effects under steady-state conditions (condition I for activity testing). SIRT3 deacetylation activity was found to be inhibited by 66.3% in the presence of 50 μM compound AA8AC40. It was also found that the level of inhibition increased with increasing compound dose. IC for the compounds 50 Values are expected to fall into the μM range (Table 2). The negative control (AA12AC02BIS) does not contribute to SIRT3 modulation. Activity results are consistent with the DEL screening output. Table 2. Potency of selected compounds on SIRT3 deacetylation activity [Table 2]

[0047] Non-steady-state activation of SIRT3 An HPLC-based assay was used to detect the deacetylation activity of SIRT3 in the presence of 10 μM of the compound AA8AC40 under non-steady-state conditions (Figure 5). In previous studies, HKL was thoroughly investigated as a model non-steady-state SIRT3 activator. Test compounds are expected to undergo three phases during the enzymatic reaction: pre-steady state, steady state, and post-steady state. The pre-steady and post-steady states can also be considered non-steady states, where the enzyme concentration versus NAD + The substrate concentration ratio is high. Depending on the initial ratio of enzyme to limiting substrate in the system (and the ratio of enzyme to test compound concentrations), the net effect of the test compound over the course of the reaction may be either inhibitory or activating; if this ratio is higher than a threshold, activation occurs.

[0048] In the current study, the top hit compounds have a larger structural volume compared to HKL (structure below). Computational studies indicate that AA8AC40 binds to the exterior of SIRT3.

[0049] Protein-compound docking Ligand docking, as described in Materials and Methods, was performed on the top-binding ligands derived from DEL, as identified by affinity-mediated selection assays. However, due to the large size and flexibility of each DNA-tagged combinatorially generated ligand, it was not possible to dock the entire complex to SIRT3. To address this issue, we decided to dock only the structures of the combinatorial ligands, particularly their C-terminally amidated forms, to SIRT3. In addition to the most active combinatorial ligands, several inactive ligands identified by the same assay were selected as negative controls to validate the selection of the binding site and docking method. The structures of some of the positive and negative controls used in our docking studies are shown in Figure 6.

[0050] Despite the considerable size difference between the DEL-derived ligands and compounds known to bind to internal sites in SIRT3, such as Ex-527 and honokiol, it was decided to first examine known internal sites in this protein. However, attempts to dock both active and inactive hits from the DEL library into internal sites in two structures of SIRT3, 4BVG and 4FVT, were unsuccessful. These results were not unexpected for two reasons: (1) the hits obtained from the DEL library and the peptides and NAD of SIRT3 were not clearly identified; + (1) there is no significant overlap between the pharmacophore or other structural features of known compounds that can bind at the internal site in the (or Carba-NAD) binding structure, such as Ex-527 and honokiol; (2) the hit is significantly longer than either Ex-527 or honokiol even without the DNA tag, and the space within the internal site available to accommodate compounds is only barely adequate to accommodate these two known ligands in the presence of the peptide and Carba-NAD. While it is possible to accommodate larger compounds at the site, doing so requires the removal of NAD from the site, as seen for some potent inhibitors of SIRT3. + Or Carba-NAD replacement may be required.

[0051] Attempts to dock the hits and controls at internal sites within both SIRT structures (4FVT and 4BVG) revealed several further problems. First, clustering of the top poses for the hits from the active ligands was poor at the internal sites within both structures, which is inevitable even though a significant proportion of these are partially in the external cavity (not shown). Furthermore, the orientation of the top docking poses for many hits was incompatible with the location of the DNA tag attachment. Inactive control compounds from the library also showed substantially better clustering than the hits when docked at the internal sites of both structures (not shown). Therefore, it was unlikely that the hits from the DEL library bound to the internal sites of SIRT3, and we decided to explore the possibility that they might bind to more exposed sites on (or near) the protein surface.

[0052] A search was then initiated for putative binding sites in SIRT3, either atypical or external. Two such sites were readily identified, one each for 5H4D and 4O8Z. Both were defined by the co-crystallized but weak ligand of SIRT known as amiodarone. However, again, the top docking poses of hits from the DEL library did not show any significant overlay with each other when docked at the external sites in either protein structure (not shown). Furthermore, the positive top docking poses had little or no hydrogen-bonding interactions with residues lining the 5H4D and 4O8Z sites.

[0053] Next, 4FVT and 4BVG were scanned to locate potential binding sites using shape-based methods. We were able to identify one atypical binding site in 4FVT (in addition to the known internal site) that was sufficient to partially accommodate the positive library hits. A similar scan of 4BVG revealed two additional atypical potential binding sites that were large enough to accommodate these compounds. Attempts to dock the positive hits into the atypical binding sites in 4FVT revealed some overlap between the top docking poses for some of the positive hits, but the degree of pharmacophore overlap for the top poses of different ligands was still poor, and no conserved patterns of interaction with the receptor were observed.

[0054] Docking at the first putative atypical binding site in 4BVG revealed a high degree of overlap for the top docking poses of most positive hits. As shown in Figure 7, only 2 / 10 positive hits (AA8-AC71, AA8-AC92) did not consistently overlap with the top docking poses of the other 8 / 10 positive hits. The top poses of most positive hits also showed a high degree of pharmacophore overlap and conserved interactions with residues lining the site. Notably, the inactive control also docked well at this site, but showed fewer interactions (hydrophobic and hydrophilic) with hits involving the lining residues.

[0055] The second putative atypical site in 4BVG is similar to that defined by amiodarone in 5H4D and 4O8Z. While the top poses of the hits show some pharmacophore overlay with each other (not shown), the degree of overlap is nowhere near that seen at atypical site 1 in 4BVG; therefore, the latter is the most likely binding site for hits from the DEL library. However, it is noteworthy that 2 / 10 tested hits docked poorly at atypical site 1. It may also be more difficult than usual to develop ligands for atypical site 1 in 4BVG because they are less deep and more solvent exposed than are typical for small molecule sites.

[0056] II. Identification of hits capable of mechanism-based activation of Sirtuin-3 using virtual screening (Formulas III-XIV) We used AWS cloud servers for virtual screening of a database of 1.2 million compounds from ChemBridge using AutoDock Vina. Two QM-MM geometry-optimized SIRT3 complexes (4FVT and 4BVG) were used as receptors in this docking exercise. Hits were identified based on their docking scores (in AutoDock Vina and MOE) with these two receptors and H-bond interactions with specific residues lining the binding site, including those in the cofactor-binding loop. Honokiol was included as a positive control in the library and subsequently identified as a hit compound according to these criteria. From the top 1,000, we selected the top 20 compounds based on their docking scores (-11.7 to -9.5, as shown in Table 3). The AutoDock Vina scores for the selected compounds were significantly better than the average (median -5.5) for the entire dataset, indicating a better fit to the binding site, demonstrating that compounds with higher scores exhibit better overall interactions with SIRT3 than those with lower scores. The residues having H-bonds with the compounds and the degree of receptor buried for each compound are also reported in Table 3 . Table 3. Docking scores of the top 20 compounds selected through virtual screening and in vitro SIRT3 modulation by these compounds (hit compounds * ). [Table 3]

[0057] Comparison of the binding modes of top hit compounds to HKL The interactions of residues in the binding site involved in docking, especially the flexible loop, were analyzed for the top hit compounds (see Table 4). The top hit compounds had AutoDock scores superior to those of the reference ligand (HKL). Pro155, Arg158, and Ile230 interact with the top hit compounds through hydrogen bonds in a pattern similar to HKL. Our previous study showed that HKL forms hydrogen bonds with Pro155 in the SIRT3 open loop conformation and with Arg158 in the SIRT3 closed loop conformation. Interestingly, the hit compounds 6068318 and 5761865 have the same binding pattern (Figure 8).

[0058] Validation of selected hit compounds for SIRT3 modulation effects In vitro validation of the hit compounds was performed in commercial Fluor de Lys enzymatic assay and HPLC assay under steady-state conditions (top 70) and non-steady-state conditions (top 20) in Figures 9 and 10, respectively.

[0059] The compounds showed inhibitory potency against SIRT3 ranging from 0 to 58.4% at a 10 μM modulator concentration (Table 3) under steady-state conditions. Two hit compounds showed excellent inhibitory potency (>50% inhibition) at a 10 μM modulator concentration. Nine of the 20 compounds were found to activate SIRT3 (3.1% to 14.0%) under non-steady-state conditions at a 10 μM modulator concentration. The hit rate (number of hits divided by the number of compounds tested) for VS was 45%, indicating the success of the current VS workflow.

[0060] Comparison of top hit compounds in terms of HKL binding mode Residues involved in docking, especially the flexible loop region, were analyzed for the top hit compounds (Table 4). The top hit compounds have AutoDock scores superior to those of the reference ligand (HKL). Pro155, Arg158, and Ile230 were found to interact with the top hit compounds through hydrogen bonds, a pattern similar to that of HKL. Our previous study showed that HKL forms hydrogen bonds with Pro155 in the SIRT3 open loop conformation and with Arg158 in the SIRT3 closed loop conformation. Interestingly, hit compounds 6068318 and 5761865 exhibit the same binding characteristics. The definition of docking sites, docking templates, and ligand queries plays an important role in ensuring the high hit rate of the current VS (45% for activators and 55% for inhibitors and activators). Table 4. Summary of ligand-protein interactions observed in docking. Color code: blue, post-docked and post-minimized (H-bonds); pink, conventional hydrogen bonds. [Table 4]

[0061] Structural similarity of top hit compounds Our docking studies of 1.2 million compounds with two SIRT3 receptor structures (4BVG and 4FVT) revealed that several hits share common structural features. While these common features are not sufficient for constructing an appropriate SAR model, they are sufficient to influence the selection of building blocks for combinatorial libraries. The most obvious recurring motif is a bicyclic ring (with aromaticity, partial aromaticity, or partial delocalization) linked to another single aromatic ring through an amide (or urea) linker (Figure 11). These criteria can be applied in designing future ligands and scaffolds for combinatorial libraries.

[0062] Compounds of Formulas III, V, VI, VII, VIII, IX, X, XI, and XIV were included in the SIRT3 docking study as follows (Tables 6-32). Initial 3D structures of the compounds were generated from SMILES using RDKit (2019 version) or, if this failed, Openbabel version 3.0. In ambiguous cases (e.g., axial vs. equatorial substitution), all conformational isomers were generated. Additionally, for racemates, all conformers were generated. For structures with non-standard ring pucker, structures were minimized using DFT to ensure a feasible geometry for docking. This was done using ORCA version 3.0.1. All compounds were docked into SIRT3 using GOLD v5.2 and three PDB files, 4BVG and 4FVT, along with an internally generated X-ray structure of human SIRT3 (designated Xtal). In each case, the binding pocket was restricted to one within 20 Å of the ligand in the original PDB file, and 30 dockings were requested for each ligand. All other options were set to default options. An in-house algorithm was used to identify and evaluate the quality of all hydrogen bonds (H-bonds) made between the ligand and the protein, and this information, along with the GOLD docking score, was used to select the optimal solution. Two dockings were selected for each compound: the highest-scoring docking containing the most H-bonds and the highest-scoring of the remaining dockings. These dockings were manually evaluated and compared to the parent reference compound used to seed the design.

[0063] In the following table of docking results, the best scoring docking was selected independent of the number of hydrogen bonds. Compounds with docking scores at least 90% higher than the docking score (GoldScore) of the reference compound are considered as potential SIRT3 modulators.

[0064] The reference compounds on which the docking studies were based are labeled as in Tables 6-32. These are, in fact, the compounds from the top 20 virtual screenings that were involved in laboratory activity testing and shown to modulate sirtuins. The results of the activity testing of the reference compounds are summarized in Table 5, and the values shown are based on tests performed in triplicate. Table 5. Activity test results for reference compounds (top 20 VS) [Table 5]

[0065] The results of docking studies using compounds of Formulas III, V-XI, and XIV are provided below in Tables 6-32. Table 6. SIRT3 docking results for compounds of formula III [Table 6] Table 7. SIRT3 docking results for compounds of formula III [Table 7] Table 8. SIRT3 docking results for compounds of formula III [Table 8] Table 9. SIRT3 docking results for compounds of formula VI [Table 9] Table 10. SIRT3 docking results for compounds of formula VI [Table 10] Table 11. SIRT3 docking results for compounds of formula VI [Table 11] Table 12. SIRT3 docking results for compounds of formula V [Table 12] Table 13. SIRT3 docking results for compounds of formula V [Table 13] Table 14. SIRT3 docking results for compounds of formula V [Table 14] Table 15. SIRT3 docking results for compounds of formula VII [Table 15] Table 16. SIRT3 docking results for compounds of formula VII [Table 16] Table 17. SIRT3 docking results for compounds of formula VII [Table 17] Table 18. SIRT3 docking results for compounds of formula VIII [Table 18] Table 19. SIRT3 docking results for compounds of formula VIII [Table 19] Table 20. SIRT3 docking results for compounds of formula VIII [Table 20] Table 21. SIRT3 docking results for compounds of formula IX [Table 21] Table 22. SIRT3 docking results for compounds of formula IX [Table 22] Table 23. SIRT3 docking results for compounds of formula IX [Table 23] Table 24. SIRT3 docking results for compounds of formula X [Table 24] Table 25. SIRT3 docking results for compounds of formula X [Table 25] Table 26. SIRT3 docking results for compounds of formula X [Table 26] Table 27. SIRT3 docking results for compounds of formula XI [Table 27] Table 28. SIRT3 docking results for compounds of formula XI [Table 28] Table 29. SIRT3 docking results for compounds of formula XI [Table 29] Table 30. SIRT3 docking results for compound of formula XIV [Table 30] Table 31. SIRT3 docking results for compound of formula XIV [Table 31] Table 32. SIRT3 docking results for compound of formula XIV [Table 32] The docking results show that the analogs have docking scores very close to or lower than those of the reference compounds (which modulate—activate or inhibit—SIRT3 and have been identified through activity tests performed in the laboratory), suggesting that these analogs also interact with SIRT3 protein and modulate its activity in a similar manner.

[0066] Further compounds belonging to formula III, V, VII and XI were tested for their activity under condition I (steady state).These activity tests show that these compounds regulate (activate or inhibit) SIRT3 activity, and confirm the results obtained from docking studies.Table 33 summarizes the results of these activity tests. Table 33. Sirtuin-modulating compounds identified by activity assays [Table 33] TIFF2025525358000066.tif36165

[0067] Steady-state SIRT3 activating compounds Importantly, our drug discovery workflow (Figure 296) identified the first reported steady-state activators of SIRT3 (Table 5), which qualitatively improve on the properties of honokiol and are ideal candidates for SIRT activator drug development. Steady-state activators 5329973 (Formula VII) and 5689785 (Formula XI) were characterized and exhibited physiologically relevant [NAD] activity under steady-state conditions, respectively. + ] = 100 μM were found to provide an increase in SIRT3 activity of 184.5% (20 μM) and 194.4% (1 μM) (Figure 297), indicating that both of these compounds significantly increased the catalytic efficiency (k cat / K m) nearly doubled (Table 34). The MST binding affinity results for compound 5329973 were in the nM range, indicating that compound 5329973 is a strong binder to SIRT3 (Figure 298F). Steady-state parameter calculations (Table 34) suggest that the K m The improved catalytic efficiency was primarily due to a reduction in the ATP content, predicting that this could be achieved through conformational modulation of the flexible loop induced by appropriately designed small molecule binding to the active site. The observed results in Table 34 and Figure 299 (Michaelis-Menten plot) closely match the properties predicted for sirtuin activators based on a steady-state mechanism. The dose-response curves (Figure 397A) for compounds 5689785 and 6068318 (Formula III) also show that these compounds achieve activation above 50% of the maximum activation effect (AC50) at concentrations of 100 nM and <1 μM, respectively, making these hits promising for further development as drug-like leads. Table 34. Model parameter estimates from Michaelis-Menten global nonlinear fits for SIRT3 in the presence and absence of 1 μM 5689785 and 20 μM 5329973. [Table 34]

[0068] SIRT3 activation by these steady-state activators is mediated by saturable NAD + (Figure 297A) and saturated peptide substrate (Figure 297B-D). + is ~200 μM in young people and can decrease by more than 50% in old age, so NAD in aging mitochondria + Similar to 100 μM NAD +By nearly doubling the deacetylation rate of SIRT3 in vivo, the level of SIRT3 activation by these compounds is sufficient to largely restore the mitochondrial deacetylation activity characteristic of young SIRT3. Furthermore, steady-state activators such as these compounds are likely to be less affected than honokiol by fluctuations in physiological conditions related to SIRT3 activity, such as enzyme expression levels.

[0069] material and method Activity Assays—Activity Tests of Reference Compounds, Table 5 Chemicals and Reagents MnSOD (KGELLEAI-(KAc)-RDFGSFDKF) was synthesized by GenScript (Piscataway, NJ). Human SIRT3 (recombinant) (His-tag), NAD + (SIRT substrate), FdL2(QPKKAC-AMC) peptide, also known as p53-AMC peptide, nicotinamide, and HDAC buffer were purchased from Enzo Life Sciences (Farmingdale, NY). Dimethyl sulfoxide and trifluoroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Test compounds were purchased from ChemBridge Corporation (San Diego, CA). Carba-NAD was synthesized by Dalton Pharma (Toronto, ON).

[0070] hSIRT3 102~399 Effect of hit compounds on deacetylase activity - HPLC assay using native peptides The enzymatic reaction was carried out in the presence of the hit compound (50 μM (condition I) or 10 μM (condition II)) in HDAC buffer (50 mM TRIS-HCl, pH 8.0, containing 137 mM sodium chloride, 2.7 mM potassium chloride, and 1 mM magnesium chloride), dimethyl sulfoxide solution, 95 / 5, containing 1000 μM NAD + and 50 μM MnSOD peptide (condition I) or 50 μM NAD +and 600 μM peptide substrate (condition II). Reactions were initiated by adding 5 U or 50 U of hSIRT3 102-399 and incubated at 37 °C for 30 min. The reactions were terminated by adding stop solution (2% TFA, 5 mM nicotinamide final concentration). The peptide products and substrates were separated using HPLC (reverse-phase C18 HPLC column). Solvent A consisted of 90% HPLC-grade water and 10% acetonitrile with 0.05% (v / v) TFA. Solvent B consisted of acetonitrile with 0.02% (v / v) TFA. A linear gradient was run from 0% B to 51% (v / v) B over 20 min. The proportion of solvent B was increased to 100% within 5 min and then returned to the starting conditions (0% B) within 5 min. The proportion of solvent A (100%) was maintained at 100% for an additional 5 minutes (total run time 36 minutes).

[0071] hSIRT3 102~399 Effect of hit compounds on deacetylase activity - a fluorescence-based assay using fluorescently labeled peptides Using FdL2 peptide, SIRT3 102~399 The modulatory effects of hit compounds on deacetylase activity were determined. Similar enzyme reactions were performed as described above. The reactions were terminated by adding 1× developer, 2 mM NAM solution, and fluorescence was measured on a TECAN microplate reader. GraphPad Prism (GraphPad Software, Inc., CA) was used to fit the raw data to the defined model equation.

[0072] Activity Assays - Activity Tests, Table 33 Chemicals and Reagents MnSOD (KGELLEAI-(KAc)-RDFGSFDKF) was synthesized by GenScript (Piscataway, NJ). Human SIRT3 (recombinant) (His-tag), NAD +(SIRT substrate), nicotinamide, and HDAC buffer were purchased from Enzo Life Sciences (Farmingdale, NY). Dimethyl sulfoxide and trifluoroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Test compounds were purchased from ChemBridge Corporation (San Diego, CA).

[0073] HPLC assay The enzymatic reaction was carried out in the presence of the hit compound (50 μM) (condition I) in HDAC buffer (50 mM TRIS-HCl, pH 8.0, containing 137 mM sodium chloride, 2.7 mM potassium chloride, and 1 mM magnesium chloride), dimethyl sulfoxide solution, 95 / 5, containing 1000 μM NAD + and 50 μM MnSOD peptide (condition I). The reaction was initiated by adding 5 U of hSIRT3 102-399 and incubated at 37 °C for 30 min. The reaction was terminated by adding stop solution (12% TFA, 30 mM nicotinamide). The peptide products and substrates were separated using HPLC (reverse-phase C18 HPLC column). Solvent A consisted of 90% HPLC-grade water and 10% acetonitrile with 0.05% (v / v) TFA. Solvent B consisted of acetonitrile with 0.02% (v / v) TFA. A linear gradient was run from 0% B to 51% (v / v) B over 20 min. The proportion of solvent B was increased to 100% within 5 min and then returned to the starting conditions (0% B) within 5 min. The proportion of solvent A (100%) was maintained at 100% for an additional 10 min (total run time 41 min).

[0074] Virtual screening of 1 million compounds (Tables 3 and 4) High-throughput batch docking A database (Chembridge) containing 1.2 million compounds was screened against two SIRT3 structures (PDB: 4FVT and 4BVG). Our previous knowledge of the bioactive conformations of HKL and its binding site in SIRT3 was applied to define the docking site and protein conformation selection. VS was performed using structure-based docking in a sequential process (Figure 299). Briefly, these 1.2 million compounds were first imported into the database and converted from 2D to 3D using the appropriate module of MOE. Next, another module (iMOE) was used to add hydrogens as needed, calculate partial charges, and perform a rapid round of energy minimization using the MMFF94x force field. Where appropriate, the most likely tautomers were generated and automatically selected.

[0075] Figure 299 illustrates the compound screening process used in this application. A database containing 1.2 million compounds was subjected to a series of operations for each compound, starting with 2D-to-3D conversion and ending with rapid energy minimization. These compounds were then independently docked with two SIRT3 receptor structures (4BVG and 4FVT). The best poses (by calculated binding energy) of the top 1,000 compounds in each docking run were selected for further post-docking analysis, which was performed using MOE as described in the post-docking analysis. The final step involved careful inspection and selection of 20 compounds from both docking runs, which allowed us to optimally cover scaffold diversity, lack repetition, and obtain the maximum number of favorable interactions with residues in the putative binding site.

[0076] These prepared ligand structures were converted back into the preferred input file format using AutoDock Vina. Our previous studies of the bioactive conformations of HKL and its binding site in SIRT3 were applied to define the docking site and selection of protein conformations. Docking was performed using AutoDock Vina on the AWS cloud server. The grid centers and volumes were defined as follows: for 4FVT, center(x,y,z)=23.19,31.19,-14.31; volume=29.6x34.8x33.8; for 4BVG, center(x,y,z)=22.24,30.39,-7.98; volume=25.0x25.0x25.0. For each ligand, 20 possible conformations were collected.

[0077] Post-docking analysis The docked ligands were then ranked by docking score (predicted affinity in kcal / mol) in AutoDock Vina.The top 1,000 compounds were then screened for (1) non-covalent interaction with the 4FVT and 4BVG binding cavities; (2) hydrogen bond formation with the residues lining the binding site; (3) interaction with specific residues in the cofactor binding loop of SIRT3; (4) predicted ADME properties, such as LogP and LogD, and (5) predictions regarding synthetic feasibility.Only the best-ranked poses of each compound from the top 1,000 were included in the list.

[0078] This list of the top 1,000 compounds was then subjected to further post-docking analysis using MOE. The ligands were imported into the database, hydrogen atoms were added, and partial charges for each structure were recalculated before being converted to the unmodified form utilized by MOE. A small percentage of these structures were carefully examined to ensure that this processing did not introduce any undesired artifacts. Next, a truncated post-docking energy minimization of hydrogen atoms from the ligand and neighboring residues was performed using the MOE script known as "analysis_dock.svl" to further optimize the interactions between the docking pose and site residues. The energy optimization step provided a new value for the compound's approximate calculated binding energy (in MOE). The optimized structure was then used for all subsequent post-docking analysis steps.

[0079] We screened for the presence of H-bonds between the optimized poses and residues lining the binding site. Based on previous docking structures of honokiol, the following residues were considered to be part of the site: 4FVT (G145, S149, G153, G163, L168, Y171, F180, K195, E198, Y204, Q228, and H248), 4BVG (D156, P176, I179, F294, S321, E323 (and NLE7 from the co-crystallized Ac-ACS peptide chain)). We also used the docking pose of honokiol to measure the relative "receptor buriedness" of the optimized poses in both receptor structures. We calculated BHB (buriedness, hydrogen bond, and binding energy) scores for each compound using the general formula originally described by Feher M. et al.

[0080] hSIRT3 118~399 Effect of hit compounds on deacetylase activity - a fluorescence-based assay using fluorescently labeled peptides Using FdL2 peptide, SIRT3 118~399The modulatory effects of hit compounds on deacetylase activity were determined. Similar enzyme reactions were performed as described above. The reactions were terminated by adding 1× developer, 2 mM NAM solution, and fluorescence was measured on a TECAN microplate reader. GraphPad Prism (GraphPad Software, Inc., CA) was used to fit the raw data to the defined model equation.

[0081] Docking studies for hit compounds MD simulation Computational studies were performed using MOE (Molecular Operating Environment from Chemical Computing Group Inc., Montreal, PQ) version MOE 2020.09 on a computer with a quad-core Intel x86-64 (Intel Core i7-8th generation) processor, 16 GB of RAM, and Windows 10 Pro. The MMFF94x force field and solvation as implemented in MOE were used for all computational studies.

[0082] Ligand docking method Two methods were used to define the ligand-binding site in the receptor protein structure. The first method, used for 4BVG, defined the site using a co-crystallized ligand (EX-527). The second method, used for 4FVT and 4BVG, utilized the "Site Finder module" in MOE to generate and place dummy atoms within the potential ligand-binding site for both structures. This step was performed after removing all molecules of minor compounds present in the receptor structure, such as sulfate ions, glycerol, and 1,2-ethanediol. Coincidentally, the main site identified by the second method for 4BVG was nearly identical to that defined by the co-crystallized ligand. Therefore, the main putative (and internal) ligand-binding site identified in 4FVT was assumed to be the best candidate for docking studies in the receptor structure.

[0083] Energy-optimized models of selected compounds were sequentially docked into these sites by applying "Dock" in MOE; for all docking studies, default settings for parameters using the "Rigid Receptor" protocol were used. The top pose showing reproducibility (RMSD < 0.2 Å) in three independent docking simulations at a given site was the most optimal pose for that site. The main settings for ligand docking were as follows: docking protocol = Rigid Receptor; configuration = Triangle Matcher; rescoring = London dG; retention = 100; fine-tuning = force field; rescoring = GBVI / WSA dG; retention = 100 top pose. The default settings for docking parameters using the "Rigid Receptor" protocol were found to be sufficient for docking honokiol and EX-527, which were used as test ligands. The top-scoring poses for the ligands from each docking run were used to calculate approximate binding energies and other inputs for calculating the final docking scores.

[0084] Ligand binding energy calculation The binding energy of the top docking pose of each ligand at each site was calculated using an updated version of a published method. This method involves calculating the energy of the docked receptor-ligand complex (Ecpx), using the equation: Ebind = Ecpx - (Elig + Eprot); the unbound ligand in solution (Elig); and the unbound solvated rigid receptor from the complex (Eprot). The top selected docking poses for each ligand, obtained from three independent docking runs, were used to calculate the approximate ligand binding energy. Optimization of the semi-rigid (fixed heteroatom) receptor-ligand complex was performed under distance-dependent dielectric conditions, and the final step was performed under solvation, using a procedure that allowed only hydrogens and cavities in the ligand to undergo further structural optimization.

[0085] Binding energies calculated using molecular mechanics should always be viewed as approximations of the actual binding energies, because any semi-rigid receptor approximation will almost always underestimate the effect of induced fit, while a fully flexible receptor approximation will usually overestimate it. Therefore, the actual binding energy would be expected to lie between the rigid and flexible receptor values. The methodology used to calculate the receptor burial degree of the top docked (and energy-optimized) ligand poses, as well as enumerate their hydrogen-bonding interactions with residues in the binding site, has been previously described; the code is available upon request.

[0086] Various embodiments of the present invention have been described in fulfillment of various objectives of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and applications thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. Formula VII and / or salts thereof: 【Chemical 1】 wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl; Ar1 and Ar2 are independently selected from aryl and heteroaryl, and the aryl and heteroaryl are optionally selected from the group consisting of (C 1 ~C 10 )-alkyl, (C 1 ~C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl of sirtuin-modulating compounds.

2. 2. The sirtuin-modulating compound of claim 1, wherein R1 and R2 are independently selected from the group consisting of hydrogen and alkyl, and Ar2 is nitrobenzene.

3. Formula VII and / or salts thereof: 【Chemistry 2】 3. The sirtuin-modulating compound of claim 2, wherein:

4. Formula VII and / or salts thereof: 【Chemistry 3】 wherein R3 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, halo, and heterocycloalkyl.

2. The sirtuin-modulating compound of claim 1, wherein:

5. The sirtuin-modulating compound of claim 1, wherein the compound is a sirtuin activator.

6. 10. The sirtuin-modulating compound of claim 1, wherein the compound is a sirtuin inhibitor.

7. The sirtuin-modulating compound of claim 1 , wherein the compound modulates SIRT3 activity.

8. Formula III and / or salts thereof: 【Chemistry 4】 In the formula, Ar 1 is aryl or heteroaryl, and R 1 is hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine, and -C(O)NR 3 R 4 and Ar 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and amine are optionally selected from the group consisting of (C 1 ~C 10 )-alkyl, (C 1 ~C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, and hydroxy; 3 and R 4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and heteroaryl; R 2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, amine, alkylamine, and O; Y is selected from the group consisting of N and CH; and X is selected from the group consisting of NH, S, and O. Sirtuin-modulating compounds.

9. R 1 is aryl and -C(O)NR 3 R 4 9. The sirtuin-modulating compound of claim 8, selected from the group consisting of:

10. Formula III and / or salts thereof: 【Chemistry 5】 9. The sirtuin-modulating compound of claim 8, wherein:

11. Formula III and / or salts thereof: 【Chemistry 6】 9. The sirtuin-modulating compound of claim 8, wherein:

12. 9. The sirtuin-modulating compound of claim 8, wherein the compound is a sirtuin activator.

13. 9. The sirtuin-modulating compound of claim 8, wherein the compound is a sirtuin inhibitor.

14. The sirtuin-modulating compound of claim 8, wherein the compound modulates SIRT3 activity.

15. Formula XI and / or salts thereof: 【Chemistry 7】 In the formula, Ar 1 and Ar 2 is independently selected from aryl and heteroaryl, and the aryl and heteroaryl are optionally selected from (C 1 ~C 10 )-alkyl, (C 1 ~C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxyl Sirtuin-modulating compounds.

16. Formula V and / or salts thereof: 【Chemistry 8】 In the formula, R 1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, and heterocycloalkyl; Ar 1 and Ar 2 is independently selected from aryl and heteroaryl, and the aryl and heteroaryl are optionally selected from (C 1 ~C 10 )-alkyl, (C 1 ~C 10 )-substituted with one or more substituents selected from the group consisting of alkenyl, alkoxy, halo, amine, sulfonyl-alkyl, and hydroxyl; X is selected from the group consisting of O, S, and N; and m and n are integers each having a value independently selected from 0 to 10. Sirtuin-modulating compounds.

17. Formula V and / or salts thereof: 【Chemistry 9】 17. The sirtuin-modulating compound of claim 16, wherein:

18. Formula V and / or salts thereof: 【Chemistry 10】 17. The sirtuin-modulating compound of claim 16, wherein:

19. Formula V and / or salts thereof: 【Chemistry 11】 17. The sirtuin-modulating compound of claim 16, wherein:

20. 17. The sirtuin-modulating compound of claim 16, wherein the compound is a sirtuin activator.

21. 17. The sirtuin-modulating compound of claim 16, wherein the compound is a sirtuin inhibitor.

22. 17. The sirtuin-modulating compound of claim 16, wherein the compound modulates SIRT3 activity.

Images