5-((3-Amidobenzyl)oxy)nicotinamides as Sirtuin 2 Inhibitors - Journal

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5-((3-Amidobenzyl)oxy)nicotinamides as Sirtuin 2 Inhibitors Teng Ai, Daniel J. Wilson, Swati S. More, Jiashu Xie, and Liqiang Chen J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01376 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 20, 2016

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Journal of Medicinal Chemistry

5-((3-Amidobenzyl)oxy)nicotinamides as Sirtuin 2 Inhibitors Teng Ai, Daniel J. Wilson, Swati S. More, Jiashu Xie, and Liqiang Chen* Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street S.E., Minneapolis, MN 55455, United States

Abstract

Derived from our previously reported human sirtuin 2 (SIRT2) inhibitors that were based on a 5aminonaphthalen-1-yloxy nicotinamide core structure, 5-((3-amidobenzyl)oxy)nicotinamides offered excellent activity against SIRT2 and high isozyme selectivity over SIRT1 and SIRT3. Selected compounds also exhibited generally favorable in vitro absorption, distribution, metabolism, and excretion (ADME) properties. Kinetic studies revealed that a representative SIRT2 inhibitor acted competitively against both NAD+ and the peptide substrate, an inhibitory modality that was supported by our computational study. More importantly, two selected compounds exhibited significant protection against α-synuclein aggregation-induced cytotoxicity in SH-SY5Y cells. Therefore, 5-((3-amidobenzyl)oxy)nicotinamides represent a new class of SIRT2 inhibitors, which are attractive candidates for further lead optimization in our continued effort to explore selective inhibition of SIRT2 as a potential therapy for Parkinson’s disease (PD).

Key words

Sirtuins, Sirtuin inhibitor, SIRT1, SIRT2, SIRT3, SIRT5, Parkinson’s disease

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INTRODUCTION

Sirtuin 2 (SIRT2) is a member of the mammalian sirtuin (NAD+-dependent histone deacetylase) family that comprises SIRT1-7.1 SIRT2 is the only sirtuin that predominantly resides in the cytosol even though it is localized in the nucleus during mitosis. Interestingly, an alternatively spliced isoform has been reported to permanently reside in the nucleus. As the most abundant sirtuin homolog in the brain,2 SIRT2 has emerged as an important regulator in brain physiology and pathology.3 Several studies have suggested that selective pharmacological inhibition of SIRT2 is a promising therapeutic approach for Parkinson’s disease (PD). Overexpression of SIRT2 induced neuronal apoptosis.4 In contrast, blocking SIRT2 protected cells from the neurotoxicity induced by α-synuclein, a risk factor associated with familial PD, in the cellular and fruit fly models of PD.5 Furthermore, SIRT2 exacerbated the nigrostriatal neurotoxicity induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) while genetic deletion6 or pharmacological inhibition7 of SIRT2 prevented MPTP-induced neurodegeneration in mice. Moreover, as a major deacetylase of α-tubulin,8 SIRT2 decreased the acetylation level of microtubules, enhancing their association with pathologically mutated leucine-rich kinase 2 (LRRK2) in the Roc-COR domain (R1441C and Y1699C). While this strengthened association impaired axonal transport, genetic knockdown of SIRT2 restored both axonal transport and locomotion in fruit flies.9

Besides PD, blocking SIRT2 may also provide protection against other neurodegenerative diseases. Inhibition of SIRT2 had a protective effect in a cellular model of multiple system atrophy,10 which is a form of synucleinopathy similar to PD. In the granule cells obtained from



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slow Wallerian degeneration (Wld(s)) mice, genetic knockdown of SIRT2 enhanced resistance to axonal degeneration while overexpression of SIRT2 abrogated the resistance.11 Furthermore, inhibiting SIRT2 offered neuroprotection in the fly, worm, and primary striatal neuron models of Huntington’s disease, likely by reducing sterol biosynthesis,12 although the therapeutic potential still remains to be unequivocally defined. Moreover, SIRT2 inhibitors have been explored for their potential therapeutic applications in the mouse models of tauopathies, such as Alzheimer’s disease (AD)13 and frontotemporal dementia.14 Lastly, a neurotoxic role of SIRT2 in AD has been recently proposed.15 Taken together, these studies suggest that optimized SIRT2 inhibitors will have a broad impact on the treatment of neurodegenerative diseases.

SIRT2’s potential therapeutic applications in neurodegenerative diseases as well as cancers16 have generated a growing interest in the discovery of potent and selective SIRT2 inhibitors,16-17 leading to compounds that are exemplified by those shown in Figure 1. AGK2 (1), a commonly used SIRT2 inhibitor, has been shown to mitigate the neurotoxicity induced by α-synuclein in models of Parkinson’s disease,5 even though it only possesses modest activity and selectivity against SIRT2. AK-1 (2) and its analogs have been tested for their therapeutic application in Huntington’s disease and PD, and further extensive structural modifications have been reported.12,

14, 18

Compound 3, which was developed by structural optimization of an

anilinobenzamide chemotype, displays high anti-SIRT2 activity and excellent (> 200-fold) selectivity over SIRT1 and SIRT3.19 Inhibitor 4 and its analogs, which are based on a chroman4-one core structure, are low micromolar inhibitors of SIRT2 with high isozyme selectivity.20 A series of thieno[3,2-d]pyrimidine-6-carboxamides such as compound 5 possessed low nanomolar activity against SIRT2; however, their selectivity over SIRT1 and SIRT3 still needs to be


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improved.21 More recently, a potent and selective SIRT2 inhibitor 6, which was built on a new 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one scaffold, has been reported to prevent neuronal cell death triggered by lactacystin in an in vitro model of PD.22 Also remarkably, a new class of highly selective SIRT2 inhibitors including SirReal2 (7, Figure 1) has been reported.23 Through crystallographic studies, SirReal2’s selectivity has been attributed to a ligand-induced structural rearrangement of the enzyme active site, which reveals a new binding pocket that accommodates SirReal2 and can be exploited for further design of selective SIRT2 inhibitors. Furthermore, varied chemotypes such as tenovins,24 macrocyclic peptides equipped with a mechanism-based warhead,25 isoxazol-5-one cambinol analogs,26 and tetrahydro-1Hpyrido[4,3-b]indoles27 have also been reported as SIRT2 inhibitors.

Recently, we have reported potent and selective SIRT2 inhibitors based on a 5-aminonaphthalen1-yloxy nicotinamide core structure as exemplified by compound 8 in Figure 2.28 In our continued efforts to discover novel SIRT2 inhibitors, we have designed inhibitors based on a 3aminobenzyloxy nicotinamide core structure as represented by compound 9 (Figure 2). Herein we focus on our structure-activity relationship (SAR) studies on ring A that have revealed key structural features that lead to enhanced enzymatic activity against SIRT2 as well as selectivity over SIRT1 and SIRT3. We also report our preliminary efforts on evaluating selected compounds for their in vitro ADME properties. Our initial testing of selected SIRT2 inhibitors in a cellular model of PD is also discussed.

RESULTS AND DISCUSSION



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SAR Studies. Compound 8 showed excellent anti-SIRT2 activity and selectivity over SIRT1 and SIRT3. However, its inefficient chemical synthesis hampered further SAR exploration. Therefore, we proceeded to search for structural replacements for the naphthalene core in compound 8. To this end, we explored new SIRT2 inhibitors, in which the 5-aminonaphthalen-1yloxy nicotinamide core structure in compound 8 was simplified to yield a scaffold that allows for expedient chemical manipulation and subsequent detailed SAR studies.

To test the viability of 3-aminobenzyloxy nicotinamide as a new core structure for SIRT2 inhibitors, we first prepared compound 10 (Table 1), in which a 3-methyl-4-nitrobenzamide functionality was appended to the 3-aminobenzyloxy nicotinamide core structure. A similar chemical modification was previously used in our investigation of SIRT2 inhibitors based on 5aminonaphthalen-1-yloxy nicotinamide. Gratifyingly, compound 10 showed nanomolar antiSIRT2 activity together with good (> 50 fold) selectivity over SIRT1 and SIRT3. Encouraged by this finding, we proceeded to conduct detailed SAR studies, which mainly focused on the benzamide functionality as shown in compound 10.

First, we investigated whether an aromatic ring and/or conjugation within the amide functionality was required for activity against SIRT2. To that end, compound 11 which contained an acetyl group showed only modest anti-SIRT2 activity; however, compounds 12 and 13 with a more bulky cyclohexyl and adamantly group, respectively, exhibited improved anti-SIRT2 activity. When a double bond that led to a conjugated carbonyl system was introduced in compound 14, its inhibitory activity against SIRT2 was significantly enhanced when compared with that of compound 12. This observation indicated that a conjugated carbonyl system was preferred, a


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SAR trend that was also observed in compound 15 which featured an unsubstituted benzamide functionality. Compound 15 possessed nanomolar anti-SIRT2 activity and good isozyme selectivity (> 40 fold), highlighting the importance of a conjugated aromatic ring system. Compound 15 is structurally identical to compound 8 except that the naphthalene ring in compound 8 has been simplified to give the benzyl linker present in compound 15 (Figure 2). Therefore, loss of activity (about 5-fold) against SIRT2 by compound 5 clearly resulted from this structural simplification. Judged by our studies on inhibition mode and computational modeling (vide infra), compound 15 was mainly competitive against NAD+ and competitive with respect to the peptide substrate; therefore, it most likely shared a binding mode very similar to compound 86 (Figure 4). In contrast, compound 8 has been shown to behave as a non-competitive inhibitor against NAD+ and a competitive inhibitor against the peptide substrate.28 These findings indicated that the structural simplification performed on compound 8 caused a shift in inhibition mode (against NAD+), which could account for the loss of activity exhibited by compound 15. This difference in inhibition mode also explained the fact that compound 15 was amenable to chemical modifications at position 4, as judged by our following SAR studies while a similar modification performed on compound 8 led to significant loss of activity.28

The importance of an aromatic ring was further confirmed in compound 16, in which the phenyl ring in compound 15 was replaced with a thiophene ring without loss of activity. To further explore the amide functionality, we prepared compounds 17 and 18, which contained a methylene and ethylene spacer, respectively; and therefore lack a conjugate system within the amide functionality. The reduced anti-SIRT2 activity of compounds 17 and 18, when compared with the parent compound 15, could be attributable to the loss of a conjugated system. When a



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conjugated system was reinstalled as seen in compound 19, its activity against SIRT2 was restored, leading to a nanomolar IC50 value against SIRT2 and an excellent selectivity profile (> 100 fold over SIRT1 and SIRT3). Taken together, our initial SAR exploration as listed in Table 1 clearly demonstrated that a conjugated aromatic system was preferred for both activity and selectivity in inhibitors based on 3-aminobenzyloxy nicotinamide.

Having established the importance of a conjugated aromatic system, we proceeded to evaluate fused aromatic rings as a potential replacement of the phenyl ring in compound 15 (Table 2). 1Naphthalene (20) resulted in a reduction of anti-SIRT activity; however, 2-naphthalene (21) led to markedly enhanced activity compared with the parent compound 15, suggesting that the position of attachment was critical. Similarly, incorporation of 2-benzo[b]thiophene (22), 2benzofuan (23), and 2-indole (24) gave rise to compounds that showed excellent inhibitory activity towards SIRT2. Nonetheless, 2-benzothiazole (25) failed to improve potency beyond that of compound 15. The position of attachment was also important in heterocyclic fused aromatic rings. For instance, 3-benzo[b]thiophene (26) was markedly less potent than its corresponding regioisomer (22) while 3-indole (27) showed anti-SIRT2 activity very similar to that of isomer 24. When 6-indole was adopted, the resulting compound 28 was slightly more active than compound 27. The anti-SIRT2 activity was further improved when 6-indazole (29) was used even though the anti-SIRT1 activity was also enhanced, leading to a modest isozyme selectivity over SIRT1. In short, we identified fused aromatic/heteroaromatic rings that elicited superb anti-SIRT2 activity even though the selectivity profile still needs to be improved.


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In addition to fused aromatic rings, we also systematically explored substituents on the phenyl ring in compound 15. As shown in Tables 3 and S1, we studied electron-donating methyl (Me) and methoxy (OMe) groups as well as electron-withdrawing chloro (Cl), nitro (NO2) and cyano (CN) groups, substituents that are structurally simple and commonly used in SAR studies. Furthermore, in order to assess the effect of each group at different positions, we installed each substituent on position 2 (R1), 3 (R2) or 4 (R4). When a methyl scan was performed on position 2, 3 or 4, the resulting compounds 30, 31 and 32, respectively, showed sequentially enhanced activity towards SIRT2, indicating that position 4 was the preferred modification site. A similar SAR trend was observed for substituents like OMe (33-35), Cl (36-38), NO2 (39-41), and CN (42-44) (Table S1). Furthermore, compounds 31, 35, 38, 41 and 44 that contained one of these substituents on position 4 showed comparable anti-SIRT2 activities, indicating that the electron– donating or –withdrawing nature of these substituents did not appear to play a critical role. Taken together, we demonstrated that substitution on position 4 was desired for augmented activity, and simple substituents of different electronic nature could all be beneficial.

Besides the above simple substituents, we also explored the addition of a fluoro (F) group (Table 3), which is frequently used in medicinal chemistry even though its effect is difficult to generalize. When a fluoro scan was performed on position 2, 3 or 4, the resulting compounds 45, 46, and 47, respectively, showed a SAR trend that mirrored the one observed for simple substituents as listed in Tables 3 and S1. Interestingly, the activity enhancement was more pronounced, leading to compounds with low nanomolar IC50 values against SIRT2 together with good isozyme selectivity. Intrigued by this finding, we retained a fluoro group at position 4 and then placed an additional fluoro group on either position 2 or 3 to give compounds 48 and 49,



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respectively (Table 3). Compound 48 showed an anti-SIRT2 activity almost identical to that of compound 45, suggesting that a fluoro group at position 2 was dispensable and was able to offset the beneficial effect elicited by a fluoro group at position 4. In contrast, an additional fluoro group at position 3 in compound 49 had minimal influence on the inhibition of SIRT2. Since we demonstrated the beneficial effect of simple substituents such as Me, OMe and Cl at position 4 (Tables 3 and S1), we also explored combining a simple substituent at position 4 in conjunction with an additional fluoro group at position 2 or 3 (50-55) (Table S2). A synergistic effect on antiSIRT2 activity and selectivity was observed for compounds 53 (vs. 35) and 55 (vs. 38), which contained a methoxy and chloro group, respectively, at position 4. In summary, our SAR study on the effect of a fluoro group at different positions revealed that a properly positioned fluoro could significantly improve anti-SIRT2 activity and selectivity over SIRT1 and SIRT3; therefore, a fluoro group especially one at position 3 should be explored in future SAR studies.

Since we demonstrated that position 4 was a constructive modification site, we opted to conduct extensive SAR studies at this position (Table 4). To expand on the beneficial effect incurred by a methyl group as seen in compound 32 (Table 3), we prepared compounds 56-59, which featured an increasingly bulky alkyl group. Compared with compound 32, there was no significant improvement in anti-SIRT2 activity. However, when a phenyl group was appended at position 4 to give compound 60, a remarkable boost in anti-SIRT2 activity was realized, leading to a low nanomolar IC50 value. A similar effect was observed for compound 61, which contained an isosteric thiophene ring. Also importantly, both compounds 60 and 61 now exhibited more than 1000 fold selectivity over SIRT1 and SIRT3. These findings suggested that an aromatic ring at position 4 conferred greatly enhanced activity and selectivity, a SAR trend that was further tested


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by introducing varied heterocyclic aromatic rings as seen in compounds 62-69. In compound 62, a 2-thioazole at position 4 resulted in a SIRT2 IC50 comparable with that of compound 61, albeit with reduced isozyme selectivity. However, an N-pyrrole in compound 63 caused a significant loss of anti-SIRT2 activity, which was regained with an N-pyrazole (64). Noticeably, both compounds 63 and 64 possessed markedly lower selectivity in comparison with compound 61. The selectivity was further eroded in compound 65, which contained an N-imidazole. As a result, compound 65 showed similar submicromolar activity against SIRT1-3, exhibiting nearly no selectivity. When an N-triazole was used, a gain of activity against SIRT2 was observed for compound 66; nonetheless, its activity against SIRT1 and SIRT3 still remained submicromolar, leading to a low selectivity. Our SAR study with heterocyclic aromatic rings in compounds 6266 suggested that nitrogen atoms in the ring had a detrimental effect on the isozyme selectivity. This intriguing SAR trend was further confirmed in pyridine-derived compounds 67-69, in which a nitrogen atom was incorporated at position 2, 3 or 4, respectively. While the anti-SIRT2 activity remained essentially identical, the selectivity deteriorated when compared with compound 60.

We also attempted to find clues for significant erosion of isozyme selectivity seen in compounds 63-69. Our studies on inhibition mode and computational modeling (vide infra) showed our new SIRT2 inhibitors most likely adopted a binding mode so that ring A and its substituent at position 4 interacted with the residues in the substrate binding site. A previous sequence analysis of SIRT1-3 has shown high similarity in the residues surrounding the substrate binding site,29 suggesting that it would be difficult to pinpoint residues that were responsible for decrease of isozyme selectivity. Nevertheless, SIRT3 substrate specificity has been examined,30 revealing



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that positively charged residues in the substrate are preferred for tight SIRT3 binding. Since the basic amines present in compounds 64-69 are expected to be at least partially protonated under physiological conditions, the resultant positive charge might account for their higher affinity for SIRT3 and hence reduced isozyme selectivity. It is also possible that selectivity against SIRT1 deteriorates via a similar mechanism. Further studies are clearly needed to pinpoint factors that affect isozyme selectivity of our new SIRT2 inhibitors.

Having recognized that nitrogen atoms decreased the selectivity, we chose to explore acetylene as an isosteric but less bulky replacement of a phenyl group. The resulting compound 70 showed excellent inhibitory activity against SIRT2 even though the selectivity profile was still suboptimal. In summary, our investigation of substituents at position 4 clearly indicated that a non-basic aromatic ring was preferred for high activity and selectivity.

We then proceeded to investigate various functional groups including ester, acid, amide, ether and amine at position 4 (Table 5). Compound 71 with a methyl ester showed high anti-SIRT2 activity even though a carboxylic acid (72) was not tolerated. A primary amide (73) led to an IC50 value of less than 50 nM. N-Methylation (74) further enhanced the activity; however, Ndimethylation (75) caused a significant loss of activity against SIRT2. On the other hand, a reversed methyl amide (76) was well tolerated while a methyl ketone (77) reduced activity. Notably, even though primary and mono-substituted amides as well as a mono-substituted reversed amide at position 4 conferred excellent anti-SIRT2 activity, compounds that contained these functional groups generally possessed relatively low selectivity especially relative to those with non-basic aromatic rings. When a phenoxy (78) or benzoxy (79) was attached, the resulting


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compounds showed anti-SIRT2 activity comparable to that of the parent compound 15; however, a significant boost in selectivity was attained, suggesting that a properly substituted ether functionality could be installed to improve the selectivity profile.

We also explored amines at position 4 (Table 5). While compound 80 harboring a methylamino group showed an anti-SIRT2 activity comparable to that of the parent compound 15, an ethylamino group (81) caused a minor increase in activity. When the amino group was dimethylated (82), a marked enhancement in the activity and selectivity (> 250 fold) was observed. Surprisingly, a bulkier diethylamino group (83) led to a significant loss of activity compared to compound 82. These findings suggested that a disubstituted amine was desirable at position 4 even though larger groups might not be tolerated. In addition to acyclic amines, we also investigated cyclic amines as potential substituents at position 4. When compared with compound 15, compound 84 with an N-pyrrolidine ring showed slightly improved anti-SIRT2 activity; however, a piperidine ring (85) had a negligible impact. When a morpholine ring was introduced, the resultant compound 86 exhibited a low nanomolar IC50 value against SIRT2 together with more than 300 and 100 fold selectivity over SIRT1 and SIRT3, respectively. In contrast, an N-methylpiperazine (87) resulted in a drastic decrease in the anti-SIRT2 activity. Also noticeably, the isozyme selectivity was significantly lowered, reminiscent of a similar loss of selectivity in compounds that contained basic heterocyclic rings as listed in Table 4. Taken together, we showed that disubstituted amines were beneficial at position 4 even though their steric and electronic nature should be taken into account to obtain desired activity and selectivity.



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Since we discovered that a properly substituted amine enhanced anti-SIRT2 activity, we continued to explore amines with an extra carbon spacer as depicted in Table S3 (compounds 8899). Briefly, we showed that cycloalkylamino groups with a methylene spacer generally had reduced anti-SIRT2 activity in comparison with their analogs without a spacer; however, introduction of fluoro groups on the cycloalkylamines might offer an opportunity to improve the activity against SIRT2 as seen in compounds 98 and 99.

As shown in Table 4, a nitrogen atom, depending on its position, appeared to have a profound impact on anti-SIRT2 activity and selectivity. In order to test whether a similar effect existed in ring A, we first investigated compounds 100-106 (Table 6). When a nitrogen atom was placed at position 2 (100), a significant reduction in anti-SIRT2 activity was observed. A nitrogen atom at position 3 (101) was tolerated while one at position 4 (102) markedly increased the anti-SIRT2 activity. Second, we investigated a combined effect of two nitrogen atoms at various positions (103-106), which again revealed the detrimental effect of a nitrogen atom at position 2 in compounds 103, 105, and 106. In addition, the beneficial effect of a nitrogen atom at position 4 was abrogated by that of a nitrogen atom at position 3 as seen in 104. In addition to compounds 100-106, which were based on compound 15, we also explored incorporating a nitrogen atom in the phenyl of the cinnamic amide moiety of compound 19 by preparing compounds 107-109 (Table 6). In those compounds, changing the location of the nitrogen atom among positions 2, 3 and 4 produced increasingly higher inhibition against SIRT2, a SAR trend that was very similar to that observed in compounds 100-102. The SAR study in Table 6 clearly indicated that a nitrogen atom at position 4 was constructive while one at position 3 was merely tolerated.


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The latter finding coupled with our previous observation that a substituent at position 4 was preferred prompted us to study compounds 110-114 (Table 7). In those compounds, an aromatic ring was introduced at position 4 because a similar modification generally offered significantly enhanced activity and selectivity as described above (Table 4). In compound 110, a phenyl group led to excellent anti-SIRT2 activity and selectivity while a 2-thiophene ring (111) resulted in similar activity and extraordinary selectivity. Similarly, 2-furan, N-pyrrole, and N-pyrazole attached at position 4 yielded SIRT2 inhibitors 112, 113 and 114, respectively, with comparable activities. Furthermore, we also probed a fluoro group at position 3 in compounds 115-117 since a fluoro group could be beneficial as demonstrated in Table S2. Compound 115 in which a phenyl group was incorporated showed excellent anti-SIRT2 activity and selectivity. A 3pyridine and 4-pyridine in compounds 116 and 117 also offered extremely high activity even though their isozyme selectivity was reduced, a SAR trend similar to the one seen with compounds 67-69 (Table 4). Taken together, for ring A, a nitrogen atom or a fluoro group was well tolerated, therefore expanding the structural diversity of our SIRT2 inhibitors.

Additional Biochemical Evaluation. In order to accurately assess the relative isozyme activity of selected SIRT2 inhibitors, we tested these compounds in our newly established in-house sirtuin inhibitory assays, in which the concentrations of NAD+ and the peptide substrate were used at the KM values determined for each enzyme. Compounds 61, 86, 101, and 102 were selected because of their excellent activity and selectivity as well as structural diversity. Compound 61 had a lipophilic thiophene group at position 4 while inhibitor 86 contained a more hydrophilic morpholino group. Compounds 101 and 102 were chosen because they contained a pyridine A ring in contrast to a phenyl A ring in compounds 61 and 86. These four selected



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compounds all showed excellent activity against SIRT2 and selectivity over SIRT1 and SIRT3 (Table 8), generally mirroring the profiles obtained using the initial sirtuin inhibitory assays. Among these selected SIRT2 inhibitors, compound 61 displayed the highest isozyme selectivity, highlighting the importance of an aromatic ring at position 4. Compounds 86, 101, and 102 were also tested against human SIRT5 and all exhibited outstanding selectivity, suggesting that our newly discovered 5-((3-amidobenzyl)oxy)nicotinamides are devoid of SIRT5 inhibition.

Inhibition Mode. In order to characterize the inhibition mode of SIRT2 inhibitors based on a 5((3-amidobenzyl)oxy)nicotinamide core structure, compound 86 was selected because of its excellent anti-SIRT2 activity and selectivity over SIRT1, SIRT3 and SIRT5. Accordingly, compound 86’s IC50 values were determined against NAD+ and the peptide substrate. A secondary plot of IC50 values versus [S]/KM was analyzed using the tight binding versions of the Cheng-Prusoff equations.31 As depicted in Figure 3, a plot of IC50 values versus the concentrations of NAD+ (A) indicated that compound 86 was competitive against NAD+. Furthermore, a plot of IC50 values versus the concentrations of the peptide substrate (B, Figure 3) showed that compound 86 was also a competitive inhibitor with respect to the peptide substrate.

To examine whether a substituent at position 4 influenced an inhibitor’s inhibition mode, we also tested compound 61, in which a hydrophobic thiophene, in contrast to compound 86’s basic and hydrophilic morpholino group, was placed at position 4. As shown in Figure S1, an initial examination of a plot of IC50 values versus the concentrations of NAD+ suggested that compound 61 was likely to be competitive against NAD+. However, when the data was fitted to a noncompetitive inhibition modality (A, IC50 = ([S] + KM)/[(KM/Ki) + ([S]/αKi)] + [E]T/2), α = 6.2


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was obtained. Since a classical noncompetitive inhibitor displays α = 1,31 our result indicated that compound 61 was mainly competitive against NAD+; however, it also exhibited certain feature of a noncompetitive inhibitor. Furthermore, a plot of IC50 values versus the concentrations of the peptide substrate (B, Figure S1) showed that compound 61 was a competitive inhibitor with respect to the peptide substrate.

Notably, the inhibition mode of compounds 86 and 61 differed from that of compound 8, a lead compound previously reported by us.28 Like compound 8, both compounds 86 and 61 were competitive against the peptide substrate. Nevertheless, compound 8 was most likely a noncompetitive inhibitor with respect to NAD+. In contrast, compound 86 was competitive against NAD+ and compound 61 mainly displayed characteristics of a competitive inhibitor with respect to NAD+. This difference suggested that compounds 86 and 61 might represent a new class of SIRT2 inhibitors distinctive from compound 8. Also remarkably, the slightly different inhibition mode exhibited by compound 86 versus compound 61 suggested that a substituent at position 4 had certain impact on their inhibition mode against NAD+. However, it was difficult to pinpoint structural features, such as lipophilicity or basicity, which led to such a discrepancy. It is expected that future structural studies of compounds in complex with SIRT2 will provide clues about different inhibition modes exhibited by these seemingly close analogs.

Computational Modeling. In an attempt to explore the potential binding modes of compound 86, it was docked into human SIRT2 (PDB: 1j8f).32 Our docking study revealed a major potential binding mode (Figure 4), in which the nicotinamide moiety (ring C, Figure 2) of compound 86 interacted with the C-site of the NAD+ binding pocket. The primary amide functionality in ring C



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formed two hydrogen bonds with the backbone NH of Ile 169 and the side chain carboxylate of Asp170. The middle ring B was engaged in π-π interactions with Phe 119 and His 187. As a result, ring A (Figure 2) extended into the peptide substrate binding pocket, forming a π-π interaction with Phe 235. The observation that compound 86 occupied both the C-site of the NAD+ binding pocket and the peptide binding channel might well account for the competitive nature of compound 86 versus both NAD+ and the peptide substrate. This binding mode also explained our finding that position 4 was the preferred modification site since position 4 was relatively less hindered. Therefore, a range of substituents including a morpholino group in compound 86 and a phenyl group in compound 61 were well tolerated. Interestingly, a similar binding mode was also envisioned for compound 8; nevertheless, compound 8 had another potential binding mode, in which the naphthalene and benzamide moieties occupied a mainly hydrophobic pocket that was almost perpendicular to the peptide binding channel. Whether this latter binding modes led to a shift in the inhibition modality of compound 8 in relation to 86 remains to be investigated.

In Vitro Metabolic Stability and Permeability. To obtain preliminary data on the ADME properties of our new SIRT2 inhibitors, selected compounds (86, 101 and 102) were evaluated for their stability in plasma and liver microsomes from both mouse and human (Table 9). These three compounds were chosen because of their confirmed activity and selectivity (Table 8). Furthermore, they also had a relatively low molecular weight and possessed a basic functionality in ring A, both of which are generally preferred for CNS-targeting drugs.33 While all three compounds exhibited excellent stability in the human plasma, their stability in the mouse plasma was compromised to varied extents. Compounds 86 and 101 remained relatively stable in the


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mouse plasma, but compound 102 showed modest albeit acceptable stability. An interspecies variance was also observed for microsomal stability. Compound 86 showed good stability in the human liver microsomes but low stability in the mouse liver microsomes. Compounds 101 and 102 displayed a similar interspecies variance. Nevertheless, they exhibited excellent human microsomal stability together with good stability in the mouse liver microsomes. To estimate whether our selected compounds have desired in vivo BBB permeation, a property that is required for a therapeutic application in PD, we tested our compounds using the Madin-Darby Canine Kidney (MDCKII) cell monolayer assay.34 While compound 86 showed moderate MDCK permeability, compounds 101 and 102 possessed very good permeability (Table 9). In comparison, compound 8 displayed relatively low MDCK permeability.

Cellular Model of α-Synuclein Toxicity. Neuroprotection mediated by SIRT2 inhibition has been investigated in various cellular models of PD.5-6 We chose to use a cellular model of PD induced by an extracellular supply of aggregated α-synuclein35 because there has been growing evidence to support the notion that these α-synuclein aggregates are capable of selfpropagation.36 Lower order aggregates of α-synuclein are soluble and capable of permeating into neuronal cells.37 Exposure of primary mesencephalic neuroglia and human neuroblastoma cells to aggregated but soluble α-synuclein leads to cytotoxicity, mirroring the mechanism of the spread of synucleinopathies such as PD.38 Furthermore, even exogenous α-synuclein fibrils are able to seed the formation of Lewy bodies (LB)-like intracellular inclusions, leading to synaptic dysfunction, neuron death, and a rapidly progressive neurodegenerative α-synucleinopathy in mice.39 More recently, in both mice and monkeys, inoculations of PD patient-derived LB extracts resulted in progressive nigrostriatal neurodegeneration.40 These observations clearly suggest that



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α-synuclein aggregation-induced cytotoxicity in SH-SY5Y cells is a reasonable model to gauge the protective effects by our SIRT2 inhibitors.

Compounds 86 and 102 were chosen because they possessed relatively good MDCK permeability. In addition, compound 61 was also included due to its remarkable isozyme selectivity. Furthermore, compound 1, a commonly used SIRT2 inhibitor, and compound 8, which was reported by us and served as a starting point for this current study, were also included as reference compounds.

Since a freshly prepared solution of α-synuclein failed to cause any significant toxicity to SHSY5Y cells, we generated oligomeric α-synuclein by maintaining the peptide stock solution at 37 °C for 7 days. Compounds 86 and 102 were chosen because they possessed excellent activity and relatively good MDCK permeability. In addition, compound 1,5 a commonly used SIRT2 inhibitor, was included as a reference compound. When evaluated for their protective effect, both compounds 86 and 102 showed statistically significant protection in the low micromolar range (Figure 5). Protection was indicated by a significant increase in cell growth when compared to the control cells in the presence of α-synuclein (black bars). Furthermore, compounds 86 and 102 were not toxic toward SH-SY5Y cells at the concentrations used in this assay (white bars). In comparison, compound 1, exhibited relatively less protection when used at the same concentration and appeared to be toxic at a higher concentration.

We also evaluated the protective effect by compounds 86, 102, and 61 with compound 8 rather than compound 1 as a reference compound. Compound 61 was included due to its remarkable


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isozyme selectivity. As shown in Figure S2, compound 86 offered statistically significant protection at both 10 µM and 1 µM while compound 102 showed significant protection only at 10 µM. Lack of a protective effect at 1 µM was also evident for compound 61 even though it elicited statistically significant protection at 10 µM. In contrast, compound 8 provided no significant protection at either concentration. These results indicated that compound 86 offered improved protection in comparison with compounds 61 or 8. Since the latter two compound possessed excellent anti-SIRT2 and isozyme selectivity, reduced protection might be due to their inferior permeability and/or in vitro metabolic stability.

Chemical Synthesis. The synthesis of 5-((3-amidobenzyl)oxy)nicotinamide SIRT2 inhibitors was straightforward (Scheme 1). Treatment of commercially available bromide 118 with methyl 5-hydroxynicotinate gave nitrate 119, which was reduced with NaBH4 in the presence of NiCl2 to afford amine 120. The methyl ester in compound 120 was converted into the corresponding primary amide 121 in excellent yield using methanolic ammonia aided by CaCl2. Key intermediate 121 was then used to prepare 5-((3-amidobenzyl)oxy)nicotinamide SIRT2 inhibitors as represented by 122. Amide formation was accomplished in varied yields using an acyl chloride, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU),

N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide

(EDC)/1-hydroxybenzotriazole

(HOBt), or EDC alone, as exemplified by the syntheses of compounds 10, 14, 22, and 42, respectively.

CONCLUSIONS



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In our continued efforts to discover novel SIRT2 inhibitors, we have designed compounds based on a 5-((3-aminobenzyl)oxy)nicotinamide core structure (Figure 2). Our extensive SAR studies have revealed several key structural features that convey excellent activity against SIRT2 and high selectivity over SIRT1 and SIRT3. We have identified position 4 of the benzamide ring A as the most rewarding modification site, leading to a number of potent and selective SIRT2 inhibitors. Selected compounds also showed selectivity over SIRT5. Kinetic studies of the representative SIRT2 inhibitor 86 revealed that it was competitive against both NAD+ and the peptide substrate, an inhibitory modality that was supported by computational modeling. When evaluated for their in vitro ADME properties, selected compounds exhibited generally favorable properties, including metabolic stability and permeability. This finding prompted us to test compounds 86 and 102 in an α-synuclein aggregation-induced cytotoxicity model in SH-SY5Y cells, in which a protective effect against α-synuclein toxicity was observed for both compounds. Taken together, excellent anti-SIRT2 activity, high isozyme selectivity, a generally favorable in vitro ADME profile, significant protection against α-synuclein toxicity, and facile syntheses make 5-((3-amidobenzyl)oxy)nicotinamides not only a new class of SIRT2 inhibitors but also attractive candidates for further lead optimization. Optimized SIRT2 inhibitors will be tested for efficacy in animal models of PD in our future studies.

EXPERIMENTAL SECTION

Chemical Synthesis. All commercial reagents were used as provided unless otherwise indicated. An anhydrous solvent dispensing system (J. C. Meyer) using 2 packed columns of neutral alumina was used for drying THF, Et2O, and CH2Cl2, while 2 packed columns of molecular


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sieves were used to dry DMF. Solvents were dispensed under argon. Flash chromatography was performed with Ultra Pure silica gel (Silicycle) or with RediSep® Rf silica gel columns on a Teledyne ISCO CombiFlash® Rf system using the solvents as indicated. Nuclear magnetic resonance spectra were recorded on a Varian 600 MHz with Me4Si or signals from residual solvent as the internal standard for 1H. Chemical shifts are reported in ppm, and signals are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet), and dd (double doublet). Values given for coupling constants are first order. High resolution mass spectra were recorded on an Agilent TOF II TOF/MS instrument equipped with a multi-mode source (ESI or APCI). Analysis of sample purity was performed on an Agilent 1200 Infinity series HPLC system with a Phenomenex Gemini C18 column (5 µ, 4.6×250 mm). HPLC conditions were the following: solvent A = water, solvent B = MeCN or MeOH; ﬂow rate = 2.0 mL/min. Compounds were eluted with a gradient of 10% to 100% MeCN/water or 10% to 100% MeOH/water in 15 min. Purity was determined by the absorbance at 254 nm. All tested compounds have a purity of ≥ 95%.

Representative procedures for the preparation of SIRT2 inhibitors using aniline 121.

5-((3-(4-Methyl-3-nitrobenzamido)benzyl)oxy)nicotinamide (10). To a solution of amine 121 (52 mg, 0.21 mmol) and DIPEA (80 µL, 0.46 mmol) in anhydrous CH2Cl2 (5 mL) and DMF (1 mL) was added 4-methyl-3-nitrobenzoyl chloride (50 µL, 0.34 mmol) and the mixture was allowed to stir at rt for 24 h. After the solvents were removed, the residue was diluted with EtOAc (30 mL), H2O (10 mL) and saturated NaHCO3 (10 mL). After separation, the organic layer was washed with brine (20 mL) and concentrated. The residue was purified by flash



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column chromatography (0-10% MeOH/CH2Cl2) to give compound 10 as a white solid (51 mg, 59%). 1H NMR (DMSO-d6, 600 MHz) δ 10.54 (s, 1H), 8.65 (s, 1H), 8.58 (s, 1H), 8.50 (d, J = 3.0 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.14 (s, 1H), 7.91 (s, 1H), 7.86 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.62 (s, 1H), 7.41 (dd, J = 7.8, 7.8 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 5.26 (s, 2H), 2.60 (s, 3H);

13

C NMR (DMSO-d6, 150 MHz) δ 166.6, 163.7, 154.7, 149.2,

141.4, 141.0, 139.4, 137.3, 136.8, 134.1, 133.5, 132.6, 130.8, 129.4, 124.0, 123.8, 120.6, 120.5, 120.1, 70.2, 20.0. HRMS (ESI+) calcd for C21H19N4O5 (M+H)+ 407.1350, found 407.1353.

5-((3-(Cyclohex-1-enecarboxamido)benzyl)oxy)nicotinamide (14). To a solution of cyclohex1-ene-1-carboxylic acid (61.8 mg, 0.49 mmol), HBTU (186 mg, 0.49 mmol) and DIPEA (0.21 mL, 1.23 mmol) in THF (10 mL) was added amine 121 (100 mg, 0.41 mmol) and the mixture was allowed to stir at rt for 12 h. After the solvents were removed, the residue was diluted with EtOAc (30 mL), H2O (10 mL) and saturated NaHCO3 (10 mL). After separation, the organic layer was washed with brine (20 mL) and concentrated. The residue was purified by flash column chromatography (0-10% MeOH/CH2Cl2) to give compound 14 as a white solid (120 mg, 84%). 1H NMR (DMSO-d6, 600 MHz) δ 9.65 (s, 1H), 8.64 (s, 1H), 8.47 (d, J = 3.0 Hz, 1H), 8.14 (s, 1H), 7.85-7.82 (m, 1H), 7.81 (s, 1H), 7.65-7.57 (m, 2H), 7.32 (dd, J = 8.1, 8.1 Hz, 1H), 7.14 (dd, J = 7.8, 7.8 Hz, 1H), 6.65 (s, 1H), 5.20 (s, 2H), 2.27-2.22 (m, 2H), 2.20-2.14 (m, 2H), 1.661.60 (m, 2H), 1.60-1.54 (m, 2H); 13C NMR (DMSO-d6, 150 MHz) δ 167.5, 166.6, 154.7, 141.4, 141.0, 141.1, 137.0, 134.4, 133.5, 130.8, 129.1, 122.9, 120.4, 120.2, 119.6, 70.2, 25.3, 24.4, 22.2, 21.7. HRMS (ESI+) calcd for C20H22N3O3 (M+H)+ 352.1656, found 352.1669.


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5-((3-(Benzo[b]thiophene-2-carboxamido)benzyl)oxy)nicotinamide (22). To a solution of benzo[b]thiophene-2-carboxylic acid (37 mg, 0.21 mmol), HOBt (28 mg, 0.21 mmol) and EDC (28 mg, 0.21 mmol) in DCM (15 mL) was added amine 121 (50 mg, 0.21 mmol) and the mixture was allowed to stir at rt for 16 h. After the solvents were removed, the residue was diluted with EtOAc (30 mL), H2O (10 mL) and saturated NaHCO3 (10 mL). After separation, the organic layer was washed with brine (20 mL) and concentrated. The residue was purified by flash column chromatography (0-10% MeOH/CH2Cl2) to give compound 22 as a pale solid (48 mg, 57%). 1H NMR (DMSO-d6, 600 MHz) δ 10.58 (s, 1H), 8.66 (s, 1H), 8.51 (d, J = 3.0 Hz, 1H), 8.38 (s, 1H), 8.14, (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.91 (s, 1H), 7.87 (s, 1H), 7.76 (d, J = 9.6 Hz, 1H), 7.61 (s, 1H), 7.52-7.45 (m, 2H), 7.42 (dd, J = 7.8, 7.8 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H) 5.27 (s, 2H);

13

C NMR (DMSO-d6, 150 MHz) δ 166.6, 160.8, 154.7,

141.5, 141.0, 141.0, 140.4, 139.6, 139.3, 137.4, 130.8, 129.4, 126.9, 126.4, 125.9, 125.5, 123.7, 123.5, 120.5, 120.4, 119.8, 70.2. HRMS (ESI+) calcd for C22H18N3O3S (M+H)+ 404.1063, found 404.1064.

5-((3-(2-Cyanobenzamido)benzyl)oxy)nicotinamide (42). To a solution of 2-cyanobenzoic acid (22 mg, 0.25 mmol) and EDC (47 mg, 0.25 mmol) in DMF/DCM (1:1, 10 mL) was added amine 121 (50 mg, 0.21 mmol) and the mixture was allowed to stir at rt for 12 h. After the solvents were removed, the residue was diluted with EtOAc (30 mL), H2O (10 mL) and saturated NaHCO3 (10 mL). After separation, the organic layer was washed with brine (20 mL) and concentrated. The residue was purified by flash column chromatography (0-10% MeOH/CH2Cl2) to give compound 42 as a pale solid (10 mg, 13%). 1H NMR (DMSO-d6, 600 MHz) δ 10.29 (s, 1H), 8.66 (s, 1H), 8.51 (d, J = 2.4 Hz, 1H), 8.27 (d, J = 7.2 Hz, 1H), 8.14 (s, 1H), 7.92-7.85 (m,



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3H), 7.82-7.78 (m, 1H), 7.64-7.52 (m, 4H), 7.45 (d, J = 7.8 Hz, 1H), 5.30 (s, 2H);

Page 26 of 62

13

C NMR

(DMSO-d6, 150 MHz) δ 167.3, 166.6, 159.7, 154.7, 141.5, 141.0, 137.3, 134.2, 133.0, 131.7, 130.8, 130.4, 129.2, 128.4, 127.9, 127.4, 123.3, 123.2, 120.5, 110.0, 69.9. HRMS (ESI+) calcd for C21H17N4O3 (M+H)+ 373.1295, found 373.1302.

Methyl 5-((3-Nitrobenzyl)oxy)nicotinate (119). To a solution of 3-nitrobenzyl bromide (118, 9.50 g, 44 mmol) in DMF (80 mL) were added methyl 5-hydroxynicotinate (6.12 g, 40 mmol) and Cs2CO3 (26.1 g, 80 mmol) and the mixture was allowed to stir at rt for 12 h. After water (200 ml) was added, the precipitate was filtered, washed with hexanes, and dried in vacuo to yield compound 119 as a light yellow solid (8.64 g, 75%). 1H NMR (CDCl3, 600 MHz) δ 8.89 (d, J = 1.2 Hz, 1H), 8.58 (d, J = 3.0 Hz, 1H), 8.35 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.87 (dd, J = 3.0, 1.8, Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 6.61 (dd, J = 7.8, 7.8 Hz, 1H), 5.25 (s, 2H), 3.97 (s, 3H);

13

C NMR (DMSO-d6, 150 MHz) δ 165.6, 149.3, 143.2, 142.5, 142.5, 137.0, 129.4, 121.3,

121.3, 115.4, 114.1, 113.2, 70.7, 52.9. HRMS (ESI+) calcd for C14H13N2O5 (M+H)+ 289.0819, found 289.0820.

Methyl 5-((3-Aminobenzyl)oxy)nicotinate (120). To a solution of compound 119 (8.64 g, 30 mmol) and NiCl2·6H2O (14.3 g, 44 mmol) in MeOH (100 mL) was slowly added NaBH4 (4.8 g, 120 mmol) and the mixture was allowed to stir at rt for 3 h. The reaction was quenched with saturated NH4Cl (50 mL) and the mixture was extracted with EtOAc. The organic phase was washed with water and brine, dried over anhydrous K2CO3, and concentrated in vacuo. The residue was purified by flash column chromatography (0-60% EtOAc/hexanes) to afford compound 120 as a light yellow solid (6.19 g, 80%). 1H NMR (CDCl3, 600 MHz) δ 8.83 (d, J =


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1.8 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.83 (dd, J = 2.4, 1.8, Hz, 1H), 7.18 (d, J = 7.8 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.66 (dd, J = 7.8, 1.8 Hz, 1H), 5.30 (s, 2H), 5.06 (s, 2H), 3.95 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) δ 165.6, 153.8, 148.3, 143.3, 142.9, 138.9, 134.7, 130.6, 130.6, 123.5, 122.7, 121.4, 69.0, 53.0. HRMS (ESI+) calcd for C14H15N2O3 (M+H)+ 259.1077, found 259.1080.

5-((3-Aminobenzyl)oxy)nicotinamide (121). A solution of methyl ester 120 (6.19 g, 24 mmol) and CaCl2 (2.66 g, 24 mmol) in NH3/MeOH (ca. 7 N, 20 mL) in a seal tube was heated at 70 ºC for 24 h. After the solvent was evaporated in vacuo, the residue was dissolved in EtOAc (150 mL) and the resulting solution was washed with H2O (150 mL) and brine (100 mL). After the organic layer was dried over Na2SO4 and filtered, the filtrate was concentrated and the residue was purified by flash column chromatography (0-5% MeOH/CH2Cl2) to afford compound 121 as a light yellow solid (5.25 g, 90%). 1H NMR (DMSO-d6, 600 MHz) δ 8.63 (s, 1H), 8.44 (d, J = 3.0 Hz, 1H), 8.12 (s, 1H), 7.81 (s, 1H), 7.60 (s, 1H), 7.02 (dd, J = 7.5, 7.5 Hz, 1H), 6.63 (s, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.52 (d, J = 7.8 Hz, 1H), 5.13 (s, 2H), 5.07 (s, 2H); 13C NMR (DMSOd6, 150 MHz) δ 166.6, 154.8, 149.3, 141.2, 141.0, 137.2, 130.8, 129.4, 120.3, 115.4, 114.0, 113.3, 70.6. HRMS (ESI+) calcd for C13H14N3O2 (M+H)+ 244.1081, found 244.1082.

Initial SIRT1-3 and SIRT5 Biochemical Assays. Initial biochemical assays against human SIRT1-3 were performed at Reaction Biology Corporation (RBC) (Malvern, PA, USA, http://www.reactionbiology.com) as reported previously.28 The activity against SIRT5 was measured under conditions similar to those used for SIRT1-3. Briefly, human SIRT5 (UniProt ID: Q9NXA8, amino acids 37-310 with N-terminal His-tag, MW = 32.3 kDa) was expressed in



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E. coli and purified. Ac-Lys(Succ.)-AMC (50 µM) was used as a fluorogenic SIRT5 substrate in conjunction with NAD+ (500 µM). After the deacetylation reaction was allowed to proceed at 30 ºC for 2 h, the mixture was developed and the fluorescence intensity was measured at 360/460 Ex/Em. Compounds were tested in a 10-dose mode with a 3-fold serial dilution starting at 200 µM (200 µM–10.2 nM) in singlet. The IC50 values were then calculated from the resulting sigmoidal dose-response curves using GraphPad Prism.

In-house SIRT1-3 Biochemical Assays. Human SIRT1-3 proteins were expressed and purified as reported previously.28 Plasmids for the expression of SIRT1 (SIRT1.1, Addgene plasmid 13735) and SIRT3 (SIRT3L-12, Addgene plasmid 13736) were gifts from John Denu.41 The enzymatic assays were performed according to the following conditions, under which the concentrations of NAD and the peptide substrate were used at the KM values determined for each enzyme in order to accurately assess a compound’s selectivity against SIRT1-3.

Enzyme

SIRT1

SIRT2

SIRT3

NAD (mM)

0.26

0.20

0.34

Peptide substrate (mM)

0.45

0.30

0.26

Enzyme (µM)

0.25

0.10

1.5

Reaction time (min)

20

20

60

Compounds were aliquoted (0.5 µL) in duplicate into a black 384-well non-binding surface plate (Corning). A master mix containing assay buffer (50 mM Tris pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mg/mL BSA) and NAD+ was added to at least two wells


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to serve as a negative control. Enzyme was added to the remaining master mix and the resulting solution was then aliquoted into the remaining wells. The plate was mixed for 30 s at 1,500 RPM on a MixMate (Eppendorf) and left at 25 ºC for 10 min. The reactions were initiated by the addition of the peptide substrate (Ac)RHKK(Ac)-AMC (to a final volume of 50 µL) followed by mixing of the plate as described above. The reaction was allowed to proceed at 37 ºC for the desired time and then quenched by the addition of 10 µL developing buffer (12 mM nicotinamide and 30,000 U/mL trypsin in water). The plate was mixed and then developed for 20 min at 37 ºC before the fluorescence intensity was measured using an excitation at 355 nm and an emission at 460 nm. The data were analysed using either the Morrison equation (SIRT2) or the four-parameter dose response equation (SIRT1 and 3) in GraphPad Prism.

Determination of Inhibition Modality by Compound 86 and 61 against SIRT2. The mode of inhibition assay was performed using the peptide substrate (Ac)RHKK(Ac)-AMC in a manner similar to that described previously.28 The assay was carried out using 100 nM recombinant SIRT2 protein (concentration determined by active site titration) in 50 µL of assay buffer. The concentration of NAD+ was either held constant at 0.35 mM with variable peptide concentrations (0.015, 0.044, 0.133, 0.400, 1.20, and 3.60 mM) or varied (0.044, 0.133, 0.400, 1.20 and, 3.60 mM ) with a constant peptide concentration (0.52 mM). The concentration of compound 86 or 61 was varied from 0.0206 to 5.00 µM (3-fold serial dilution) with 100 µM included as a negative control. Reactions were initiated by the addition of the peptide substrate and incubated at 37 °C for 20 min. The reactions were then stopped, developed and detected as described above. The IC50 value was calculated by using GraphPad Prism software to fit the data to the log (inhibitor) vs. response -- Variable slope (four parameters). Secondary plots of IC50 as a function of the



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substrate concentration over KM were used to determine binding modality by fitting to the Cheng−Prusoff tight binding equations and selecting the mode that afforded the best fit.31

Molecular Modeling. The modeling study was carried out as described previously.28

Plasma Stability Assay. The plasma stability assay was performed in triplicate by incubating a compound in normal mouse and human plasma at 37 °C. At 0, 1, 3, 6, and 24 h, aliquots of the plasma mixture were taken and quenched with 3 volumes of acetonitrile containing an appropriate internal standard. The samples were then vortexed and centrifuged at 14,000 rpm for 5 min. The supernatants were collected and analyzed by LC-MS/MS to determine the remaining percentage at various time points.

Microsomal Stability Assay. The in vitro microsomal stability assay was conducted in triplicate in mouse and human liver microsomal systems. In a typical incubation, a compound (typically 1 µM final concentration) was spiked into the reaction mixture containing 0.5 mg/mL of liver microsomal protein and 1 mM of NADPH in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C. At various time points, 1 volume of reaction aliquot was taken and quenched with 3 volumes of acetonitrile with an appropriate internal standard. The samples were then vortexed and centrifuged at 14,000 rpm for 5 min. The supernatants were collected and analyzed by LCMS/MS to determine the in vitro metabolic half-life (t1/2) and intrinsic clearance (CLint). Verapamil was used as a positive control.


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LC-MS/MS Bioanalysis. Quantification and analysis of compounds in biological samples were carried out on an AB Sciex QTrap 5500 mass spectrometer coupled with an Agilent 1260 Infinity HPLC. The chromatographic separation of compounds was achieved using a Phenomenex Kinetex C18 column (50 × 2.1 mm, 2.6 µm), and MS/MS analysis was conducted using an ESI ion source with MRM detection. The MS/MS detection parameters including declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP) were optimized for each compound.

Directional Transport across MDCKII Cell Monolayers. The transport experiments were performed under conditions described previously.28 The recovery of compound was > 90% at the end of the experiment.

Protection against α-Synuclein Toxicity. SH-SY5Y cells were seeded in a 96-well plate at a density of 20,000 cells/well. On the next day, the cells were treated with SIRT2 inhibitors at varied concentrations at 37 °C for 30 min, followed by addition of media containing oligomeric α-synuclein to a final concentration of 10 µM. The oligomeric α-synuclein was generated by maintaining an α-synuclein (rPeptide) stock solution (100 µM in water) at 37 °C for 7 days. The cells were then incubated for 72 h. The media containing compounds was replaced with fresh media and cell viability was then determined through MTT assay.35 Data were analyzed by oneway analysis of variance (ANOVA) followed by Dunnett’s post-hoc comparison test with significance level held at 0.05 (95% confidence intervals).



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Supporting Information Available: The chemical characterization data of compounds 11-13, 15-21, 23-41, and 43-117, Table S1-S3 and Figure S1-S2.

Acknowledgment. This research was supported by the Center for Drug Design in the Academic Health Center of the University of Minnesota. We thank Dr. Huaqing Cui for the preparation of compounds 11-13, 15-21, 28-41, 45-55, 56-58, 60, 79, 84-87 and 100-106 from intermediate 121. The University of Minnesota Supercomputing Institute provided all the necessary computational resources.

Corresponding Author Information

L.C. Tel.: 612-624-2575; Fax: 612-624-8154; E-mail address: [email protected]

Abbreviations Used

SIRT2, Sirtuin 2; NAD+, nicotinamide adenine dinucleotide; SAR, structure-activity relationship; AMC, 7-amino-4-methylcoumarin; ADME, absorption, distribution, metabolism, and excretion; MDCK, Madin-Darby Canine Kidney; A to B, from the apical to basolateral side; B to A, from the basolateral to apical side; Papp, apparent permeability coefficient; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; EDC, N-ethyl-N′(3-dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzotriazole


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Figure 1. Selected SIRT2 inhibitors.



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Figure 2. Design of SIRT2 inhibitors.


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Figure 3. Inhibition of SIRT2 by compound 86. IC50 values were plotted against either NAD+ (A) or the peptide substrate (B).



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Figure 4. Proposed binding mode and interactions of compound 86 docked into the crystal structure of human SIRT2 (PDB: 1j8f). A. A potential binding mode of compound 86 (green) in the active site of SIRT2 (surface representation). B. Potential interactions between compound 86 and SIRT2.


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Figure 5. Protection against oligomeric α-synuclein toxicity by selected SIRT2 inhibitors. ≠ p < 0.001 vs. control cells without α-synuclein treatment; *** p < 0.0001, ** p < 0.001, * p < 0.05 vs. control cells with α-synuclein treatment.



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Table 1. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Aromatic Rings.a

IC50 (µM) Compound

R SIRT1

SIRT2

SIRT3

7.14

0.107

6.39

> 200

15.1

22.3

12

42.4

2.05

28.4

13

> 200

0.505

> 200

14

21.8

0.546

17.9

15

12.5

0.252

11.5

16

9.50

0.235

7.86

17

50.8

4.80

6.03

10

11

Me


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18

44.6

1.37

4.08

19

19.7

0.156

18.5

1 (AGK2)b

--

135

1.56

52.8

8

--

12.0

0.0483

44.2

a

Inhibitory activities were determined as described in Initial SIRT1-3 and SIRT5 Biochemical Assays in the Experimental Section. The IC50 values were determined in a 10-dose mode with a 3-fold serial dilution starting at 200 µM in singlet. bIC50 values reported in ref. 28.



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Table 2. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Fused Aromatic Rings.a

IC50 (µM) Compound

R SIRT1

SIRT2

SIRT3

20

192

0.572

1.08

21

52.1

0.0234

1.59

22

1.62

0.0331

3.29

23

9.93

0.0647

2.37

24

7.33

0.0714

1.77

25

65.2

0.437

36.2


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26

8.09

0.313

2.71

27

1.81

0.0831

1.56

28

2.23

0.0354

1.39

29

0.483

0.0157

1.22

a

Inhibitory activities were determined as described in Initial SIRT1-3 and SIRT5 Biochemical Assays in the Experimental Section. The IC50 values were determined in a 10-dose mode with a 3-fold serial dilution starting at 200 µM in singlet.



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Table 3. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Substituents at Different Positions.a

IC50 (µM) Compound

R1

R2

R3 SIRT1

SIRT2

SIRT3

15

H

H

H

12.5

0.252

11.5

30

Me

H

H

192

4.15

19.5

31

H

Me

H

30.4

0.580

9.06

32

H

H

Me

8.54

0.111

5.84

45

F

H

H

30.2

0.288

2.94

46

H

F

H

10.1

0.0754

3.39

47

H

H

F

4.19

0.0368

2.57

48

F

H

F

31.4

0.277

15.5

49

H

F

F

7.97

0.0418

1.56

a



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Table 4. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Substituents at Position 4.a

IC50 (µM) R

Compound

SIRT1

SIRT2

SIRT3

15

H

12.5

0.252

11.5

56

Et

21.0

0.151

8.24

57

i

Pr

13.5

0.0947

2.50

58

t

Bu

15.3

0.113

4.89

59

cyclohexyl

71.5

0.133

3.50

60

Ph

15.6

0.0135

> 200

61

106

0.0263

66.4

62

13.4

0.0460

8.15

63

14.7

0.476

6.50

64

3.51

0.0447

4.18

65

0.237

0.251

0.827



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66

0.367

0.0190

0.843

67

2.55

0.0227

0.503

68

1.55

0.0281

0.829

69

0.974

0.0229

0.697

70

3.01

0.0415

3.20

a



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Table 5. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Functional Groups at Position 4.a

IC50 (µM) Compound

R SIRT1

SIRT2

SIRT3

15

H

12.5

0.252

11.5

71

C(O)OMe

8.02

0.102

8.90

72

C(O)OH

142

2.58

106

73

C(O)NH2

1.88

0.0478

7.35

74

C(O)NHMe

1.39

0.0281

2.64

75

C(O)N(Me)2

6.27

0.519

5.58

76

NHC(O)Me

0.672

0.0486

1.88

77

C(O)Me

24.1

0.465

17.7

78

OPh

> 200

0.196

85.8

79

OBn

> 200

0.138

> 200

80

NHMe

2.82

0.216

5.62

81

NHEt

3.03

0.104

5.59

82

N(Me)2

10.5

0.0267

7.51

83

N(Et)2

11.5

0.366

8.19

5.66

0.0982

0.910

84



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

85

6.32

0.251

3.33

86

6.56

0.0167

1.87

87

2.08

0.112

0.580

a



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 6. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Heteroaromatic Rings.a

IC50 (µM) Compound

R SIRT1

SIRT2

SIRT3

15

12.5

0.252

11.5

100

20.3

1.54

4.79

101

18.7

0.193

13.4

102

2.46

0.0341

2.40

103

15.5

0.500

9.66

104

7.92

0.171

6.86

105

33.3

1.95

9.04

106

90.9

4.46

7.32



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

107

48.5

0.450

1.81

108

11.3

0.157

2.70

109

3.47

0.0436

0.723

a



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Table 7. Inhibitory Activity of 5-((3-Amidobenzyl)oxy)nicotinamides: Pyridine Ring and Fluorine.a

IC50 (µM) Compound

R SIRT1

SIRT2

SIRT3

110

18.8

0.0468

5.85

111

145

0.0419

> 200

14.9

0.0289

7.12

113

1.23

0.0224

1.62

114

11.3

0.0333

4.11

115

115

0.0293

11.4

116

2.27

0.0297

0.776

112

O



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

117

2.23

0.0460

5.02

a



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Table 8. Inhibitory Activity of Selected Compounds.a Kiapp (µM) Compound

SIRT5 IC50 (µM)

SIRT1

SIRT2

SIRT3

61

> 100

0.14 ± 0.02

> 100

86

5.0 ± 1.2

0.073 ± 0.011

5.4 ± 1.1

> 200

101

20 ± 1

0.26 ± 0.03

3.4 ± 0.9

> 200

102

3.0 ± 0.1

0.14 ± 0.02

7.2 ± 0.9

> 200

a

Inhibitory activities against SIRT1-3 were determined in duplicate as described in In-house SIRT1-3 Biochemical Assays while the inhibitory activity against SIRT5 was determined as described in Initial SIRT1-3 and SIRT5 Biochemical Assays in the Experimental Section.



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Page 60 of 62

Table 9. In vitro ADME properties of Selected Compounds.a

Compound

Plasma Stability (% remaining, 6 h/24 h)

Microsomal Stability t1/2 (min)

MDCK Papp (×10-6 cm/sec)

Mouse

Human

Mouse

Human

A to B

B to A

86

74/35

98/98

14

55

5.7

6.8

101

78/25

99/97

51

330

23

25

102

63/7.9

97/99

64

408

16

12

3.8

4.4

8 a

Plasma and microsomal stabilities were determined in triplicate as described in Plasma Stability Assay and Microsomal Stability Assay, respectively while the MDCK apparent permeability coefficients were measured as described in Directional Transport Across MDCKII Cell Monolayers in the Experimental Section. Papp, apparent permeability coefficient.


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Scheme 1a

a

Reagents and conditions: (a) methyl 5-hydroxynicotinate, Cs2CO3, DMF, 75%; (b) NaBH4, NiCl2·6H2O, MeOH, 80%; (c) NH3, CaCl2, MeOH, 90%; (d) RCOCl, base; or RCOOH, coupling agents, 1-94%.



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Graphic Abstract


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5-((3-Amidobenzyl)oxy)nicotinamides as Sirtuin 2 Inhibitors - Journal

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