-biv

m3, Ree Sc

f Sin

Sirtuins are protein deacylases with regulatory roles in metabolism and stress response. Functionalized

hyperacetylation of p53 and a-tubulin in treated HepG2 and MDA-MB-231 cells. Molecular dockingshowed that the tetrahydropyridoindole scaffold was positioned in the NAD pocket and the acetylated

activities such as thieno [3,2-d]pyrimidine-6-carboxamides [3],macrocyclic peptides [4], chroman-4-ones [5], 2-hydroxy-1-naphthalenes [6,7] and functionalized indoles [8e11]. Interest-ingly, certain scaffolds were observed to be more widely associated

7 and the caprofenin that both com-amide functional-t differed in theirgrowth and prolif-etylation status ofon experimentalanalog 1 induceditory activity was

uated a series of 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles forsirtuin inhibition (Fig. 1B). The indole ring of this scaffold is fused toa basic piperidine ring and not benzene or cyclohexane as in 1 andEX527. Our results showed that tetrahydropyridoindole is a novelchemotype associated with preferential Sirt2 inhibitory activity. Arepresentative member 18 inhibited sirtuin-mediated deacetyla-tion of physiological substrates p53 and a-tubulin in liver (HepG2)and breast (MDA-MB-231) cancer cells at low micromolar

* Corresponding author.

Contents lists availab

European Journal of M

w

European Journal of Medicinal Chemistry 92 (2015) 145e155E-mail address: phagoml@nus.edu.sg (M.-L. Go).screening have identied novel chemotypes with sirtuin inhibitory To further explore the potential of the indole scaffold, we eval-with important roles in diverse and interrelated cellular processessuch as stress response, gene expression, DNA damage repair andmetabolism [1]. They are implicated in age-related diseases such ascancer, neurodegeneration and metabolic disorders and hence areconsidered as prospective therapeutic targets for these conditions.However, perplexing gaps remain in our understanding of theirmodes of action. In carcinogenesis, sirtuins have bifurcated rolesand may function as tumor suppressors and promoters dependingon contextual variables such as the stage of the malignancy and thetumor microenvironment [2]. High-throughput and in silico

propanamide) [11] (Fig. 1A). We noted that EX52analog 1 bore a striking structural resemblancepounds were ring-fused indoles with primaryities. They were also selective Sirt1 inhibitors bucell-based activities. EX527 did not abolish celleration [12,13] and had varied effects on the acp53, a substrate of Sirt1 and Sirt2, dependingconditions [11e13]. In contrast, the carprofenapoptosis in leukemic cells and its sirtuin inhibconrmed in functional assays [11].Sirtuins are a class of evolutionary conserved nicotinamideadenine dinucleotide (NAD) -dependent protein lysine deacylases

[8], the oxindole GW 5074 [8,9], the tetrahydrocarbazole EX527[10] and the carprofen analog 1 (2-(6-chloro-9H-carbazol-2-yl)Accepted 17 December 2014Available online 18 December 2014

Keywords:TetrahydropyridoindolesSirtuin 2 inhibitionMolecular dockingHyperacetylation of p53 and a-tubulinApoptosisGrowth inhibition

1. Introductionhttp://dx.doi.org/10.1016/j.ejmech.2014.12.0270223-5234/ 2014 Elsevier Masson SAS. All rights resubstrate channel of the sirtuin 2 protein by van der Waals/hydrophobic, H bonding and stacking in-teractions. Functionalized tetrahydropyridoindoles represent a novel class of sirtuin 2 inhibitors thatcould be further explored for its therapeutic potential.

2014 Elsevier Masson SAS. All rights reserved.

with sirtuin inhibition than others. The indole ring is a case in point- it is embedded in the bisindolylmaleimide derivative Ro 31-8220Received in revised form1 December 2014 didates. The functional relevance of sirtuin inhibition was corroborated in western blots that showedReceived 22 August 2014 tetrahydro-1H-pyrido[4,3-b]indoles were identied as preferential sirtuin 2 inhibitors, with in vitroinhibitory potencies in the low micromolar concentrations (IC50 3e4 mM) for the more promising can-Original article

Functionalized tetrahydro-1H-pyrido[4,3chemotype with Sirtuin 2 inhibitory act

Tianming Yang a, Xiao Chen a, Hai-xiao Jin b, Gautaa Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 11754b Key Laboratory of Applied Marine Biotechnology, Ministry of Education, School of MarinNingbo, Zhejiang 315211, People's Republic of Chinac Department of Pharmacology, Yong Loo Lin School of Medicine, National University o

a r t i c l e i n f o

Article history:

a b s t r a c t

journal homepage: http: / /wwserved.]indoles: A novelity

Sethi c, Mei-Lin Go a, *

public of Singaporeiences, Ningbo University, Fenghua Road 818, Jiangbei District,

gapore, 10 Medical Drive, 117597, Republic of Singapore

le at ScienceDirect

edicinal Chemistry

.e lsevier .com/locate/ejmech

resulting compounds. The lipophilicity of 8, as seen from its logP

substituted aromatic moieties at position 8. This sequence wasbroadly followed throughout, except for analogs with 30-tolyl at

ole

edi(7.55) and logDpH 7.4 (5.93) values, were high. Hence to avoidexacerbating the greasiness of the target compounds, a deliberateattempt was made to introduce polar residues at R1 and R3. To thatend, several polar and sterically limited entities were introduced atconcentrations (2e 4 mM). Molecular docking showed that thetetrahydropyridoindole ring was positioned at the NAD pocketand substrate channel of the Sirt2 protein, with binding afnitymodulated by the side chains attached to the scaffold.

2. Results and discussion

2.1. Design and synthesis of target compounds

The lead compound for this series was 8 (Fig. 1B) which wasserendipitously found to occupy the NAD binding pocket of hu-man Sirt2 (PDB 3ZGV) [12]. As shown in Fig. 2A, the tetrahy-dropyridoindole ring of 8 straddled the nicotinamide-ribose site B(Arg 97, Phe 96), substrate (acetyl lysine) channel (Phe 119, His 187,Val 233, Phe 235) and nicotinamide site C (lle169). Several stackinginteractions (pp, p-cation) were identied, namely Phe 96, His 187and the aromatic ring of the scaffold (pp, pH respectively, withdistances of 3.81 and 3.74 ), Arg 97 and m-tolyl ring (pH, 4.01 )and Phe 235 and piperidine NH (pH, 3.60 ). The n-octyl sidechain of 8 occupied a hydrophobic channel that extended from siteC (Fig. 2B).

When 8 was screened for inhibition of Sirt1 and Sirt2 (asdescribed later), it was found to inhibit Sirt2 (43%, 10 mM) to agreater extent than Sirt1 (5%, 10 mM). With this lead on hand, wemodied 8 at positions 2 (R1), 5 (R2) and 8 (R3) with the intent ofestablishing structure-activity relationships (SAR) and optimizinginhibition. To that end, thirty analogs that were structurally relatedto 8 were synthesized. Having observed that the n-octyl side chainof 8 was positioned in a lipophilic region of the NAD pocket, wereasoned that it was an essential structural feature. However, itspresence would invariably impart considerable lipophilicity to the

Fig. 1. (A) Structures of known indole-based sirtuin inhibitors (B) Tetrahydropyridoindcompound 8 are indicated.

T. Yang et al. / European Journal of M146R1, such as hydroxyethyl, aminocarbonyl, methylsulfonyl, methyl,ethyl and hydrogen. In the case of R3, an arbitrary selection of 30-tolyl, 40-methylsulfonylphenyl, 20-aminopyrimidin-50-yl, 20-uo-ropyridin-40-yl and uoro was made, prompted in part by thecommercial availability of reagents. These R3 groups formed thebasis of classifying the synthesized compounds to their respectiveseries A-E (Table 1). As shown later, series D and E which had u-oropyridinyl and uoro as R3 respectively, were weak sirtuin in-hibitors, further emphasizing the need to deploy small or polargroups at R1. Analogs with shorter (n-butyl, isoprenyl) or no R2 sidechains were also synthesized to conrm the necessity of retainingthe n-octyl side chain.

Most of the compounds (except series E) were derived from acommon intermediate ethyl 8-bromo-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (2) which was obtained from aposition 8 (9, 10, 11, and 12) where higher yields were obtainedwhen the 30-tolyl ring was introduced rst (Scheme 1). The 8-uorosubstituted analogs of series E (35, 36)were obtained by the Fischerindole reaction between 4-uorophenylhydrazine hydrochlorideand 1-carbethoxy-4-piperidone, followed by the usual reactions(Supplementary information, Section 9). In the same way, theindole nitrogen of 2 was reacted with 1-bromobutane to give n-butyl analogs (16, 22, and 32). Alternatively, the indole nitrogen of 2was left in its unsubstituted state and functionalized at positions 2and 8 as described earlier to give 14, 15, 21 and 31 (Supplementaryinformation, Section 6).

2.2. Sirtuin inhibitory activity of test compounds

The tetrahydropyridoindoles were evaluated against recombi-nant human Sirt1 and Sirt2 in an assay that monitored the rate atwhich the enzyme deacetylated an N-acetyl lysine residue in apeptide substrate bearing the amino acid residues 317e320 of p53and conjugated to aminoluciferin. The deacetylation reaction trig-gered a protease mediated hydrolysis which released amino-luciferin. The latter was oxidized by luciferase to oxyluciferinwhichis luminescent. Inhibition of sirtuin resulted in less oxyluciferinbeing generated and hence a reduction in the luminescent signal.Fischer indole reaction between 4-bromophenylhydrazine hydro-chloride and 1-carbethoxy-4-piperidone (Scheme 1). The indolenitrogen of 2was N-alkylated with 1-bromooctane in the presenceof sodium hydride, followed by alkaline hydrolysis and decarbox-ylation of the carbamate moiety at position 2 to give 4a. The latterwas reacted with formaldehyde, acetaldehyde or acetone in thepresence of sodium triacetoxyborohydride to give the N-methyl(5a), N-ethyl (5b) or N-isopropyl (5c) intermediate. 4a was alsoreacted with methanesulfonyl chloride, 2-bromoethanol or potas-sium cyanate to give 5d, 5e or 6 respectively. In the next step,palladium catalyzed Suzuki coupling was carried out to introduce

scaffold with functionalization at positions 2 (R1), 5 (R2) and 8 (R3). R1,R2, R3 of lead

cinal Chemistry 92 (2015) 145e155The compounds were rst evaluated at a xed concentration of10 mM on Sirt1 and Sirt2 (Table 1). Of the 30 compounds evaluated,8 analogs (9, 12, 13, 18, 19, 24, 26, 28) inhibited Sirt2 to a greaterextent (50% inhibition) than Sirt1 (50% inhibition) while 2compounds (10, 20) inhibited both Sirt1 and Sirt2 by more than50%. The remaining compounds were weak inhibitors (

teracientac cha

T. Yang et al. / European Journal of Medicinal Chemistry 92 (2015) 145e155 147We also explored the possible involvement of aggregate for-mation as a source of non-specic inhibition [13,14]. Some com-pounds are known to form submicrometer aggregates capable ofsequestering enzymes onto their surfaces, resulting in an artefac-tual reduction of enzyme activity. Such compounds (aggregators)are generally characterized by poor solubility, high lipophilicity andextended conjugation in their structures [13]. As some of thesefeatures were present in our compounds, the involvement ofaggregate formation in the observed inhibition was investigated.Aggregators normally form particles of 30e100 nm diameter thatwere capable of scattering light. To that end, wemonitored the light

Fig. 2. Docking pose of 8 in the NAD binding pocket of Sirt2 (PDB 3ZGV). (A) Ligand in8. Polar and lipophilic residues are depicted in magenta and green respectively. (B) Orresidues Phe 119, Phe 131, lle232, lle169 which were found in site C and the hydrophobilegend, the reader is referred to the web version of this article.)scattering properties of selected compounds (9,10,12,13,18,19, 20,24, 26 and 28) at 1 mM and 10 mM in phosphate buffer pH 7.4(Supplementary information, Section 14). Negligible light scat-tering was observed at 1 mM but higher levels were observed at10 mM of 9, 12 and 13. This raised the question as to whether ag-gregation could have contributed to the inhibitory activities ofthese compounds (Table 1). The likelihood was deemed to be slightbecause 9, 12 and 13 (at a xed concentration of 10 mM) wereparadoxically weak inhibitors of Sirt1 although reasonably stronginhibitors of Sirt2. We reasoned that if aggregate formationcontributed to artefactual inhibition of sirtuins, strong inhibitionshould be observed for both and not just one enzyme.

We then proceeded to determine the IC50 (concentrationrequired to reduce basal oxyluciferin luminescence by 50%) of thosecompounds that inhibited Sirt2/Sirt1 by more than 50% at 10 mM.Some less inhibitory compounds were included for comparison.The results are presented in Table 1.

The test compounds were found to be more potent inhibitors ofSirt2 than Sirt1, with a preference for Sirt2 inhibition that variedfrom 2-fold (8) to more than 40 fold (19) based on IC50 values. TheSirt2 selective inhibitor AGK2 was used as a positive control and itwas gratifying to note that some of the Series A and B analogs weremore inhibitory than AGK2 on this assay.

Based on the inhibitory data at 10 mM and IC50 values, astructure-activity relationship (SAR) for Sirt2 inhibition is pro-posed. First, it is important to maintain an alkyl substituted indolenitrogen at R2. For compounds with the same R1 and R3, inhibitionwas most pronounced when R2 was n-octyl. n-Octyl analogs wereconsistently more inhibitory than their n-butyl or unsubstituted(NH) counterparts in Series A (8 > 14, 16), B (18 > 22) and C(24 > 32). There were however some n-octyl analogs with weakSirt2 inhibition (at 10 mM). For example, 11 and 17 had similargroups at R1 (40-hydroxybenzyl) and R3 (30-tolyl) but different R2groups (isoprenyl in 17 and n-octyl in 11). Stronger Sirt2 inhibitionwas anticipated for the n-octyl bearing 11 but as seen from Table 1,both compounds weakly inhibited Sirt2 (20e40% inhibition at10 mM). Evidently, one or both substituents at R1 and R3 of 11 werenot optimal for activity. Thus, R2 notwithstanding, groups at R1 and

tion map depicting amino acid residues in the NAD pocket which are in contact withtion of 8 in the NAD binding pocket. The n-octyl side chain is anked by non-polarnnel extending from site C. (For interpretation of the references to colour in this gureR3 should not be ignored as they may have modulatory roles.That the groups at R1 and R3 were important was further sup-

ported by examining the actives (compounds that inhibited Sirt2by 50% or more at 10 mM). By this measure, small polar groups werepreferred at R1 as seen from the recurring presence ofR1 hydrogen, methyl and aminocarbonyl among these com-pounds. On the other hand, compounds bearing ethoxycarbonyl(polar, bulky) and 40-hydroxybenzyl (bulky, less polar) side chainswere noticeably absent. In the case of R3, a key observationwas thatactives were only found in series A (R3 30-tolyl), B (R3 40-methylsulfonylphenyl) and C (R3 20-aminopyrimidin-50-yl), andnot series D (R3 30-uoropyridin-4-yl) and E (R3 F). A com-parison of IC50 values of compounds across Series A, B and C withthe same R1 and R2 (n-octyl) groups revealed the following trends:R1 H: 18 (series B, 3.8 mM) > 8 (series A, 10.1 mM)z 24 (series C,11.8 mM); R1 CH3: 19 (series B, 6.0 mM) z 10 (series A,6.2 mM) > 26 (series C, 17.9 mM); R1 CONH2: 20 (series B,4.0 mM) > 13 (series A, 7.8 mM) > 25 (series C, 14.0 mM). Takentogether, the SAR suggests that small (uoro in series E) and het-eroaromatic azines (series C aminopyrimidinyls, series D uo-ropyridinyls) were not favored at R3.

There is concern that most of the identied actives were foundin series A or B which had more lipophilic substituted-phenyl ringsat R3, as compared to the polar heteroaromatic azines at R3 of seriesC. Excessive lipophilicity would promote non-specic binding andspurious cytotoxicity to normal tissues. Thus, we evaluated thecytotoxic IC50 of 18 and 20, two of the more potent Sirt2 inhibitors,

Table 1Structures of synthesized compounds and their in vitro inhibitory activities on Sirt1 and Sirt2.

N

NR3

R2

R1

Cpd R1 R2 Sirt1 Sirt 2

% Inhibition (10 mM)a IC50 (mM)a % Inhibition (10 mM)a IC50 (mM)a

Series A: R3

7 eCOeOeC2H5 C8H17n Nilb Not done 11.2 3.9 Not done8 H C8H17n 5.1 1.0 20 11 43.1 3.2 10.1 0.89 C8H17n 100 57.5 3.6 8.2 0.1

10 eCH3 C8H17n 66.4 2.5 21 1 64.2 2.4 6.2 0.211 C8H17n Nilb Not done 19.7 13.8 Not done

12 eCH(CH3)2 C8H17n 2.4 1.9 26 4 56.3 4.3 11.2 0.713 eCONH2 C8H17n Nilb >100 49.1 9.5 7.8 0.714 H H Not done Not done Nilb 100 1115 eCH3 H Not done Not done Nilb 83 416 H C4H9n Nilb Not done Nilb 96 117 Nilb Not done 38.1 11.4 Not done

Series B: R3

18 H C8H17n 48.5 2.4 16.7 0.4 82.5 3.3 3.8 0.319 eCH3 C8H17n 12.8 3.9 >250 79.1 7.4 6.0 0.420 eCONH2 C8H17n 51.8 11.4 28 3 76.5 4.4 4.0 0.221 eCH3 H Not done Not done Nilb >25022 H C4H9n Nilb Not done Nilb 20 1Series C: R3

23 eCOeOeC2H5 C8H17n Nilb Not done 23.0 3.3 Not done24 H C8H17n 2.4 1.1 38 2 70.0 8.8 11.8 1.825 eCONH2 C8H17n Nilb 63 4 34.0 1.5 14.0 0.826 eCH3 C8H17n Nilb >100 61.9 11.4 17.9 2.227 eC2H5 C8H17n Nilb Not done 29.1 1.6 Not done28 eCH(CH3)2 C8H17n 15.3 7.6 56 2 62.3 7.9 20 629 eSO2CH3 C8H17n Nilb Not done 35.0 3.5 Not done30 eC2H4OH C8H17n Nilb Not done 31.1 2.4 Not done31 eCH3 H Not done Not done Nilb >25032 H C4H9n Nilb Not done Nilb >100Series D: R3

33 H C8H17n 1.1 0.8 Not done 27.7 3.3 Not done34 eCONH2 C8H17n Nilb Not done 18.5 8.4 Not doneSeries E: R3 F35 eCH3 C8H17n Nilb Not done 17.4 4.9 Not done36 C8H17n Nilb Not done Nilb Not done

AGK2c Not done 13.9 1.0EX527d 0.33 0.03 Not doneNicotinamidee 149 12 6.3 1.4

a Mean SD for n 3 separate determinations.b No inhibition was observed at 10 mM test compound.c Selective Sirt2 inhibitor [15].d Selective Sirt1 inhibitor [10].e Sirtuin 1 and 2 inhibitor [10].

T. Yang et al. / European Journal of Medicinal Chemistry 92 (2015) 145e155148

T. Yang et al. / European Journal of Medion non-malignant human lung broblast IMR90 cells.18was foundto be more cytotoxic (IC50 2.72mM 0.32) than 20 (IC5014.5mM 1.5). The estimated log P of 18was 6.1 as compared to 5.5for 20 (ACD/Labs Version 12.0). However, 18 is a secondary cyclicamine and its lipophilicity would be better represented by logDpH7.4 which takes into account its ionization state. Log D (pH7.4)was found to be 3.4 for 18 whereas the corresponding value for 20,which has no ionizable group, was higher at 5.5 (ACD/Labs Version12.0). Thus, the cytotoxicities of 18 and 20 on IMR90 cannot besolely attributed to their (estimated) lipophilicities. Follow up in-vestigations on other non-malignant cell lines should be carried outfor conrmation.

Besides cytotoxicity, excessive lipophilicity would adverselyaffect drug-like character, in particular solubility. To that end, weassessed the solubilities of selected compounds (10, 12, 18, 19, 20,24) from Series A, B and C by a turbimetric method [16]. As seenfrom Table 2, 24 from series C (R3 2-aminopyridimidin-5-yl)which had moderate Sirt2 inhibitory activity (IC50 11.8 mM), had a

Scheme 1. Reagents and conditions: (a) Ethanol, reux, 12 h; (b) NaH (60%), 1-bromooctane,ester, Pd(PPh3)4, K2CO3 (aqueous solution), 1, 4-dioxane, N2 atmosphere, reux, 10 h; (d) KOHketone, NaBH(OAc)3, acetic acid, 1, 2-dichloroethane, rt, overnight; (f) For 5d: CH3SO2Cl, Et3Npinacol ester, Pd(PPh3)4, potassium carbonate (K2CO3, aqueous solution), 1, 4-dioxane, micro80 C, 60 min.

Table 2Solubilities of selected compounds as determined by turbimetry at pH 7.4, 25 C.

Series Compound Solubilitya Series Compound Solubilitya

A 10 3.1e6.3 mM B 19 3.1e6.3 mMA 12 1.6e3.1 mM B 20 1.6e3.1 mMB 18 3.1e6.3 mM C 24 6.3e12.5 mM

a From at least n 3 separate determinations.cinal Chemistry 92 (2015) 145e155 149more favorable solubility prole than compounds from series A andB which were more potent Sirt2 inhibitors. At this stage of leadoptimization, moderately active but less lipophilic compounds likethose in series C should not be entirely ignored as they may yetyield more potent and drug-like analogs upon structuralmodications.

2.3. Kinetics of Sirt2 inhibition by 18

The kinetics of Sirt2 inhibition was investigated for a repre-sentative compound 18. The activity of Sirt2 as a function of theacetylated substrate (Fig. 3A) or NAD (Fig. 3B) was monitored inthe presence of varying concentrations of 18. Both plots showed adose-dependent decrease in the maximal enzyme activity withincreasing 18 that was not overcome at high substrate concentra-tions. Such a prole was indicative of non-competitive inhibitionwhich meant that 18 binds to both the enzyme and the enzy-meesubstrate complex (ES) or subsequent species [17]. To deter-mine the comparative afnity of 18 for the enzyme or the EScomplex, the double reciprocal plots of 18 for the two substrateswere examined. As seen from Fig. 3C and D, both plots displayed anest of lines that converged below the x-axis, indicating that 18 hadgreater afnity for the ES complex than the free enzyme [17]. Toobtain the Ki of 18, the slopes of the double reciprocal plots ob-tained in presence of different concentrations of 18were re-plottedagainst inhibitor concentration (Supplementary information,Section 13) [18]. Apparent Ki of 18 was found to be 2.28mM 0.42

DMF, 0 C to rt; (c) m-Tolylboronic acid or (2-aminopyrimidin-5-yl)boronic acid pinacol(aqueous solution), ethanol, reux, 16 h; (e) For 5a, 5b and 5c: appropriate aldehyde or, rt; (g) For 5e: 2-BrC2H4OH, K2CO3, 80 C; (h) Appropriate boronic acid or boronic acidwave, 110 C, 15e30 min; (i) potassium cyanate (KNCO), 4 M HCl, ethanol, microwave

2.5. 18 increased levels of acetylated p53 and alpha-tubulin in

), 1.1the

AD

ediIn our preliminary investigations, we examined the docking(for acetylated substrate) and 3.70mM 0.35 (for NAD).

2.4. Molecular docking of series AeC compounds in the Sirt2binding pocket

Fig. 3. Kinetics of inhibition of SIRT2 by 18 at concentrations of 10 mM ( ), 3.3 mM (function of acetylated substrate at a xed NAD concentration (5 mM). Panel (B) depictssubstrate. The double reciprocal plot of 1/rate versus 1/[acetylated substrate] and 1/[N

T. Yang et al. / European Journal of M150pose of lead compound 8 in the Sirt2 pocket (PDB 3ZGV) and foundthat its tetrahydropyridoindole ring straddled several sites in theNAD pocket of Sirt2, namely site B, site C and the substratechannel (Fig. 2). We found that 18, 19, 20 of Series B and 24, 25, 26,28 of Series C were similarly positioned in the NAD bindingpocket as 10. Fig. 4 depicts the docking poses of 18 (Series B) and 24(Series C) in the Sirt2 pocket. These poses were representative ofother compounds in the respective series. Signicant interactionswere the positioning of the n-octyl side chain along a hydrophobicchannel which extended from site C, and pp/p-cation interactionsbetween the scaffold and Phe 96 (site B), His 187 (substrate chan-nel) and Phe 235 (substrate channel). The p-cation (pH) interactionwith Phe235 was observed only when the piperidine nitrogen wasprotonated and was absent in 20 and 25 which had an amino-carbonyl substituent at R1. We noted that the aromatic rings atposition 8 of 18 and 24 were involved in H bonding but this was atthe expense of p-cation stacking interactions that were character-istic of the series A analog 8 (Fig. 2). As shown in Fig. 4, the sulfonylmoiety of 18 was H bonded to Ser 263 and the amino group of 24was H bonded to Gly 261. These residues were located in the polarregion that accommodates the phosphoryl residues of the co-crystalized ligand ADP-ribose. The docking pose of ADP-riboseshowed that its phosphate residues were H bonded to Ser 263.The involvement of Ser 263 in H bonding with the sulfonyl moietyof the series B analogs 18, 19, 20may account for the stronger Sirt2inhibition observed for these compounds.

Although 18 exhibited non-competitive inhibition of Sirt2,molecular docking showed that it occupied the NAD and substratebinding sites in the enzyme. While this may seem paradoxical, wenoted that EX-527 which inhibited several sirtuins non-cancer cell lines

Having shown that 18 inhibited the Sirt2 mediated deacetyla-competitively or uncompetitively, was also reported to occupythe nicotinamide binding site of the enzyme [10,19].

mM ( ) and 0 mM ( ). Panel (A) shows the rate (luminescence per min) of catalysis as arate of catalysis as function of NAD at a xed concentration (35 mM) of the acetylated] at different concentrations of 18 are depicted in Panels C and D respectively.cinal Chemistry 92 (2015) 145e155tion of a luminogenic peptide, we proceeded to determine if itinhibited deacetylation of physiological substrates of Sirt2. To thatend, HepG2 and MDA-MB-231 cells were incubated with varyingconcentrations of 18 (2e8 mM) after which acetylated p53 (acety-lated at Lys 382) and acetylated a-tubulin were probed by immu-noblotting (Fig. 5A, B). p53 is a substrate of both Sirt1 and Sirt2whereas a-tubulin is a Sirt2 substrate [2]. 18 had a growth inhibi-tory IC50 of 2.1 mM on HepG2 and MDA-MB-231 (Supplementaryinformation, Section 15). As seen in Fig. 5A, B, 18 increased thelevels of acetylated p53 and a-tubulin in these cells at 2e4 mM.

Acetylation of p53 increased its stability by limiting ubiquiti-nation of key lysine residues and subsequent proteasomal degra-dation [20,21]. The stabilized and hence activated p53 is thus ableto increase transcription of genes involved in cell-cycle arrest, DNArepair and apoptosis. Having shown that 18 increased levels ofacetylated p53, we proceeded to determine if this would translateto apoptotic cell death. Hence, two apoptotic marker proteins(caspase 3 and PARP) were monitored by immunoblotting. In theevent of apoptosis, elevated levels of cleaved caspase 3 and cleavedPARP would be observed and this were duly noted in 18- treatedMDA-MB-231 cells (Fig. 5C).

3. Conclusion

We have shown that functionalization of the tetrahydropyr-idoindole scaffold resulted in analogs with low micromolar Sirt2inhibitory activity. There was a modest preference for Sirt2 inhi-bition (2e40 fold) among the potent members, unlike the struc-turally related EX527 and caprofen analog 1 which were Sirt1

ediT. Yang et al. / European Journal of Mselective inhibitors. SAR analysis revealed structure specic re-quirements for inhibition, of which substitution at the indole ni-trogen (R2) and position 8 (R3) of the scaffold were deemedimportant. Of the groups explored at the indole nitrogen, Sirt2 in-hibition decreased in the order n-octyl > n-butyl > H, implicatinglipophilicity and steric bulk as essential features for optimal

Fig. 4. Docking poses of 18 (A,B) and 24 (C,D) in the NAD binding pocket of Sirt2 (PDBNAD pocket in contact with 18 and 24. (B) and (D) depict the key residues anking 18 a

Fig. 5. Compound 18 induced hyperacetylation of p53 (Ac p53) and a-tubulin (Ac a-tubulin)and MDA-MB-231 cells respectively. For the detection of p53, cell lysate protein was loadedprotein was loaded at 1.5 mg (HepG2) and 2.0 mg (MDA-MB-231). (C) 18 induced apoptosiscaspase 3 and cleaved PARP (89 kDa) after 24 h incubation. Cell lysate protein was loadedcinal Chemistry 92 (2015) 145e155 151activity. In the case of substitution at position 8 (R3), there was anapparent preference for Series B which had a 40-methyl-sulfonylphenyl ring at the position 8. Inhibitory potency wasaffected to a lesser degree by R1 as inferred from the laxity in termsof size and polarity permitted at this site. Nonetheless, small groupswere preferred at R1 to avoid exacerbating lipophilicity of the

3ZGV). (A) and (C) are ligand interaction maps depicting amino acid residues in thend 24 in the binding pocket.

in (A) HepG2 and (B) MDA-MB-231 cells. Incubation times were 6 h and 24 h for HepG2at 20 mg (HepG2) and 100 mg (MDA-MB-231). For the detection of a-tubulin, cell lysatein MDA-MB-231 cells as seen from the increases in apoptotic marker proteins cleavedat 100 mg. b-actin was the loading control.

Sirt2 inhibition by 18 was corroborated by Western blot analyseswhich showed that 18 increased levels of acetylated p53 and a-tubulin in MDA-MB-231 and HepG2 cell lysates. Levels of apoptotic

itive ion mode using electro-spray ionization (ESI) (Applied Bio-system, Q-Trap 2000 LC/MS) or high-resolution LC-MS (IT TOF:Waters-Micromass QTOF premier mass spectrometer). Synthetic

ediprotocols and spectral data of synthesized intermediates/targetcompounds are provided in Supplementary information (Sections1e9). Purity of nal compounds were determined by reversephase HPLC and found to be 95% (Supplementary information,Section 10).

4.2. Ethyl 8-bromo-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (2)

The mixture of 4-bromophenylhydrazine hydrochloride (10.0 g,44.7 mmol) and 1-carbethoxy-4-piperidone (7.66 g, 44.7 mmol) inabsolute EtOH (20 mL) was reuxed for 12 h. On cooling to roomtemperature (25 C), the solid product was ltered, washed with50% aqueous EtOH, and recrystallized from 95% EtOH to give 2 as anoff-white solid: 12.71 g, yield: 88.0%. 1H NMR (400 MHz, DMSO-d6)d 11.23 (s, 1H), 7.60 (d, 1H, J 2.0 Hz), 7.26 (d, 1H, J 8.6 Hz),7.14e7.11 (m, 1H), 4.54 (s, 2H), 4.08 (q, 2H, J 7.2 Hz), 3.72 (t, 2H,J 5.6 Hz), 2.78 (t, 2H, J 5.6 Hz),1.20 (t, 3H, J 7.2 Hz). 13C NMR(100 MHz, DMSO-d6) d 155.09, 134.48, 134.34, 126.83, 122.95,marker proteins (cleaved PARP, cleaved caspase 3) were alsoelevated in MDA-MB-231 cell lysates, implicating induction ofapoptosis by 18. Further exploration of this scaffold may yielduseful compounds that could be used to interrogate the biology ofSirt2.

4. Experimental

4.1. General conditions for organic synthesis

Reagents were purchased from SigmaeAldrich Chemical(Singapore) or Alfa Aesar (Ward Hill, MA) and used without furtherpurication. Microwave reactions were carried out on the BiotageInitiator Microwave Synthesizer. 1H NMR (300 MHz or 400 MHz)and 13C NMR (75 MHz or 100 MHz) spectra were measured on aBruker Spectrospin 300 or 400 Ultrashield magnetic resonancespectrometer. Chemical shifts (d) were reported in ppm and refer-enced to residual solvents: CDCl3 (d7.26), DMSO-d6 (d2.50), CD3OD(d 3.31) (for 1H spectra) or CDCl3 (d 77.00), DMSO-d6 (d 39.43),CD3OD (d 49.05) (for 13C spectra). Coupling constants (J) were re-ported in Hz. Reactions were monitored by TLC on Silica Gel 60F254 (Merck). Column chromatography was carried out on MerckSilica Gel 60 (0.04e0.06 mm). Mass spectra were recorded in pos-resulting compounds. The more promising Sirt2 inhibitors werefound in Series B, notably 18 and 20 (IC50 3e4 mM) which weremore inhibitory than the Sirt2 selective inhibitor AGK2 (14 mM).However, the moderately active members in series C (24,25 IC5011e14 mM) should also be pursued for lead optimization as theyhave more favorable solubility proles than series B. Kinetic anal-ysis of Sirt2 inhibition by 18 revealed non-competitive inhibitionagainst both NAD and the acetylated substrate. Molecular dockingshowed the functionalized scaffold of 18 (and other analogs)positioned at both the NAD binding pocket (sites B, C) and theacetylated substrate channel by stacking, van der Waals/hydro-phobic and H bonding interactions. The functional relevance of

T. Yang et al. / European Journal of M152119.59, 112.84, 111.06, 105.43, 60.85, 40.76, 23.03, 22.87, 14.59.4.3. Ethyl 8-bromo-5-n-octyl-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (3a)

To a mixture of 2 (2.39 g, 7.40 mmol) and sodium hydride (60%,740 mg, 18.5 mmol, 2.5 equiv.) was added dry DMF (10 mL) underN2 at 0 C. The reaction mixture was stirred at room temperaturefor 15 min after which a solution of 1-bromooctane (1.72 g,8.9 mmol, 1.2 equiv.) in dry DMF (5 mL) was added drop wise to thestirred mixture at 0 C. The reaction mixture was stirred at roomtemperature (z3 h), poured into an ice-water and extracted withDCM (15 3 mL). The combined DCM layer was sequentiallywashed with water (20 3 mL) and brine, dried (Na2SO4), ltered,and concentrated under reduced pressure and the residue waspuried by silica gel column chromatography (hexane: EtOAc 3:1)to give 3a as yellow oil (2.70 g, yield: 83.5%). 1H NMR (300 MHz,CDCl3) d 7.57 (d, 1H, J 2.0 Hz), 7.25e7.22 (m, 1H), 7.15e7.12 (m,1H), 4.65 (br, 2H), 4.20 (q, 2H, J 7.2 Hz), 3.97 (t, 2H, J 7.2 Hz), 3.87(br, 2H), 2.80 (br, 2H), 1.74e1.67 (m, 2H), 1.33e1.25 (m,13H), 0.87 (t,3H, J 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 155.96, 134.97, 128.98,128.17, 126.71, 123.69, 120.23, 112.20, 110.53, 61.53, 53.39, 43.21,41.08, 41.02, 31.71, 30.24, 29.22, 29.09, 26.95, 22.54, 14.70, 14.02.

4.4. 8-Bromo-5-n-octyl-2, 3, 4, 5-tetrahydro-1H-pyrido[4,3-b]indole (4a)

To the solution of 3a (1.66 g, 3.82 mmol) in 15 mL ethanol wasadded an aqueous solution of KOH (4.28 g,76.4 mmol, in 5 mLwater) and reuxed for 16 h. Ethanol was removed under reducedpressure and the residual mixture was extracted with DCM(15 3 mL). The combined DCM layer was sequentially washedwith water (15 3 mL) and brine, dried (Na2SO4), ltered, andconcentrated under reduced pressure to give a brown residuewhich was puried by silica gel column chromatography (DCM:methanol 25:1) to give 4a as a brown oil, 1.03 g, yield: 74.5%. 1HNMR (400 MHz, CDCl3) d 7.49 (d, 1H, J 1.6 Hz), 7.21e7.18 (m, 1H),7.11 (d, 1H, J 8.8 Hz), 4.00 (br, 2H), 3.93 (t, 2H, J 7.2 Hz), 3.24 (t,2H, J 5.6 Hz), 2.73 (t, 2H, J 5.6 Hz), 2.68 (br, 1H), 1.68 (t, 2H,J 7.2 Hz), 1.28e1.24 (m, 10H), 0.87 (t, 3H, J 6.8 Hz). 13C NMR(100 MHz, CDCl3) d 134.69, 134.56, 127.12, 123.26, 120.15, 111.93,110.33, 107.74, 43.17, 42.98, 41.91, 31.68, 30.19, 29.20, 29.06, 26.94,23.19, 22.51, 13.98. MS (ESI), [MH], Calcd for C19H28BrN2, 363.1;Found 363.3, 365.5.

4.5. 8-(40-(Methylsulfonyl)phenyl)-5-n-octyl-2, 3, 4, 5-tetrahydro-1H-pyrido[4,3-b]indole (18)

To the mixture of 4a (200 mg, 0.55 mmol), 4-(methanesulfonyl)phenylboronic acid (132.6 mg, 0.66 mmol) and Pd(PPh3)4 (32.0 mg,0.028 mmol) in 4 mL 1, 4-dioxane was added 0.5 mL aqueous so-lution of K2CO3 (229.0 mg, 1.66 mmol). The mixture was heated(110 C, 0.5 h) in a microwave reactor with stirring. On cooling, thesolvent was evaporated and the resulting residue was extractedwith DCM (10mL 3), the DCM layer was washed with brine, dried(Na2SO4) and ltered. The residue obtained on removal of the sol-vent was puried by silica gel column chromatography (DCM:methanol 20:1) to give the target compound.18was obtained as anoff-yellow oil (120 mg, yield: 49.6%). 1H NMR (400 MHz, CDCl3)d 7.96 (d, 2H, J 8.4 Hz), 7.79 (d, 2H, J 8.4 Hz), 7.63 (br, 1H),7.43e7.41 (m, 1H), 7.37e7.35 (m, 1H), 4.29 (br, 2H), 4.02 (t, 2H,J 7.2 Hz), 3.44 (br, 2H), 3.09 (s, 3H), 2.96 (br, 2H), 1.76e1.71 (m,2H), 1.31e1.25 (br, 10H), 0.87 (t, 3H, J 6.8 Hz). 13C NMR (100 MHz,CDCl3) d 147.91, 137.92, 136.43, 133.45, 130.44, 127.81, 127.76, 127.70,125.84, 120.93, 116.84, 109.82, 48.77, 48.13, 44.68, 43.38, 31.74,30.32, 29.25, 29.12, 29.05, 27.04, 22.56, 14.03. MS (ESI), [MH],

cinal Chemistry 92 (2015) 145e155calcd for C26H35N2O2S, 439.2, found, 439.2. HRMS (ESI), [MH],

edicalcd for C26H35N2O2S, 439.2419; found 439.2415.

4.6. In vitro assay for Sirt1 and Sirt2 activities

Sirt1 and Sirt2 activities were determined using the Sirt-GloAssay Kit (G6450, Promega, Madison, WI, USA) following manu-facturer's instructions. Compounds were dissolved in DMSO andtested over a 103 fold concentration range (nal DMSO concentra-tion 2.5%). IC50 values were determined in triplicate and from 2 ormore independent experiments. Briey, assays were carried out ona white wall 384-well plate with human recombinant Sirt1 (BML-SE239) and Sirt2 (BML-SE251) enzymes from Enzo Life Sciences(NY, USA). The amount of enzyme used in the assaywas determinedby measuring the signal-to-noise ratio of serially diluted enzyme inSirt-Glo Buffer solution. The concentration of enzyme corre-sponding to themid-range linear portion of the signal to noise ratioversus concentration plot was selected and was 0.1 unit Sirt1 perwell and 0.2 unit Sirt2 per well. Serially diluted inhibitor solutions(10 mL in Sirt-Glo Buffer solution, with 1 mL of DMSO) were addedto each well followed by the sirtuin enzyme (10 mL in Sirt-GloBuffer solution). The plate was agitated (400 rpm, 30 min, 25 C,Tecan Innite 200 plate reader) after which 20 mL of Sirt-GloReagent solution was added per well, the contents mixed byshaking (400 rpm) for another 30 min, 25 C, after which lumi-nescence was read on the plate reader. Enzyme activity (%) wasmeasured by the following expression:

Enzyme Activity% hLumCompound

i Lum Blank

Lum Control Lum Blank 100%

where Lum_Compound luminescence of wells containingenzyme and test compound in vehicle (SIRT Glo Buffer solution),Lum_Control luminescence of well containing enzyme only invehicle; and Lum_Blank luminescence of well containing vehicleonly. The IC50 of test compoundwas determined from the sigmoidalcurve obtained by plotting % enzyme activity versus logarithmicconcentration of test compound (GraphPad Prism, Version 5, SanDiego, USA). EX527 (Sigma LifeScience, MO, USA), AGK2 (TocrisBioscience, Bristol, UK) and nicotinamide (SIRT-Glo Assay Kitprovided, Promega, Madison, WI, USA) were used as positive con-trols. Representative dose response curves are presented inSupplementary information (Section 11).

4.7. In vitro assay for evaluating inhibition of protease andluciferase

Stock solutions (10 mM) of test compounds (9, 10, 12, 13, 18, 19,20, 22, 24 and 26) were prepared in DMSO and serially diluted withSirt-Glo Buffer solution.10 mL of the diluted solutionwas added toeach well in a whiteewall 384 well plate followed by 10 mL of Sirt-Glo Buffer solution and 20 mL of Sirt-Glo Reagent solutioncontaining 1 mM SIRT-Glo Control Substrate (non-acetylatedpeptide, a gift from Promega, Madison, WI, USA). The nal con-centration of test compound in the well was 10 mM and DMSOcontent per well was 2.5% v/v. The contents of thewells weremixedby shaking (400 rpm) for 30 min, 25 C, after which luminescencewas read on the plate reader. Luminescence (%) was measured bythe following expression:

Luminescence % hLumCompound

i Lum Blank

Lum Control Lum Blank 100%

T. Yang et al. / European Journal of Mwhere Lum_Compound luminescence of wells containing non-acetylated peptide, Sirt-Glo Reagent and test compound invehicle (SIRT Glo Buffer solution), Lum_Control luminescenceof wells containing non-acetylated peptide, Sirt-Glo Reagent invehicle Sirt-Glo Reagent and Lum_Blank luminescence of wellcontaining vehicle only. Results are given in Supplementaryinformation, Section 12.

4.8. Kinetic study of Sirt2 inhibition by 18

The kinetics of Sirt2 inhibition by 18 was investigated using adeconstructed Sirt-Glo Kinetics Assay Kit which was a gift fromPromega (Madison, WI, USA). Human recombinant SIRT2 (BML-SE251, Enzo Life Sciences, NY, USA) (0.2 unit/well in 5 mL of assaybuffer) was aliquoted into each well of a 384-well plate whichcontained test compound (18) at 0, 1.1, 3.3 or 10 mM in 5 mL of assaybuffer per well. The contents of the platewere incubated for 60minat 25 C with agitation (400 rpm). Final concentration of DMSO perwell was kept at 2.5%. At the same time, the Luciferin DetectionReagent (LDR) solution was prepared by combining the acetylatedsubstrate (Z-QPK(Me)2K(Ac)-aminoluciferin) (4.12, 12.4, 37.0, 111.1or 333.3 mM), NAD (1 mM) and developer reagent. The mixturewas incubated for 60min at 37 C, after which aliquots (10 mL) weredispensed to each well containing the 18-Sirt2 enzyme mixture.Incubation was continued for another 30 min, 25 C with agitation(400 rpm). Luminescence readings were then taken. The data fromthese experiments were used to obtain the double-reciprocal plotfrom which Km of Sirt2 for acetylated substrate was obtained. Theslope and intercept of the lines obtained in presence of 18 werereplotted against concentration of 18 according to Equation (1) togive apparent Ki of 18 (Supplementary information, Section 13,Fig. S2). The Km of Sirt2 for acetylated peptide was also obtainedfrom EadieeHofstee plot (Supplementary information, Section 13,Fig. S3) which is based on Equation (2) and involved plotting re-action rate against rate/(substrate concentration).

Slope Km=Vmax Km=Vmax I=Ki (1)

Rate Km Rate=Subtrate Vmax (2)The above procedure was repeated at a xed concentration of

the acetylated substrate (38.5 mM) and varying amounts of NAD(185, 556, 1667, 5000 mM). The resulting double reciprocal plot gavethe Km of Sirt2 for NAD and the secondary plots gave apparent Kiof 18with NADwas substrate. Plots and kinetic parameters Km, Ki,Vmax were obtained with GraphPad Prism (Version 5, San Diego,CA). From the double reciprocal plots, the Km values of Sirt2 foracetylated substrate and NADwere found to be 38.0 2.0 mM and1317 208 mM respectively. Km values from EadieeHofstee plots ofthe same data were comparable at 29.9 1.6 mM (acetylated sub-strate) and 1298 218 mM (NAD) (Supplementary information,Section 13, Fig. S3).

4.9. Molecular docking

The human Sirt2 enzyme was retrieved from the RCSB proteindata bank (PDB 3ZGV) [12]. Water molecules were removed and themonomeric enzyme was processed for docking using LigX in Mo-lecular Operating Environment (MOE, version 2011, ChemicalComputing Group, Montreal, Canada). The structures of the testcompounds were separately prepared for docking onMOE. Dockingwas carried out on GOLD v 5.2 (Cambridge Crystallographic DataCentre Software Ltd, Cambridge, UK) with default genetic algorithmsettings. The binding pocket was dened by the atoms within 10 radius of the co-crystallized ligand (ADP-ribose). Docking was

cinal Chemistry 92 (2015) 145e155 153carried out without the reference ligand. GOLD uses a genetic

edialgorithm for docking exible ligands into the binding pocket toexplore the full range of ligand conformational exibility [22]. TheGOLD Scorewas used as the tness function for selection of the bestdocked conformations of test compounds in the binding pocket. Foreach molecule, the top 5 docked conformations were retained andvisualized on MOE.

4.10. Assessment of aggregation tendency by light scattering

Stock solutions (10 mM) of test compounds prepared in DMSO,diluted to 1 mM with DMSO and then serially diluted with potas-sium phosphate buffer (5 mM, pH 7.4, preltered before use) to givenal concentrations of 1 mM and 10 mM. Final concentration ofDMSO was 1% v/v. Measurements were carried out on the MalvernInstrument Zetasizer Nano ZS system equipped with a 4 mWHeeNe laser at 633 nm and detector angle of 90. Three or moredeterminations of derived count rates (kilocounts per second, kcps)were obtained from each concentration of test compound, usingtwo separately prepared stock solutions. Data collection was car-ried out using the software supplied with the instrument. Resultsare represented as mean standard deviation. The positive controlwas benzyl benzoate which gave a count rate of 1148kcps 37(250 mM) and 76 5 (25 mM). The vehicle (phosphate buffer, 1%DMSO) gave a reading of 15.5 0.4. The results are presented inSupplementary information, Section 14.

4.11. Determination of cell growth inhibition

Human breast cancer MDA-MB-231, hepatocellular carcinomaHepG2 and human lung broblast IMR90 cells were purchasedfrom ATCC (Rockville, MD). All cells were grown in DMEM (Invi-trogen, High Glucose.) at 37 C, 5% CO2. DMEM was supplementedwith 10% fetal bovine serum (Hyclone, heat treated at 65 C for30 min before use), 50 units/L penicillin (Gibco) and 50 mg/mLstreptomycin (Gibco). Cells were subcultured at 85e90% conuencyand used within 15 passages. Cell viability was assessed usingCellTitre 96 Aqueous One Solution (Promega, Madison, WI) con-taining the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS).Seeding densities were 2500 cells/well in the 96-well plates. Cellswere grown in media (10% FBS) in 96-well plates for 24 h, at 37 C,5% CO2 after which aliquots of 18 (stock solution in DMSO, dilutedwith media containing 10% FBS) were added to each well and theplates were incubated for 72 h, at 37 C, 5% CO2. Final concentrationof DMSO per well was 0.5% v/v. At the end of the incubation period,10 mL of the MTS solution was added to each well, the plates wereincubated for 4 h in the dark after which absorbance readings wereread at 490 nm (Tecan Innite M200 Microplate reader). Cellviability was determined from the expression:

Cell viability% Absorbancecellscpd Absorbancecpd

Absorbancecellsvc Absorbancevc 100%

where Absorbancecellscpd absorbance of wells containing cellsand test compound in vehicle (media 0.5% DMSO),Absorbancecellsvc absorbance of wells containing cells in vehicle(vc) only; Absorbancevc absorbance of wells containing vehicle;Absorbancecpd absorbance of wells containing test compound.The % viability readings were plotted against log concentration onGraphPad Prism (Version 5.0, San Diego, CA) to give a sigmoidalcurve from which IC50 (concentration required to reduce viabilityby 50% compared to control/untreated cells) was obtained. The plot

T. Yang et al. / European Journal of M154was constrained to 0 and 100%. At least 3 separatedeterminations of IC50 were determined. Representative doseresponse curves are presented in Supplementary information,Section 15.

4.12. Western blot

Rabbit monoclonal antibodies to PARP and caspase 3, mousemonoclonal antibodies to p53, goat anti-rabbit-horse radishperoxidase (HRP) conjugate and goat anti-mouse HRP were fromSanta Cruz Biotechnology (CA, USA). Rabbit monoclonal antibodiesto cleaved caspase 3 and acetylated p53 (K382) were purchasedfrom Cell Signaling (MA, USA). Mouse monoclonal antibodies toacetylated a-tubulin and a-tubulinwere from Sigma Aldrich (SigmaLifescience, MO, USA). Mouse monoclonal antibodies to b-actinwere purchased from Invitrogen Life Technologies (CA, USA).HepG2 and MDA-MB-231 cells were seeded at a cell density of in a10 cm petri dish at 50,000 cells/mL (total 10 mL in DMEM, highglucose, Invitrogen) containing a known concentration of testcompound. Final concentration of DMSO in the ask was main-tained at 0.5% v/v. Treated cells were incubated at 37 C in a hu-midied 5% CO2 atmosphere for 6 h (for HepG2 cells) or 24 h (forMDA-MB-231 cells) after which they were transferred to a 15 mLFalcon tube, pelleted at 500 g (5 min) and washed twice with ice-cold 1x PBS. The cells were transferred to a microcentrifuge tubecontaining 70 mL lysis buffer (20 mM Tris pH 7.4, 250 mM NaCl,2 mM EDTA pH 8.0, 0.1% Triton X-100, 0.01 mg/ml aprotinin,0.005 mg/ml leupeptin, 0.4 mM PMSF, and 4 mM NaVO4) andincubated at 0 C for at least 20 min. After the incubation, the ly-sates were spun at 13,300 g for 10 min to remove cell debris. Analiquot (3 mL) was retained for protein determination (BradfordProtein Assay Kit, Bio-Rad Laboratories Inc, CA, USA) while theremaining supernatant was diluted with 4 SDS solution (0.2 MTris pH 6.8, 0.28 M SDS, 40% v/v glycerol, 0.59 M b-mercaptoetha-nol, 50 mM EDTA, 1.1 mM bromophenol blue) to give 1 SDS so-lution which was then deactivated at 100 C, 5 min andsubsequently stored for no more than 1 week at80 C. Cell lysateswere separated on the SDS-PAGE Bio-Rad Mini-Protean II system(Bio-Rad Laboratories Inc, CA, USA). Membranes were blocked withblocking buffer for at least 60 min at 25 C and then incubated withprimary antibodies at appropriate dilutions in 2.5% BSA in TBST(50 mM Tris, 150 mM NaCl, 0.1% Tween 20) solution overnight(4 C). Membranes were than washed thrice with TBST (10 min perwash), incubated for 1 h with secondary antibodies in TBST at 25 Cfollowed by washing in TBST (thrice, 10 min per wash). Theimmunoreactive bands were detected by the ECL reagent (GEHealthcare, Little Chalfont, UK) using Bio-Rad Universal Hood II GelDoc. If needed, the membranes were stripped with stripping buffer,blocked with blocking buffer for 30 min 25 C, and re-probed withother antibodies. Western blotting for each compound at statedconcentrations were repeated at least 3 times.

4.13. Determination of solubility

The solubilities of 10, 12, 18, 19, 20 and 24 were determined inphosphate buffer pH 7.4 by a turbimetric method [16]. Briey, stocksolutions of test compound were prepared in DMSO and aliquotswere added to a 96-well plate containing phosphate buffer in eachwell to give a range of concentrations (1e50 mM). DMSO concen-tration was kept at 1% v/v. After shaking the plate for 30 min at25 C, absorbance readings were taken at 620 nm. Absorbancereadings were plotted against concentration. When the concen-tration exceeded compound solubility, the occurrence of precipi-tation led to an increase in absorbance. Solubility was then assignedto the range of concentrations where a two-fold or greater increase

cinal Chemistry 92 (2015) 145e155in absorbance was observed from the absorbance-concentration

plot. Results were obtained from 3 separate determinations.

Acknowledgments

This work was supported by grants from the BiomedicalResearch Council (10/1/21/19/664) and Ministry of Health FacultyResearch Grant (R148000171112) to M.L.G. We thank Promega forproviding the SIRT-Glo Kinetics Assay Kit and SIRT-Glo ControlSubstrate, and Dr. Andrew Niles for helpful advice and discussionon the assay details.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.12.027.

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Functionalized tetrahydro-1H-pyrido[4,3-b]indoles: A novel chemotype with Sirtuin 2 inhibitory activity1. Introduction2. Results and discussion2.1. Design and synthesis of target compounds2.2. Sirtuin inhibitory activity of test compounds2.3. Kinetics of Sirt2 inhibition by 182.4. Molecular docking of series AC compounds in the Sirt2 binding pocket2.5. 18 increased levels of acetylated p53 and alpha-tubulin in cancer cell lines

3. Conclusion4. Experimental4.1. General conditions for organic synthesis4.2. Ethyl 8-bromo-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (2)4.3. Ethyl 8-bromo-5-n-octyl-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (3a)4.4. 8-Bromo-5-n-octyl-2, 3, 4, 5-tetrahydro-1H-pyrido[4,3-b]indole (4a)4.5. 8-(4-(Methylsulfonyl)phenyl)-5-n-octyl-2, 3, 4, 5-tetrahydro-1H-pyrido[4,3-b]indole (18)4.6. In vitro assay for Sirt1 and Sirt2 activities4.7. In vitro assay for evaluating inhibition of protease and luciferase4.8. Kinetic study of Sirt2 inhibition by 184.9. Molecular docking4.10. Assessment of aggregation tendency by light scattering4.11. Determination of cell growth inhibition4.12. Western blot4.13. Determination of solubility

AcknowledgmentsAppendix A. Supplementary dataReferences