Functionalized tetrahydro-1 H-pyrido[4,3-b]indoles: A novel chemotype with Sirtuin 2 inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2014.12.027

Sirtuins are protein deacylases with regulatory roles in metabolism and stress response. Functionalized tetrahydro-1H-pyrido[4,3-b]indoles were identified as preferential sirtuin 2 inhibitors, with in vitro inhibitory potencies in the low micromolar conce

Full text of DOI:10.1016/j.ejmech.2014.12.027

Accepted Manuscript
Functionalized tetrahydro-1H-pyrido[4,3-b]indoles: A novel chemotype with Sirtuin 2
inhibitory activity
Tianming Yang, Xiao Chen, Hai-xiao Jin, Gautam Sethi, Mei-Lin Go
PII:
S0223-5234(14)01136-2
10.1016/j.ejmech.2014.12.027
EJMECH 7587
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 August 2014
Revised Date: 1 December 2014
Accepted Date: 17 December 2014
Please cite this article as: T. Yang, X. Chen, H.-x. Jin, G. Sethi, M.-L. Go, Functionalized tetrahydro-1H-
pyrido[4,3-b]indoles: A novel chemotype with Sirtuin 2 inhibitory activity, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2014.12.027.
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Graphical Abstract
CH3SO2
N
N
H
> 250 M
Sirt2 IC50
CH3SO2
NH
N
20 M
Sirt2 IC50
C4H9-n
CH3SO2
NH
N
C8H17-n
Sirt2 IC50
4 M

ACCEPTED MANUSCRIPT
Functionalized tetrahydro-1H-pyrido[4,3-b]indoles: A novel chemotype with Sirtuin 2 inhibitory
activity.
b
Tianming Yang, a Xiao Chen, a Hai-xiao Jin, Gautam Sethi, c Mei-Lin Go a, *
a Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Republic of
b
Singapore 117543.
Key Laboratory of Applied Marine Biotechnology, Ministry of Education,
School of Marine Sciences, Ningbo University, Ningbo, Fenghua Road 818, Jiangbei District,
Zhejiang, 315211, People's Republic of China. c Department of Pharmacology, Yong Loo Lin
School of Medicine, National University of Singapore, 10 Medical Drive, Republic of Singapore
*
117597. Corresponding Author: Mei Lin Go
,
Department of Pharmacy, National University of
Singapore, 18 Science Drive 4, Republic of Singapore 117543. Tel: 65-65162654. Fax: 65-
68721610. Email: phagoml@nus.edu.sg,
Abstract
Sirtuins are protein deacylases with regulatory roles in metabolism and stress response.
Functionalized tetrahydro-1H-pyrido[4,3-b]indoles were identified as preferential sirtuin 2
inhibitors, with in vitro inhibitory potencies in the low micromolar concentrations (IC50 3-4 µM) for
the more promising candidates. The functional relevance of sirtuin inhibition was corroborated in
western blots that showed hyperacetylation of p53 and α-tubulin in treated HepG2 and MDA-
MB-231 cells. Molecular docking showed that the tetrahydropyridoindole scaffold was positioned
in the NAD+ pocket and the acetylated substrate channel of the sirtuin 2 protein by van der
Waals/hydrophobic, H bonding and stacking interactions. Functionalized
tetrahydropyridoindoles represent a novel class of sirtuin 2 inhibitors that could be further
explored for its therapeutic potential.
Keywords
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tetrahydropyridoindoles, sirtuin 2 inhibition, molecular docking, hyperacetylation of p53 and α-
tubulin, apoptosis, growth inhibition.
1. Introduction
Sirtuins are a class of evolutionary conserved nicotinamide adenine dinucleotide (NAD+) -
dependent protein lysine deacylases with important roles in diverse and interrelated cellular
processes such as stress response, gene expression, DNA damage repair and metabolism [1].
They are implicated in age-related diseases such as cancer, neurodegeneration and metabolic
disorders and hence are considered as prospective therapeutic targets for these conditions.
However, perplexing gaps remain in our understanding of their modes of action. In
carcinogenesis, sirtuins have bifurcated roles and may function as tumor suppressors and
promoters depending on contextual variables such as the stage of the malignancy and the
tumor microenvironment [2]. High-throughput and in silico screening have identified novel
chemotypes with sirtuin inhibitory 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 [8-11]. Interestingly, certain scaffolds were observed to be more widely
associated with sirtuin inhibition than others. The indole ring is a case in point - it is embedded
in the bisindolylmaleimide derivative Ro 31-8220 [8], the oxindole GW 5074 [8,9], the
tetrahydrocarbazole EX527 [10] and the carprofen analog 1 (2-(6-chloro-9H-carbazol-2-
yl)propanamide) [11] (Figure 1A). We noted that EX527 and the caprofen analog 1 bore a
striking structural resemblance in that both compounds were ring-fused indoles with primary
amide functionalities. They were also selective Sirt1 inhibitors but differed in their cell-based
activities. EX527 did not abolish cell growth and proliferation [12, 13] and had varied effects on
the acetylation status of p53, a substrate of Sirt1 and Sirt2, depending on experimental
conditions [11-13]. In contrast, the carprofen analog 1 induced apoptosis in leukemic cells and
its sirtuin inhibitory activity was confirmed in functional assays [11].
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Figure 1
To further explore the potential of the indole scaffold, we evaluated a series of 2,3,4,5-
tetrahydro-1H-pyrido[4,3-b]indoles for sirtuin inhibition (Figure 1B). The indole ring of this
scaffold is fused to a basic piperidine ring and not benzene or cyclohexane as in 1 and EX527.
Our results showed that tetrahydropyridoindole is a novel chemotype associated with
preferential Sirt2 inhibitory activity. A representative member 18 inhibited sirtuin-mediated
deacetylation of physiological substrates p53 and α-tubulin in liver (HepG2) and breast (MDA-
MB-231) cancer cells at low micromolar concentrations (2-5 µM). Molecular docking showed
that the tetrahydropyridoindole ring was positioned at the NAD+ pocket and substrate channel
of the Sirt2 protein, with binding affinity modulated 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 (Figure 1B) which was serendipitously found to occupy
the NAD+ binding pocket of human Sirt2 (PDB 3ZGV) [12]. As shown in Figure 2A, the
tetrahydropyridoindole 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 stacking interactions (ππ, π-cation) were identified, namely Phe96, His 187
and the aromatic ring of the scaffold (ππ, πH respectively, with distances of 3.81 Å and 3.74 Å),
Arg97 and m-tolyl ring (πH, 4.01 Å) and Phe235 and piperidine NH+ (πH, 3.60 Å). The n-octyl
side chain of 8 occupied a hydrophobic channel that extended from site C (Figure 2B).
Figure 2
When 8 was screened for inhibition of Sirt1 and Sirt2 (as described later), it was found to inhibit
Sirt2 (43%, 10 µM) to a greater extent than Sirt1 (5%, 10 µM). With this lead on hand, we
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modified 8 at positions 2 (R1), 5 (R2) and 8 (R3) with the intent of establishing structure-activity
relationships (SAR) and optimizing inhibition. To that end, thirty analogs that were structurally
related to 8 were synthesized. Having observed that the n-octyl side chain of 8 was positioned
in a lipophilic region of the NAD+ pocket, we reasoned that it was an essential structural feature.
However, its presence would invariably impart considerable lipophilicity to the resulting
compounds. The lipophilicity of 8, as seen from its log P (7.55) and logD
(5.93) values,
pH 7.4
were high. Hence to avoid exacerbating the “greasiness” of the target compounds, a deliberate
attempt was made to introduce polar residues at R1 and R3. To that end, several polar and
sterically limited entities were introduced at R1, such as hydroxyethyl, aminocarbonyl,
methylsulfonyl, methyl, ethyl and hydrogen. In the case of R3, an arbitrary selection of 3’-tolyl,
4’-methylsulfonylphenyl, 2’-aminopyrimidin-5’-yl, 2’-fluoropyridin-4’-yl and fluoro was made,
prompted in part by the commercial availability of reagents. These R3 groups formed the basis
of classifying the synthesized compounds to their respective series A-E (Table 1). As shown
later, series D and E which had fluoropyridinyl and fluoro as R3 respectively, were weak sirtuin
inhibitors, further emphasizing the need to deploy small or polar groups at R1. Analogs with
shorter (n-butyl, isoprenyl) or no R2 side chains were also synthesized to confirm the necessity
of retaining the n-octyl side chain.
Most of the compounds (except series E) were derived from a common intermediate ethyl 8-
bromo-3,4-dihydro-1H-pyrido[4,3-b]indole-2(5H)-carboxylate (2) which was obtained from a
Fischer indole reaction between 4-bromophenylhydrazine hydrochloride and 1-carbethoxy-4-
piperidone (Scheme 1). The indole nitrogen of 2 was N-alkylated with 1-bromooctane in the
presence of sodium hydride, followed by alkaline hydrolysis and decarboxylation of the
carbamate moiety at position 2 to give 4a. The latter was reacted with formaldehyde,
acetaldehyde or acetone in the presence of sodium triacetoxyborohydride to give the N-methyl
(5a), N-ethyl (5b) or N-isopropyl (5c) intermediate. 4a was also reacted with methanesulfonyl
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chloride, 2-bromoethanol or potassium cyanate to give 5d, 5e or 6 respectively. In the next
step, palladium catalyzed Suzuki coupling was carried out to introduce substituted aromatic
moieties at position 8. This sequence was broadly followed throughout, except for analogs with
3’-tolyl at position 8 (9, 10, 11, and 12) where higher yields were obtained when the 3’-tolyl ring
was introduced first (Scheme 1). The 8-fluoro substituted analogs of series E (35, 36) were
obtained by the Fischer indole reaction between 4-fluorophenylhydrazine hydrochloride and 1-
carbethoxy-4-piperidone, followed by the usual reactions (Supplementary Information, Section
9). In the same way, the indole nitrogen of 2 was reacted with 1-bromobutane to give n-butyl
analogs (16, 22, and 32). Alternatively, the indole nitrogen of 2 was left in its unsubstituted state
and functionalized at positions 2 and 8 as described earlier to give 14, 15, 21 and 31
(Supplementary information, Section 6).
Scheme 1
2.2. Sirtuin inhibitory activity of test compounds
The tetrahydropyridoindoles were evaluated against recombinant human Sirt1 and Sirt2 in an
assay that monitored the rate at which the enzyme deacetylated an N-acetyl lysine residue in a
peptide substrate bearing the amino acid residues 317-320 of p53 and conjugated to
aminoluciferin. The deacetylation reaction triggered a protease mediated hydrolysis which
released aminoluciferin. The latter was oxidized by luciferase to oxyluciferin which is
luminescent. Inhibition of sirtuin resulted in less oxyluciferin being generated and hence a
reduction in the luminescent signal. The compounds were first evaluated at a fixed
concentration of 10 µM 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 greater extent (≥ 50% inhibition) than Sirt1 (
50% inhibition) while 2 compounds (10, 20) inhibited both Sirt1 and Sirt2 by more than 50%.
The remaining compounds were weak inhibitors (< 50% inhibition) of both enzymes.
≤
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To ensure that the reduction in luminescence was not due to aberrant inhibition of the protease
or luciferase enzymes, the assay was repeated using deacetylated substrate in the absence of
sirtuin for selected compounds (9, 10, 12, 13, 18, 19, 20, 22, 24, 26) at 10 µM (Supplementary
Information, Section 12). We found luminescence readings to be comparable to that of control
readings (no test compound) indicating the absence of non-specific inhibition of the protease
and luciferase enzymes.
We also explored the possible involvement of aggregate formation as a source of non-specific
inhibition [13,14]. Some compounds are known to form submicrometer aggregates capable of
sequestering enzymes onto their surfaces, resulting in an artefactual reduction of enzyme
activity. Such compounds (“aggregators”) are generally characterized by poor solubility, high
lipophilicity and extended conjugation in their structures [13]. As some of these features were
present in our compounds, the involvement of aggregate formation in the observed inhibition
was investigated. Aggregators normally form particles of 30-100 nm diameter that were
capable of scattering light. To that end, we monitored the light scattering properties of selected
compounds (9, 10, 12, 13, 18, 19, 20, 24, 26 and 28) at 1 µM and 10 µM in phosphate buffer pH
7.4 (Supplementary Information, Section 14). Negligible light scattering was observed at 1 µM
but higher levels were observed at 10 µM of 9, 12 and 13. This raised the question as to
whether aggregation could have contributed to the inhibitory activities of these compounds
(Table 1). The likelihood was deemed to be slight because 9, 12 and 13 (at a fixed
concentration of 10 µM) were paradoxically weak inhibitors of Sirt1 although reasonably strong
inhibitors of Sirt2. We reasoned that if aggregate formation contributed to artefactual inhibition
of sirtuins, strong inhibition should be observed for both and not just one enzyme.
We then proceeded to determine the IC50 (concentration required to reduce basal oxyluciferin
luminescence by 50%) of those compounds that inhibited Sirt 2 /Sirt1 by more than 50% at 10
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µM. Some less inhibitory compounds were included for comparison. The results are presented
in Table 1.
Table 1
The test compounds were found to be more potent inhibitors of Sirt2 than Sirt1, with a
preference for Sirt2 inhibition that varied from 2-fold (8) to more than 40 fold (19) based on IC50
values. The Sirt2 selective inhibitor AGK2 was used as a positive control and it was gratifying to
note that some of the Series A and B analogs were more inhibitory than AGK2 on this assay.
Based on the inhibitory data at 10 µM and IC50 values, a structure-activity relationship (SAR) for
Sirt2 inhibition is proposed. First, it is important to maintain an alkyl substituted indole nitrogen
at R2. For compounds with the same R1 and R3, inhibition was most pronounced when R2 was n-
octyl. n-Octyl analogs were consistently 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 weak Sirt2 inhibition (at 10 µM). For example, 11 and 17 had similar groups
at R1 (4’-hydroxybenzyl) and R3 (3’-tolyl) but different R2 groups (isoprenyl in 17 and n-octyl in
11). Stronger Sirt2 inhibition was anticipated for the n-octyl bearing 11 but as seen from Table 1,
both compounds weakly inhibited Sirt2 (20-40% inhibition at 10 µM). Evidently, one or both
substituents at R1 and R3 of 11 were not optimal for activity. Thus, R2 notwithstanding, groups at
R1 and R3 should not be ignored as they may have modulatory roles.
That the groups at R1 and R3 were important was further supported by examining the “actives”
(compounds that inhibited Sirt2 by 50% or more at 10 µM). By this measure, small polar groups
were preferred at R1 as seen from the recurring presence of R1 = hydrogen, methyl and
aminocarbonyl among these compounds. On the other hand, compounds bearing
ethoxycarbonyl (polar, bulky) and 4’-hydroxybenzyl (bulky, less polar) side chains were
noticeably absent. In the case of R3, a key observation was that “actives” were only found in
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series A (R3 = 3’-tolyl), B (R3 = 4’-methylsulfonylphenyl) and C (R3 = 2’-aminopyrimidin-5’-yl), and
not series D (R3 = 3’-fluoropyridin-4-yl) and E (R3 = F). A comparison of IC50 values of
compounds across Series A, B and C with the same R1 and R2 (n-octyl) groups revealed the
following trends: R1 = H: 18 (series B , 3.8 µM) > 8 (series A, 10.1 µM) ≈ 24 (series C, 11.8 µM);
R1 = CH3: 19 (series B, 6.0 µM) ≈ 10 (series A, 6.2 µM) > 26 (series C, 17.9 µM); R1 = CONH2:
20 (series B, 4.0 µM) > 13 (series A, 7.8 µM) > 25 (series C, 14.0 µM). Taken together, the SAR
suggests that small (fluoro in series E) and heteroaromatic azines (series C aminopyrimidinyls,
series D fluoropyridinyls) were not favored at R3.
There is concern that most of the identified actives were found in series A or B which had more
lipophilic substituted-phenyl rings at R3, as compared to the polar heteroaromatic azines at R3 of
series C. Excessive lipophilicity would promote non-specific binding and spurious cytotoxicity to
normal tissues. Thus, we evaluated the cytotoxic IC50 of 18 and 20, two of the more potent Sirt2
inhibitors, on non-malignant human lung fibroblast IMR90 cells. 18 was found to be more
cytotoxic (IC50 2.72 µM ± 0.32) than 20 (IC50 14.5 µM ± 1.5). The estimated logP of 18 was 6.1
as compared to 5.5 for 20 (ACD/Labs Version 12.0). However, 18 is a secondary cyclic amine
and its lipophilicity would be better represented by log DpH7.4 which takes into account its
ionization state. LogD (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 Version 12.0). Thus, the
cytotoxicities of 18 and 20 on IMR90 cannot be solely attributed to their (estimated)
lipophilicities. Follow up investigations on other non-malignant cell lines should be carried out for
confirmation.
Besides cytotoxicity, excessive lipophilicity would adversely affect drug-like character, in
particular solubility. To that end, we assessed the solubilities of selected compounds (10,
12,18,19,20,24) from Series A,B and C by a turbimetric method [16]. As seen from Table 2, 24
from series C (R3 = 2-aminopyridimidin-5-yl) which had moderate Sirt2 inhibitory activity (IC50
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11.8 µM), had a more favorable solubility profile than compounds from series A and B which
were more potent Sirt2 inhibitors. At this stage of lead optimization, moderately active but less
lipophilic compounds like those in series C should not be entirely ignored as they may yet yield
more potent and drug-like analogs upon structural modifications.
Table 2
2.3 Kinetics of Sirt2 inhibition by 18
The kinetics of Sirt2 inhibition was investigated for a representative compound 18. The activity
of Sirt2 as a function of the acetylated substrate (Figure 3A) or NAD+ (Figure 3B) was
monitored in the presence of varying concentrations of 18. Both plots showed a dose-dependent
decrease in the maximal enzyme activity with increasing 18 that was not overcome at high
substrate concentrations. Such a profile was indicative of non-competitive inhibition which
meant that 18 binds to both the enzyme and the enzyme-substrate complex (ES) or subsequent
species [17]. To determine the comparative affinity of 18 for the enzyme or the ES complex, the
double reciprocal plots of 18 for the two substrates were examined. As seen from Figures 3C
and 3D, both plots displayed a nest of lines that converged below the x-axis, indicating that 18
had greater affinity for the ES complex than the free enzyme [17]. To obtain the Ki of 18, the
slopes of the double reciprocal plots obtained in presence of different concentrations of 18 were
re-plotted against inhibitor concentration (Supplementary Information, Section 13) [18].
Apparent Ki of 18 was found to be 2.28 µM ± 0.42 (for acetylated substrate) and 3.70 µM ± 0.35
(for NAD+).
Figure 3
2.4. Molecular docking of series A-C compounds in the Sirt2 binding pocket
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In our preliminary investigations, we examined the docking pose of lead compound 8 in the Sirt2
pocket (PDB 3ZGV) and found that its tetrahydropyridoindole ring straddled several sites in the
NAD+ pocket of Sirt2, namely site B, site C and the substrate channel (Figure 2). We found that
18, 19, 20 of Series B and 24, 25, 26, 28 of Series C were similarly positioned in the NAD+
binding pocket as 10. Figure 4 depicts the docking poses of 18 (Series B) and 24 (Series C) in
the Sirt2 pocket. These poses were representative of other compounds in the respective series.
Significant interactions were the positioning of the n-octyl side chain along a hydrophobic
channel which extended from site C, and ππ /π-cation interactions between the scaffold and Phe
96 (site B), His187 (substrate channel) and Phe 235 (substrate channel). The π-cation (πH)
interaction with Phe235 was observed only when the piperidine nitrogen was protonated and
was absent in 20 and 25 which had an aminocarbonyl substituent at R1. We noted that the
aromatic rings at position 8 of 18 and 24 were involved in H bonding but this was at the expense
of π-cation stacking interactions that were characteristic of the series A analog 8 (Figure 2). As
shown in Figure 4, the sulfonyl moiety of 18 was H bonded to Ser 263 and the amino group of
24 was H bonded to Gly 261. These residues were located in the polar region that
accommodates the phosphoryl residues of the co-crystalized ligand ADP-ribose. The docking
pose of ADP-ribose showed that its phosphate residues were H bonded to Ser 263. The
involvement of Ser263 in H bonding with the sulfonyl moiety of the series B analogs 18, 19, 20
may account for the stronger Sirt2 inhibition observed for these compounds.
Figure 4
Although 18 exhibited non-competitive inhibition of Sirt2, molecular docking showed that it
occupied the NAD+ and substrate binding sites in the enzyme. While this may seem
paradoxical, we noted that EX-527 which inhibited several sirtuins non-competitively or
uncompetitively, was also reported to occupy the nicotinamide binding site of the enzyme [10,
19].
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2.5. 18 increased levels of acetylated p53 and alpha-tubulin in cancer cell lines
Having shown that 18 inhibited the Sirt2 mediated deacetylation of a luminogenic peptide, we
proceeded to determine if it inhibited deacetylation of physiological substrates of Sirt2. To that
end, HepG2 and MDA-MB-231 cells were incubated with varying concentrations of 18 (2-8 µM)
after which acetylated p53 (acetylated at Lys 382) and acetylated
α
-tubulin were probed by
-tubulin is a
immunoblotting (Figure 5A,B). p53 is a substrate of both Sirt1 and Sirt2 whereas
α
Sirt2 substrate [2]. 18 had a growth inhibitory IC50 of 2.1 µM on HepG2 and MDA-MB-231
(Supplementary Information, Section 15). As seen in Figure 5A-B, 18 increased the levels of
acetylated p53 and
α-tubulin in these cells at 2 -4 µM.
Figure 5
Acetylation of p53 increased its stability by limiting ubiquitination of key lysine residues and
subsequent proteasomal degradation [20, 21]. The stabilized and hence activated p53 is thus
able to increase transcription of genes involved in cell-cycle arrest, DNA repair and apoptosis.
Having shown that 18 increased levels of acetylated p53, we proceeded to determine if this
would translate to apoptotic cell death. Hence, two apoptotic marker proteins (caspase 3 and
PARP) were monitored by immunoblotting. In the event of apoptosis, elevated levels of cleaved
caspase 3 and cleaved PARP would be observed and this were duly noted in 18- treated MDA-
MB- 231 cells (Figure 5C).
3. Conclusion
We have shown that functionalization of the tetrahydropyridoindole scaffold resulted in analogs
with low micromolar Sirt2 inhibitory activity. There was a modest preference for Sirt2 inhibition
(2- 40 fold) among the potent members, unlike the structurally related EX527 and caprofen
analog 1 which were Sirt1 selective inhibitors. SAR analysis revealed structure specific
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requirements for inhibition, of which substitution at the indole nitrogen (R2) and position 8 (R3) of
the scaffold were deemed important. Of the groups explored at the indole nitrogen, Sirt2
inhibition decreased in the order n-octyl > n-butyl > H, implicating lipophilicity and steric bulk as
essential features for optimal activity. In the case of substitution at position 8 (R3), there was an
apparent preference for Series B which had a 4’-methylsulfonylphenyl ring at the position 8.
Inhibitory potency was affected to a lesser degree by R1 as inferred from the laxity in terms of
size and polarity permitted at this site. Nonetheless, small groups were preferred at R1 to avoid
exacerbating lipophilicity of the resulting compounds. The more promising Sirt2 inhibitors were
found in Series B, notably 18 and 20 (IC50 3-4 µM) which were more inhibitory than the Sirt2
selective inhibitor AGK2 (14 µM). However, the moderately active members in series C (24,25
IC50 11-14 µM) should also be pursued for lead optimization as they have more favorable
solubility profiles than series B. Kinetic analysis of Sirt2 inhibition by 18 revealed non-
competitive inhibition against both NAD+ and the acetylated substrate. Molecular docking
showed the functionalized scaffold of 18 (and other analogs) positioned at both the NAD+
binding pocket (sites B, C) and the acetylated substrate channel by stacking, van der Waals /
hydrophobic and H bonding interactions. The functional relevance of Sirt2 inhibition by 18 was
corroborated by Western blot analyses which showed that 18 increased levels of acetylated p53
and
α-tubulin in MDA-MD-231 and HepG2 cell lysates. Levels of apoptotic marker proteins
(cleaved PARP, cleaved caspase 3) were also elevated in MDA-MB-231 cell lysates, implicating
induction of apoptosis by 18. Further exploration of this scaffold may yield useful compounds
that could be used to interrogate the biology of Sirt2.
4. Experimental
4.1. General Conditions for Organic Synthesis.
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Reagents were purchased from Sigma-Aldrich Chemical (Singapore) or Alfa Aesar (Ward Hill,
MA) and used without further purification. Microwave reactions were carried out on the Biotage
Initiator ® Microwave Synthesizer. 1H NMR (300 MHz or 400 MHz) and 13C NMR (75 MHz or
100 MHz) spectra were measured on a Bruker Spectrospin 300 or 400 Ultrashield magnetic
resonance spectrometer. Chemical shifts (δ) were reported in ppm and referenced to residual
solvents: CDCl3 (δ7.26), DMSO-d6 (δ2.50), CD3OD (δ 3.31) (for 1H spectra) or CDCl3 (δ 77.00),
DMSO-d6 (δ 39.43), CD3OD (δ 49.05) (for 13C spectra). Coupling constants (J) were reported in
Hz. Reactions were monitored by TLC on Silica Gel 60 F254 (Merck). Column chromatography
was carried out on Merck Silica Gel 60 (0.04–0.06 mm). Mass spectra were recorded in positive
ion mode using electro-spray ionization (ESI) (Applied Biosystem, Q-Trap 2000 LC/MS) or high-
resolution LC-MS (IT TOF: Waters-Micromass QTOF premier mass spectrometer). Synthetic
protocols and spectral data of synthesized intermediates / target compounds are provided in
Supplementary Information (Sections 1-9). Purity of final compounds were determined by
reverse phase 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) in absolute EtOH (20 mL) was refluxed for 12 h. On cooling to
room temperature (25oC), the solid product was filtered, washed with 50% aqueous EtOH, and
1
recrystallized from 95% EtOH to give 2 as an off-white solid: 12.71 g, yield: 88.0%. H NMR
(400MHz, DMSO-d6)
δ 11.23 (s, 1 H), 7.60(d, 1 H, J=2.0 Hz), 7.26 (d, 1 H, J=8.6 Hz), 7.14-
7.11(m, 1 H), 4.54 (s, 2 H), 4.08 (q, 2H, J=7.2 Hz), 3.72 (t, 2 H, J=5.6 Hz), 2.78 (t, 2 H, J=5.6
Hz),1.20 (t, 3H, J=7.2 Hz). 13C NMR (100MHz, DMSO-d6)
δ
155.09, 134.48, 134.34, 126.83,
122.95, 119.59, 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).
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To a mixture of 2 (2.39 g, 7.40 mmol) and sodium hydride (60 %, 740mg, 18.5 mmol, 2.5 equiv.)
was added dry DMF (10 mL) under N2 at 0°C. The reaction mixture was stirred at room
temperature for 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 the stirred mixture at 0°C. The reaction mixture was
stirred at room temperature (≈ 3h), poured into an ice-water and extracted with DCM (15 ×3
mL). The combined DCM layer was sequentially washed with water (20 ×3 mL) and brine, dried
(Na2SO4), filtered, and concentrated under reduced pressure and the residue was purified by
silica gel column chromatography (hexane : EtOAc 3:1) to give 3a as yellow oil (2.70 g, yield:
1
83.5%). H NMR (300MHz, CDCl3)
δ 7.57 (d, 1H, J=2.0 Hz), 7.25-7.22(m, 1H), 7.15-7.12(m,
1H), 4.65(br, 2H), 4.20(q, 2H, J=7.2Hz), 3.97 (t, 2H, J=7.2 Hz), 3.87(br, 2H), 2.80(br, 2H), 1.74-
1.67(m, 2H), 1.33-1.25(m, 13H), 0.87(t, 3H, J=6.6Hz). 13C NMR (75MHz, CDCl3)
δ
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 was added an aqueous solution of
KOH (4.28 g,76.4 mmol, in 5 mL water) and refluxed for 16 h. Ethanol was removed under
reduced pressure and the residual mixture was extracted with DCM (15 × 3 mL). The combined
DCM layer was sequentially washed with water (15 × 3 mL) and brine, dried (Na2SO4), filtered,
and concentrated under reduced pressure to give a brown residue which was purified by silica
gel column chromatography (DCM: methanol 25:1) to give 4a as a brown oil, 1.03g, yield:
74.5%. 1H NMR (400MHz, CDCl3)
δ 7.49 (d, 1 H, J=1.6 Hz), 7.21-7.18 (m, 1 H), 7.11 (d, 1 H,
J=8.8 Hz), 4.00 (br, 2 H), 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), 1.28-1.24 (m, 10 H), 0.87 (t, 3H, J=6.8Hz).13C NMR (100
MHz, CDCl3)
δ
134.69, 134.56, 127.12, 123.26, 120.15, 111.93, 110.33, 107.74, 43.17, 42.98,
14

ACCEPTED MANUSCRIPT
41.91, 31.68, 30.19, 29.20, 29.06, 26.94, 23.19, 22.51, 13.98. MS (ESI), [M+H]+, Calcd for
C19H28BrN2, 363.1; Found 363.3, 365.5.
4.5. 8-(4’-(Methylsulfonyl)phenyl)-5- n-octyl-2, 3, 4, 5-tetrahydro-1H-pyrido[4,3-b]indole
(18). To the mixture of 4a (200mg, 0.55 mmol), 4-(methanesulfonyl)phenylboronic acid
(132.6mg,0.66 mmol) and Pd(PPh3)4 (32.0mg, 0.028 mmol) in 4 mL 1,4-dioxane was added 0.5
mL aqueous solution 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, the solvent was evaporated and the resulting
residue was extracted with DCM (10 mL×3), the DCM layer was washed with brine, dried
(Na2SO4) and filtered. The residue obtained on removal of the solvent was purified by silica gel
column chromatography (DCM: methanol 20:1) to give the target compound. 18 was obtained
1
as an off-yellow oil (120 mg, yield: 49.6%). H NMR (400MHz, CDCl3)
δ 7.96(d, 2H, J=8.4Hz),
7.79(d, 2H, J=8.4Hz), 7.63(br, 1H), 7.43- 7.41(m, 1H), 7.37-7.35(m, 1H), 4.29(br, 2H), 4.02(t,
2H, J=7.2Hz), 3.44(br, 2H), 3.09(s, 3H), 2.96(br, 2H), 1.76-1.71(m, 2H), 1.31-1.25(br, 10H),
0.87(t, 3H, J=6.8Hz). 13C NMR (100MHz, CDCl3)
δ 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), [M+H]+, calcd for C26H35N2O2S,
439.2, found, 439.2. HRMS (ESI), [M+H]+, calcd 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-Glo™ Assay Kit (G6450, Promega,
Madison, WI, USA) following manufacturer’s instructions. Compounds were dissolved in DMSO
and tested over a 103 fold concentration range (final DMSO concentration 2.5 %). IC50 values
were determined in triplicate and from 2 or more independent experiments. Briefly, assays were
carried out on a white wall 384-well plate with human recombinant Sirt 1 (BML-SE239) and Sirt
2 (BML-SE251) enzymes from Enzo Life Sciences (NY, USA). The amount of enzyme used in
15

ACCEPTED MANUSCRIPT
the assay was determined by measuring the signal-to-noise ratio of serially diluted enzyme in
Sirt-Glo™ Buffer solution. The concentration of enzyme corresponding to the mid-range linear
portion of the signal to noise ratio versus concentration plot was selected and was 0.1 unit Sirt1
per well and 0.2 unit Sirt2 per well. Serially diluted inhibitor solutions (10 µL in Sirt-Glo™ Buffer
solution, with 1 µL of DMSO) were added to each well followed by the sirtuin enzyme (10 µL in
o
Sirt-Glo™ Buffer solution). The plate was agitated (400 rpm, 30 min, 25 C, Tecan Infinite 200
plate reader) after which 20 µL of Sirt-Glo™ Reagent solution was added per well, the contents
mixed by shaking (400 rpm) for another 30 min, 25oC, after which luminescence was read on
the plate reader. Enzyme activity (%) was measured by the following expression:
ꢂLumꢃꢄꢅꢆꢇꢅꢈꢉꢊꢋ − [Lum_ꢌꢍꢎꢉꢏ
]
ꢀ ꢁ
Enzyme Activity % =
× 100%
ꢂLum_ꢄꢅꢉꢐꢑꢅꢍꢋ − [Lum_ꢌꢍꢎꢉꢏ
]
where Lum_Compound = luminescence of wells containing enzyme and test compound in
vehicle (SIRT Glo™ Buffer solution), Lum_Control = luminescence of well containing enzyme
only in vehicle; and Lum_Blank = luminescence of well containing vehicle only. The IC50 of test
compound was determined from the sigmoidal curve obtained by plotting % enzyme activity
versus logarithmic concentration of test compound (GraphPad Prism, Version 5, San Diego,
USA). EX527 (Sigma LifeScience, MO, USA), AGK2 (Tocris Bioscience, Bristol, UK) and
nicotinamide (SIRT-Glo™ Assay Kit provided, Promega, Madison, WI, USA) were used as
positive controls. Representative dose response curves are presented in Supplementary
Information (Section 11).
4.7. In vitro assay for evaluating inhibition of protease and luciferase
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 with Sirt-Glo™ Buffer solution. 10 µL of the diluted
solution was added to each well in a white-wall 384 well plate followed by 10 µL of Sirt-Glo™
16

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Buffer solution and 20 µL of Sirt-Glo™ Reagent solution containing 1 µM SIRT-Glo™ Control
Substrate (non-acetylated peptide, a gift from Promega, Madison, WI, USA). The final
concentration of test compound in the well was 10 µM and DMSO content per well was 2.5%
v/v. The contents of the wells were mixed by shaking (400 rpm) for 30 min, 25oC, after which
luminescence was read on the plate reader. Luminescence (%) was measured by the following
expression:
ꢂLumꢃꢄꢅꢆꢇꢅꢈꢉꢊꢋ − [Lum_ꢌꢍꢎꢉꢏ
]
ꢀ ꢁ
Luminescence % =
× 100%
ꢂLum_ꢄꢅꢉꢐꢑꢅꢍꢋ − [Lum_ꢌꢍꢎꢉꢏ
]
where Lum_Compound = luminescence of wells containing non-acetylated peptide, Sirt-Glo™
Reagent and test compound in vehicle (SIRT Glo™ Buffer solution), Lum_Control =
luminescence of wells containing non-acetylated peptide, Sirt-Glo™ Reagent in vehicle Sirt-
Glo™ Reagent and Lum_Blank = luminescence of well containing vehicle only. Results are
given in Supplementary Information, Section 12.
4.8. Kinetic study of Sirt2 inhibition by 18
The kinetics of Sirt2 inhibition by 18 was investigated using a deconstructed Sirt-Glo™ Kinetics
Assay Kit which was a gift from Promega (Madison, WI, USA). Human recombinant SIRT2
(BML-SE251, Enzo Life Sciences, NY, USA) (0.2 unit/well in 5 ꢀL of assay buffer) was aliquoted
into each well of a 384-well plate which contained test compound (18) at 0, 1.1, 3.3 or 10 µM in
L of assay buffer per well. The contents of the plate were incubated for 60 min at 25oC with
5
ꢀ
agitation (400 rpm). Final concentration of DMSO per well was kept at 2.5 %. At the same time,
the Luciferin Detection Reagent (LDR) solution was prepared by combining the acetylated
substrate (Z-QPK(Me)2K(Ac)-aminoluciferin) (4.12, 12.4, 37.0, 111.1 or 333.3
ꢀM), NAD+ (1
mM) and developer reagent. The mixture was incubated for 60 min at 37oC, after which aliquots
(10 ꢀL) were dispensed 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
17

ACCEPTED MANUSCRIPT
taken. The data from these experiments were used to obtain the double-reciprocal plot from
which Km of Sirt2 for acetylated substrate was obtained. The slope and intercept of the lines
obtained in presence of 18 were replotted against concentration of 18 according to Equation 1
to give apparent Ki of 18 (Supplementary Information, Section 13, Figure S2). The Km of Sirt2
for acetylated peptide was also obtained from Eadie–Hofstee plot (Supplementary Information,
Section 13, Figure S3) which is based on Equation 2 and involved plotting reaction rate against
rate / (substrate concentration).
Equation 1: Slope = (Km/ Vmax) + (Km/ Vmax) ( [I] / Ki)
Equation 2: Rate = - Km ( Rate/ [Subtrate]) + Vmax
The above procedure was repeated at a fixed concentration of the acetylated substrate (38.5
ꢀ
M) and varying amounts of NAD+ (185, 556, 1667, 5000 ꢀM). The resulting double reciprocal
plot gave the Km of Sirt2 for NAD+ and the secondary plots gave apparent Ki of 18 with NAD+
was 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 for
acetylated substrate and NAD+ were found to be 38.0±2.0 µM and 1317±208 µM respectively.
Km values from Eadie-Hofstee plots of the same data were comparable at 29.9±1.6µM
(acetylated substrate) and 1298 ± 218 µM (NAD+) (Supplementary Information, Section 13,
Figure S3).
4.9. Molecular Docking
The human Sirt2 enzyme was retrieved from the RCSB protein data bank (PDB 3ZGV) [12].
Water molecules were removed and the monomeric enzyme was processed for docking using
LigX in Molecular Operating Environment (MOE, version 2011, Chemical Computing Group,
Montreal, Canada). The structures of the test compounds were separately prepared for docking
on MOE. Docking was carried out on GOLD v 5.2 (Cambridge Crystallographic Data Centre
Software Ltd, Cambridge, UK) with default genetic algorithm settings. The binding pocket was
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defined by the atoms within 10 Å radius of the co-crystallized ligand (ADP-ribose). Docking was
carried out without the reference ligand. GOLD uses a genetic algorithm for docking flexible
ligands into the binding pocket to explore the full range of ligand conformational flexibility [22].
The GOLD Score was used as the fitness function for selection of the best docked
conformations of test compounds in the binding pocket. For each molecule, the top 5 docked
conformations were retained and visualized 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 potassium phosphate buffer (5mM, pH 7.4, prefiltered before use) to
give final concentrations of 1 µM and 10 µM. Final concentration of DMSO was 1% v/v.
Measurements were carried out on the Malvern Instrument Zetasizer Nano ZS system equipped
with a 4mW He-Ne laser at 633nm and detector angle of 90o. Three or more determinations of
derived count rates (kilocounts per second, kcps) were obtained from each concentration of test
compound, using two separately prepared stock solutions. Data collection was carried out using
the software supplied with the instrument. Results are represented as mean ± standard
deviation. The positive control was benzyl benzoate which gave a count rate of 1148kcps ±37
(250 µM) and 76 ± 5 (25 µM). The vehicle (phosphate buffer, 1% DMSO) gave a reading of 15.5
± 0.4. The results are presented in Supplementary Information, Section 14.
4.11. Determination of cell growth inhibition
Human breast cancer MDA-MB-231, hepatocellular carcinoma HepG2 and human lung
fibroblast IMR90 cells were purchased from ATCC (Rockville, MD). All cells were grown in
DMEM (Invitrogen, High Glucose.) at 37oC, 5% CO2. DMEM was supplemented with 10% fetal
bovine serum (Hyclone, heat treated at 65 o C for 30 min before use), 50 units/ L penicillin
(Gibco) and 50 µg/mL streptomycin (Gibco). Cells were subcultured at 85-90% confluency and
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used within 15 passages. Cell viability was assessed using CellTitre 96 Aqueous One Solution
(Promega, Madison, WI) containing the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Seeding densities were 2 500
cells/well in the 96-well plates. Cells were grown in media (10% FBS) in 96-well plates for 24 h,
at 37oC, 5% CO2 after which aliquots of 18 (stock solution in DMSO, diluted with media
containing 10% FBS) were added to each well and the plates were incubated for 72 h, at 37oC,
5% CO2. Final concentration of DMSO per well was 0.5% v/v. At the end of the incubation
period, 10 µL of the MTS solution was added to each well, the plates were incubated for 4 h in
the dark after which absorbance readings were read at 490 nm (Tecan Infinite M200 Microplate
reader). Cell viability was determined from the expression:
Absorbance cells + cpd − Absorbance
Cell viabilityꢀ%ꢁ =
cpd × 100%
Absorbance cells + vc − Absorbance
vc
Where Absorbance cells + cpd = absorbance of wells containing cells and test compound in vehicle
(media + 0.5% DMSO), Absorbance = absorbance of wells containing cells in vehicle
cells + vc
(vc) only; Absorbance vc = absorbance of wells containing vehicle; Absorbance cpd = absorbance
of wells containing test compound. The % viability readings were plotted against log
concentration on GraphPad Prism (Version 5.0, San Diego, CA) to give a sigmoidal curve from
which IC50 (concentration required to reduce viability by 50% compared to control/untreated
cells) was obtained. The plot was constrained to
≥0 and ≤100%. At least 3 separate
determinations of IC50 were determined. Representative dose response curves are presented in
Supplementary Information, Section 15.
4.12. Western Blot
Rabbit monoclonal antibodies to PARP and caspase 3, mouse monoclonal antibodies to p53,
goat anti-rabbit-horse radish peroxidase (HRP) conjugate and goat anti-mouse HRP were from
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Santa Cruz Biotechnology (CA, USA). Rabbit monoclonal antibodies to cleaved caspase 3 and
acetylated p53 (K382) were purchased from Cell Signaling (MA, USA). Mouse monoclonal
antibodies to acetylated
α
-tubulin and
α
-tubulin were from Sigma Aldrich (Sigma Lifescience,
-actin were purchased from Invitrogen Life
MO, USA). Mouse monoclonal antibodies to
β
Technologies (CA, USA). HepG2 and MDA-MB-231 cells were seeded at a cell density of in a
10 cm petri dish at 50 000 cells/mL (total 10 mL in DMEM, high glucose, Invitrogen) containing a
known concentration of test compound. Final concentration of DMSO in the flask was
maintained at 0.5% v/v. Treated cells were incubated at 37 ˚C in a humidified 5 % CO2
atmosphere for 6h (for HepG2 cells) or 24 h (for MDA-MB-231 cells) after which they were
transferred to a 15 mL Falcon tube, pelleted at 500 g (5 min) and washed twice with ice-cold 1x
PBS. The cells were transferred to a microcentrifuge tube containing 70 ꢀL 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) and incubated at 0oC for at least 20 min.
After the incubation, the lysates were spun at 13 300 g for 10 min to remove cell debris. An
aliquot (3
Laboratories Inc, CA, USA) while the remaining supernatant was diluted with 4×SDS solution
(0.2 M Tris pH 6.8, 0.28 M SDS, 40 % v/v glycerol, 0.59 M -mercaptoethanol, 50 mM EDTA,
ꢀL) was retained for protein determination (Bradford Protein Assay Kit, Bio-Rad
β
1.1 mM bromophenol blue) to give 1×SDS solution which was then deactivated at 100 oC, 5 min
and subsequently stored for no more than 1 week at -80 oC. Cell lysates were separated on the
SDS-PAGE Bio-Rad Mini-Protean II system (Bio-Rad Laboratories Inc, CA, USA). Membranes
o
were blocked with blocking buffer for at least 60 min at 25 C and then incubated with primary
antibodies at appropriate dilutions in 2.5% BSA in TBST (50 mM Tris, 150 mM NaCl, 0.1%
Tween 20) solution overnight (4oC). Membranes were than washed thrice with TBST (10 min
per wash), incubated for 1 h with secondary antibodies in TBST at 25oC followed by washing in
TBST (thrice, 10 min per wash). The immunoreactive bands were detected by the ECL reagent
(GE Healthcare, Little Chalfont, UK) using Bio-Rad Universal Hood II Gel Doc. If needed, the
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membranes were stripped with stripping buffer, blocked with blocking buffer for 30 min 25 oC,
and re-probed with other antibodies. Western blotting for each compound at stated
concentrations were repeated at least 3 times
4.13 Determination of solubility
The solubilities of 10, 12, 18, 19, 20 and 24 were determined in phosphate buffer pH 7.4 by a
turbimetric method [16]. Briefly, stock solutions of test compound were prepared in DMSO and
aliquots were added to a 96-well plate containing phosphate buffer in each well to give a range
of concentrations (1-50 µM). DMSO concentration was kept at 1% v/v. After shaking the plate
for 30 min at 25oC, absorbance readings were taken at 620 nm. Absorbance readings were
plotted against concentration. When the concentration exceeded compound solubility, the
occurrence of precipitation led to an increase in absorbance. Solubility was then assigned to the
range of concentrations where a two-fold or greater increase in absorbance was observed from
the absorbance-concentration plot. Results were obtained from 3 separate determinations.
5. Acknowledgements
This work was supported by grants from the Biomedical Research Council (10/1/21/19/664) and
Ministry of Health Faculty Research Grant (R148000171112) to M.L.G. We thank Promega for
providing the SIRT-Glo™ Kinetics Assay Kit and SIRT-Glo™ Control Substrate, and Dr. Andrew
Niles for helpful advice and discussion on the assay details.
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Tables :
Table 1: Structures of synthesized compounds and their in vitro inhibitory activities on Sirt1
and Sirt2.
R1
N
R3
N
R2
Sirt 1
Sirt 2
% Inhibition
Cpd
R1
R2
% Inhibition
(10 µM) a
IC50 (µM) a
(10 µM) a
IC50 (µM) a
Series A :
R3 =
7
8
-CO-O-C2H5
H
C8H17-n
C8H17-n
C8H17-n
C8H17-n
C8H17-n
Nil b
5.1±1.0
< 5
Not done
20 ± 11
>100
11.2±3.9
43.1±3.2
57.5±3.6
64.2±2.4
Not done
10.1 ± 0.8
8.2 ± 0.1
6.2 ± 0.2
Not done
9
10
11
-CH3
66.4±2.5
Nil b
21 ± 1
Not done 19.7±13.8
12
13
14
15
16
-CH(CH3)2
C8H17-n
C8H17-n
H
2.4±1.9
Nil b
26 ± 4
>100
56.3±4.3
49.1± 9.5
Nil b
11.2 ± 0.7
7.8 ± 0.7
100 ± 11
83 ± 4
-CONH2
H
-CH3
H
Not done
Not done
Nil b
Not done
Not done
Not done
H
Nil b
C4H9-n
Nil b
96 ± 1
1

ACCEPTED MANUSCRIPT
Nil b
Not done 38.1±11.4
Not done
17
Series B :
R3 =
18
19
20
21
22
H
-CH3
-CONH2
-CH3
H
C8H17-n
C8H17-n
C8H17-n
H
48.5±2.4
12.8±3.9
51.8 ±11.4
Not done
Nil b
16.7 ±0.4
>250
82.5±3.3
79.1±7.4
76.5±4.4
Nil b
3.8 ± 0.3
6.0 ± 0.4
4.0 ± 0.2
>250
28 ± 3
Not done
Not done
C4H9-n
Nil b
20 ± 1
Series C : R3 =
23
24
25
26
27
28
29
30
31
-CO-O-C2H5
H
C8H17-n
Nil b
2.4±1.1
Nil b
Not done
38 ± 2
23.0±3.3
70.0±8.8
34.0±1.5
61.9±11.4
29.1±1.6
62.3±7.9
35.0±3.5
31.1±2.4
Not done
11.8 ± 1.8
14.0 ± 0.8
17.9 ± 2.2
Not done
20 ± 6
C8H17-n
C8H17-n
C8H17-n
C8H17-n
C8H17-n
C8H17-n
C8H17-n
H
-CONH2
-CH3
63 ± 4
Nil b
>100
-C2H5
Nil b
Not done
56 ± 2
-CH(CH3)2
-SO2CH3
-C2H4OH
-CH3
15.3±7.6
Nil b
Not done
Not done
Not done
Not done
>250
Nil b
Not done
Not done Nil b
2

ACCEPTED MANUSCRIPT
Nil b
Not done Nil b
>100
32
H
C4H9-n
Series D : R3 =
33
34
H
C8H17-n
1.1 ±0.8
Nil b
Not done
Not done
27.7±3.3
18.5±8.4
Not done
Not done
-CONH2
C8H17-n
Series E : R3 = F
Nil b
Nil b
Not done
17.4 ± 4.9
Nil b
Not done
Not done
35
36
-CH3
C8H17-n
C8H17-n
Not done
AGK2 c
Not done
0.33±0.03
149 ± 12
13.9 ± 1.0
Not done
6.3± 1.4
EX527d
Nicotinamide e
a Mean ± SD for n = 3 separate determinations. b No inhibition was observed at 10 µM test
c
compound. Selective Sirt2 inhibitor.[15]. d Selective Sirt 1 inhibitor [10]. e Sirtuin 1 and 2
inhibitor [10].
Table 2: Solubilities of selected compounds as determined by turbimetry at pH 7.4, 25oC
Series
A
Compound
Solubility a
3.1 -6.3 µM
Series
B
Compound
Solubility a
3.1 -6.3 µM
10
19
A
B
12
18
1.6 – 3.1µM
3.1 -6.3 µM
B
C
20
24
1.6 – 3.1µM
6.3-12.5 µM
a Mean ± SD for at least n = 3 separate determinations.
3