Hit-to-lead optimization on aryloxybenzamide derivative virtual screening hit against SIRT

Article abstract of DOI:10.1016/j.bmc.2020.115961

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD⁺)-dependent protein histone deacetylases (HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD⁺ as a cosubstrate. Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory effect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 inhibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01, ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this study provides structural insights for further inhibitor improvement.

Full text of DOI:10.1016/j.bmc.2020.115961

Bioorg. Med. Chem. 30 (2021) 115961
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Hit-to-lead optimization on aryloxybenzamide derivative virtual screening
hit against SIRT
,
,
Semih Yagcia 1, Mahmut Gozellea 1, Selen Gozde Kayaa, Yesim Ozkanb, Ahmet Bugra Aksela,
Filiz Bakar-Atesc, Yasemin Dundara, Gokcen Erena
,
*
a SIRTeam Group, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
c Department of Biochemistry, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein histone deacetylases
(HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the
reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD+ as a cosubstrate.
Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various
diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-
based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory ef-
fect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was
explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have
been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By
the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for
ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed
for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 in-
hibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01,
ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line
to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on
the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with
their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this
study provides structural insights for further inhibitor improvement.
Keywords:
SIRT1
SIRT2
Aryloxybenzamide
Hit-to-lead
Pharmacophore
Molecular docking
1. Introduction
are the best studied members of sirtuin family at present, and several
SIRT1/2 inhibitors have been described, including nicotinamide, spli-
Sirtuins (SIRTs) as nicotinamide adenine dinucleotide (NAD+)-
dependent histone deacetylases (HDACs) are known to catalyze the
reversible deacetylation of acetylated lysine residues resulting in the
formation of deacetylated protein substrate, O-acetyl-ADP-ribose, and
free nicotinamide, the endogenous inhibitor of the SIRTs.1 Mammalian
SIRTs consist of seven isoforms (SIRT1-7), which show different sub-
cellular localizations and enzymatic functions.2 The SIRT enzymes have
been engaged in various pathologies such as cancer, neurodegeneration,
diabetes, inflammation, cardiovascular diseases and therefore, the
modulation of SIRTs is of therapeutic importance.3–12 SIRT1 and SIRT2
tomicin and its analogues, sirtinol, tenovin, suramin and its analogues,
cambinol, salermide, toxoflavin, thiobarbiturates, and other com-
pounds.1,13–19 Despite the synthetic efforts to develop potent inhibitors,
none has been approved for the market.
In our previous study20, we performed a multi-step pharmacophore-
based virtual screening campaign to discover new SIRT2 inihibitors. (S-
3-[(6-methylpyridazin-3-yl)oxy]-N-(1-phenylethyl)benzamide
(1),
which affected the levels of SIRT1/2 activity, was one of the hits ob-
tained through this campaign (Fig. 1). Further hit-to-lead optimization
studies were then performed on this compound to enhance the SIRT
* Corresponding author.
E-mail address: gokcene@gazi.edu.tr (G. Eren).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2020.115961
Received 11 September 2020; Received in revised form 27 November 2020; Accepted 16 December 2020
Available online 25 December 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
inhibitory potency.
again, the problem of reversed binding mode by the introduction of the
cyclic amines was overcome by replacement of methylpyridazine with
phenyl or pyrimidine rings allowing the cyclic amide moiety to prefer-
entially direct towards the selectivity pocket.
The structural modifications of 1 were focused on the regions A-D as
depicted in Fig. 2. The design of aryloxybenzamide derivatives was
guided by the binding modes in SIRT1 and SIRT2 active sites achieved
from molecular docking studies. At the end, fourteen aryloxybenzamide
derivatives (ST01-14), ten of which were new, have been synthesized
and evaluated for their activity as SIRT inhibitors. The compounds
which exhibited SIRT1/2 inhibitory potency were further tested for their
in vitro cytotoxic effects against MCF-7 human breast cancer cell line.
We aimed to retain the aryloxybenzamide scaffold, but the substi-
tution pattern was modified for some derivatives by shifting the amide
moiety from meta to para position at the central phenyl ring represented
by region C in Fig. 2.
Finally, based on the biological evaluation results of the compounds
designed up to this stage, benzamide scaffold was modified to N-
hydroxybenzimidamide bearing a further hydrophilic function to pro-
vide all the expected interactions with key residues in SIRT2 active site
as well as to improve water solubility. According to molecular docking
studies the only compound showing a significant improvement in SIRT2
binding after this modification, thus promising to display higher SIRT2
inhibition compared to corresponding analogue was prepared and sub-
jected to biological evaluation.
2. Results and discussion
2.1. Design
The hit-to-lead optimization phase of the virtual screening hit (1)
was mainly carried out through structure-based drug design approaches
such as molecular docking and pharmacophore modeling for better
binding to the enzyme active site.21 The influence of the chemical
modifications considering the feasibility of organically synthesizing and
reasonable physicochemical properties has been evaluated.
2.2. Chemistry
Our initial effort was based on removing the chirality to facilitate
synthesis of target compounds. The 1-/2-naphthyl, 4-acetylphenyl, and
4-carbamoylphenyl rings, which allow to eliminate the chirality were
introduced instead of 1-phenylethyl group denoted by region A while
keeping other regions fixed.
The synthetic route for the preparation of compounds ST01-14 is
outlined in Scheme 1. The coupling reaction of commercially available
bromobenzene (1a) or 4-bromoanisole (1b) with ethyl 3-/4-hydrox-
ybenzoate catalyzed by CuI and N,N-dimethylglycine gave the ethyl
aryloxybenzoates (2a, 2b, 2d) in 48–85% yields, while 2-bromopyrimi-
dine (1c) was coupled with ethyl 3-/4-hydroxybenzoate using Pd(dba)2
and t-BuXPhos to generate the corresponding ethyl aryloxybenzoates
(2c, 2e) in 79–81% yields. Further basic hydrolysis of compounds 2a-e
led to the aryloxybenzoic acid derivatives 3a-e, respectively (Scheme
1A). The resulting compounds 3a-e were subsequently reacted with
appropriate amines in the presence of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) to
give the desired compounds ST01-13 in 40–92% yields (Scheme 1B).
The reaction between commercially available 3-phenoxybenzaldehyde
and NH2OH.HCl produced the crude 3-phenoxybenzaldehyde oxime
intermediate followed by treating with N-chlorosuccinimide (NCS)
without further purification to obtain 3-phenoxybenzohydroximinoyl
chloride intermediate. The compound ST14 was obtained by the
condensation of commercially available 1,2,3,4-tetrahydroisoquinoline
and 3-phenoxybenzohydroximinoyl chloride intermediate in the pres-
ence of triethylamine (TEA) in 72% yield (Scheme 1C).
The methylpyridazine moiety of the new compounds obtained by
introducting these groups, shifted to the selectivity pocket of SIRT2
active site, in contrast to this moiety of the hit compound occupied the
active site entrance. The H-bonding between carbonyl oxygen of the
amide and the conserved water HOH549 which was reported as crucial
for ligand affinity in SIRT222 as well as the matching to H-bond acceptor
feature of pharmacophore model generated previously20 were lost since
the new compounds adopted a reversed binding mode. A similar situa-
tion was observed for also SIRT1 binding. The reversed binding
conformation also prevented carbonyl oxygen of the amide from H-
bonding with Asn346 which was reported for SIRT1 inhibition.23
Thereupon, the methylpyridazine ring denoted by region D, was
replaced with phenyl ring and the desired binding conformation was
thus achieved. In some derivatives whose LogP values increase with the
introduction of the phenyl ring in region D, methylpyridazine was
replaced with pyrimidine ring in order to decrease the lipophilicity.
The another type of modification performed on the hit compound 1
was focused on region B yielding cyclic amide moieties with the purpose
of searching new fragments for the SIRT2 selectivity pocket and as well
as eliminating the chirality. The cyclic amide moieties were derived
from 1,2,3,4-tetrahydroisoquinoline, 4-phenylpiperazine, and 4-isopro-
pylpiperazine groups with the potential to form key interactions in
SIRT2 selectivity pocket according to molecular docking results. Here
2.3. Biological evaluation
The synthesized compounds were evaluated in a fluorescence-based
assay24 for their inhibitory activity against SIRT1-3 at an inhibitor
concentration of 100 μM. Chemical structures and bioactivities of the
compounds tested are summarized in Table 1.
Fig. 1. Chemical structure of 1 and the relative fold change in SIRT activity in the presence of 1 and the reference compounds (NAM: Nicotinamide, Suramin, EX-
527) tested at 300 M.
μ
2

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Fig. 2. Structural modifications performed on the hit compound 1.
Scheme 1. Synthesis of compounds ST01-14. Reagents and conditions: (a) 2a, 2b, 2d: CuI, Cs2CO3, N,N-dimethylglycine, 1,4-dioxane, DMF, 100 ◦C, overnight; (b)
2c, 2e: Pd(dba)2, t-BuXPhos, K3PO4, toluen, reflux, 6–8 h; (c) i. NaOH, H2O, THF, EtOH, reflux, 1–2 h, ii. conc. HCl; (d) appropriate amine, EDC, DMAP, DCM, rt,
overnight; (e) i. NH2OH.HCl, NaOH, H2O, EtOH, rt, 1 h, ii. NCS, DMF, rt, 4 h; (f) 1,2,3,4-tetrahydroisoquinoline, TEA, DCM, 0 ◦C → rt, 1 h.
The compounds obtained by the replacement of 1-phenylethyl moi-
improvement in potency and isoform selectivity against SIRT1 with
respect to the hit compound 1. While ST10 displayed low but selective
inhibitory effect on SIRT1, for ST05 with corresponding substituents at
ety of 1 with 4-acetylphenyl (ST10) and 4-carbamoylphenyl (ST11),
which have phenyl ring at 4-position exhibited
a substantial
3

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Table 1
Inhibitory activities of compounds ST01-14, the hit compound 1 and the reference compounds against SIRT1-3 enzymes.
ID
Type
R1
X
Po
R2
Inhibition (%)a,b,c
SIRT1
SIRT2
SIRT3
ST01
ST02
ST03
ST04
ST05
ST06
ST07
ST08
ST09
ST10
ST11
ST12
ST13
ST14
A
A
A
A
B
A
B
B
A
B
B
A
B
H
CH
CH
CH
CH
CH
N
3
3
3
3
3
3
3
3
4
4
4
4
4
3,4-dihydroisoquinolin-2(1H)-yl
4-isopropylpiperazine-1-yl
4-phenylpiperazine-1-yl
4-phenylpiperazine-1-yl
4-(COCH3)phenyl
56.53 ± 3.84
48.15 ± 3.68
n.d.
10.80 ± 1.62
n.d.
H
n.d.
n.d.
H
n.d.
n.d.
OCH3
H
n.d.
n.d.
n.d.
n.d.
11.06 ± 0.91
5.79 ± 1.16
n.d.
H
3,4-dihydroisoquinolin-2(1H)-yl
1-naphthyl
n.d.
n.d.
H
N
n.d.
n.d.
n.d.
H
N
2-naphthyl
36.08 ± 2.74
21.50 ± 5.13
36.12 ± 1.01
46.04 ± 7.31
n.d.
17.12 ± 2.05
n.d.
H
CH
CH
CH
N
3,4-dihydroisoquinolin-2(1H)-yl
4-(COCH3)phenyl
n.d.
n.d.
H
n.d.
n.d.
H
4-(CONH2)phenyl
n.d.
n.d.
H
4-isopropylpiperazine-1-yl
1-naphthyl
n.d.
n.d.
H
N
n.d.
n.d.
n.d.
47.16 ± 1.49
51.61 ± 0.14
n.d.
1d
31.45 ± 1.73
42.47 ± 1.42
18.23 ± 2.61
EX-527b
97.56 ± 2.89
94.52 ± 0.92
76.39 ± 2.49
98.37 ± 0.25
44.61 ± 3.81
96.82 ± 3.26
Suramineb
a
SD, standard deviation (n = 3).
b
c
Inhibition at 100
μM concentration.
n.d.: not determined.
d
Inhibition at 300 μM concentration.
3-position no significant inhibition was detected.
41.05
μ
M) and 72 h (IC50 = 33.12
μ
M) of treatment compared with the
The compounds bearing 1-naphthyl and pyrimidine (ST07, ST13)
instead of 1-phenylethyl and methylpyridazine moieties in the hit
compound were found to be inactive against all SIRT isoforms tested.
However, the corresponding 2-naphthyl analogue (ST08) showed
inhibitory effect on SIRT1 and SIRT2, even if it was slightly poor.
The introduction of 1,2,3,4-tetrahydroisoquinoline and 4-isopropyl-
piperazine, thus changing a secondary with tertiary and cyclic amide led
to increased selective inhibition on SIRT1 in case of the compounds
ST01, ST02 containing phenoxy at 3-position compared to the hit
compound while the corresponding analogues ST06 and ST12 bearing
pyrimidine ring at 3- and 4-position, respectively, were found to be
inactive against SIRT1 and SIRT2. Moreover, the compounds substituted
with 4-phenylylpiperazine moiety (ST03, ST04) displayed no inhibitory
effect on the SIRT isoforms evaluated.
hit compound 1 (IC50 values 327.76
μ
M and > 1 mM at 48 and 72 h,
respectively). ST14 was found to inhibit cell viability at concentrations
where it effectively inhibited SIRT1/2 enzyme activities. This was also
observed for ST01 which displayed a better inhibition profile (IC50
values 159.32 μM for 48 h; 146.54 μM for 72 h) than the hit compound,
though not as potent as ST14. Comparing the inhibition rates of ST01
and ST14 on cell viability, it might be suggested that the presence of
SIRT2 inhibition enhanced the cytotoxic activity since both compounds
have similar SIRT1 inhibition value. In case of ST02 and ST11 exhibiting
a moderate selective SIRT1 inhibition, much lower cytotoxic activity
was observed which was similar to that of the hit compound after 48 h of
treatment (IC50 values 464.94
μM for ST02; 523.84 μM for ST11), but
slightly greater than the hit compound at 72 h (IC50 values 320.98
μM
for ST02; 356.18 μM for ST11). When the selective SIRT1 inhibitors
The compound ST14 which was derived from ST01, exhibited a
significant improvement in potency against SIRT1 and SIRT2 compared
to hit compound 1 and by the modification of amide functional group of
ST01 to N-hydroxyimidamide moiety, a significantly increased SIRT2
inhibition was achieved as expected, while retaining the SIRT1 inhibi-
tory activity.
ST01, ST02, and ST11 were examined, a correlation was observed be-
tween cell viability and SIRT1 inhibition values.
2.4. Molecular modeling
Binding patterns of ST01, ST02, ST11, and ST14 in the active sites of
SIRT1 (Fig. 4) and SIRT2 (Fig. 5) were investigated compared to co-
crystallized ligands and the hit compound by the Glide module imple-
To explore the isoform selectivity, the synthesized compounds were
also tested for their inhibitory activity against SIRT3 and no inhibition
was observed in any of the compounds.
¨
mented in Schrodinger Small-Molecule Drug Discovery Suite. Molecular
Given the fact that the higher expression pattern of SIRT1 signifi-
cantly linked to increased invasiveness and metastasis of breast cancer
cells,25,26 while several pharmacological studies were reported demos-
trating anticancer activity of the selective SIRT2 inhibitors against
breast cancer cell lines,27–29 the inhibition on MCF-7 cell viability was
evaluated for ST01, ST02, ST11, and ST14 in a dose dependent manner
for assayed time periods (Fig. 3).
docking studies in conjunction with the bioactivity results illustrated the
importance of key interaction in SIRT1 and SIRT2 active sites.
As depicted in Fig. 4A, the co-crystallized EX-527 analogue (PDB:
4I5I) was clamped in a hydrophobic cage of SIRT1 by π-π stacking with
Phe273, Phe297 and H-bonding with Ala262, Ile270, Gln345, and
Asp348,23 while the hit compound 1 occupied the same region by
interacting with only Phe297. The compounds ST01, ST02, ST11, and
ST14 showing inhibition against SIRT1 with inhibition% values ranging
According to the results, ST14 significantly reduced the viability of
MCF-7 cells in a concentration dependent manner after 48 h (IC50
=
from 46.04 to 56.53 at 100 μM inhibitor concentration, exhibited a
4

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Fig. 3. The cell viability% in MCF-7 cell line treated with different concentrations of ST01, ST02, ST11, and ST14 for 48 h and 72 h. Bars represent means ± SD.
Each experiment was done in triplicate. Significant differences are presented as *p < 0.05, #p < 0.0001.
similar binding conformation to the hit compound 1, albeit slightly
bended. The replacement of methylpyridazine moiety in 1 that oriented
towards the entrance of the binding groove without any interaction, by
contacts with Tyr139 and Phe190, which was already observed for co-
crystallized SirReal inhibitor with 4,6-dimethylpyrimidine ring. In
addition, a H-bond was formed between Pro94 and its N-hydroxyl group
through the structural water HOH549. The fact that ST14 showed a
moderate SIRT2 inhibition even though it completely formed the in-
teractions observed for co-crystallized SirReal inhibitor might be
explained by the lower compliance with the pharmacophore model
compared to SirReal inhibitor. In case of ST01, the H-bond was formed
between carbonyl group of amide and structural water HOH549 forcing
the compound to get a bended conformation, thus resulting the lack of
crucial hydrophobic interactions. It was suggested that modification of
carbonyl oxygen to N-hydroxyl moiety in H-bond acceptor region
allowing the compound to be better oriented due to the introduction of
an additional atom towards water molecule, would probably improve
SIRT2 inhibitory activity.
phenyl ring led to π-cation contact with Arg282. The carbonyl oxygen of
amide of ST01 and ST02 mimicked the carboxamide of co-crystallized
EX-527 analogue interacting with Ile347 and Asp348 (observed for
only ST02) via H-bonding what was observed for ST14 with its N-hy-
droxyl group of imidamide. The extended conformation of aryl groups
adjacent to amide moiety in docked compounds allowed to be posi-
tioned favorably for mimicking 5-chlorophenyl of co-crystallized EX-
527 analogue which sterically inhibited catalytic placement of acety-
lated substrate. In case of ST11, the substitution of carboxamide group
to the phenyl ring adjacent to amide provided an additional H-bonding
with Val412 employing as an anchor with π-π contacts by Phe273 and
Phe297 in hydrophobic cage. The moderate inhibition value of the
compounds might be explained by the lack of critical H-bonding
network obtained by indole nitrogen and carboxamide group of co-
crystallized EX-527 analogue.
3. Conclusion
Among the compounds synthesized, SIRT2 inhibition was detected
To identify lead compounds with improved SIRT inhibition and
binding, a typical hit-to-lead optimization involving chemical modifi-
cations around the starting hit was employed with the guidance of
molecular docking and pharmacophore modeling. A series of arylox-
ybenzamide derivatives were synthesized as a result of optimization
approach. The inhibition assays on these synthesized derivatives against
SIRT1-3 led to the identification of promising selective SIRT1 inhibitors
for only ST14 with an inhibition% value of 51.61 at 100 μM concen-
tration, while ST01 from which ST14 was derivatized displayed no in-
hibition towards SIRT2. To get a better understanding of the relationship
between bioactivity and interaction pattern as well as the fit to the
pharmacophore model developed for SIRT2 inhibitors previously,20
ST01 and ST14 were docked into SIRT2 active site. Through molecular
docking analyses, we observed that, ST14 positioned in the extended C-
site to make good contacts with the key residues (Fig. 5D), which was
the same interaction pattern reported for co-crystallized SirReal inhib-
itor (PDB: 5DY4).22 The terminal phenyl ring of ST14 extended beyond
the substrate channel interacting with Phe119, Phe131, and Phe234.
ST01 (56.53% inhibition @100
M), and ST11 (46.04% inhibition @100
of the hit compound from 31.45% inhibition at 300
μ
M), ST02 (48.15% inhibition @100
M). The increase in the effect
M to 56.53% at
μ
μ
μ
100 μM is indicative of a successful optimization run. Moreover, further
optimization of ST01 based on crucial interactions for SIRT2 affinity
yielded ST14 with an improved SIRT2 inhibition (51.61% inhibition
The selectivity pocket was filled by its isoquinoline moiety forming π-π
5

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Fig. 4. Active site of SIRT1 (PDB: 4I5I). (A) Binding
conformation of EX-527 analogue in the x-ray struc-
ture. (B) Predicted binding conformation of the hit
compound 1 (XP Glidescore: ꢀ 6.789 kcal/mol). (C)
Predicted binding conformation of ST01 (XP Glide-
score: ꢀ 10.018 kcal/mol). (D) Predicted binding
conformation of ST02 (XP Glidescore: ꢀ 7.280 kcal/
mol). (E) Predicted binding conformation of ST11 (XP
Glidescore: ꢀ 7.785 kcal/mol). (F) Predicted binding
conformation of ST14 (XP Glidescore: ꢀ 8.650 kcal/
mol). H-bonds, aromatic H-bonds,
π-cation and π-π
stacking contacts are represented by yellow, red, or-
ange, and cyan dashed lines, respectively.
@100
μ
M). These compounds were also tested for their ability to inhibit
resolution mass spectra data (HRMS) were acquired on a Waters LCT
Premier XE Mass Spectrometer (high sensitivity orthogonal acceleration
time-of-flight instrument) operating in electrospray ionization (ESI)
mode, also coupled to an AQUITY Ultra Performance Liquid Chroma-
tography system (Waters Corporation, Milford, MA, USA) using a UV
detector monitoring at 254 nm. Purity for all target compounds were >
95%, according to the UPLC/MS method using (A) water + 0.1% Formic
Acid and (B) acetonitrile + 0.1% Formic Acid; flow rate = 0.3 ml/min,
Column: Aquity BEH C18 column (2.1 × 100 mm, 1.7 mm, Waters
Corporation, Milford, MA, USA). Melting points were determined by
Schmelzpunktbestimmer SMP-II Digital Melting Point Apparatus
(Schorpp Geraetetechnik, Überlingen, Germany) automatic melting
point apparatus without correction.
the cell viability against MCF-7 cell line in order to determine the
reflection of structural modifications and consequently improvement in
SIRT1/2 inhibition to their cytotoxic effects. The results were in
agreement with SIRT1/2 inhibitory profiles suggesting that improved
SIRT1 inhibition might be responsible for the observed cancer cell
growth inhibition; besides, the cytotoxicity was enhanced in the pres-
ence of SIRT2 inhibition. Taken together, this study will aid further ef-
forts to design and develop new potent and selective SIRT inhibitors.
4. Experimental
4.1. Synthesis
All chemicals were obtained through commercial purchase and used
without further purification. Reactions were monitored by thin layer
chromatography (TLC) on silica plated aluminum sheets (Silica gel 60
4.1.1. General procedure for the synthesis of ethyl aryloxybenzoates 2a-e
Method 130: A mixture of appropriate bromobenzene (2 equiv) and
ethyl hydroxybenzoate derivative (1 equiv) in 1,4-dioxane/DMF (8:2)
was stirred overnight at 100 ◦C under argon atmosphere in the presence
of CuI (0.3 equiv), N,N-dimethylglycine (1 equiv), and Cs2CO3 (2 equiv).
After the reaction completed, the cooled mixture was partitioned be-
tween ethyl acetate and water. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (10 ml × 3). The com-
bined organic layers were washed with brine (10 ml × 3), dried over
Na2SO4, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel with appropriate eluents.
F
254, Merck) with spots visualized by UV light (254 or 365 nm). Puri-
fication by flash column chromatography was performed on RediSep®
prepacked disposable silica gel columns using Teledyne Isco Combiflash.
1H NMR and 13C NMR spectra were recorded on a Varian Mercury 400
MHz High Performance Digital FT-NMR (Agilent Technologies, Santa
Clara, CA, USA) and a Bruker Avance Neo 500 MHz FT spectrometers
using tetramethylsilane as the internal standard. Chemical shifts were
given in δ (ppm). All coupling constants are reported as Hertz. High
6

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Fig. 5. Active site of SIRT2 (PDB: 5DY4). (A) Binding conformation of SirReal inhibitor in the x-ray structure. (B) Predicted binding conformation of the hit
compound 1 (XP Glidescore: ꢀ 9.032 kcal/mol). (C) Predicted binding conformation of ST01 (XP Glidescore: ꢀ 9.787 kcal/mol). (D) Predicted binding conformation
of ST14 (XP Glidescore: ꢀ 11.877 kcal/mol). H-bonds and π-π stacking contacts are represented by yellow and cyan dashed lines, respectively. The pharmacophore
model developed for SIRT2 inhibitors with four features: hollow rings R1, R2 and R3 are aromatic and sphere with vectors A1 is H-bond acceptor feature.
Method 231: A mixture of Pd(dba)2 (0.01 equiv), t-BuXPhos (0.015
equiv), and K3PO4 (2 equiv) in toluene was stirred at room temperature
for 15 min. Then, the solution of 2-bromopyrimidine (1c, 1 equiv) and
ethyl hydroxybenzoate derivative (1.2 equiv) in toluene was added. The
reaction mixture was refluxed for 6–8 h. After the reaction completed,
the cooled mixture was concentrated in vacuo, residue was diluted with
ethyl acetate and extracted with NaOH 5% aqueous solution (10 ml × 3)
and brine (10 ml × 3). The combined organic layers were dried over
Na2SO4, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel with appropriate eluents.
(498 mg, 3 mmol). The crude product was purified by column chro-
matography on silica gel, eluting with a mixture of ethyl acetate/pe-
troleum ether (1:30) to afford pure 2b as a colorless oil (394 mg, 48%
yield). CAS#: 773134–61-5. HRMS (m/z) calculated for C16H16O4 [M +
H]+ 373.1127, found 273.1137.
Ethyl 3-(pyrimidin-2-yloxy)benzoate 2c
The compound was synthesized according to the general procedure
Method 2 from 1c (795 mg, 5 mmol) and ethyl 3-hydroxybenzoate (997
mg, 6 mmol). The crude product was purified by column chromatog-
raphy on silica gel, eluting with a mixture of ethyl acetate/n-hexane
(20:80) to afford pure 2c as a yellowish oil (988 mg, 81% yield). CAS#:
1159822-51-1. 1H NMR (CDCl3, 400 MHz): δ 1.38 (3H, t, J = 7.2 Hz),
4.37 (2H, q, J = 7.2 Hz), 7.06 (1H, t, J = 4.8 Hz), 7.40 (1H, ddd, J = 8.0,
2.4, 1.2 Hz), 7.50 (1H, t, J = 8.0 Hz), 7.87 (1H, t, J = 2.2 Hz), 7.95 (1H,
dt, J = 8.0, 1.4 Hz), 8.56 (2H, d, J = 5.2 Hz) ppm. 13C NMR (CDCl3, 100
MHz): δ 14.28, 61.19, 116.45, 122.89, 126.25, 126.64, 129.60, 132.25,
152.83, 159.77, 165.16, 165.76 ppm. HRMS (m/z) calculated for
Ethyl 3-phenoxybenzoate 2a31
The compound was synthesized according to the general procedure
Method 1 from 1a (632 µl, 6 mmol) and ethyl 3-hydroxybenzoate (499
mg, 3 mmol). The crude product was purified by column chromatog-
raphy on silica gel, eluting with a mixture of ethyl acetate/petroleum
ether (1:30) to afford pure 2a as a yellowish oil (618 mg, 85% yield).
CAS#: 60677–14-7. 1H NMR (CDCl3, 400 MHz): δ 1.38 (3H, t, J = 7.2
Hz), 4.37 (2H, q, J = 7.2), 7.00–7.04 (2H, m), 7.11–7.16 (1H, m), 7.20
(1H, ddd, J = 8.4, 2.8, 1.2 Hz), 7.33–7.42 (3H, m), 7.68–7.70 (1H, m),
7.79 (1H, dt, J = 8.0, 1.2 Hz) ppm. 13C NMR (CDCl3, 100 MHz): δ 14.28,
61.15, 118.99, 119.72, 123.21, 123.66, 124.31, 129.68, 129.89, 132.32,
156.80, 157.36, 166.03 ppm. HRMS (m/z) calculated for C15H14O3 [M
+ H]+ 243.1021, found 243.1028.
C
13H12N2O3 [M + H]+ 245.0926, found 245.0933.
Ethyl 4-phenoxybenzoate 2d31
The compound was synthesized according to the general procedure
Method 1 from 1a (632 µl, 6 mmol) and ethyl 4-hydroxybenzoate (498
mg, 3 mmol). The crude product was purified by column chromatog-
raphy on silica gel, eluting with a mixture of ethyl acetate/petroleum
ether (1:30) to afford pure 2d as a yellowish oil (393 mg, 54% yield).
CAS#: 31994–68-0. HRMS (m/z) calculated for C15H14O3 [M + H]+
243.1021, found 243.1019.
Ethyl 3-(4-methoxyphenoxy)benzoate 2b
The compound was synthesized according to the general procedure
Method 1 from 1b (1122 mg, 6 mmol) and ethyl 3-hydroxybenzoate
7

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
Ethyl 4-(pyrimidin-2-yloxy)benzoate 2e
m), 7.03–7.20 (9H, m), 7.33–7.40 (3H, m) ppm. 13C NMR (CDCl3, 100
MHz): δ 29.55, 44.82, 45.27, 49.71, 116.67, 117.13, 119.40, 119.77,
121.32, 123.87, 125.89, 126.62, 128.66, 128.98, 129.92, 130.07,
132.83, 133.75, 137.74, 156.46, 157.67 ppm. HRMS (m/z) calculated
for C22H19NO2 [M + H]+ 330.1494, found 330.1494.
The compound was synthesized according to the general procedure
Method 2 from 1c (79 mg, 0.5 mmol) and ethyl 4-hydroxybenzoate (100
mg, 0.6 mmol). The crude product was purified by column chromatog-
raphy on silica gel, eluting with a mixture of ethyl acetate/n-hexane
(30:70) to afford pure 2e as a white solid (97 mg, 79% yield). CAS#:
1484643-26-6. Mp 86.8–87.5 ◦C. 1H NMR (CDCl3, 400 MHz): δ 1.39 (3H,
q, J = 7.2 Hz), 4.38 (2H, q, J = 7.2 Hz), 7.08 (1H, t, J = 4.8 Hz), 7.27
(2H, d, J = 8.8 Hz), 8.13 (2H, d, J = 8.4 Hz), 8.58 (2H, d, J = 5.2 Hz)
ppm. 13C NMR (CDCl3, 100 MHz): δ 14.34, 60.99, 116.66, 121.45,
127.64, 131.39, 156.50, 159.80, 164.90, 165.86 ppm. HRMS (m/z)
calculated for C13H12N2O3 [M + H]+ 245.0926, found 245.0920.
(4-Isopropylpiperazin-1-yl)(3-phenoxyphenyl)methanone ST02
The compound was synthesized according to the general procedure
from 3a (171 mg, 0.8 mmol) and 1-isopropylpiperazine (115 µl, 0.8
mmol). The crude product was purified by column chromatography on
silica gel, eluting with a mixture of MeOH/DCM (2:98) to afford pure
ST02 as a yellowish oil (190 mg, 73% yield). 1H NMR (CDCl3, 400 MHz):
δ 1.04 (6H, d, J = 6.4 Hz), 2.44 (2H, br s), 2.57 (2H, br s), 2.71–2.75 (1H,
m), 3.42 (2H, br s), 3.76 (2H, br s), 7.01–7.04 (4H, m), 7.10–7.15 (2H,
m), 7.35 (3H, t, J = 8.0 Hz) ppm. 13C NMR (CDCl3, 100 MHz): δ 18.27,
42.31, 47.89, 48.24, 49.00, 54.64, 117.01, 119.35, 119.55, 121.54,
123.83, 129.89, 129.96, 137.51, 156.46, 157.56, 169.35 ppm. HRMS
(m/z) calculated for C20H24N2O2 [M + H]+ 325.1916, found 325.1914.
(3-Phenoxyphenyl)(4-phenylpiperazin-1-yl)methanone ST03
4.1.2. General procedure for the synthesis of aryloxybenzoic acids 3a-e
To appropriate ethyl aryloxybenzoate (1 equiv.) was added 10 ml of
THF/EtOH (2:1) and 1 ml of NaOH 1 M solution. The solution was
refluxed for 1–2 h. The mixture was then acidified using conc. HCl to pH
6. The product was extracted with ethyl acetate (10 ml × 3), the com-
bined organic layers were dried over Na2SO4 and concentrated in vacuo
to yield aryloxybenzoic acids as a pure product.
The compound was synthesized according to the general procedure
from 3a (171 mg, 0.8 mmol) and 1-phenylpiperazine (122 µl, 0.8 mmol).
The crude product was purified by column chromatography on silica gel,
eluting with a mixture of ethyl acetate/n-hexane (40:60) to afford pure
3-Phenoxybenzoic acid 3a32
The compound was synthesized according to the general procedure
from 2a (606 mg, 2.5 mmol) as a white solid (503 mg, 94% yield). CAS#:
1
ST03 as a colorless oil (248 mg, 92% yield). CAS#: 863761-56-2. H
◦
3739-38-6. Mp 147.9–148.3 C. HRMS (m/z) calculated for C13H10O3
NMR (CDCl3, 400 MHz): δ 3.11 (2H, br s), 3.23 (2H, br s), 3.58 (2H, br s),
3.90 (2H, br s), 6.89–6.93 (3H, m), 7.03–7.08 (4H, m), 7.12–7.16 (2H,
m), 7.26–7.30 (2H, m), 7.34–7.40 (3H, m) ppm. 13C NMR (CDCl3, 100
MHz): δ 42.05, 47.45, 49.89, 116.75, 116.99, 119.40, 119.73, 120.67,
121.54, 123.92, 129.26, 129.93, 130.07, 137.27, 150.91, 156.41,
157.70, 169.58 ppm. HRMS (m/z) calculated for C23H22N2O2 [M + H]+
359.1760, found 359.1772.
[M + ACN + H]+ 256.0974, found 256.0973.
3-(4-Methoxyphenoxy)benzoic acid 3b30
The compound was synthesized according to the general procedure
from 2b (335 mg, 1.23 mmol) as a white solid (267 mg, 89% yield).
◦
CAS#: 117423-75-3. Mp 143.8–144.1 C. HRMS (m/z) calculated for
C
14H12O4 [M + ACN + H]+ 286.1079, found 286.1084.
3-(Pyrimidin-2-yloxy)benzoic acid 3c33
(3-(4-Methoxyphenoxy)phenyl)(4-phenylpiperazin-1-yl)methanone
ST04
The compound was synthesized according to the general procedure
from 2c (892 mg, 3.65 mmol) as a white solid (442 mg, 56% yield).
The compound was synthesized according to the general procedure
from 3b (171 mg, 0.7 mmol) and 1-phenylpiperazine (107 µl, 0.7 mmol).
The crude product was purified by flash chromatography on silica gel
(12 g), eluting with a gradient of 0–10% ethyl acetate in n-hexane to
afford pure ST04 as a yellowish oil (239 mg, 88% yield). 1H NMR
(CDCl3, 400 MHz): δ 3.10 (2H, br s), 3.23 (2H, br s), 3.57 (2H, br s), 3.80
(3H, s), 3.90 (2H, br s), 6.88–6.93 (5H, m), 6.97–7.01 (4H, m), 7.08 (1H,
d, J = 7.6 Hz), 7.26–7.30 (2H, m), 7.34 (1H, t, J = 8.0 Hz) ppm. 13C NMR
(CDCl3, 100 MHz): δ 42.12, 47.56, 49.81, 55.64, 115.02, 115.80,
116.74, 118.60, 120.65, 120.78, 121.21, 129.25, 129.93, 137.11,
149.31, 150.92, 156.32, 158.88, 169.69 ppm. HRMS (m/z) calculated
for C24H24N2O3 [M + H]+ 389.1865, found 389.1866.
◦
CAS#: 176034-07-4. Mp 180.4–181.0 C. HRMS (m/z) calculated for
C
11H8N2O3 [M + H]+ 217.0613, found 217.0612.
4-Phenoxybenzoic acid 3d34
The compound was synthesized according to the general procedure
from 2d (392 mg, 1.62 mmol) as a white solid (305 mg, 89% yield).
CAS#: 2215-77-2. Mp 162.7–163.2 ◦C. HRMS (m/z) calculated for
C
13H10O3 [M + ACN + H]+ 256.0974, found 256.0975.
4-(Pyrimidin-2-yloxy)benzoic acid 3e34
The compound was synthesized according to the general procedure
from 2e (782 mg, 3.2 mmol) as a white solid (436 mg, 63% yield). CAS#:
855423-33-5. Mp 167.3–167.6 ◦C. HRMS (m/z) calculated for
C
11H8N2O3 [M + H]+ 217.0613, found 217.0612.
N-(4-Acetylphenyl)-3-phenoxybenzamide ST0535
The compound was synthesized according to the general procedure
from 3a (107 mg, 0.5 mmol) and 4′-aminoacetophenone (68 mg, 0.5
mmol). The crude product was purified by crystallization from EtOH to
afford pure ST05 as a white solid (110 mg, 67% yield). CAS#: 733034-
83-8. Mp 155.0–155.4 ◦C. 1H NMR (DMSO‑d6, 400 MHz): δ 2.52 (3H, s),
7.04–7.07 (2H, m), 7.17 (1H, t, J = 7.4 Hz), 7.23 (1H, dd, J = 8.0 and
2.4 Hz), 7.39–7.43 (2H, m), 7.55 (1H, t, J = 8.0 Hz), 7.58 (1H, t, J = 2.0
Hz), 7.75 (1H, dt, J = 8.4 and 1.4 Hz), 7.89–7.96 (4H, m), 10.55 (1H, br
s) ppm. 13C NMR (DMSO‑d6, 100 MHz): δ 26.38, 117.57, 118.79,
119.45, 121.81, 122.68, 123.81, 129.17, 130.12, 130.16, 132.03,
136.27, 143.27, 156.12, 156.70, 164.98, 196.51 ppm. HRMS (m/z)
calculated for C21H17NO3 [M + H]+ 332.1287, found 332.1291.
(3,4-Dihydroisoquinolin-2(1H)-yl)(3-(pyrimidin-2-yloxy)phenyl)meth-
anone ST06
4.1.3. General procedure for the synthesis of aryloxybenzamides ST01-
ST13
A mixture of appropriate aryloxybenzoic acid (1 equiv), EDC (1.2
equiv) and DMAP (0.2 equiv) in DCM was stirred at ambient tempera-
ture for 30 min. Then, to the solution of the mixture, appropriate amine
(1 equiv) was added, and the reaction mixture was continued to stir
overnight at room temperature. Upon completion of the reaction, the
mixture was diluted with DCM and extracted with HCl 1 M (10 ml × 3),
and NaHCO3 5% aqueous solution (10 ml × 3), and brine (10 ml × 3).
The combined organic layers were dried over Na2SO4, and concentrated
in vacuo. The residue was purified by flash or column chromatography
on silica gel or crystallization with appropriate eluents.
(3,4-Dihydroisoquinolin-2(1H)-yl)(3-phenoxyphenyl)methanone ST01
The compound was synthesized according to the general procedure
from 3a (171 mg, 0.8 mmol) and 1,2,3,4-tetrahydroisoquinoline (100 µl,
0.8 mmol). The crude product was purified by column chromatography
on silica gel, eluting with a mixture of ethyl acetate/n-hexane (15:85) to
afford pure ST01 as a yellowish oil (200 mg, 76% yield). CAS#: 380211-
07-4. 1H NMR (CDCl3, 400 MHz): δ 2.83 (1H, br s), 2.96 (1H, br s), 3.63
(1H, br s), 3.96 (1H, br s), 4.57 (1H, br s), 4.86 (1H, br s), 6.91–6.94 (1H,
The compound was synthesized according to the general procedure
from 3c (160 mg, 0.74 mmol) and 1,2,3,4-tetrahydroisoquinoline (93 µl,
0.74 mmol). The crude product was purified by column chromatography
on silica gel, eluting with a mixture of ethyl acetate/DCM (20:80) to
afford pure ST06 as a yellowish oil (194 mg, 79% yield). 1H NMR
(CDCl3, 400 MHz): δ 2.88 (1H, br s), 2.96 (1H, br s), 3.70 (1H, br s), 3.97
(1H, br s), 4.64 (1H, br s), 4.87 (1H, br s), 7.06 (1H, t, J = 4.6 Hz),
8

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
7.15–7.36 (7H, m), 7.49 (1H, t, J = 7.8 Hz), 8.55 (2H, d, J = 4.8 Hz)
ppm. 13C NMR (CDCl3, 100 MHz): δ 29.68, 44.96, 45.35, 49.76, 116.50,
120.51, 123.03, 123.95, 126.64, 128.71, 129.88, 132.85, 137.43,
152.79, 159.72, 165.03 ppm. HRMS (m/z) calculated for C20H17N3O2
[M + H]+ 332.1399, found 332.1403.
ST12
The compound was synthesized according to the general procedure
from 3e (179 mg, 0.83 mmol) and 1-isopropylpiperazine (119 µl, 0.83
mmol). The crude product was purified by flash chromatography on
silica gel (12 g), eluting with a gradient of 0–10% MeOH in DCM to
afford pure ST12 as a yellowish oil (203 mg, 75% yield). 1H NMR
(CDCl3, 400 MHz): δ 1.04 (6H, d, J = 6.4 Hz), 2.50 (4H, br s), 2.69–2.76
(1H, m), 3.51 (2H, br s), 3.77 (2H, br s), 7.06 (1H, t, J = 4.6 Hz),
7.22–7.25 (2H, m), 7.47–7.50 (2H, m), 8.56 (2H, d, J = 5.2 Hz) ppm. 13C
NMR (CDCl3, 100 MHz): δ 18.35, 42.54, 48.27, 48.70, 54.54, 116.53,
121.55, 128.89, 132.79, 153.87, 159.74, 164.96, 169,46 ppm. HRMS
(m/z) calculated for C18H22N4O2 [M + H]+ 327.1821, found 327.1820.
N-(Naphthalen-1-yl)-4-(pyrimidin-2-yloxy)benzamide ST13
N-(Naphthalen-1-yl)-3-(pyrimidin-2-yloxy)benzamide ST07
The compound was synthesized according to the general procedure
from 3c (173 mg, 0.8 mmol) and 1-naphtylamine (108 mg, 0.8 mmol).
The crude product was purified by crystallization from 2-propanol to
afford pure ST07 as a white solid (109 mg, 40% yield). Mp
132.0–132.5 ◦C. 1H NMR (DMSO‑d6, 400 MHz): δ 7.29 (1H, t, J = 4.8
Hz), 7.46–7.48 (1H, m), 7.52–7.65 (5H, m), 7.85 (1H, d, J = 7.6 Hz),
7.90 (1H, t, J = 2.0 Hz), 7.95–8.00 (3H, m), 8.67 (2H, d, J = 4.4 Hz),
10.46 (1H, br s) ppm. 13C NMR (DMSO‑d6, 100 MHz): δ 117.15, 120.98,
123.30, 123.96, 124.67, 125.14, 125.52, 125.99, 126.07, 126.39,
128.04, 129.16, 129.88, 133.63, 133.74, 136.03, 152.84, 160.13,
164.65, 165.17 ppm. HRMS (m/z) calculated for C21H15N3O2 [M + H]+
342.1243, found 342.1245.
The compound was synthesized according to the general procedure
from 3e (151 mg, 0.7 mmol) and 1-naphtylamine (94 mg, 0.7 mmol).
The crude product was purified by flash chromatography on silica gel
(12 g), eluting with a gradient of 0–10% ethyl acetate in DCM to afford
pure ST13 as a white solid (164 mg, 48% yield). Mp 189.9–190.1 ◦C. 1H
NMR (DMSO‑d6, 400 MHz): δ 7.31 (1H, t, J = 4.6 Hz), 7.38 (2H, d, J =
8.4 Hz), 7.52–7.61 (4H, m), 7.86 (1H, d, J = 8.0 Hz), 7.95–8.01 (2H, m),
8.15 (2H, d, J = 8.8 Hz), 8.68 (2H, d, J = 4.4 Hz), 10.45 (1H, br s) ppm.
13C NMR (DMSO‑d6, 100 MHz): δ 117.32, 121.58, 123.34, 123.89,
125.55, 125.98, 126.07, 126.29, 128.06, 129.20, 129.58, 131.35,
133.76, 133.82, 155.41, 160.17, 164.47, 165.48 ppm. HRMS (m/z)
calculated for C21H15N3O2 [M + H]+ 342.1243, found 342.1244.
N-(Naphthalen-2-yl)-3-(pyrimidin-2-yloxy)benzamide ST08
The compound was synthesized according to the general procedure
from 3c (173 mg, 0.8 mmol) and 2-naphtylamine (108 mg, 0.8 mmol).
The crude product was purified by crystallization from 2-propanol to
afford pure ST08 as a white solid (166 mg, 61% yield). Mp
173.2–175.2 ◦C. 1H NMR (DMSO‑d6, 400 MHz): δ 7.29 (1H, t, J = 5.0
Hz), 7.39–7.49 (3H, m), 7.61 (1H, t, J = 7.8 Hz), 7.79–7.92 (6H, m), 8.43
(1H, d, J = 1.6 Hz), 8.67 (2H, d, J = 4.8 Hz), 10.46 (1H, br s) ppm. 13C
NMR (DMSO‑d6, 100 MHz): δ 116.71, 117.17, 120.86, 120.93, 124.65,
124.84, 125.12, 126.39, 127.41, 127.44, 128.15, 129.87, 130.04,
133.27, 136.41, 136.58, 152.75, 160.15, 164.63, 164.68 ppm. HRMS
(m/z) calculated for C21H15N3O2 [M + H]+ 342.1243, found 342.1238.
(3,4-Dihydroisoquinolin-2(1H)-yl)(4-phenoxyphenyl)methanone ST09
The compound was synthesized according to the general procedure
from 3d (150 mg, 0.7 mmol) and 1,2,3,4-tetrahydroisoquinoline (88 µl,
0.7 mmol). The crude product was purified by column chromatography
on silica gel, eluting with a mixture of ethyl acetate/n-hexane (15:85) to
afford pure ST09 as a colorless oil (196 mg, 85% yield). CAS#: 476298-
18-7. 1H NMR (CDCl3, 400 MHz): δ 2.93 (2H, br s), 3.69–3.95 (2H, m),
4.69–4.85 (2H, m), 7.01–7.07 (4H, m), 7.14–7.19 (5H, m), 7.36–7.40
(2H, m), 7.45 (2H, d, J = 8.8 Hz) ppm. 13C NMR (CDCl3, 100 MHz): δ
29.68, 45.69, 46.62, 118.05, 119.60, 124.03, 126.50, 126.71, 129.07,
129.93, 130.48, 133.01, 156.23, 158.98 ppm. HRMS (m/z) calculated
for C22H19NO2 [M + H]+ 330.1494, found 330.1490.
4.1.4. (E/Z)-(3,4-Dihydroisoquinolin-2(1H)-yl)(3-phenoxyphenyl)
methanone oxime ST14
The solution of (E/Z)-N-hydroxy-3-phenoxybenzimidoyl chloride
(247 mg, 1 mmol) that prepared according to the previously reported
method36 in 10 ml of DCM was added dropwise, while cooling at 0 ◦C, to
the mixture of 1,2,3,4-tetrahydroisoquinoline (126 µl, 1 mmol) and TEA
(139 µl, 1 mmol) in 10 ml of DCM. The reaction mixture was stirred at
0 ◦C for 15 min and then at room temperature for 1 h. Upon completion
of the reaction, the mixture was extracted with HCl 5% aqueous solution
(15 ml × 3). The combined organic layers were dried over Na2SO4, and
concentrated in vacuo. The residue was purified by preparative thin-
layer chromatography (PTLC) eluting with a mixture of ethyl acetate/
n-hexane (28:72) to give pure ST14 as a colorless oil (248 mg, 72%
yield). 1H NMR (CDCl3, 500 MHz): δ 2.83 (2H, t, J = 5.8 Hz), 3.28 (2H, t,
J = 5.9 Hz), 4.37 (2H, s), 4.76 (1H, br s), 7.00–7.23 (10H, m), 7.32–7.46
(3H, m) ppm. 13C NMR (CDCl3, 125 MHz): δ 28.89, 45.01, 48.79,
119.11, 119.18, 119.58, 123.64, 126.11, 126.33, 126.54, 128.84,
129.81, 129.85, 132.47, 133.69, 134.23, 156.74, 157.50, 159.85 ppm.
HRMS m/z calculated for C22H21N2O2 [M + H]+ 345.1603, found:
345.1591.
N-(4-Acetylphenyl)-4-phenoxybenzamide ST10
The compound was synthesized according to the general procedure
from 3d (101 mg, 0.47 mmol) and 4′-aminoacetophenone (64 mg, 0.47
mmol). The crude product was purified by crystallization from acetone
to afford pure ST10 as a white solid (78 mg, 50% yield). CAS#: 723755-
90-6. Mp 212.4–212.8 ◦C. 1H NMR (DMSO‑d6, 400 MHz): δ 2.53 (3H, s),
7.07–7.11 (4H, m), 7.21 (1H, t, J = 7.4 Hz), 7.44 (2H, t, J = 8.0 Hz),
7.90–8.00 (6H, m), 10.47 (1H, s) ppm. 13C NMR (DMSO‑d6, 100 MHz): δ
26.46, 117.40, 119.38, 119.62, 124.47, 129.04, 129.28, 130.15, 130.30,
131.92, 143.67, 155.44, 160.07, 165.15, 196.58 ppm. HRMS (m/z)
calculated for C21H17NO3 [M + H]+ 332.1287, found 332.1284.
N-(4-Carbamoylphenyl)-4-phenoxybenzamide ST11
4.2. In vitro SIRT1-3 inhibition assay
The SIRT inhibition assays were performed using Fluor de Lys SIRT1-
3 Fluorometric Drug Discovery Kits (BML-AK555-0001 for SIRT1; BML-
AK556-0001 for SIRT2; BML-AK557-0001 for SIRT3) according to the
supplier’s protocol (Enzo Life Sciences). Briefly, test compounds in 1%
(v/v) final DMSO concentration and/or SIRT assay buffer (10
μ
l/well)
l/well, BML-SE239
for SIRT1; BML-SE25 for SIRT2; BML-SE270 for SIRT3) were incu-
The compound was synthesized according to the general procedure
from 3d (214 mg, 1 mmol) and 4-aminobenzamide (136 mg, 1 mmol).
The crude product was purified by washing with water, ethyl acetate
and DCM to afford pure ST11 as a white solid (272 mg, 82% yield).
and human recombinant SIRT1/2/3 enzymes (15
μ
+
◦
bated at 37 C for 30 min. Then, NAD and Fluor de Lys deacetylase
substrate (25 μl/well, BML-KI177 for SIRT1; BML-KI179 for SIRT2 and
CAS#: 794577-49-4. Mp > 300 C. 1H NMR (DMSO‑d6, 400 MHz): δ
◦
SIRT3) were added to the mixture. The reaction mixture was incubated
at 37 ◦C for 1 h. After that, the reaction was stopped by adding Fluor de
Lys Developer II (BML-KI176) and nicotinamide 2 mM in SIRT assay
7.07–7.10 (4H, m), 7.19–7.22 (2H, m), 7.43 (2H, t, J = 7.6 Hz),
7.81–7.87 (5H, m), 7.99 (2H, d, J = 8.8 Hz), 10.33 (1H, br s) ppm. 13
C
NMR (DMSO‑d6, 100 MHz): δ 117.42, 119.29, 119.59, 124.43, 128.21,
129.06, 129.24, 130.06, 130.29, 141.89, 155.49, 159.95, 164.99,
167.40 ppm. HRMS (m/z) calculated for C20H16N2O3 [M + H]+
333.1239, found 333.1243.
buffer (total volume 50 μl), and the incubation was continued for 45 min
at 37 ◦C. Fluorescence readings were obtained using a SpectraMax i3x
Multi-mode microplate reader with excitation wavelength 360 nm and
emission 460 nm. The value of inhibition% was calculated from the
fluorescence readings of inhibited wells relative to those of control
(4-Isopropylpiperazin-1-yl)(4-(pyrimidin-2-yloxy)phenyl)methanone
9

S. Yagci et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115961
wells.24
Acknowledgements
This work was financially supported by the Scientific and Techno-
logical Research Council of Turkey (TUBITAK 118S673). We also thank
Serkan Levent (Anadolu University, Faculty of Pharmacy) for his valu-
able support.
4.3. In vitro cytotoxicity assay
The MCF-7 human breast cancer cells were purchased from ATCC
(Germany) and the cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% FBS and 1% pen-
icillin–streptomycin until 70% confluency is reached. The cytotoxic ef-
fects of synthesized compounds on MCF-7 cells were evaluated by MTT
assay.37,38 A 180 µl of 5 × 104 cell/ml cell suspension was seeded on 96-
Appendix A. Supplementary material
1H NMR, 13C NMR and HRMS spectra of compounds 2a, 2c, 2e,
ST01-14. Supplementary data to this article can be found online at htt
ps://doi.org/10.1016/j.bmc.2020.115961.
◦
well plate and incubated for 24 h at 37 C in 5% CO2 humidified at-
mosphere. Then, the cells were treated with the compounds at different
concentrations (1, 10, 25, 50, 100 and 200 µM) and incubated for 48 and
72 h. Following incubation the cells were treated with MTT reagent (5
mg/ml) for 4 h and the absorbance of reduced formazan crystals was
measured by a spectrophotometer (Thermo, Germany) at 540 nm. The
untreated cells were used as control. Each experiment was performed in
triplicate and the IC50 values were calculated by using linear regression
equations obtained from the concentration vs viable cell amount%
graphs. Statistical analyses were performed using GraphPad Prism 6.0
version (GraphPad Software Inc.). Data obtained from the cell culture
experiments were expressed as mean ± SD, and one-way ANOVA test
was applied for multiple comparisons.
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