Synthesis and evaluation of substituted chroman-4-one and chromone derivatives as sirtuin 2-selective inhibitors

Article abstract of DOI:10.1021/jm3005288

A series of substituted chromone/chroman-4-one derivatives has been synthesized and evaluated as novel inhibitors of SIRT2, an enzyme involved in aging-related diseases, e.g., neurodegenerative disorders. The analogues were efficiently synthesized in a on

Full text of DOI:10.1021/jm3005288

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Substituted Chroman-4-one and
Chromone Derivatives as Sirtuin 2‑Selective Inhibitors
Maria Friden-Saxin,†,‡ Tina Seifert,†,‡ Marie Ryden Landergren,§ Tiina Suuronen,∥
́
́
Maija Lahtela-Kakkonen,⊥ Elina M. Jarho,*,⊥ and Kristina Luthman*,†
†Department of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE-412 96 Goteborg, Sweden
̈
§Department of Medicinal Chemistry, R&I iMed, AstraZeneca R&D, SE-431 83 Molndal, Sweden
̈
⊥School of Pharmacy and Department of Neurology, Institute of Clinical Medicine, University of Eastern Finland, P.O. Box 1627,
∥
70211 Kuopio, Finland
S
* Supporting Information
ABSTRACT: A series of substituted chromone/chroman-4-one derivatives has been synthesized and evaluated as novel
inhibitors of SIRT2, an enzyme involved in aging-related diseases, e.g., neurodegenerative disorders. The analogues were
efficiently synthesized in a one-step procedure including a base-mediated aldol condensation using microwave irradiation. The
most potent compounds, with inhibitory concentrations in the low micromolar range, were substituted in the 2-, 6-, and 8-
positions. Larger, electron-withdrawing substituents in the 6- and 8-positions were favorable. The most potent inhibitor of SIRT2
was 6,8-dibromo-2-pentylchroman-4-one with an IC50 of 1.5 μM. The synthesized compounds show high selectivity toward
SIRT2 over SIRT1 and SIRT3 and represent an important starting point for the development of novel SIRT2 inhibitors.
INTRODUCTION
■
Sirtuins (SIRTs) belong to class III histone deacetylases
Chart 1. Selective SIRT2 Inhibitors
(HDACs) and function as deacetylating enzymes on lysine
residues of various histones and nonhistone substrates with the
special requirement for nicotinamide adenine dinucleotide
(NAD+) as cosubstrate for their activity.1,2 The SIRTs are a
conserved class of enzymes from bacteria to humans. The
mammalian sirtuin family consists of seven enzymes (SIRT1−
SIRT7) localized in different parts of the cell.3 The sirtuins have
become highly interesting targets in drug design as they are
involved in important cellular processes3−5 such as aging6 and
hence in neurodegenerative disorders such as Parkinson’s,
Alzheimer’s, or Huntington’s disease.7−11 SIRTs are also
considered to be involved in other age-related diseases such
as diabetes mellitus12 and cancer.13,14 SIRT215,16 in particular is
involved in cell cycle regulation; inhibition of SIRT2 leads to
hyperacetylation of α-tubulin and as a consequence to an
inhibition of tumor growth.17 Other studies have linked SIRT2
activity to Parkinson's disease,18 and reports have shown that
SIRT2 inhibition appears to lead to a decreased neuronal cell
death.19 Research particularly focused on SIRT2 has resulted in
the discovery of a number of potent SIRT2-selective inhibitors
such as bis(indolyl)maleimide-based kinase inhibitors,20
coumarin-based compounds,21 the benzothiazole AC-93253,22
cambinol derivatives,23 and the sulfobenzoic acid AK-724
(Chart 1).
Received: April 13, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Chromones and chroman-4-ones constitute a naturally
occurring class of substances25 which are classified as privileged
structures,26 as compounds based on these scaffolds display a
wide range of biological activities defined by the substitution
pattern of the scaffold.27 We have put considerable effort into
the development of synthetic strategies for this class of
compounds resulting, for example, in an efficient synthetic
route to 2-alkyl-substituted chroman-4-ones.28 The incorpo-
ration of various functional groups to furnish highly substituted
structures have successfully been conducted through different
Pd-mediated cross-coupling reactions,29,30 through Mannich
reactions,31 and via a SmI2−KHMDS-mediated Reformatsky
type reaction.32 Recently, we have also developed chromone/
chroman-4-one-based β-turn peptidomimetics.31,33 In the
present study we report substituted chromone and chroman-
4-one derivatives as potent and highly selective SIRT2
inhibitors.
deacetylation of three different substrates: an artificial substrate
with a fluorophore and a peptide and a protein substrate
without a fluorophore. On the basis of these results, a series of
analogues of 1a was synthesized and evaluated as SIRT2
inhibitors.
Chemistry. The synthetic pathways toward the test
compounds 1a−p, 3a,b, and 4−6 are presented in Scheme 1.
The chroman-4-ones 1a−p were synthesized according to a
procedure previously reported by our group.28 Commercially
available 2′-hydroxyacetophenones were reacted with appro-
priate aldehydes in a base-promoted crossed aldol condensation
followed by an intramolecular oxa-Michael addition. The
reactions were conducted by heating ethanolic mixtures to
160−170 °C using microwave (MW) irradiation for 1 h in the
presence of DIPA as base. The desired 2-alkyl-chroman-4-ones
(1a−p) were isolated in low to high yields (17−88%). The
outcome of the reaction was strongly depended on the
substitution pattern of the acetophenones. In general,
electron-deficient 2′-hydroxyacetophenones gave high yields
of the desired chroman-4-ones whereas electron-donating
groups led to higher amounts of byproduct originating from
self-condensation of the used aldehyde. This caused purification
problems, lowering the yields; for example, the 6,8-dimethyl-
and 6-methoxy-substituted 2-pentylchroman-4-one derivatives
1d and 1h were obtained in only 17% yield. The enantiomers of
1a were separated from the racemic mixture by preparative
HPLC on a chiral stationary phase.37
RESULTS AND DISCUSSION
■
Characterization of the Lead Compound. In an initial
study, a set of compounds based on the chromone and
chroman-4-one scaffolds were tested against human SIRT2 to
see if these privileged structures could serve as scaffolds for
sirtuin inhibitors or activators (data not shown). Interestingly,
8-bromo-6-chloro-2-pentylchroman-4-one 1a gave excellent
inhibition (88%) in a preliminary test at 200 μM concentration
in a fluorescence-based assay. A more detailed determination of
the inhibitory activity gave an IC50 value of 4.5 μM. Compound
1a was also tested against SIRT1 and SIRT3 at 200 μM
concentration resulting in less than 10% inhibition of these
sirtuin subtypes (see Supporting Information). As 1a turned
out to be a novel potent and highly selective SIRT2 inhibitor, it
was chosen for further structure−activity studies.
For the synthesis of the 2-pentylchromone 3a, chroman-4-
one 1a was converted to the corresponding 3-bromo-2-
pentylchroman-4-one 2 using Py·Br3 as the brominating
agent.38 Interestingly, 2 was isolated as a diastereomeric
1
mixture of 80:20 according to H NMR spectroscopy with
the cis-isomer as the major product. Computational studies
confirmed the higher stability of the cis-product.28 Hydro-
bromide elimination of 2 by CaCO3 in DMF in a microwave-
assisted reaction provided the desired chromone 3a in 84%
yield. Flavone 3b was prepared from 3′-bromo-5′-chloro-2′-
hydroxyacetophenone via esterification with benzoyl chloride
followed by a Baker−Venkataraman rearrangement yielding a
diketo intermediate that was cyclized in an acid-catalyzed
reaction.30 To obtain compounds 5 and 6, the carbonyl group
in 1a was reduced by NaBH4 in MeOH, providing 8-bromo-6-
chloro-2-pentylchroman-4-ol 4 in almost quantitative yield and
in 96:4 diastereomeric ratio. With the chroman-4-ol 4 in hand,
the hydroxyl group could be removed either by dehydroxylation
or dehydration. Dehydroxylation was carried out using
triethylsilane as hydrogen source in the presence of BF3·Et2O
yielding 8-bromo-6-chloro-2-pentylchroman 5 in moderate
yield (44%). Dehydration was performed using a catalytic
amount of p-toluenesulfonic acid with MgSO4 present as drying
agent. The corresponding 2H-chromene 6 could be isolated in
a yield of 63%.
Structure−Activity Relationship (SAR) Studies. The set
of synthesized substituted chroman-4-one and chromone
derivatives was used to explore the SAR (Table 1). The
inhibitory activities of the synthesized compounds 1a−p, 3a,b,
and 4−6 were determined using in vitro SIRT1, SIRT2, and
SIRT3 assays.39 It is noteworthy that the most potent inhibitors
(over 70% inhibition at 200 μM) are completely SIRT2
selective. They show less than 10% inhibition of SIRT1 and
SIRT3 at 200 μM, the only exception being 16% inhibition of
SIRT3 by compound 1m. The results from the SIRT2 assay are
Compound 1a structurally also resembles some naturally
occurring polyphenolic flavones, such as fisetin and quercetin,
with reported SIRT1-activating properties (Chart 2).34 There
Chart 2. Putative SIRT1 Activators
has been controversy whether resveratrol, another polyphenolic
SIRT1 activator, directly activates SIRT1 or not. It has been
shown that in vitro, resveratrol activates SIRT1-mediated
deacetylation of substrates that have a fluorophore covalently
attached but not substrates lacking this fluorophore.35,36
To confirm that the detected SIRT2 inhibition by 1a was not
caused by interaction with an artificial fluorophore, we further
verified SIRT2 inhibition with two different methods. First, a
Western blot analysis of the SIRT2-mediated deacetylation of
acetylated α-tubulin was carried out and inhibition of the
SIRT2-catalyzed reaction by 1a was observed (Figure 1A).
Second, a SIRT2 activity assay based on the release of
radioactive 14C-nicotinamide was performed in the presence of
an acetylated peptidic substrate (RSTGGK(Ac)APRKQ)
without a fluorophore (Figure 1B). In this assay 1a gave 66%
inhibition. Taken together, 1a was able to inhibit the
B
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Inhibition of SIRT2-mediated deacetylation reactions by compound 1a. (A) Western blot analysis of the inhibition of SIRT2-mediated α-
tubulin deacetylation by 1a. The concentration of 1a was 200 μM, and measurements were performed at 30 min and 1 h. (B) Inhibition by 1a of the
SIRT2-mediated deacetylation of the acetylated peptide RSTGGK(Ac)APRKQ. The reaction was detected by formation of the reaction product 14C-
nicotinamide.
a
Scheme 1. General Methods for the Syntheses of Compounds 1a−p, 2, 3a,b, and 4−6
a
Reagents and conditions: (a) appropriate aldehyde, DIPA, EtOH, MW, 160−170 °C, 1 h, 17−88%; (b) Py·Br3, CH2Cl2, room temp, 2.5 h, 81%,
cis/trans ratio 80:20; (c) CaCO3, DMF, MW, 100 °C, 20 min, 84%; (d) i. benzoyl chloride, pyridine, room temp, 2 h; ii. KOH, pyridine, 50 °C, 4 h;
iii. HCl, AcOH, reflux, 14 h, 89% (over three steps); (e) NaBH4, MeOH/THF, 0 °C→rt, 15 min, 98%, 95:5 ratio of diastereomers; (f) Et3SiH,
BF3·Et2O, CH2Cl2, −78 °C→rt, 19 h, 44%; (g) p-TSA (cat.), MgSO4, toluene, 90 °C, 1.5 h, 63%.
summarized in Table 1 (see Supporting Information for results
enantiomers had only slightly different inhibitory activities.
Enantiomer (−)-1a was a more potent inhibitor with an IC50
value of 1.5 μM compared to (+)-1a with an IC50 of 4.5 μM.
The unsaturated analogue of 1a, chromone 3a, was
from the SIRT1 and SIRT3 assays).
In the initial SAR investigation the individual enantiomers of
the lead compound 1a were tested. It turned out that the
C
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Results from Evaluation of Compounds 1a−p, 3a,b, and 4−6 in a SIRT2 Activity Assay
a
bc
,
compd
R2
R6
Cl
R7
R8
Br
inhibition SD at 200 μM (%)
IC50 (μM)
1a
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)4CH3
(CH2)2CH3
(CH2)6CH3
CH2CH2Ph
CH(CH3)2
H
H
H
H
H
H
H
H
H
H
H
F
88 0.9
70 0.8
91 0.8
4.9 4.8
92 1.2
83 0.7
30 1.3
55 2.4
58 0.7
20 4.1
28 1.1
18 1.0
76 1.8
57 2.5
81 0.7
52 1.0
53 1.7
27 1.6
82 0.4
20 1.4
31 3.0
38 1.3
38 1.2
4.3 (3.5−5.4)
4.5 (3.5−5.9)
1.5 (1.3−1.7)
(+)-1a
(−)-1a
1b
1c
Cl
Br
Br
H
Cl
H
n.d.
Br
Br
CH3
F
1.5 (1.3−1.7)
6.2 (4.7−8.1)
1d
1e
CH3
F
n.d.
1f
Cl
H
n.d.
1g
1h
1i
NO2
OCH3
H
H
n.d.
H
n.d.
Br
H
n.d.
1j
H
n.d.
1k
1l
Cl
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
10.6 (9.0−12.5)
Cl
n.d.
1m
1n
1o
1p
3a
Cl
6.8 (5.8−8.0)
Cl
n.d.
CH2CH2(3-indolyl)
CH2CH2(N-Ts)(3-indolyl)
(CH2)4CH3
Cl
n.d.
Cl
n.d.
Cl
5.5 (4.8−6.2)
3b
4
Ph
Cl
n.d.
n.d.
n.d.
n.d.
(CH2)4CH3
Cl
5
(CH2)4CH3
Cl
6
(CH2)4CH3
Cl
a
b
SD, standard deviation (n = 3). IC50 (95% confidence interval). IC50 values were determined for compounds showing >70% inhibition of SIRT2 at
c
200 μM concentration. n.d. = not determined
insignificantly less active than rac-1a with an IC50 value of 5.5
μM.
To further probe the importance of the 6- and 8-substituents
for inhibitory potency, derivatives lacking one of these groups
were synthesized (1f−i). Compound 1f that only contains the
6-chloro substituent showed a decrease in activity. No change
in activity was observed when the 6-chloro substituent was
replaced with an electron-withdrawing nitro group (1g).
Interestingly, with an electron-donating methoxy group in the
same position (1h) the inhibitory activity decreased to 20%
(the inhibitory activity was determined at a concentration of
200 μM). This particular example shows that the activity can be
dramatically altered by the electronic nature of the substituent.
Compound 1i lacking a substituent in the 6-position was
significantly less potent than the lead compound 1a. Thus, the
substituent in the 6-position is more important for activity than
that in the 8-position. It was also clarified that electron-rich
chroman-4-ones generally are less potent inhibitors than
electron-poor compounds. One example of substitution in the
7-position was explored with the fluorinated 1j which showed
only weak inhibitory activity (18%).
The influence of the 2-substituent is reflected by compounds
1k−p and 3b in which the length and branching of the alkyl
chain have been altered and also aromatic groups of different
sizes have been introduced. It was shown that the inhibitory
effect is dependent on the length of the alkyl chain. Chroman-
4-one 1k with an n-propyl chain gave slightly better activity
(76%, IC50 10.6 μM) than the n-heptyl substituted 1l (57%).
However, the originally chosen pentyl group had the most
optimal length among the studied alkyl derivatives. Branching
of the chain in the vicinity of the chroman-4-one ring system
Thereafter, the effect of variations of the substituents in the
6- and 8-position on the inhibitory activity of 1a was studied.
The unsubstituted 2-pentylchroman-4-one 1b lost all inhibitory
activity, indicating that substituents in the aromatic system are
necessary to achieve any inhibition. To reveal whether the
inhibitory effect of 1a is caused by steric or electrostatic
properties of the substituents, various disubstituted 2-
pentylchroman-4-ones (1c−e) were synthesized. The change
of the 6-chloro substituent in 1a to the larger but less
electronegative 6-bromo substituent in 1c was tolerated and
resulted in an IC50 value (1.5 μM) identical to that of the
(−)-1a enantiomer. Interestingly, the difluorinated 1e with
smaller but more electronegative substituents was considerably
less active than the other dihalogenated derivatives (1a and 1c)
but more potent than the unsubstituted 1b. This suggests that
electron-withdrawing groups in general enhance activity but
that electrostatic properties are not exclusively responsible for
strong inhibition. Replacement of the halogens with methyl
groups (1d) caused a slight decrease in activity compared to the
chloro- and bromo-substituted 1a and 1c but a clear increase in
activity compared to the difluorinated 1e. This supports the
previous finding that larger substituents in the 6- and 8-
positions are necessary to achieve significant inhibition.
Altogether these results revealed that the size of the 6- and
8-substituents is important although electron-withdrawing
properties can further improve the inhibitory activity.
D
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
decreased the inhibitory activity; the isopropyl analogue 1n was
less active (52%) than the n-propyl derivative 1k (76%, IC50
10.6 μM). Introduction of a phenyl group as in flavone 3b
resulted also in decreased inhibition (20%) compared to the n-
pentyl-substituted chromone 3a (82%, IC50 5.5 μM). These
results showed that bulky groups directly connected to the ring
system diminish the inhibitory effect against SIRT2. However,
the phenethyl-substituted derivative 1m gave 81% inhibition
(IC50 6.8 μM), which showed that substituents other than
aliphatic chains are allowed if a spacer is introduced between
the scaffold and the aromatic ring. Replacement of the phenyl
group in 1m with sterically more demanding indole groups
resulted in decreased activity. Compound 1o with an
unsubstituted indole showed 53% inhibitory activity and was
more active than the N-tosyl-substituted 1p (27% inhibitory
activity). These results indicate a space limitation for the
substituents in the 2-position and showed that more bulky
groups in this position lower the activity considerably.
Finally, the impact of the carbonyl group was investigated
and is demonstrated by compounds 4−6. Any modification of
the carbonyl group resulted in a significant loss of inhibitory
activity compared to 1a. Interestingly, a simple manipulation as
a reduction of the ketone to a hydroxyl moiety (4) resulted in
the same decrease of activity as the removal of the group (5 and
6). The lack of activity of 4−6 could be due to that an
important hydrogen bonding interaction could no longer be
formed with the enzyme. In case of 4, the low activity is likely
to be due to conformational changes in the ring system
resulting in changes in the orientation of the oxygen atom.
These results showed that the presence of the carbonyl group is
an essential feature to attain potent inhibitors.
We can conclude that there is an exceptionally close
relationship between the molecular structure of the compounds
and their SIRT2 inhibitory activity because even minor
alterations in one of the four positions in the chroman-4-one
ring system markedly affect the activity of the compounds.
Determination of the Absolute Configuration of the
Enantiomers of 1a. The enantiomers of the lead compound
1a were separated by preparative chiral HPLC. The absolute
configuration was intended to be determined by means of X-ray
crystallography. Unfortunately, all attempts to obtain useful
crystals of the enantiomers failed. A valid alternative for the
determination of the absolute configuration of small molecules
is to compare experimental and calculated vibrational circular
dichroism (VCD) spectra.40,41 To predict the VCD spectra of
low energy conformers of a molecule, density functional theory
(DFT) calculations can be used. Highly flexible groups in a
molecule lead to many conformers which have to be considered
in the calculation of the VCD data. Therefore, to facilitate the
configurational determination, the calculations were done on a
slightly truncated structure (2-ethyl instead of 2-pentyl, Figure
2), as the change in the alkyl group is not expected to alter the
VCD spectra to any greater extent. The experimental spectra of
the two enantiomers of 1a were compared with the calculated
spectra of the R- and the S-enantiomers of the modified
structure.
As can be seen in the VCD spectra (Figure 3), several bands
in the frequency region from 1500 to 1100 cm−1 show good
alignment between the experimental spectrum of (−)-1a and
the calculated spectrum of the S-enantiomer of the 2-ethyl
analogue. Equally good alignment in the same region is
obtained when comparing the experimental spectrum of (+)-1a
and the calculated spectrum of the R-enantiomer. Thus, from
this study it is concluded that (−)-1a most likely has the S-
configuration and (+)-1a the R-configuration.
CONCLUSIONS
■
A series of compounds based on the chromone and chroman-4-
one scaffolds have been synthesized using an efficient one-step
procedure. The derivatives were selective and potent SIRT2
inhibitors with IC50 values in the low micromolar range. Among
the investigated modifications, it was found that an alkyl chain
with three to five carbons in the 2-position, larger, electron-
withdrawing groups in the 6- and 8-positions, and an intact
carbonyl group were crucial for high potency. Synthesis and
evaluation of additional chroman-4-one analogues is ongoing in
our laboratory. Special emphasis is directed toward the
introduction of functional groups that can enhance the polarity
of the derivatives.
EXPERIMENTAL SECTION
■
General. All reactions were carried out using magnetic stirring
under ambient atmosphere if not otherwise noted. Room temperature
corresponds to a temperature interval from 20 to 21 °C. All starting
materials and reagents were obtained from commercial producers and
were used without prior purification. Solvents were generally used as
supplied by the manufacturer. Microwave reactions were carried out
using a Biotage Initiator Sixty with fixed hold time modus in 2−5 mL
or 10−20 mL capped microwave vials. All reactions were monitored by
thin-layer chromatography (TLC) on silica-plated aluminum sheets
(Silica gel 60 F254, E. Merck). Spots were detected by UV (254 or 365
nm). Purification by flash column chromatography was performed
using an automatic Biotage SP4 Flash+ instrument. Prefabricated
columns of three different cartridge sizes (surface area 500 m2/g,
porosity 60 Å, particle size 40−63 μm) were used. The NMR spectra
were measured with a JEOL JNM-ECP 400 or a Varian 400-MR
spectrometer. 1H and 13C NMR spectra were measured at 400 and 100
MHz, respectively. Chemical shifts are reported in ppm with the
solvent residual peak as internal standard [CHCl3 δH 7.26, CDCl3 δC
77.16]. All NMR experiments were measured at ambient temperature.
VCD spectra were recorded with a ChiralIR-2X from BioTools. The
optical rotation was measured with an automatic polarimeter AA-5
(Optical Activity Ltd.). Melting points were measured with a Buchi B-
̈
545 melting point apparatus or a Mettler FP82 hot stage equipped
with a FP80 temperature controller and are uncorrected. Positive ion
mass spectra (ESI-MS) were acquired with an LCQ quadrupole ion
trap mass spectrometer (Finnigan LTQ) equipped with an electro-
spray ionization source or on a Perkin-Elmer API 150EX mass
spectrometer. Combustion analyses for CHN were measured on a
Thermo Quest CE Instruments EA 1110 CHNS-O elemental analyzer.
The syntheses of compounds 1a, 1m−p, and 3b have previously been
described by our group, and the characterization data were in
agreement with those reported in the literature.28,29
General Procedure for Synthesis of Chroman-4-ones 1b−n.
The appropriate aldehyde (1.1 equiv) and DIPA (1.1 equiv) were
added to a 0.4 M solution of the appropriate 2′-hydroxyacetophenone
in EtOH. The mixture was heated by microwave irradiation at 160−
170 °C for 1 h (fixed hold time, normal absorption), diluted with
Figure 2. Structures used for the DFT calculations of VCD spectra
used to determine the absolute configuration of the enantiomers of 1a.
To simplify the ab initio calculations, the 2-pentyl group in 1a was
truncated to an ethyl group.
E
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Comparison of the experimental VCD spectra of (−)-1a (blue) and (+)-1a (green) with the calculated spectra of the S-enantiomer
(purple) and the R-enantiomer (red) of 8-bromo-6-chloro-2-ethylchroman-4-one, respectively.
CH2Cl2, and washed with NaOH (aq, 10%), HCl (aq, 1 M), water,
and finally brine. The organic phase was dried over MgSO4, filtered,
and concentrated under reduced pressure. Purification by flash column
chromatography gave chroman-4-ones 1b−n.
3H). 13C NMR δ 192.7, 161.8, 136.0, 127.0, 121.2, 121.1, 118.0, 78.0,
43.1, 35.0, 31.6, 24.6, 22.6, 14.1. Anal. (C16H22O2) C, H, N.
6,8-Difluoro-2-pentylchroman-4-one (1e). The title compound
was synthesized according to the general procedure from 3′,5′-
difluoro-2′-hydroxyacetophenone (0.20 g, 1.16 mmol), hexanal (0.16
mL, 1.28 mmol), and DIPA (0.18 mL, 1.28 mmol). The crude product
was purified by flash column chromatography using EtOAc:hexane
(3→6%) to afford 1e (70 mg, 23%) as an off-white oil which solidified
in the refrigerator. 1H NMR δ 7.35 (ddd, J = 8.0, 3.0, 1.8 Hz, 1H), 7.06
(ddd, J = 10.1, 8.0, 3.1 Hz, 1H), 4.49 (dddd, J = 10.4, 7.4, 5.1, 5.1 Hz,
1H), 2.79−2.68 (m, 2H), 1.98−1.89 (m, 1H), 1.78−1.69 (m, 1H),
1.60−1.31 (m, 6H), 0.94−0.89 (m, 3H).13C NMR δ 190.8 (dd, J =
3.3, 2.1 Hz), 155.7 (dd, J = 244.2, 9.7 Hz), 151.9 (dd, J = 252.6, 10.9
Hz), 147.0 (dd, J = 11.6, 2.8 Hz), 122.8 (dd, J = 7.4, 1.7 Hz) 110.9
(dd, J = 28.0, 21.4 Hz), 107.3 (dd, J = 23.0, 4.2 Hz), 79.3, 43.0 (d, J =
1.3 Hz), 34.8, 31.6, 24.6, 22.6, 14.1. Anal. (C14H16 F2O2) C, H, N.
6-Chloro-2-pentylchroman-4-one (1f). The title compound was
synthesized according to the general procedure from 5′-chloro-2′-
hydroxyacetophenone (0.17 g, 1.00 mmol), hexanal (0.14 mL, 1.14
mmol) and DIPA (0.16 mL, 1.14 mmol). Purifications by flash column
chromatography using EtOAc:hexane (3%), EtOAc:hexane (1.5−
1.8%) and finally THF:hexane (1%) gave 1f (0.13 g, 51%) as a white
solid. Mp 42−44 °C. 1H NMR δ 7.82 (d, J = 2.7 Hz, 1H), 7.40 (dd, J =
8.8, 2.7 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 4.46−4.39 (dddd, J = 10.4,
7.4, 5.3, 5.3 Hz, 1H), 2.7−2.62 (m, 2H), 1.92−1.82 (m, 1H), 1.74−
1.65 (m, 1H), 1.60−1.25 (m, 6H), 0.93−0.88 (m, 3H). 13C NMR δ
191.6, 160.3, 135.9, 126.8, 126.4, 121.9, 119.8, 42.8, 34.9, 31.7, 24.7,
22.7, 14.1. Anal. (C14H17ClO2) C, H, N.
2-Pentylchroman-4-one (1b). The title compound was synthe-
sized according to the general procedure from 2′-hydroxyacetophe-
none (1.00 mL, 8.31 mmol), hexanal (1.12 mL, 9.11 mmol), and DIPA
(1.28 mL, 9.13 mmol). The crude product was purified by flash
chromatography using EtOAc:heptane (5%) as eluent to obtain 1b
(0.99 g, 55%) as a pale yellow viscous liquid. 1H NMR δ 7.85 (ddd, J =
7.8, 1.7, 0.4 Hz, 1H), 7.44 (ddd, J = 8.3, 7.2, 1.8, 1H), 7.02−6.91 (m,
2H), 4.49−4.35 (m, 1H), 2.72−2.60 (m, 2H), 1.94−1.78 (m, 1H),
1.76−1.62 (m, 1H), 1.61−1.18 (m, 6H), 0.90 (t, J = 7.0 Hz, 3H). 13C
NMR δ 192.7, 161.7, 135.9, 126.9, 121.1, 121.0, 117.9, 77.9, 43.0, 34.9,
31.6, 24.6, 22.5, 14.0. Anal. (C14H18O2) C, H, N.
6,8-Dibromo-2-pentylchroman-4-one (1c). The title com-
pound was synthesized according to the general procedure from
3′,5′-dibromo-2′-hydroxyacetophenone (0.29 g, 1.10 mmol), hexanal
(0.14 mL, 1.14 mmol), and DIPA (0.16 mL, 1.14 mmol). Purification
by flash column chromatography EtOAc:hexane (2%) and recrystal-
lization from hexane gave 1c (0.21 g, 56%) as an off-white solid. Mp
1
80−82 °C. H NMR δ 7.94 (d, J = 2.4 Hz, 1H), 7.83 (d, J = 2.4 Hz,
1H), 4.54−4.47 (m, 1H), 2.78−2.66 (m, 2H), 1.99−1.89 (m, 1H),
1.78−1.43 (m, 3H), 1.41−1.31 (m, 4H), 0.93−0.89 (m, 3H). 13C
NMR δ 190.5, 157.1, 140.9, 128.8, 122.8, 113.6, 113.0, 79.0, 42.3, 34.6,
31.5, 24.6, 22.5, 13.9. Anal. (C14H16Br2O2) C, H, N.
6,8-Dimethyl-2-pentylchroman-4-one (1d). The title com-
pound was synthesized according to the general procedure from
3′,5′-dimethyl-2′-hydroxyacetophenone (0.49 g, 2.98 mmol), hexanal
(1.35 mL, 10.98 mmol), and DIPA (1.05 mL, 7.49 mmol). The crude
product was purified twice by flash column chromatography using
EtOAc:hexane (5%) to afford 1d (0.12 g, 17%) as an off-white solid.
6-Nitro-2-pentylchroman-4-one (1g). The title compound was
synthesized according to the general procedure from 2′-hydroxy-5′-
nitroacetophenone (0.50 g, 2.76 mmol), hexanal (0.37 mL, 3.04
mmol), and DIPA (0.43 mL, 3.07 mmol). Flash column chromatog-
raphy of the crude product using EtOAc:hexane (5%) gave 1g as a
slightly yellow solid (0.42 g, 58%). Mp 81−83 °C. 1H NMR δ 8.77 (d,
J = 2.8 Hz, 1H), 8.32 (dd, J = 9.1, 2.8 Hz, 1H), 7.10 (d, J = 9.1 Hz,
1
Mp 35−37 °C. H NMR δ 7.53−7.50 (m, 1H), 7.14−7.17 (m, 1H),
4.38 (app dq, J = 8.0, 4.7 Hz, 1H), 2.60−2.70 (m, 2H), 2.56 (s, 3H),
2.21 (s, 3H), 1.83−1.94 (m, 1H), 1.74−1.28 (m, 7H), 0.94−0.86 (m,
F
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 4.60−4.54 (m, 1H), 2.83−2.71 (m, 2H), 1.97−1.89 (m, 1H),
1.80−1.73 (m, 1H), 1.63−1.30 (m, 6H), 0.94−0.90 (m, 3H). 13C
NMR δ 190.5, 165.7, 142.1, 130.4, 123.78 120.6, 119.4, 79.2, 42.5,
34.7, 31.6, 24.6, 22.6, 14.1. Anal. (C14H17NO4) C, H, N.
4.48 (d, J = 5.8 Hz, 0.2H), 4.40 (d, J = 1.7 Hz, 0.8H), 4.21 (ddd, J =
8.0, 5.4, 1.7 Hz, 0.8H), 2.16 (m, 0.8H), 1.84−1.76 (m, 1.2H), 1.67−
1.29 (m, 6H), 0.95−0.88 (m, 3H). 13C NMR δ 184.4, 183.9, 155.7,
139.3, 139.0, 127.7, 127.6, 126.9, 126.5, 120.0, 119.6, 112.9, 112.6,
82.7, 79.4, 49.4, 48.4, 32.4, 31.6, 31.3, 31.0, 24.9, 24.2, 22.43, 22.37,
13.91, 13.87.
8-Bromo-6-chloro-2-pentylchromone (3a). Chroman-4-one 2
(0.18 g, 0.43 mmol) was dissolved in DMF (3 mL), and CaCO3 (0.13
g, 1.30 mmol) was added. The mixture was heated by microwave
irradiation at 100 °C for 20 min. The mixture was diluted with EtOAc,
filtered, and washed with water. Finally, the filtrate was dried over
Na2SO4 and concentrated under vacuum. Purification by flash column
chromatography using EtOAc:heptane (5%) gave 3a (0.12 g, 84%) as
a white solid. Mp 73−75 °C. 1H NMR δ 8.08 (d, J = 2.4 Hz, 1H), 7.83
(d, J = 2.4 Hz, 1H), 6.19 (s, 1H), 2.67 (t, J = 7.6 Hz, 2H), 1.85−1.70
(m, 2H), 1.45−1.31 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR δ
176.5, 170.5, 151.8, 136.6, 131.1, 125.5, 124.7, 112.7, 109.9, 34.2, 31.2,
26.4, 22.4, 14.0. Anal. (C14H14BrClO2) C, H, N.
6-Methoxy-2-pentylchroman-4-one (1h). The title compound
was synthesized according to the general procedure from 2′-hydroxy-
5′-methoxyacetophenone (1.00 g, 6.02 mmol), hexanal (0.81 mL, 6.62
mmol), and DIPA (0.93 mL, 6.63 mmol). Purification by flash column
chromatography EtOAc:hexane (5%) and recrystallization from
hexane gave 1h (0.24 g, 17%) as an off-white solid. Mp 55−56 °C.
1H NMR δ 7.30 (d, J = 3.2 Hz, 1H), 7.08 (dd, J = 9.0, 3.2 Hz, 1H),
6.91 (d, J = 9.0 Hz, 1H), 4.42−4.35 (m, 1H), 3.81 (s, 3H), 2.74−2.60
(m, 2H), 1.95−1.85 (m, 1H), 1.73−1.63 (m, 1H), 1.61−1.25 (m, 6H),
0.93−0.89 (m, 3H). 13C NMR δ 193.0, 156.6, 154.0, 125.3, 119.4,
107.4, 78.2, 43.1, 35.0, 24.7, 22.7, 14.1. Anal. (C15H20O3) C, H, N.
8-Bromo-2-pentylchroman-4-one (1i). The title compound was
synthesized according to the general procedure from 3′-bromo-2′-
hydroxyacetophenone (0.18 g, 0.82 mmol), hexanal (0.11 mL, 0.90
mmol), and DIPA (0.13 mL, 0.92 mmol). Purification by flash column
chromatography using EtOAc:hexane (5%) gave 1i (0.10 g, 42%) as a
8-Bromo-6-chloro-2-pentylchroman-4-ol (4). A solution of 1a
(0.40 g, 1.2 mmol) in MeOH:THF (20:5 mL) was cooled to 0 °C with
an ice bath, and NaBH4 (55 mg, 1.5 mmol) was added. The mixture
was allowed to warm to room temperature and stirred for 15 min.
Water was added, and the aqueous phase was separated and extracted
three times with EtOAc. The combined organic phases were washed
with brine, dried over MgSO4, filtered, and concentrated under
reduced pressure. Chroman-4-ol 4 (0.39 g, 98%) was obtained as a
1
slightly yellow solid. Mp 44−47 °C. H NMR δ 7.83 (dd, J = 7.8, 1.6
Hz, 1H), 7.72 (dd, J = 7.8, 1.6 Hz, 1H), 6.89 (dd, J = 7.8, 7.8 Hz, 1H),
4.56−4.48 (m, 1H), 2.77−2.67 (m, 2H), 2.00−1.91 (m, 1H), 1.77−
1.32 (m, 7H), 0.94−0.90 (m, 3H). 13C NMR δ 192.0, 158.1, 139.3,
126.4, 122.4, 122.0, 112.1, 78.7, 42.7, 34.9, 31.6, 24.6, 22.6, 14.1. Anal.
(C14H17BrO2) C, H, N.
1
white solid consisting of a 96:4 mixture of diastereomers. H NMR δ
7-Fluoro-2-pentylchroman-4-one (1j). The title compound was
synthesized according to the general procedure from 4′-fluoro-2′-
hydroxyacetophenone (1.00 g, 6.49 mmol), hexanal (0.88 mL, 7.16
mmol), and DIPA (1.00 mL, 7.13 mmol). The crude product was
purified twice by flash column chromatography using EtOAc:hexane
7.42−7.39 (m, 2H), 4.95−4.85 (m, 0.96H), 4.76−4.71 (m, 0.04H),
4.22−4.11 (m, 1H), 2.32 (ddd, J = 13.0, 6.2, 1.9 Hz, 1H), 1.90−1.33
(m, 9H), 0.94−0.89 (m, 3H). δ 13C NMR δ 150.2, 132.1, 128.5, 126.3,
125.5, 111.1, 77.5, 77.2, 76.8, 76.2, 65.6, 37.6, 35.4, 31.7, 24.9, 22.7,
14.1. Anal. (C14H18BrClO2) C, H, N.
1
(5%) to afford 1j (0.31 g, 20%) as an off-white oil. H NMR δ 7.89
(dd, J = 8.8, 6.7 Hz, 1H), 6.74−6.63 (m, 2H), 4.56 (dddd, J = 8.7, 7.4,
6.6, 5.1 Hz, 1H), 2.72−2.59 (m, 2H), 1.95−1.80 (m, 1H), 1.76−1.22
(m, 7H), 0.96−0.84 (m, 3H). 13C NMR δ 191.3, 167.6 (d, J = 255.8
Hz) 163.5 (d, J = 13.7 Hz), 129.5 (d, J = 11.4 Hz), 118.0 (d, J = 2.4
Hz), 109.6 (d, J = 22.8 Hz), 104.7 (d, J = 24.3 Hz), 78.6, 42.6, 34.7,
31.5, 24.5, 22.5, 13.9. Anal. (C14H17FO2) C, H, N.
8-Bromo-6-chloro-2-pentylchromane (5). A solution of chro-
man-4-ol 4 (0.20 g, 0.60 mmol) in CH2Cl2 (10 mL) was cooled to
−78 °C. Triethylsilane (1.4 mL, 8.76 mmol) was added dropwise,
followed by boron trifluoride etherate (0.3 mL, 2.43 mmol), and the
mixture was stirred for 19 h at room temperature. The mixture was
filtered, and the filtrate was concentrated under reduced pressure.
Purification by flash column chromatography using heptane gave 5 (84
mg, 44%) as a white solid. Mp 33−36 °C. 1H NMR δ 7.32 (d, J = 2.5
Hz, 1H), 6.97 (d, J = 2.6 Hz, 1H), 4.09−4.01 (m, 1H), 2.87−2.69 (m,
2H), 2.04−1.96 (m, 1H), 1.85−1.29 (m, 9H), 0.94−0.89 (m, 3H). 13C
NMR δ 150.6, 130.4 128.4, 124.9, 124.7, 111.5, 77.5, 77.3, 77.2, 76.8,
35.1, 31.8, 27.0, 25.1, 25.0, 22.7, 14.2. Anal. (C14H18BrClO) C, H, N.
8-Bromo-6-chloro-2-pentyl-2H-chromene (6). Chroman-4-ol 4
(0.13 g, 0.39 mmol) was dissolved in toluene (20 mL) and heated to
90 °C for 1.5 h in the presence of a catalytic amount of p-
toluenesulfonic acid and anhydrous MgSO4 (50 mg). The mixture was
filtered, and the filtrate was concentrated under reduced pressure.
Purification by flash column chromatography using EtOAc:heptane
(5%) gave 6 (0.12 g, 94%) as a slightly yellow oil which solidified in
8-Bromo-6-chloro-2-propylchroman-4-one (1k). The title
compound was synthesized according to the general procedure from
3′-bromo-5′-chloro-2′-hydroxyacetophenone (0.50 g, 2.00 mmol),
butanal (0.20 mL, 2.23 mmol), and DIPA (0.4 mL, 2.85 mmol).
Flash column chromatography of the crude product using EtOAc:hex-
1
ane (5%) gave 1k (0.43 g, 71%) as a white solid. Mp 84−86 °C. H
NMR δ 7.80 (d, J = 2.6 Hz, 1H), 7.70 (d, J = 2.6 Hz, 1H), 4.52 (dddd,
J = 10.8, 8.8, 4.4, 4.4 Hz, 1H), 2.78−2.66 (m, 2H), 1.99−1.89 (m,
1H), 1.72−1.53 (m, 3H), 1.01 (t, J = 7.2 Hz, 3H). 13C NMR δ 190.8,
156.8, 138.5, 126.9, 125.9, 122.5, 112.9, 79.2, 42.5, 34.8, 31.9, 29.3,
29.3, 29.3, 25.1, 22.8, 14.2. Anal. (C12H12BrClO2) C, H, N.
8-Bromo-6-chloro-2-heptylchroman-4-one (1l). The title
compound was synthesized according to the general procedure from
3′-bromo-5′-chloro-2′-hydroxyacetophenone (0.50 g, 2.00 mmol),
octanal (0.34 mL, 2.20 mmol), and DIPA (0.30 mL, 2.20 mmol).
Purification by flash column chromatography EtOAc:hexane (10%)
1
the refrigerator. H NMR δ 7.30 (d, J = 2.5 Hz, 1H), 6.88 (d, J = 2.5
Hz, 1H), 6.29 (dd, J = 9.9, 1.6 Hz, 1H), 5.78 (dd, J = 9.9, 3.6 Hz, 1H),
4.98−4.92 (m, 1H), 1.84−1.73 (m, 1H), 1.67−1.24 (m, 7H), 0.92−
0.88 (m, 3H). 13C NMR δ 148.8, 131.5, 128.0, 125.8, 125.1, 124.0,
122.4, 110.4, 76.4, 35.2, 31.5, 24.5, 22.5, 14.0. Anal. Calcd for
C14H16BrClO: C, 53.27; H, 5.11. Found: C, 53.72 H, 5.05.
1
gave 1l (0.52 g, 56%) as a slightly yellow solid. Mp 79−81 °C. H
NMR δ 7.79 (d, J = 2.6 Hz, 1H), 7.69 (d, J = 2.6 Hz, 1H), 4.54−4.47
(m, 1H), 2.77−2.65 (m, 2H), 1.98−1.89 (m, 1H), 1.78−1.19 (m,
11H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR δ 190.8, 156.8, 138.5, 126.9,
125.9, 122.5, 112.9, 79.2, 42.5, 34.8, 31.9, 29.3, 29.3, 25.1, 22.8, 14.2.
Anal. (C16H20BrClO2) C, H, N.
Biochemistry. In Vitro Fluor de Lys Assay for SIRT1, SIRT2,
and SIRT3 Activities. The Fluor de Lys fluorescence assays were
based on the method described in the BioMol product sheet (Enzo
Life Sciences) using the BioMol KI177 substrate for SIRT1 and the
KI179 substrate for SIRT2 and SIRT3. The determined Km value of
SIRT1 for KI177 was 58 μM, and the Km of SIRT2 for KI179 was 198
μM.42 The Km of SIRT3 for KI179 was reported by BioMol to be 32
μM. The Km values of SIRT1, SIRT2 and SIRT3 for NAD+ were
reported by BioMol to be 558 μM, 547 μM, and 2 mM, respectively.
Briefly, assays were carried out using the Fluor de Lys acetylated
peptide substrate at a concentration corresponding to 0.7 Km and
NAD+ (N6522, Sigma) at a concentration corresponding to 0.9 Km,
recombinant GST-SIRT1/2-enzyme or recombinant His-SIRT3 and
3,8-Dibromo-6-chloro-2-pentylchroman-4-one (2).28 Chro-
man-4-one 1a (0.20 mg, 0.6 mmol) in CH2Cl2 (5 mL) was added
to Py·Br3 (0.21 g, 0.66 mmol) in CH2Cl2 (5 mL), and the mixture was
stirred for 2.5 h at room temperature. After dilution with CH2Cl2, the
organic phase was washed with brine and water. The organic phase
was dried over Na2SO4, filtered, and concentrated under vacuum.
Purification by flash column chromatography using toluene:hexane
(5→50%) gave 2 (0.20 g, 81%) as an off-white solid consisting of a
1
80:20 mixture of cis/trans-isomers. H NMR δ 7.87 (d, J = 2.6 Hz,
0.8H), 7.84 (d, J = 2.6 Hz, 0.2H), 7.77−7.76 (m, 1H), 4.71 (m, 0.2H),
G
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
SIRT assay buffer (HDAC assay buffer, KI143, supplemented with 1
mg/mL BSA, A3803, Sigma). GST-SIRT1 and GST-SIRT2 were
produced as described previously.43,44 His-SIRT3 (BML-SE270) was
purchased from Enzo Life Sciences. The buffer, Fluor de Lys
acetylated peptide substrate, NAD+, and DMSO/compounds in
DMSO (2.5 μL in 50 μL total reaction volume; DMSO from Sigma,
D2650) were preincubated for 5 min at room temperature. The
reaction was started by adding the enzyme. The reaction mixture was
incubated for 1 h at 37 °C. After that, Fluor de Lys developer (KI176)
and nicotinamide (2 mM in HDAC assay buffer giving total volume of
50 μL) were added, and the incubation was continued for 45 min at 37
°C. Fluorescence readings were obtained using a VictorTM 1420
Multilabel Counter (Wallac, Finland) with excitation wavelength 355
nm and emission 460 nm.
spectra for each enantiomer were acquired for 20 h for (+)-1a) and 15
h for (−)-1a in a BioTools ChiralIR-2X instrument equipped with a
single photoelastic modulator. The resolution was 4 cm−1. DFT
calculations: A Monte Carlo molecular mechanics search for low
energy geometries was conducted for both enantiomers of the
truncated structure using MacroModel within the Maestro graphical
interface (Schrodinger Inc.). All conformers within 5 kcal/mol of the
̈
lowest energy conformer were used as starting points for DFT
minimizations within Gaussian09. Optimized structures and VCD
spectra were determined for each conformer. In these calculations, the
B3LYP generalized gradient approximation (GGA) exchange-correla-
tion density functional was used. The 6-31G* basis set was used.
The IC50 values were based on nine-point dose−response
determinations (2000 μM, 1000 μM, 100 μM, 10 μM, 1 μM, 0.1
μM, 0.01 μM, 0.001 μM, and 0.0001 μM) where more dose points
were added between the critical concentrations depending on the
compound. Each experiment was repeated at least three times, and
values were calculated using the Graph Pad Prism Software version
4.03 ( 19922005 GraphPad Software, Inc.).
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures (1a, 1n−p, 3b), characterization data and
elemental analysis for all tested compounds, complete list of
SIRT1−3 activity assay results, separation of enantiomers, IR
1
spectrum of 1a, and H NMR and 13C NMR spectra of all
In Vitro 14C-NAD+ Assay for SIRT2 Activity. Inhibition of SIRT2
by compound 1a was verified by a protocol based on release of 14C-
nicotinamide from NAD+ during the reaction.45 Radioactive
nicotinamide was detected using a TLC-based technique.46 The
assay was carried out using [carbonyl-14C]NAD+ (43.2 nCi)
(Amersham Pharmacia, CFA372), acetylated RSTGGK(Ac)APRKQ
peptide (196 μM) (purchased from Caslo Laboratory ApS), and SIRT
assay buffer and DMSO (0.7 μL) or compound 1a in DMSO. The
final reaction concentration of 1a was 200 μM. The total reaction
volume was 9.7 μL, and reactions were started by adding GST-
SIRT2.43,44 The reaction mixture was incubated for 1 h at 37 °C. The
entire volume of the reaction mixture was spotted on a TLC plate
(Whatman, PE SIL G). Plates were eluted with ethanol/ammonium
acetate (2.5 M) (80:20), dried, and exposed overnight to a medical X-
ray film (SuperRX-film, Fujifilm Corporation, Tokyo, Japan),
developed with a Kodak X-OMAT 2000 developing machine
(Eastman Kodak Company, Rochester, NY) and scanned with an
Epson Perfection V750 Pro scanner (Epson America Inc., Long Beach,
CA) using the Jasc Paint Shop Pro 8 software (Jasc Software Inc.,
Eden Prairie, MN). Images were processed with the use of the UN-
SCAN-IT gelTM software (Silk Scientific Corporation, Orem, UT).
Western Blot Analysis for Acetylated α-Tubulin. Western blot
analysis was carried out as described previously.47 Briefly, acetylated α-
tubulin (0.5 μg) isolated from rat stem cells was incubated 30 or 60
min at 37 °C (total volume of reaction mixture 50 μL) with
recombinant GST-SIRT2 enzyme (3 μL), NAD+ (500 μM), SIRT-
assay buffer (41 μL), and DMSO (2.5 μL) or 1a in DMSO. The final
reaction concentration of 1a was 200 μM. The GST-SIRT2 enzyme
was prepared as described previously.43,44
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(K.L.) Tel: +46 31 786 9031; fax: +46 31 772 1394; e-mail:
luthman@chem.gu.se. (E.M.J.) Tel: +358 40 355 2460; fax:
+358 17 162424; e-mail: Elina.Jarho@uef.fi.
Author Contributions
‡These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Swedish Research Council (project no. 2010-
4846), the Academy of Finland (grants no. 127062 and
132780) and the Department of Chemistry and Molecular
Biology, University of Gothenburg, for financial support and
Biocenter Kuopio for providing facilities. We also thank Karin
Nilsson for skillful assistance in the lab and Susanne Olsson for
help with the VCD spectra. This work is part of COST Action
TD09056: “Epigenetics: Bench to Bedside”.
Samples were solubilized in NuPAGE LDS sample buffer (NP0007,
Invitrogen, Carlsbad, CA), boiled for 4 min, resolved at 200 V on a
10% SDS-PAGE gel, and electrophoretically transferred to an ECL-
nitrocellulose membrane (Amersham, GE Healthcare, London, UK).
Blots were blocked for 1 h with nonfat dry milk (3%) in PBS
(phosphate-buffered saline) and Tween-20 (0.05%). Membranes were
probed for 2 h at room temperature with monoclonal antiacetylated
tubulin antibody (Clone 6-11B-1, Sigma, T 6793, 1:8000), washed,
and incubated for 2 h with horseradish peroxidase-conjugated donkey-
antimouse (1:50000, Amersham, NA934OV) secondary antibody.
Results were detected by Immobilon Western Chemiluminescent HRP
Substrate (ECL-kit, Millipore, Billerica, MA). Filters were exposed to a
medical X-ray film (SuperRX-film), developed using a Kodak X-
OMAT 2000 developing machine, and scanned with the Epson
Perfection V750 Pro scanner using the Jasc Paint Shop Pro 8 software.
Images were processed with the use of the UN-SCAN-IT gel software.
Determination of Absolute Configuration of (+)- and (−)-1a
Using VCD. Experimental: Each sample was prepared by dissolving 12
mg of the solid material in 150 μL of CDCl3. Subsequently, the
solutions were transferred to a 0.75 mm BaF2 cell, and the VCD
ABBREVIATION USED
■
BSA, bovine serum albumin; DFT, density functional theory;
DIPA, N,N-diisopropylamine; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; ECL, enhanced chemilumines-
cence; ESI-MS, electrospray ionization mass spectrometry;
GST, glutathione-S-transferase; HDAC, histone deacetylase;
HPLC, high performance liquid chromatography; HRP,
horseradish peroxidase; IC50, the half maximal inhibitory
concentration; KHMDS, potassium hexamethyldisilazane;
LDS, lithium dodecyl sulfate; MW, microwave; NAD+,
nicotinamide adenine dinucleotide; NMR, nuclear magnetic
resonance; PBS, phosphate-buffered saline; Py, pyridine; SAR,
structure−activity relationship; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; SIRT, silent
information regulator human type; THF, tetrahydrofuran;
TLC, thin layer chromatography; p-TSA, p-toluenesulfonic
acid; UV, ultraviolet; VCD, vibrational circular dichroism
H
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
selective tumor cytotoxicity. Biochem. Biophys. Res. Commun. 2009,
386, 729−733.
(23) Medda, F.; Russell, R. J. M.; Higgins, M.; McCarthy, A. R.;
Campbell, J.; Slawin, A. M. Z.; Lane, D. P.; Lain, S.; Westwood, N. J.
Novel Cambinol Analogs as Sirtuin Inhibitors: Synthesis, Biological
Evaluation, and Rationalization of Activity. J. Med. Chem. 2009, 52,
2673−2682.
(24) Taylor, D. M.; Balabadra, U.; Xiang, Z. M.; Woodman, B.;
Meade, S.; Amore, A.; Maxwell, M. M.; Reeves, S.; Bates, G. P.; Luthi-
Carter, R.; Lowden, P. A. S.; Kazantsev, A. G. A Brain-Permeable Small
Molecule Reduces Neuronal Cholesterol by Inhibiting Activity of
Sirtuin 2 Deacetylase. ACS Chem. Biol. 2011, 6, 540−546.
(25) Harborne, J. B.; Williams, C. A. Advances in flavonoid research
since 1992. Phytochemistry 2000, 55, 481−504.
(26) Horton, D. A.; Bourne, G. T.; Smythe, M. L. The combinatorial
synthesis of bicyclic privileged structures or privileged substructures.
Chem. Rev. 2003, 103, 893−930.
REFERENCES
■
(1) Imai, S.; Armstrong, C. M.; Kaeberlein, M.; Guarente, L.
Transcriptional silencing and longevity protein Sir2 is an NAD-
dependent histone deacetylase. Nature 2000, 403, 795−800.
(2) Sauve, A. A. Sirtuin chemical mechanisms. Biochim. Biophys. Acta
2010, 1804, 1591−1603.
(3) Guarente, L.; Nakagawa, T. Sirtuins at a glance. J. Cell. Sci. 2011,
124, 833−838.
(4) Taylor, D. M.; Maxwell, M. M.; Luthi-Carter, R.; Kazantsev, A. G.
Biological and Potential Therapeutic Roles of Sirtuin Deacetylases.
Cell. Mol. Life Sci. 2008, 65, 4000−4018.
(5) Sinclair, D.; Michan, S. Sirtuins in mammals: insights into their
biological function. Biochem. J. 2007, 404, 1−13.
(6) Guarente, L. Sirtuins, Aging, and Medicine. N. Engl. J. Med. 2011,
364, 2235−2244.
(7) Luthi-Carter, R.; Taylor, D. M.; Pallos, J.; Lambert, E.; Amore, A.;
Parker, A.; Moffitt, H.; Smith, D. L.; Runne, H.; Gokce, O.; Kuhn, A.;
Xiang, Z. M.; Maxwell, M. M.; Reeves, S. A.; Bates, G. P.; Neri, C.;
Thompson, L. M.; Marsh, J. L.; Kazantsev, A. G. SIRT2 inhibition
achieves neuroprotection by decreasing sterol biosynthesis. Proc. Natl.
Acad. Sci. U.S.A. 2010, 107, 7927−7932.
(27) Saengchantara, S. T.; Wallace, T. W. Chromanols, Chroma-
nones, and Chromones. Nat. Prod. Rep. 1986, 3, 465−475.
(28) Friden-Saxin, M.; Pemberton, N.; Andersson, K. D.; Dyrager,
́
C.; Friberg, A.; Grøtli, M.; Luthman, K. Synthesis of 2-Alkyl-
Substituted Chromone Derivatives Using Microwave Irradiation. J.
Org. Chem. 2009, 74, 2755−2759.
(8) Kazantsev, A.; Outeiro, T. F.; Marques, O. Therapeutic role of
sirtuins in neurodegenerative disease. Biochim. Biophys. Acta 2008,
1782, 363−369.
(9) Superti-Furga, G.; Huber, K. After the grape rush: Sirtuins as
epigenetic drug targets in neurodegenerative disorders. Bioorg. Med.
Chem. 2011, 19, 3616−3624.
(10) Zhu, X. W.; Bonda, D. J.; Lee, H. G.; Camins, A.; Pallas, M.;
Casadesus, G.; Smith, M. A. The sirtuin pathway in ageing and
Alzheimer disease: mechanistic and therapeutic considerations. Lancet
Neurol. 2011, 10, 275−279.
(11) Guarente, L.; Donmez, G.; Wang, D.; Cohen, D. E. SIRT1
Suppresses beta-Amyloid Production by Activating the alpha-Secretase
Gene ADAM10. Cell 2010, 142, 320−332.
(12) Milne, J. C.; Denu, J. M. The Sirtuin family: therapeutic targets
to treat diseases of aging. Curr. Opin. Chem. Biol. 2008, 12, 11−17.
(13) Dryden, S. C.; Nahhas, F. A.; Nowak, J. E.; Goustin, A. S.;
Tainsky, M. A. Role for human SIRT2 NAD-dependent deacetylase
activity in control of mitotic exit in the cell cycle. Mol. Cell. Biol. 2003,
23, 3173−3185.
(14) Inoue, T.; Hiratsuka, M.; Osaki, M.; Oshimura, M. The
molecular biology of mammalian SIRT proteins - SIRT2 in cell cycle
regulation. Cell Cycle 2007, 6, 1011−1018.
(15) North, B. J.; Marshall, B. L.; Borra, M. T.; Denu, J. M.; Verdin,
E. The human Sir2 ortholog, SIRT2, is an NAD(+)-dependent tubulin
deacetylase. Mol. Cell 2003, 11, 437−444.
(16) Pavletich, N. P.; Finnin, M. S.; Donigian, J. R. Structure of the
histone deacetylase SIRT2. Nat. Struct. Biol. 2001, 8, 621−625.
(17) Schemies, J.; Sippl, W.; Jung, M. Histone deacetylase inhibitors
that target tubulin. Cancer Lett. 2009, 280, 222−232.
(18) Garske, A. L.; Smith, B. C.; Denu, J. M. Linking SIRT2 to
Parkinson’s disease. ACS Chem. Biol. 2007, 2, 529−532.
(19) Outeiro, T. F.; Kontopoulos, E.; Altmann, S. M.; Kufareva, I.;
Strathearn, K. E.; Amore, A. M.; Volk, C. B.; Maxwell, M. M.; Rochet,
J. C.; McLean, P. J.; Young, A. B.; Abagyan, R.; Feany, M. B.; Hyman,
B. T.; Kazantsev, A. G. Sirtuin 2 inhibitors rescue alpha-synuclein-
mediated toxicity in models of Parkinson’s disease. Science 2007, 317,
516−519.
́ ́
(29) Dahlen, K.; Wallen, E. A. A.; Grøtli, M.; Luthman, K. Synthesis
of 2,3,6,8-tetrasubstituted chromone scaffolds. J. Org. Chem. 2006, 71,
6863−6871.
́
(30) Dahlen, K.; Grøtli, M.; Luthman, K. A scaffold approach to
3,6,8-trisubstituted flavones. Synlett 2006, 897−900.
(31) Wallen, E. A. A.; Dahlen, K.; Grøtli, M.; Luthman, K. Synthesis
́
́
of 3-aminomethyl-2-aryl-8-bromo-6-chlorochromones. Org. Lett. 2007,
9, 389−391.
́
(32) Ankner, T.; Friden-Saxin, M.; Pemberton, N.; Seifert, T.; Grøtli,
M.; Luthman, K.; Hilmersson, G. KHMDS Enhanced SmI2-Mediated
Reformatsky Type α-Cyanation. Org. Lett. 2010, 12, 2210−2213.
(33) Unpublished results.
(34) Howitz, K. T.; Bitterman, K. J.; Cohen, H. Y.; Lamming, D. W.;
Lavu, S.; Wood, J. G.; Zipkin, R. E.; Chung, P.; Kisielewski, A.; Zhang,
L. L.; Scherer, B.; Sinclair, D. A. Small molecule activators of sirtuins
extend Saccharomyces cerevisiae lifespan. Nature 2003, 425, 191−196.
(35) Borra, M. T.; Smith, B. C.; Denu, J. M. Mechanism of human
SIRT1 activation by resveratrol. J. Biol. Chem. 2005, 280, 17187−
17195.
(36) Pacholec, M.; Bleasdale, J. E.; Chrunyk, B.; Cunningham, D.;
Flynn, D.; Garofalo, R. S.; Griffith, D.; Griffor, M.; Loulakis, P.; Pabst,
B.; Qiu, X. Y.; Stockman, B.; Thanabal, V.; Varghese, A.; Ward, J.;
Withka, J.; Ahn, K. SRT1720, SRT2183, SRT1460, and Resveratrol
Are Not Direct Activators of SIRT1. J. Biol. Chem. 2010, 285, 8340−
8351.
(37) First eluting enantiomer: [α]D +82.5 (c = 0.2, hexane, 23 °C),
second eluting enantiomer: [α]D −92.5 (c = 0.2, hexane, 23 °C)).
(38) Reddy, N. J.; Sharma, T. C. Synthesis and Reduction of 3-
Bromoflavanones. Bull. Chem. Soc. Jpn. 1979, 52, 647−648.
(39) Compound 2 has not been tested, as the present study is
focusing on changes in the substitution pattern in the aromatic ring
and the 2-position of the chroman-4-one scaffold.
(40) Freedman, T. B.; Cao, X. L.; Dukor, R. K.; Nafie, L. A. Absolute
configuration determination of chiral molecules in the solution state
using vibrational circular dichroism. Chirality 2003, 15, 743−758.
(41) Stephens, P. J.; Devlin, F. J.; Pan, J. J. The determination of the
absolute configurations of chiral molecules using vibrational circular
dichroism (VCD) spectroscopy. Chirality 2008, 20, 643−663.
(20) Jung, M.; Trapp, J.; Jochum, A.; Meier, R.; Saunders, L.;
Marshall, B.; Kunick, C.; Verdin, E.; Goekjian, P.; Sippl, W. Adenosine
mimetics as inhibitors of NAD(+)-dependent histone deacetylases,
from kinase to sirtuin inhibition. J. Med. Chem. 2006, 49, 7307−7316.
(21) Altucci, L.; Rotili, D.; Carafa, V.; Tarantino, D.; Botta, G.;
Nebbioso, A.; Mai, A. Simplification of the tetracyclic SIRT1-selective
inhibitor MC2141: Coumarin- and pyrimidine-based SIRT1/2
inhibitors with different selectivity profile. Bioorg. Med. Chem. 2011,
19, 3659−3668.
́
(42) Kiviranta, P. H.; Suuronen, T.; Wallen, E. A. A.; Leppanen, J.;
̈
Tervonen, J.; Kyrylenko, S.; Salminen, A.; Poso, A.; Jarho, E. M. N-ε-
Thioacetyl-Lysine-Containing Tri-, Tetra-, and Pentapeptides as
SIRT1 and SIRT2 Inhibitors. J. Med. Chem. 2009, 52, 2153−2156.
(43) Kiviranta, P. H.; Leppanen, J.; Rinne, V. M.; Suuronen, T.;
̈
Kyrylenko, O.; Kyrylenko, S.; Kuusisto, E.; Tervo, A. J.; Jarvinen, T.;
(22) Zhang, Y. J.; Au, Q. Y.; Zhang, M. H.; Barber, J. R.; Ng, S. C.;
Zhang, B. Identification of a small molecule SIRT2 inhibitor with
̈
́
Salminen, A.; Poso, A.; Wallen, E. A. A. N-(3-(4-hydroxyphenyl)-
I
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
propenoyl)-amino acid tryptamides as SIRT2 inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 2448−2451.
(44) Tervo, A. J.; Kyrylenko, S.; Niskanen, P.; Salminen, A.;
Leppanen, J.; Nyronen, T. H.; Jarvinen, T.; Poso, A. An in silico
̈
̈
approach to discovering novel inhibitors of human sirtuin type 2. J.
Med. Chem. 2004, 47, 6292−6298.
(45) McDonagh, T.; Hixon, J.; DiStefano, P. S.; Curtis, R.; Napper,
A. D. Microplate filtration assay for nicotinamide release from NAD
using a boronic acid resin. Methods 2005, 36, 346−350.
(46) Tanny, J. C.; Moazed, D. Coupling of histone deacetylation to
NAD breakdown by the yeast silencing protein Sir2: Evidence for
acetyl transfer from substrate to an NAD breakdown product. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 415−420.
(47) Huhtiniemi, T.; Suuronen, T.; Rinne, V. M.; Wittekindt, C.;
Lahtela-Kakkonen, M.; Jarho, E.; Wallen
́
, E. A. A.; Salminen, A.; Poso,
A.; Leppanen, J. Oxadiazole-carbonylaminothioureas as SIRT1 and
̈
SIRT2 inhibitors. J. Med. Chem. 2008, 51, 4377−4380.
J
dx.doi.org/10.1021/jm3005288 | J. Med. Chem. XXXX, XXX, XXX−XXX