Novel cambinol analogs as sirtuin inhibitors: Synthesis, biological evaluation, and rationalization of activity

Article abstract of DOI:10.1021/jm8014298

The tenovins and cambinol are two classes of sirtuin inhibitor that exhibit antitumor activity in preclinical models. This report describes modifications to the core structure of cambinol, in particular by incorporation of substitutents at the N1-position, which lead to increased potency and modified selectivity. These improvements have been rationalized using molecular modeling techniques. The expected functional selectivity in cells was also observed for both a SIRT1 and a SIRT2 selective analog.

Full text of DOI:10.1021/jm8014298

J. Med. Chem. 2009, 52, 2673–2682
2673
Novel Cambinol Analogs as Sirtuin Inhibitors: Synthesis, Biological Evaluation, and
Rationalization of Activity
Federico Medda,† Rupert J. M. Russell,‡ Maureen Higgins,§ Anna R. McCarthy,†,§ Johanna Campbell,§ Alexandra M.
Z. Slawin,† David P. Lane,§ Sonia Lain,*,§ and Nicholas J. Westwood*,†
School of Chemistry and Centre for Biomolecular Sciences and School of Biology and Centre for Biomolecular Sciences, UniVersity of St
Andrews, St Andrews, Fife, KY16 9ST, Scotland, U.K., and Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical
School, UniVersity of Dundee, Dundee DD1 9SY, Scotland, U.K.
ReceiVed NoVember 13, 2008
The tenovins and cambinol are two classes of sirtuin inhibitor that exhibit antitumor activity in preclinical
models. This report describes modifications to the core structure of cambinol, in particular by incorporation
of substitutents at the N1-position, which lead to increased potency and modified selectivity. These
improvements have been rationalized using molecular modeling techniques. The expected functional selectivity
in cells was also observed for both a SIRT1 and a SIRT2 selective analog.
Introduction
modifications to the structure of 1 that lead to an increase in
potency. Additional studies aimed at exploring the effect of
structural modifications on the ability of 1 to inhibit selectively
SIRT1 over other members of the sirtuin family, in particular
SIRT2, were planned. SIRT2 is a predominantly cytoplasmic
protein that colocalizes with microtubules and has been shown
to promote deacetylation of R-tubulin at Lys40 both in vitro
and in vivo.6a,6b In addition, knockdown of SIRT2 via siRNA
results in tubulin hyperacetylation, and SIRT2 overexpression
abrogated microtubule hyperacetylation and resistance to axonal
degeneration.6b Conversely, SIRT2 knockdown by using a
lentiviral vector expressing small interfering RNA enhanced
microtubule acetylation and resistance to axonal degeneration.6c
Small molecule inhibition of SIRT2 has been linked to potential
treatments for Parkinson’s disease.7
The central role that p53 plays in preventing tumor develop-
ment is clear although ongoing research continues to dissect
the details of how exactly this is achieved. Furthermore, a role
for p53 in development, longevity, and overall fitness of an
organism is starting to emerge.1 p53 acts as a transcriptional
regulator, inducing the expression of a range of antiproliferative
target genes. More than 50% of adult human tumors are
characterized by inactivating mutations or deletions of the p53
gene. Other tumor types in which p53 is wild-type frequently
have alterations in the mechanisms that control p53 activation.
It is widely accepted that activation of the p53 tumor suppressor
protein through the use of nongenotoxic compounds may prove
therapeutically important. To date, our approach for identifying
novel p53 activators has been based on a cell-based high-
throughput reporter gene assay2 that we have used to identify a
novel class of nongenotoxic p53 activators known as the
tenovins (Scheme 1B).3
The splitomicin8,3 derivative 1 (see Scheme 1B for structure
of splitomicin) inhibits SIRT1 and SIRT2 in vitro with IC50
values of 56 and 59 µM, respectively, demonstrating moderate
potency and no selectivity.5 Only one analog of 1 has been
reported to date in which replacement of the ꢀ-naphthol ring in
1 by a phenol ring led to the loss of inhibitory activity against
both SIRT1 and SIRT2.5
Tenovins function through the inhibition of a group of NAD+-
dependent protein deacetylases known as the sirtuins (HDACa class
III).3 To date, one sirtuin family member, SIRT1, is known to
regulate p53 activity by deacetylating this protein at Lys382.4a-c
Cells derived from SIRT1 deficient mice and cells treated with
siRNAs against SIRT1 show high levels of hyperacetylated
p53.4d,e Acetylation of p53 is known to augment its DNA
binding ability.4f In addition, a dominant-negative SIRT1 mutant
increases p53-dependent transcriptional activity.3,4c Partly due
to its ability to decrease p53 function, SIRT1 is believed to
represent an important target for cancer treatment with two
reported sirtuin inhibitors, 1 (cambinol)5 and tenovin-6, showing
antitumor activity in preclinical models.3,5 Here we report
optimization studies on 1 (Scheme 1). The goal was to identify
Synthesis of 1 and Its Analogs. An authentic sample of 1
was prepared using a previously reported route, giving 1 in 42%
yield over three steps (Scheme 1).9 Initial Knoevenagel con-
densation of 2 (2-hydroxy-1-naphthaldehyde) with 3a (ethyl
benzoylacetate) afforded 4a (3-benzoyl-5,6-benzocoumarin).
Subsequent conjugate reduction of 4a using sodium borohydride
in pyridine10 gave the saturated lactone 5a, which was then
converted to 1 upon treatment with excess thiourea in the
presence of sodium ethoxide. An analogous approach was used
to prepare a set of analogs of 1 in which (i) the thiocarbonyl
functionality in 1 was replaced by a carbonyl (6a); (ii) electron-
withdrawing (e.g., bromine in 6b) or electron-donating (e.g.,
methoxy in 6c) substituents were incorporated in the para-
position of the phenyl ring in 1; (iii) the ꢀ-naphthol ring in 1
was replaced by a biphenyl-4-ol group (6d), and (iv) a methyl
group (R2 ) Me, 6e) was incorporated at the N1 position in 1.
In brief, replacement of thiourea with urea enabled the conver-
sion of 5a to 6a, and analogs 6b and 6c were prepared from
the corresponding commercially available ethyl benzoylacetates
3b and 3c, respectively. In order to prepare analog 6d, 7 (3-
benzoyl-6-phenylchroman-2-one) (Scheme 1) was synthesized
* To whom correspondence should be addressed. Phone: +44-1334-
463816. Fax: +44-1334-462595. E-mail: njw3@st-andrews.ac.uk. Current
address for Dr. S. Lain: Department of Microbiology, Tumour and Cell
Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden. Phone: +46-
852-487219. Fax: +46-830-304276. E-mail: sonia.lain@ki.se.
†
School of Chemistry and Centre for Biomolecular Sciences, University
of St Andrews.
‡
School of Biology and Centre for Biomolecular Sciences, University
of St Andrews.
§
University of Dundee.
a Abbreviations: SIRT1, sirtuin 1; SIRT2, sirtuin 2; HDAC, histone
deacetylase; HMBC, heteronuclear multiple-bond correlation.
10.1021/jm8014298 CCC: $40.75
2009 American Chemical Society
Published on Web 04/01/2009

2674 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Medda et al.
Scheme 1. (A) Synthesis of 1 and 6a–ja and (B) Structural
from 5-bromo-2-hydroxybenzaldehyde in 55% yield over 2
steps.11 Conversion of 7 to the desired analog 6d was carried
out by conjugate reduction and subsequent reaction with thiourea
in the presence of base.
Comparison of Tenovin-6 and Splitomicin
Synthesis of N1 Substituted Analogs of 1. Analog 6e was
prepared by reaction of 5a with N-methylthiourea and sodium
ethoxide. Although the yield for formation of 6e was low due
to the formation of additional side products,11 no evidence could
be found to support formation of the N3-methyl isomer of 6e.
Structural assignment of 6e was achieved using 2D [1H-13C]
HMBC correlation analysis.11 The observed regiochemistry may
be rationalized by initial reaction of the most nucleophilic
nitrogen atom in N-methylthiourea with the most reactive
carbonyl group, the aryl ketone, in 5a. The encouraging
inhibition and selectivity data for SIRT2 shown by 6e (Table
1) led to the synthesis of further analogs in which different N1-
aliphatic chains were incorporated (R2 in Scheme 1). Initial plans
included analogs with alkyl, benzyl, and aromatic substituents
at N1. However, limitations in the synthetic method restricted
the set of analogs that could be prepared rapidly to four analogs
(6f, g, h, and j).11
Parallel Synthesis Approach. Encouraged by the yields and
straightforward workup associated with the synthesis of analogs
of 1 and the initial observation that incorporation of substituents
into the phenyl ring of 1 resulted in tuning of the required
biological activity (Table 1), it was decided to prepare additional
analogs using a parallel synthesis approach. The commercial
availability of a range of ethyl benzoylacetates enabled the rapid
incorporation of different electron-withdrawing or electron-
donating substituents at the required positions (Table 1).
Nineteen ethyl benzoylacetates (3i-ix) were loaded with 2 into
different vessels of a parallel synthesis apparatus. Ethanol and
piperidine were then added, and in 18 of the 19 cases, the desired
products (4i-xviii) formed upon heating in good yields
(45-98%) as yellow precipitates. The 2-pyridyl-containing
substrate 3xix (Scheme 1) failed to react. Subsequent reduction
of 4i-xviii afforded 15 1,2-dihydroketocoumarins 5i-xv,
following precipitation of the products upon addition of aqueous
hydrochloric acid (2 M) to the crude reaction mixture. Analyti-
cally pure samples of 5i-xv were obtained by recrystallization
from ethanol in a wide range of yields (35-95%). Interestingly,
1H NMR analysis of the ortho-substituted analog 5vii (R1 )
o-Br) showed that it exists predominantly as the enol-lactone
isomer.11 In the next step, a modified protocol for substrates
5i-xv was used compared to that employed in the preparation
of 1. Addition of the sodium ethoxide solution as the final step
and heating under reflux for 18 h was followed by removal of
the solvent in parallel and dissolution of the resulting solids in
water. Subsequent acidification of the aqueous solutions afforded
11 new analogs 6i-xi in moderate yields after purification by
column chromatography.
a Reagents and conditions: (i) piperidine, ethanol, reflux, 2 h (4a, 86%;
4b, 87%; 4c, 95%); (ii) NaBH4, pyridine, rt, 2 h (5a, 95%; 5b, 93%; 5c,
76%); 7 was reduced in 95%; (iii) Na, ethanol, thiourea (for 1, 51%; 6b,
43%; 6c, 42%; 6d, 40%) or urea (6a, 20%) or N-methylthiourea (6e, 16%),
reflux, 18 h. For substituents in compounds i-xi and 6f, g, h, and j, see
Table 1; for compounds xii-xix, R1 ) p-F (xii); m-I (xiii); m-NO2 (xiv);
phenyl ring replaced by 2-furyl (xv); o-Cl (xvi); o-I (xvii); o-NO2 (xviii);
phenyl ring replaced by 2-pyridyl (xix).
(6i-6iii), with these three analogs clearly demonstrating selec-
tive inhibition of SIRT1. The p-bromo-analog 6b also exhibits
relatively selective inhibition of SIRT1 (IC50 ) 13 µM)
compared to SIRT2 (IC50 > 90 µM). The IC50 value attributed
to 6b represents a 3-fold improvement in activity against SIRT1
compared to 1. Replacement of the p-bromo-substituent in 6b
with chlorine, iodine, or trifluoromethyl (6iv, 6v, or 6vi,
respectively) led to a significant decrease in activity against both
enzymes. The position of the bromine atom in 6b was also
important, as SIRT1 activity was lost when this substituent was
placed at either the o- or m-position of the aromatic ring (analogs
6vii and 6viii). Incorporation of a m-chloro-substituent in analog
6ix also reduced activity against SIRT1 and SIRT2. The use of
a fluorine substituent to reduce electron density without increas-
ing steric bulk led to analogs 6x and 6xi, which have comparable
activity to 1 against SIRT1. This implies that the improved
activity of the p-bromo-analog 6b results from an increase in
additional hydrophobic interactions rather than through a
reduction in electron density associated with the aromatic ring.
Previous studies have reported that when the ꢀ-naphthol ring
in 1 is replaced by a phenol ring, activity against SIRT1 and
SIRT2 is lost.5 The observed reduction in activity of the
In Vitro Inhibition of SIRT1 and SIRT2 by Analogs
6i-xi and 6a-6j. 1 and its analogs were tested for in vitro
activity against SIRT1 and SIRT2 using an established fluo-
rescent assay system (Table 1). The IC50 values determined from
our experiments with 1 were similar to those previously
reported5 (41 vs 56 µM5 for SIRT1 and 48 vs 595 µM for
SIRT2). One of the key structural modifications we explored
was the incorporation of functional groups into the phenyl ring
of 1. In brief, the introduction of a p-methoxy substituent in
the phenyl ring of 1 to give 6c led to a loss of activity against
both SIRT1 and SIRT2. Interestingly, only activity against
SIRT2 was lost when a methyl substituent was incorporated at
the para-position or any other position in the aromatic ring

NoVel Cambinol Analogs as Sirtuin Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2675
Table 1. Inhibition (at 60 µM ( SEa (%)) of Sirtuins by the Novel
Analogs 6i-xi and 6a-6j
Recently reported docking studies proposed that 1 binds in
the nicotinamide C-subpocket of the catalytic domain of
SIRT2.16 While no direct evidence to support this binding mode
was provided, our attempts to identify a binding mode for 1
using GOLD led to an analogous result. In our hands, 1 gives
the lowest energy solutions when bound in the C-pocket,
oriented such that the ꢀ-napthol ring is sandwiched between
the two aromatic residues Phe119 and His187 with π-stacking
interactions probably contributing to the calculated stability of
this binding mode. It is in this pocket that the acetylated lysine
of SIRT2 substrates binds. In addition, we observed that the
polar component of 1, represented by the carbonyl and thio-
carbonyl functionalities and the two nitrogen atoms, has the
potential to form hydrogen bonds to the active site. Although
our model did not include water molecules in the active site,
the predicted orientation of this part of the ligand is in close
agreement with that proposed previously. It is of interest that
our small molecule X-ray structure of 1 in the absence of protein
identifies the existence of an intramolecular hydrogen bond
between the phenolic OH and the carbonyl functionality.11 When
this conformation of 1 was used as the starting point for the
docking studies, rather than the lowest energy conformation
predicted by the PRODRG server, analogous results were
obtained suggesting that loss of this hydrogen bond can be
accommodated for on binding to the protein. In general, visual
analysis of the highest scoring docking poses for the new analogs
of 1 showed a very similar situation to that previously reported
and found by us for 1 itself, with all the new inhibitors showing
the same preferred binding mode with the ꢀ-naphthol ring
sandwiched in the previously identified hydrophobic channel
between Phe119 and His187. Modest differences in the con-
formation of the ꢀ-naphthol moiety of the inhibitors were
observed.
b
b
compd
R1
R2
X
SIRT1
IC50
SIRT2
IC50
1
H
H
p-Br
p-CH3O
H
o-CH3
m-CH3
p-CH3
p-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
S
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
59.5 ( 1
15.5 ( 2
82.3 ( 1
6.4 ( 1
37.8 ( 1
79.6 ( 1
83.2 ( 4
79.0 ( 2
13.7 ( 1
7.7 ( 1
15.1 ( 1
19.7 ( 1
4.8 ( 1
11.3 ( 1
89.0 ( 1
87.8 ( 1
40.7 ( 11 51.9 ( 1 47.9 ( 12
6.5 ( 4
6a
6b
6c
6d
6i
6ii
6iii
6iv
6v
6vi
6vii
12.7 ( 2
9.4 ( 1 >90
9.2 ( 2
52.7 ( 2
43.0 ( 2 29.1 ( 2
44.2 ( 2 12.7 ( 2
44.5 ( 1 13.4 ( 3
20.1 ( 1
p-I
p-CF3
o-Br
7.7 ( 1
6.7 ( 1
6viii m-Br
6.6 ( 3
7.6 ( 6
50.0 ( 1 19.5 ( 1
38.3 ( 1 52.1 ( 1
6ix
6x
6xi
6e
6f
6g
6h
6j
m-Cl
o-F
m-F
H
H
H
Me
Et
allyl
n-prop
n-But
29.4 ( 1 > 90
31.9 ( 1
80.4 ( 1 20.1 ( 5
86.8 ( 1 10.5 ( 3
88.3 ( 1 22.2 ( 1
37.5 ( 1
H
H
25.0 ( 2
94.7 ( 1
97.6 ( 1
4.8 ( 2
1.0 ( 1
16.9 ( 1
a SE, standard error (n ) 2). b IC50 values were determined for
compounds that had over 60% inhibition at 60 µM for SIRT1 and SIRT2
(repeated at least twice).
biphenyl-4-ol-containing analog 6d against SIRT1 compared to
1 further supports the view that the ꢀ-naphthol ring in 1 is
important for activity against this enzyme, although the observed
activity against SIRT2 suggests that this isoform is more tolerant
to change at this position in 1. Substitution of the thiocarbonyl
functionality in 1 for a carbonyl (6a) resulted in significantly
reduced activity against both enzymes.
The second key structural question that we looked to address
involved the requirement for the N1-hydrogen. The presence
of a N1-methyl substituent in analog 6e led to an increase in
activity against SIRT2 (IC50 ) 20 µM, cf. 48 µM for 1) and to
a decrease in potency against SIRT1 (IC50 > 90 µM for 6e). As
a result of these changes in activity compared to 1, 6e is a
selective SIRT2 inhibitor in vitro. This result inspired the
synthesis of four other analogs 6f, g, h, and j with different
aliphatic chains at the N1-position. Interestingly, a steady
improvement in potency against SIRT2 was observed as the
chain length increased, culminating in the N1-butyl analog 6j
being the most potent inhibitor of SIRT2 identified to date in
this inhibitor class with an IC50 ) 1.0 µM for SIRT2, while
demonstrating poor inhibition of SIRT1 (Table 1). The majority
of the synthetic intermediates prepared en route to analogs of 1
were also tested against SIRT1 at 60 µM concentration due to
the structural similarity with splitomicin.11 No active compounds
were identified.
Identification of a Potential Binding Mode. In order to
provide a potential rationalization for the observed in vitro
activity and selectivity of 1 and its analogs, automated ligand
docking studies were carried out using the software GOLD.12
The reported structure of human SIRT213 was used in this study.
All the SIRT2 crystal structures so far determined are character-
ized by a highly conserved catalytic domain of 270 amino acids
that consists of a large classical Rossman-fold domain and a
small zinc binding subdomain. The active site is situated at the
interface between the two domains and is commonly divided
into A, B, and C subpockets into which the substrate and
cofactor bind. No ligand bound structures of human SIRT2 have
been solved, but its homologs from yeast14 and Archaeoglobus
fulgidus15 have been extensively characterized with regard to
the mode of substrate and cofactor binding. To date, no crystal
structure has been reported for SIRT1.
Rationalization of the Observed Increase in SIRT2 Se-
lectivity and Activity Associated with an N1-Substituent. The
improvement in the inhibitory activity and selectivity observed
for analogs 6e and 6f-j against SIRT2 compared to 1 can be
rationalized by the formation of additional hydrophobic interac-
tions between the N1-aliphatic substituent and a previously
unoccupied narrow lipophilic channel around Phe96, Leu138,
and Ile169 in the SIRT2 active site (Figure 1A). The proposed
binding mode of this subset of analogs was observed consistently
during the docking studies. It is interesting to note that a
structure of the Archaeoglobus fulgidus enzyme has been solved
in which this channel is occupied by a pentaethylene glycol
molecule.15 While increasing aliphatic chain lengths lead to
increased SIRT2 activity at least up to a C4 chain, it is difficult
to predict from computational studies what the optimal N1 side
chain will be. For example, it is difficult to explain why the
N1-allyl analog 6g has reduced but still significant activity.
Visual analysis of this newly identified hydrophobic channel
suggests that there is insufficient space to accommodate larger
substituents, such as phenyl or benzyl groups in the human
SIRT2 active site. However, a structure of the yeast enzyme
has been elucidated which shows a nicotinamide molecule
occupying this hydrophobic channel.17a Further studies are
required to fully explore the range of substituents that can
be accommodated.
In the absence of a crystal structure for SIRT1, the selectivity
of the N1-substituted series for SIRT2 over SIRT1 was
addressed via the generation of a homology model of human
SIRT118 based on the crystal structure of human SIRT213 using
the PHYRE server. While the majority of the active site is
predicted to be highly conserved, notable differences in the loop

2676 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Medda et al.
Figure 2. Levels of p53 and acetylated p53 in MCF-7 breast
adenocarcinoma cells treated with different concentrations of cambinol
(1) and 6b in the presence of etoposide. The black line represents the
position of the 51KDa molecular weight marker. Samples were first
analyzed with the K382-acetylated p53 antibody and subsequently
reloaded and analyzed with the antibody against total p53.
Figure 1. (A) Docking solution obtained for N1 substituted analog
6j. The N1-aliphatic carbon chain in 6j is proposed to insert into a
narrow lipophilic channel delimited by Phe96, Leu 138, and Ile169.
(B) A comparison of the amino acid sequence for the 96-loop in human
SIRT1 and SIRT2.
at the base of the site potentially underlie the observed
specificity. Figure 1B shows a sequence alignment of the
residues which comprises this loop in the human SIRT2 and
SIRT1 enzymes. This loop (the 96-loop) contains Phe96, which
lines the lipophilic channel into which the N1-substituent packs.
An amino acid insertion and a two amino acid deletion in SIRT2
compared to SIRT1 subsequent to Phe96 is likely to cause a
structural alteration of this loop and thus cause a difference in
the size and nature of the lipophilic channel. This loop also
contains Arg97, which interacts with the phosphate of an NAD+
analog in the yeast enzyme.17a The yeast and human SIRT2
enzymes, despite having near identical sequences of this loop,
do show conformational differences, reinforcing the concept that
structural differences of this loop between human SIRT2 and
SIRT1 could explain the observed selectivity.
Rationalization of the Observed Variation in Activity
for Analogs Incorporating a Substituent in the Phenyl
Ring of 1. A key conclusion from the SAR data (Table 1) is
the increase in potency and net SIRT1 selectivity associated
with the incorporation of a p-bromo-substituent (6b) into the
phenyl ring of 1. Visual analysis of the docking poses for N1-
substituted analogs suggested that the active site of SIRT2 is
too small to accommodate a phenyl ring bearing a large
p-substituent, consistent with the observed relative lack of
activity against SIRT2 of the p-bromo (6b) and p-iodo (6v)
analogs. Although the majority of the amino acids in this region
are conserved between SIRT2 and SIRT1, again the structural
variation of the 96-loop could account for the observed
selectivity. Within this loop, Tyr104 points directly into the
pocket that is predicted to accommodate the phenyl ring of 1.
Due to the predicted structural alteration of the 96-loop between
SIRT2 and SIRT1, this pocket has the potential to be larger in
SIRT1 compared to SIRT2, with the pocket now being able to
accommodate the p-bromo-substituent.
Figure 3. Western blot analysis of the levels of R-tubulin acetylated
at K40 and total R-tubulin in H1299 cells treated with increasing
concentrations of cambinol (1) and analog 6j. All samples were also
treated with trichostatin A, an inhibitor of class I and II HDACs, to
reduce the background effect of these deacetylases. The black line
represents the position of the 51KDa molecular weight marker. Samples
were first analyzed with the K40-acetylated R-tubulin antibody and
subsequently reloaded and analyzed with the antibody against total
R-tubulin. For a more detailed statistical analysis of this experiment,
see Supporting Information Figure S14.
increased levels of p53 and acetylated p53 in NCI H460 cancer
cells treated with 1 in the presence of the genotoxic agent
etoposide (10 µM). Here we tested our more selective SIRT1
inhibitor 6b for this effect in MCF-7 breast adenocarcinoma
cells (p53 wild type) and compared it to 1. As previously
observed for 1, in the presence of etoposide (10 µM), the levels
of p53, and especially the levels of Lys382 acetylated p53,
increased with 20 and 50 µM concentrations of 1 (Figure 2).
The levels of p53 and acetylated p53 decreased again upon
exposure to 1 at higher concentrations of 200 µM, probably as
a result of toxicity. Treatment of MCF-7 cells with different
concentrations of 6b alone did not increase the levels of p53 in
the absence of etoposide, as was the case for all the analogs of
1 we have tested in this assay to date.11 However, in the presence
of etoposide (10 µM), compound 6b increased total p53 levels
and the levels of p53 acetylated at Lys382 more clearly than 1
(Figure 2).
As expected for a selective SIRT1 inhibitor, 6b did not lead
to an increase in the levels of acetylated R-tubulin (Figure
S1311). As a control, and in agreement with previous reports,
treatment of H1299 cells with the unselective inhibitor 1 did
lead to an increase in levels of acetylated tubulin (Figure 3).
When this experiment was repeated, this time using our
improved and SIRT2 selective inhibitor 6j, an increase in the
levels of acetylated R-tubulin was achieved compared to the
effect of 1 across a concentration range of 10-50 µM (Figure
3 and Figure S14). This experiment could not be carried out at
higher concentrations due to toxicity associated with the use of
6j at these concentrations. While an improvement in the ability
During the course of these studies, we demonstrated that it
was possible to convert the essentially unselective 1 into analogs
that possess either SIRT1 (6b) or SIRT2 (e.g., 6j) selectivity in
vitro. Further evidence to support the selective targeting of
SIRT1 and SIRT2 by these analogs comes from cell-based
studies. In a previous report,5 Bedalov and co-workers detected

NoVel Cambinol Analogs as Sirtuin Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2677
Synthesis of 1 and Analogs 6b and 6c. 1 was prepared via 4a
and 5a as described in Scheme 1 according to the following
generalized protocols. Analytical data for additional compounds
made using these protocols are also provided.
of 6j to increase levels of acetylated R-tubulin in cells compared
to 1 was observed, the effect was less than may have been
expected given the 50-fold difference in IC50 of 6j and 1 against
SIRT2 observed in vitro. Issues relating to cell permeability,
compartmentalization of the compound, or its metabolism may
explain this observation and are currently being assessed.
General Procedure for Preparation of 4a-4c. To a solution
of 2-hydroxy-1-naphthaldehyde (2) (0.86 g, 5 mmol) in ethanol
(10 mL) were added ethyl benzoyl acetate analogs (3a-c, 1.06 g,
5 mmol). Piperidine (15 drops) was added, and the reaction was
heated under reflux for 2 h. After cooling to room temperature, the
product was collected by filtration and washed with ethanol.
2-Benzoyl-3H-benzo[f]chromen-3-one (4a).19,20 Yielded 1.30 g
(4.33 mmol, 86%) as a yellow powder. Mp 208-210 °C (lit.19,20
209 °C). 1H NMR (CDCl3, 300 MHz): δ ) 8.93 (s, 1H, H-1), 8.27
(d, 1H, J ) 8.2 Hz, H-10), 8.12 (d, 1H, J ) 9.0 Hz, H-6), 7.98-7.90
(m, 3H, H-7 + 2 × ArH), 7.77-7.70 (m, 1H, H-9), 7.67-7.59
(m, 2H, H-8, ArH), 7.56-7.47 (m, 3H, H-5 + 2 × ArH). LRMS
[ES]+: m/z 323.06 [M + Na]+ (100%).
Conclusion
A recent report has described 1 as a potential lead for cancer
chemotherapy due to its ability to inhibit the function of the
class III histone deacetylases known as the sirtuins. Here we
describe the results of a SAR study we have carried out on 1.
These studies have indicated that inhibition of the sirtuins is
sensitive to changes in the phenyl ring of 1 leading to the
identification of an analog 6b with improved activity and
increased selectivity for SIRT1. In addition, we have demon-
strated that N1-functionalization leads to an increase in potency
against SIRT2 in vitro that is not seen for SIRT1, resulting in
the identification of a range of SIRT2 selective analogs. In both
cases, we have rationalized the observed selectivity through the
use of computational methods providing evidence in support
of a previously proposed binding mode for 1. The identification
of an interesting lipophilic channel, the targeting of which may
aid selective SIRT2 inhibitor development, is also reported.
Studies on the activity of 6b and 6j in cells are also consistent
with the observed in vitro selectivity, implying that the novel
analogs identified in this work may also be of interest in the
treatment of cancer and neurodegenerative diseases.
2-(5′-Bromobenzoyl)-3H-benzo[f]chromen-3-one (4b).20 Yielded
600 mg (1.58 mmol, 93%) as a yellow powder. Mp 245-248 °C
(lit.20 246-247 °C). H NMR (CDCl3, 400 MHz): δ ) 8.98 (s,
1
1H, H-1), 8.29 (d, 1H, J ) 8.3 Hz, H-10), 8.13 (d, 1H, J ) 9.0 Hz,
H-6), 7.96 (d, 1H, J ) 8.2 Hz, H-7), 7.81-7.73 (m, 3H, H-9,
AA′BB′ system, J ) 8.7 Hz, H-3′, H-7′), 7.64 (m, 3H, H-8, AA′BB′
system, J ) 8.7 Hz, H-4′, H-6′), 7.51 (d, 1H, J ) 9.0 Hz, H-5).
LRMS [ES]+: m/z 401.07 [M + Na]+ (100%).
2-(5′-Methoxybenzoyl)-3H-benzo[f]chromen-3-one (4c).19,20
Yielded 1.45 g (4.39 mmol, 95%) as a white-red powder. Mp
207-210 °C (lit.19,20 209-210 °C). 1H NMR (400 MHz, CDCl3):
δ ) 8.86 (s, 1H, H-1), 8.26 (d, 1H, J ) 8.4 Hz, H-10), 8.10 (d,
1H, J ) 9.0 Hz, H-6), 7.98-7.89 (m, 3H, H-7, AA′BB′ system, J
) 8.8 Hz, H-3′, H-7′), 7.76-7.70 (m, 1H, H-9), 7.65-7.58 (m,
1H, H-8), 7.53 (d, 1H, J ) 9.0 Hz, H-5), 6.97 (d, 2H, AA′BB′
system, J ) 8.8 Hz, H-4′, H-6′), 3.83 (s, 3H, CH3O). LRMS [ES]+:
m/z 353.07 [M + Na]+ (100%).
General Experimental Procedures
Chemistry. All chemicals and solvents were purchased from
Aldrich (U.K.) or Alfa-Aesar and used without further purification.
All the reactions were carried out under a positive pressure of
nitrogen or argon in flame or oven-dried glassware. Ethanol was
dried over Mg/I2; pyridine was dried over KOH pellets. Thin layer
chromatography (TLC) analysis was performed on silica precoated
SIL G-25 UV254 sheets (layer: 0.25 mm silica gel with fluorescent
indicator UV254, Alugram, U.K.). Compounds were visualized by
UV light (UV lamp, model UVGL-58, Mineralight LAMP, Multi-
band UV-254/365 nm) and stained with potassium permanganate.
Flash column chromatography was carried out on silica gel (40-63
µm, Fluorochem, U.K.). Melting points were measured with an
electrothermal 9100 capillary melting point apparatus and are
uncorrected. Fourier Transform infrared spectra (FT IR) were
acquired on a Perkin-Elmer Paragon 1000 FT spectrometer.
Absorption maxima are reported in wavenumbers (cm -1). Unless
General Procedure for Preparation of 5a-5c. To a stirring
solution of 2-benzoylketocoumarin (4a-c, 500 mg, 1.56 mmol) in
dry pyridine (10 mL) was added NaBH4 (59 mg, 1.56 mmol). The
reaction was stirred at room temperature for 2 h. The reaction
mixture was poured into cold 2 M hydrochloric acid (90 mL). The
resulting white precipitate was collected by filtration, washed with
HCl (aq, 2 M), and recrystallized from ethanol.
2-Benzoyl-1,2-dihydrobenzo[f]chromen-3-one (5a).21 Yielded
440 mg (1.45 mmol, 95%) as a yellow powder. Mp 158-160 °C
(lit.21 158-160 °C). 1H NMR (CDCl3, 300 MHz): δ ) 8.03-7.98
(m, 2H, ArH), 7.90-7.85 (m, 2H, H-10, H-7), 7.81 (d, 1H, J )
9.0 Hz, H-6), 7.67-7.61 (m, 1H, H-9), 7.59-7.52 (m, 1H, H-8),
7.54-7.45 (m, 3H, ArH), 7.29 (d, 1H, J ) 9.0 Hz, H-5), 4.82 (dd,
1H, 3J ) 10.2 Hz, 3′J ) 6.8 Hz, H-2), 3.85 (dd, 1H, 2J ) 16.7 Hz,
2
3
3J ) 10.2 Hz, H-1), 3.62 (dd, 1H, J ) 16.7 Hz, J ) 6.8 Hz,
H-1). LRMS [ES]+: m/z 325.10 [M + Na]+ (100%).
1
otherwise stated, H NMR spectra were measured at room tem-
perature (298 K) on Bruker DPX 400 (1H ) 400 MHz) and Bruker
Avance 300 (1H ) 300.1 MHz) instruments. Deuterated solvents
were used and 1H NMR chemical shifts were internally referenced
to CHCl3 (7.26 ppm) in chloroform-d1 solution, to CD2HSO2CHD2
(2.50 ppm) in dimethylsulfoxide-d6 solution, and to CD3COCHD2
(2.09 ppm) in acetone-d6. Chemical shifts are expressed as δ in
units of ppm. 13C NMR spectra were recorded in the same
conditions and in the same solvents using the PENDANT sequence
mode on a Bruker DPX 400 (13C ) 100 MHz). Data processing
was carried out using TOPSPIN 2 NMR version (Bruker UK, Ltd.).
2-(5′-Bromobenzoyl)-1,2-dihydrobenzo[f]chromen-3-one (5b).
Yielded 892 mg (2.35 mmol, 94%) as a white powder. Mp 221-223
1
°C. H NMR (CDCl3, 400 MHz): δ ) 7.91-7.84 (m, 4H, H-7,
H-10, AA′BB′ system, J ) 8.6 Hz, H-3′, H-7′), 7.82 (d, 1H, J )
8.8 Hz, H-6), 7.65 (d, 2H, AA′BB′ system, J ) 8.6 Hz, H-2′, H-4′),
7.62-7.54 (m, 1H, H-9), 7.52-7.45 (m, 1H, H-8), 7.28 (d, 1H, J
3
) 8.8 Hz, H-5), 4.74 (dd, 1H, J ) 10.5 Hz, 3′J ) 6.7 Hz, H-2),
2
3
3
3.83 (dd, 1H, J ) 16.6 Hz, J ) 10.5 Hz, H-1), 3.62 (dd, 1H, J
) 16.6 Hz, 2J ) 6.7 Hz, H-1). LRMS [CI]+: m/z 337.06 [M + H]+
(100%).
1
In H NMR assignment the multiplicity used is indicated by the
2-(5′-Methoxybenzoyl)-1,2-dihydrobenzo[f]chromen-3-one (5c).
Yielded 400 mg (1.20 mmol, 76%) as a white-red powder. Mp
175-176 °C. 1H NMR (400 MHz, CDCl3): δ ) 7.99 (d, 2H,
AA′BB′ system, J ) 9.0 Hz, H-3′, H-7′), 7.90-7.84 (m, 2H, H-10,
H-7), 7.80 (d, 1H, J ) 9.0 Hz, H-6), 7.60-7.53 (m, 1H, H-9),
7.51-7.44 (m, 1H, H-8), 7.27 (d, 1H, J ) 9.0 Hz, overlapped with
solvent signal, H-5), 6.97 (d, 2H, AA′BB′ system, J ) 9.0 Hz,
following abbreviations: s ) singlet, d ) doublet, t ) triplet, q )
quartet, m ) multiplet, br ) broad, br s ) broad singlet. Signals
of protons and carbons were assigned, as far as possible, by using
the following two-dimensional NMR spectroscopy techniques: [1H,
1H] COSY, [1H, 13C] COSY (HSQC: heteronuclear single quantum
coherence) and long-range [1H, 13C] COSY (HMBC: heteronuclear
multiple bond connectivity). Mass spectra (electrospray mode, ES;
chemical ionization mode, CI) were recorded on a high performance
orthogonal acceleration reflecting TOF mass spectrometer operating
in positive and negative mode, coupled to a Waters 2975 HPLC.
3
H-4′, H-6′), 4.77 (dd, 1H, J ) 10.5 Hz, 3′J ) 6.8 Hz, H-2), 3.88
2
3
(s, 3H, CH3O), 3.83 (dd, 1H, J ) 16.6 Hz, J ) 10.5 Hz, 1-H),
3.60 (dd, 1H, 3J ) 16.6 Hz, 2J ) 6.8 Hz, H-1). LRMS [ES]+: m/z
355.12 [M + Na]+ (100%).

2678 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Medda et al.
General Procedure for the Synthesis of 1 and Its Analogs
6b and 6c. To a solution of NaOEt (10 mL, 2 M), prepared by
dissolving Na metal in dry ethanol, were added thiourea (15.6 equiv)
and different 2-benzoyl-1,2-dihydrocoumarins (5a-c, 200 mg, 1
equiv), and the reaction was heated under reflux for 24 h. After
removing the solvent in vacuo, the residue was dissolved in the
minimum amount of distilled water and the products were
precipitated upon addition of 2 M aqueous HCl. The solid was
collected by filtration and purified by recrystallization from ethanol.
LR MS [ES]-: m/z 388.73 [M - H]- (100%). HRMS [ES]-: m/z
calcd for C22H17N2O3S 389.0960 [M - H]-, found 389.0965 (+1.4
ppm).
Synthesis of 6a. To a solution of NaOEt (10 mL, 2 M), prepared
by dissolving Na metal in dry ethanol, were added urea (15.6 equiv)
and 2-benzoyl-1,2-dihydrocoumarins 5a (200 mg, 1 equiv), and the
reaction was heated under reflux for 24 h. After removing the
solvent in vacuo, the mixture was dissolved in distilled water, and
the products precipitated upon addition of 2 M aqueous HCl. The
solid was collected by filtration and purified by recrystallization
from ethanol or chloroform.
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-thioxo-2,3-di-
hydro-1H-pyrimidin-4-one (1, Cambinol).9 Yielded 120 mg
(51%) as a white powder after recrystallization from ethanol. Mp
253-255 °C (lit.S4 253 °C). IR (NaCl, thin layer) νmax/cm-1: 3111
(OH), 2804 (CH2), 1628 (CdO), 1556 (NH), 1494 (C-N), 1212
(CdS, CSNH), 877 and 766 (C-HAr). 1H NMR (400 MHz, DMSO-
d6): δ ) 12.55 (br s, 1H, NH), 12.30 (br s, 1H, NH), 9.44 (s, 1H,
OH), 7.64 (d, 1H, J ) 7.7 Hz, H-5′′), 7.48 (d, 1H, J ) 8.8 Hz,
H-4′′), 7.44-7.11 (m, 8H, H-6′′, H-7′′, H-8′′ + 5 × ArH), 6.90 (d,
1H, J ) 8.8 Hz, H-3′′), 3.90 (s, 2H, CH2). 13C NMR (100 MHz,
DMSO-d6): δ ) 173.8 (CdS), 162.2 (CdO), 152.7 (C2′′), 150.1
(C6), 133.1 (C8′′a), 131.6 (C1′), 129.4 (C4′), 128.5 (C5′′, C3′),
128.0 (C5′′), 128.0 (C4′′a), 127.8 (C2′, C6′), 127.5 (C4′′), 125.5
(C7′′), 122.8 (C6′′), 121.9 (C8′′), 118.3 (C3′′), 116.4 (C1′′a), 115.2
(C5), 21.5 (CH2). LRMS [ES+]: m/z 382.97 [M + Na]+ (100%).
LRMS [ES]-: m/z 358.78 [M - H]- (100%). HRMS [ES]-: m/z
calcd for C21H15N2O2S 359.0854 [M - H]-, found 359.0858 (-0.9
ppm). A sample of 1 was further recrystallized from ethanol to
give crystals of sufficient size for small molecule X-ray crystal-
lographic analysis. Crystallographic data for cambinol was deposited
at the Cambridge Crystallographic Data Centre and allocated the
deposit number CCDC 704011.
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-1H-pyrimidine-
2,4-dione (6a). Yielded 40 mg (0.11 mmol, 20%) as a white powder
after recrystallization from CHCl3. Mp 292-295 °C. IR (NaCl, thin
layer) νmax/cm-1: 3417 and 3105 (OH), 2923 (CH2), 1653 (CdO),
1576 (NH), 1456 (C-N), 1119 (C-O, CdS). 1H NMR (400 MHz,
DMSO-d6): δ ) 11.33 (br s, 1H, NH), 10.90 (br s, 1H, NH), 9.64
3
4
(s, 1H, OH), 7.65 (dd, 1H, J ) 7.8 Hz, J ) 1.8 Hz, H-5′′), 7.50
(d, 1H, J ) 8.8 Hz, H-4′′), 7.46-7.35 (m, 3H, H-8, 2 × ArH),
7.35-7.28 (m, 2H, 2 × ArH), 7.19-7.08 (m, 3H, H-7′′, H-6′′ + 1
× ArH), 6.92 (d, 1H, J ) 8.8 Hz, H-3′′), 3.90 (s, 2H, CH2). 13C
NMR (100 MHz, DMSO-d6): δ ) 166.0 (CdO), 152.8 (C2′′), 150.3
(CdO), 133.0 (C1′), 132.6 (C8′′a), 129.4 (5′′), 128.4 (C5′, C3′),
128.2 (C4′′a), 128.1, 127.5 (C4′′), 125.4 (C7′′), 122.8 (C8′′), 121.9
(C6′′), 118.8 (C3′′), 117.3 (C1′′a), 109.3 (C5), 21.2 (CH2). LRMS
[ES]+: m/z 367.04 [M + Na]+ (100%). LR MS [ES]-: m/z 342.81
[M - H]- (100%). HRMS [ES]-: m/z calcd for C21H16N2O3Na
367.1059 [M - H]-, found 367.1056 (-0.7 ppm).
2-Benzoyl-6a-phenylchromen-2-one (7). To a solution of
2-hydroxy-5-phenylbenzaldehyde22,23 (220 mg, 1.1 mmol) in
ethanol (6 mL) was added ethyl benzoyl acetate 3a (211 mg, 1.1
mmol). Piperidine (5 drops) was added, and the reaction was heated
under reflux for 2 h. After cooling to room temperature, the product
was collected by filtration and washed with ethanol. The reaction
yielded 235 mg (0.72 mmol, 66%) as a white-yellow powder. Mp
166-168 °C. 1H NMR (400 MHz, CDCl3): δ ) 8.14 (s, 1H, H-1),
7.93-7.84 (m, 3H, ArH), 7.77 (s, 1H, J ) 2.1 Hz, H-7), 7.66-7.56
(m, 3H, ArH), 7.54-7.45 (m, 5H, ArH), 7.45-7.37 (m, 1H, ArH).
LRMS [ES]+: m/z 349.07 [M + Na]+ (100%).
6-(4′-Bromophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6b). Yielded 100 mg
(0.22 mmol, 43%) as a white powder after recrystallization from
ethanol. Mp 282-287 °C. IR (NaCl, thin layer) νmax/cm-1: 3413
(OH), 1699 and 1634 (CdO), 1547 (NH), 1445 (C-N), 1189 (CdS,
CSNH), 1158, 1124, and 1112 (C-O and CdS), 1070 (C-O), 971,
827, and 742 (C-HAr), 668 (C-Br). 1H NMR (400 MHz, DMSO-
d6): δ ) 12.56 (br s, 1H, NH), 12.22 (br s, 1H, NH), 9.42 (s, 1H,
OH), 7.71 (d, 1H, J ) 7.5 Hz, H-5′′), 7.47 (d, 1H, J ) 8.8 Hz,
H-4′′), 7.44 (d, 1H, J ) 8.7 Hz, H-8′′), 7.32 (d, 2H, AA′BB′ system,
J ) 8.4 Hz, H-2′, H-6′), 7.30-7.22 (m, 1H, H-7′′), 7.20-7.14 (m,
1H, H-6′′), 6.98 (d, 2H, AA′BB′ system, J ) 8.4 Hz, H-3′, H-5′),
6.87 (d, 1H, J ) 8.7 Hz, H-3′′), 3.91 (s, 2H, CH2). 13C NMR (100
MHz, DMSO-d6): δ ) 173.8 (CdS), 161.9 (CdO), 152.6 (C2′′),
148.8 (C6), 132.9 (C1′), 130.6 (C8′′a), 130.4 (C3′), 130.1 (C2′),
128.0 (C5′′), 127.9 (C4′′a), 127.4 (C4′′), 125.5 (C7′′), 122.7 (C6′′),
121.8 (C8′′), 117.9 (C3′′), 116.3 (C1′′), 115.7 (C5), 20.9 (CH2).
LRMS [ES]+: m/z 462.96 [M + Na]+ (100%). LRMS [ES]-: m/z
436.93 [M - H]- (100%). HRMS [ES]-: m/z calcd for
C21H12N2O2S81Br 436.9960 [M - H]-, found 436.9970 (+ 0.2
ppm); m/z calcd for C21H12N2O2S79Br 438.9945 [M - H]-, found
438.9946 (+ 0.1 ppm).
6-Phenyl-5-[2′′-hydroxybiphenyl-(1′′)-methyl]-2-thioxo-2,3-di-
hydro-1H-pyrimidin-4-one (6d). To a stirring solution of 7 (213
mg, 0.6 mmol) in dry pyridine (6 mL) was added NaBH4 (25 mg,
0.65 mmol). The reaction was stirred at room temperature for 2 h.
The reaction mixture was poured into cold 2 M hydrochloric acid
(20 mL). The resulting white precipitate was collected by filtration,
washed with HCl (aq, 2 M), and recrystallized from ethanol. The
reaction yielded 200 mg (0.61 mmol, 94%) as a white powder. 1H
NMR (400 MHz, CDCl3): δ ) 8.00-7.94 (m, 2H, ArH), 7.67-7.60
(m, 1H, ArH), 7.56-7.47 (m, 5H, ArH), 7.47-7.38 (m, 3H, ArH),
7.38-7.30 (m, 1H, ArH), 7.18 (d, 1H, J ) 8.4 Hz, ArH), 4.75 (dd,
1H, 3J ) 10.5 Hz, 3′J ) 6.4 Hz, H-2), 3.60 (dd, 1H, 2J ) 16.6 Hz,
3J ) 9.6 Hz, H-1), 3.25 (dd, 1H, 2J ) 16.6 Hz, 3J ) 6.4 Hz, H-1).
LRMS [ES]+: m/z 350.12 [M + Na]+ (100%). To a solution of
NaOEt (10 mL, 2 M), prepared by dissolving Na metal in dry
ethanol, were added thiourea (15.6 equiv) and the white solid (170
mg, 1 equiv), and the reaction was heated under reflux for 24 h.
After removing the solvent in vacuo, the residue was dissolved in
the minimum amount of distilled water and the product was
precipitated upon addition of 2 M aqueous HCl. The solid was
collected by filtration. The reaction yielded 80 mg (40%) as a white
powder after recrystallization from ethanol. Mp 253-255 °C. IR
(NaCl, thin layer) νmax/cm-1: 3417 (OH), 1628 (CO), 1119 (CdS).
1H NMR (400 MHz, DMSO-d6): δ ) 12.59 (br s, 1H, NH), 12.46
(br s, 1H, NH), 9.49 (s, 1H, OH), 7.49-7.34 (m, 9H, ArH),
7.31-7.22 (m, 2H, ArH), 6.99 (d, 1H, J ) 2.1 Hz, ArH), 6.80 (d,
1H, J ) 8.3 Hz, H-3′′), 3.41 (s, 2H, CH2). 13C NMR (100 MHz,
DMSO-d6): 174.5 (CdS), 161.9 (CdO), 154.5 (C2′′), 151.4 (C6),
140.4, 131.6, 130.8, 129.9, 128.8, 128.3, 128.2, 126.2, 126.0, 126.0,
125.2, 115.1, 113.0, 25.3 (CH2). LRMS [ES]-: m/z 385.06 [M -
H]- (100%). HRMS [ES]-: m/z calcd for C23H17N2O2S 385.1011
[M - H]-, found 385.1013 (+ 0.6 ppm).
6-(4′-Methoxyphenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6c). Yielded 95 mg (0.24
mmol, 42%) as a white powder after recrystallization from ethanol.
Mp 262-265 °C. IR (NaCl, thin layer) νmax/cm-1: 3425 (OH), 2930
(CH2), 1652 and 1628 (CdO), 1576 (NH), 1457 (C-N), 1253 (OH),
1
1213, 1186, and 1023 (C-O and CdS), 736 (C-HAr). H NMR
(400 MHz, DMSO-d6): δ ) 12.52 (br s, 1H, NH), 12.25 (br s, 1H,
NH), 9.50 (s, 1H, OH), 7.70-7.62 (m, 1H, H-8′′), 7.51 (d, 1H, J
) 8.8 Hz, H-4′′), 7.35 (d, 1H, J ) 8.3 Hz, H-5′′), 7.26-7.14 (m,
4H, H-6′′, H-7′′, AA′BB′ system, J ) 8.7 Hz, H-2′, H-6′), 6.94 (d,
1H, J ) 8.8 Hz, H-3′′), 6.86 (d, 2H, AA′BB′ system, J ) 8.7 Hz,
H-3′, H-5′), 3.92 (s, 2H, CH2), 3.75 (s, 3H, CH3O). 13C NMR (100
MHz, DMSO-d6): δ ) 173.8 (CdS), 162.4 (CdO), 160.1 (C4′),
152.7 (C2′′), 150.0 (C6), 133.1 (C1′), 130.2 (C2′, C7′), 128.1 (C4′′),
127.5 (C5′′), 125.5 (C7′′), 123.8 (C8′′a), 122.8 (C6′′), 121.9 (C8′′),
118.5 (C3′′), 116.6 (C1′′a), 115.0 (C5), 113.3 (C3′, C5′), 55.2
(CH3O), 21.7 (CH2). LRMS [ES]+: m/z 412.99 [M + Na]+ (100%).

NoVel Cambinol Analogs as Sirtuin Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2679
General Protocol for Synthesis of Analogs 6e-j. To a stirring
solution of N-substituted thiourea (15.6 equiv) in NaOEt (10, mL,
2 M) was added 5a (1 equiv) in one portion, and the reaction was
stirred at reflux for 18 h. After cooling to room temperature, the
solvent was removed in vacuo and the remaining residue was
dissolved in the minimum amount of distilled water and acidified
with 2 M aq HCl. The aqueous layer was extracted with DCM (3
× 50 mL), the organic layers were collected and dried over MgSO4,
and the solvent was removed in vaquo. The products were purified
by flash column chromatography (EtOAc/hexane) and recrystallized
from ethanol.
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-1-propyl-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6h). Yielded 16 mg (0.04
mmol, 10%) as a white powder after column chromatography
(EtOAc/hexane, 1:4) and recrystallization from ethanol. Mp 227-229
°C. IR (KBr) ν max/cm-1: 3676 and 2988 (OH), 2969, and 2902
(CH3, CH2), 1630 (CdO), 1482 (CSNH), 1432 (C-N), 1206, 1131,
and 1108 (CdS), 1075, 1066, and 1057 (C-O), 880, 820, and 767
1
(C-HAr), 745, 701, and 666 (C-H). H NMR (DMSO-d6, 400
MHz): δ ) 12.77 (br s, 1H, NH), 9.21 (s, 1H, OH), 7.65 (d, 1H,
J ) 8.8 Hz, H-5′′), 7.55-7.45 (m, 2H, H-4′′, H-8′′), 7.29-7.12
(m, 5H, H-6′′, H-7′′, H-3′, H-4′, H-5′), 6.97 (d, 2H, J ) 7.0 Hz,
H-2′, H-6′), 6.84 (d, 1H, J ) 8.8 Hz, H-3′′), 3.78 (s, 2H, H-1′′),
3.69 (br, 2H, H-1′′′), 1.41 (m, 2H, H-2′′′), 0.43 (t, 3H, J ) 7.4 Hz,
H-3′′′). 13C NMR (DMSO-d6, 100 MHz): δ ) 174.8 (CdS), 160.4
(CdO), 152.7 (C2′′), 151.7 (C6), 133.1 (C8′′a), 131.7 (C1′), 129.3
(C4′), 128.9 (C5′, C3′), 128.6 (C2′, C6′), 128.1 (C5′′), 128.0 (C4′′),
127.8 (C4′′a), 125.6 (C7′′), 122.6 (C8′′), 121.8 (C6′′), 119.0 (C1′′a),
117.9 (C3′′), 116.2 (C5), 52.1 (C1′′′), 22.1 (C1′′), 20.3 (C2′′′), 10.5
(C3′′′). HRMS [ES]+: m/z calcd for C24H22N2O2NaS 425.1300 [M
+ Na]+, found 425.1307 (+0.2 ppm).
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-1-methyl-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6e). Yielded 40 mg (0.10
mmol, 16%) as a white powder after recrystallization from CHCl3.
Mp 254-257 °C. IR (KBr) νmax/cm-1: 3348 (OH), 2804 (CH2),
1628 (CdO), 1556 (NH), 1431 (CSNH), 1212, (CdS), 877, 766
and 702 (C-HAr). 1H NMR (400 MHz, DMSO-d6): δ ) 12.80 (br
s, 1H, NH), 9.22 (br, 1H, OH), 7.65 (d, 1H, J ) 7.8 Hz, H-5′′),
7.51-7.47 (m, 2H, H-4′′, H-8′′), 7.30-7.11 (m, 5H, H-6′′, H-7′′
+ 3 × ArH, H-3′, H-4′, H-5′), 6.97 (d, 2H, J ) 7.0 Hz, ArH, H-2′,
H-6′), 6.85 (d, 1H, J ) 8.8 Hz, H-3′′), 3.82 (s, 2H, CH2), 3.16 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ ) 175.2 (CdS),
160.6 (CdO), 152.7 (C2′′), 152.0 (C6), 133.1 (C8′′a), 132.2 (C1′),
128.9 (C3′, C5′), 128.3 (C2′, C6′), 128.0 (C4′), 127.9 (C5′′), 127.7
(C4′′a), 127.2 (C4′′), 125.5 (C7′′), 122.7 (C8′′), 121.7 (C6′′), 118.5
(C3′′), 117.9 (C1′′a), 116.2 (C5), 40.2 (CH3), 22.0 (CH2). LRMS
[ES]+: m/z 397.08 [M+Na]+ (100%); LRMS [ES]-: m/z 373.05
[M - H]- (100%); HRMS [ES]-: m/z calcd for C22H17N2O2S
373.1011 [M - H]-, found 373.1010 (-0.2 ppm).
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-1-ethyl-2-thioxo-
2,3-dihydro-1H-pyrimidin-4-one (6f). Yielded 30 mg (0.07 mmol,
20%) as a white powder after column chromatography (EtOAc/
hexane, 1:4) and recrystallization from ethanol. Mp 256-258 °C.
IR (KBr) ν max/cm-1: 3676, 2988, and 2972 (OH), 2902 (CH2, CH3),
1633 (CdO), 1437 (C-N), 1394 and 1241 (OH), 1217 and 1103
(CdS), 1076, 1057, and 1028 (C-O), 812, 767, and 741 (C-HAr).
1H NMR (DMSO-d6, 400 MHz): δ ) 12.75 (br, 1H, NH), 9.20
(br, 1H, OH), 7.65 (d, 1H, J ) 7.1 Hz, H-5′′), 7.51-7.45 (m, 2H,
H-4′′, H-8′′), 7.27-7.17 (m, 5H, H-6′′, H-7′′, H-3′, H-4′, H-5′),
6.98 (d, 2H, J ) 7.1 Hz, H-2′, H-6′), 6.84 (d, 1H, J ) 8.6 Hz,
H-3′′), 3.86 (br, 2H, CH2, H-1′′′), 3.77 (s, 2H, CH2, H-1′′), 0.92 (t,
3H, J ) 7.0 Hz, CH3). 13C NMR (DMSO-d6, 100 MHz): 175.5
(CdS), 161.4 (CdO), 153.6 (C2′′), 152.6 (C6), 132.7 (C8′′a), 131.6
(C1′), 129.1 (C4′), 128.9 (C3′, C5′), 128.9 (C4′′a), 128.3 (C2′, C6′),
127.9 (C5′′), 127.2 (C4′′), 126.5 (C7′′), 123.6 (C8′′), 122.8 (C6′′),
118.9 (C3′′), 117.2 (C1′′a), 116.1 (C5), 46.9 (C1′′′), 23.0 (C1′′),
13.7 (C2′′′). LRMS [ES]+: m/z 411.33 [M + Na]+ (100%). LRMS
[ES]-: m/z 387.16 [M - H]- (100%). HRMS [ES]+: m/z calcd for
C23H20N2O2SNa 411.1143 [M + Na]+, found 411.1149 (+1.3 ppm).
6-Phenyl-5-[2′′-hydroxynaphthalyl-(1′′)-methyl]-1-butyl-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6j). Yielded 29 mg (0.07
mmol, 7%) as a white powder after column chromatography
(EtOAc/hexane 1:9) and recrystallization from ethanol. Mp 201-203
°C. IR (KBr) ν max/cm-1: 3667 and 3213 (OH), 2972 and 2902
(CH3, CH2), 1645 (CdO), 1487 (CSNH), 1428 (C-N), 1394 and
1233 (OH), 1204, 1179, 1129, and 1104 (CdS), 1067 and 1057
1
(C-O), 823 and 747 (C-HAr), 705 and 756 (C-H). H NMR
(DMSO-d6, 400 MHz): δ ) 12.75 (s, 1H, NH), 9.20 (s, 1H, OH),
7.65 (d, 1H, J ) 8.2 Hz, H-5′′), 7.51-7.45 (m, 2H, H-8′′, H-4′′),
7.24-7.14 (m, 5H, H-7′′, H-6′′, H-3′, H-4′, H-5′), 6.98 (d, 2H, J
) 7.0 Hz, H-2′, H-6′), 6.84 (d, 1H, J ) 8.7 Hz, H-3′′), 3.79 (s
overlapped with br, 4H, H-1′′, H-1′′′), 1.40 (br, 2H, H-2′′′),
0.90-0.80 (m, 2H, H-3′′′), 0.50 (t, 3H, J ) 7.3 Hz, H-4′′′). 13C
NMR (DMSO-d6, 100 MHz): δ ) 174.6 (CdS), 160.4 (CdO),
152.7 (C2′′), 151.7 (C6), 133.1 (C8′′a), 131.6 (C1′), 128.9 (C3′,
C5′), 128.1 (C2′, C6′), 128.0 (C4′), 128.0 (C5′′), 127.9 (C4′′a), 127.6
(C4′′), 125.6 (C7′′), 122.6 (C8′′), 121.8 (C6′′), 119.1 (C3′′), 117.9
(C1′′a), 116.2 (C5), 50.3 (C1′′′), 28.6 (C2′′′), 22.0 (C1′′), 18.9
(C3′′′), 12.9 (C4′′′). LRMS [ES]+: m/z 439.05 [M + Na]+ (100%).
LRMS [ES]-: m/z 415.16 [M - H]- (100%). HRMS [ES]+: m/z
calcd for C25H24N2O2SNa 439.1456 [M + Na]+, found 439.1473
(+3.8 ppm).
General Procedure for the Parallel Synthesis of Analogs
6i-6xi. Thiourea (15.6 equiv) and intermediates 5i-xv (100 mg,
1 equiv) were mixed into different vessels of the parallel synthesis
apparatus. 2 M NaOEt stock solution (5 mL), previously prepared
by dissolving Na metal in dry ethanol, was added to each reaction
vessel and the reaction was heated under reflux for 18 h. After
removing the solvent in vacuo, the crude reaction mixtures were
dissolved in the minimum amount of distilled water and the products
precipitated upon addition of 2 M aqueous HCl. The solids were
collected by filtration and purified by column chromatography
(EtOAc/hexane) and recrystallized from CHCl3. Reactions using
5xii-5xv were unsuccessful.
6-(2′-Methylphenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6i). Yielded 115 mg
(0.30 mmol, 57%) as a white powder after column chromatography
(EtOAc/hexane, 1:4) and recrystallization from CHCl3. Mp 170-172
°C. IR (KBr) νmax/cm-1: 2988 (OH), 2902 (CH3, CH2), 1627 (CdO),
1554 (NH), 1453 (CSNH), 1394 and 1255 (OH), 1211 (CdS), 1075
and 1057 (C-O), 819, 808, and 744 (C-HAr), 729 (C-H). 1H NMR
(DMSO-d6, 300 MHz): δ ) 12.60 (br s, 1H, NH), 12.17 (br s, 1H,
NH), 9.21 (br s, 1H, OH), 7.63 (d, 1H, J ) 8.1 Hz, H-5′′), 7.43 (d,
1H, J ) 8.8 Hz, H-4′′), 7.39 (d, 1H, J ) 7.4 Hz, H-8′′), 7.26-7.08
(m, 3H, H-6′′, H-7′′ + ArH), 7.03-6.99 (m, 2H, ArH), 6.90 (d,
1H, J ) 7.1 Hz, ArH), 6.78 (d, 1H, J ) 8.8 Hz, H-3′′), 3.88 (q,
2H, CH2), 1.62 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ
) 173.3 (CdS), 162.4 (CdO), 152.6 (C2′′), 150.0 (C6), 135.6 (C2′),
132.9 (C1′), 131.0 (C8′′a), 129.2 (CAr), 129.0 (CAr), 128.4 (C5′′),
128.0 (C4′′), 127.8 (C4′′a), 127.2 (CAr), 125.4 (C7′′), 125.1 (CAr),
6-Phenyl-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-1-allyl-2-thioxo-
2,3-dihydro-1H-pyrimidin-4-one (6g). Yielded 20 mg (0.05 mmol,
15%) as a white powder after column chromatography (EtOAc/
hexane, 1:4) and recrystallization from ethanol. Mp 175-177 °C.
IR (KBr) ν max/cm-1: 3676 and 2988 (OH), 2902 (CH2), 1626
(CdO), 1479 (CSNH), 1406, 1394, and 1242 (OH), 1075, 1057,
and 1028 (C-O), 892, 823, and 747 (C-HAr), 701 (C-H). 1H NMR
(DMSO-d6, 300 MHz): δ ) 12.82 (br s, 1H, NH), 9.22 (br, 1H,
OH), 7.65 (d, 1H, J ) 7.1 Hz, H-5′′), 7.52-7.43 (m, 2H, H-4′′,
H-8′′), 7.28-7.18 (m, 5H, H-6′′, H-7′′, H-3′, H-4′, H-5′), 6.93 (d,
2H, J ) 7.0, H-2′, H-6′), 6.85 (d, 1H, J ) 8.8 Hz, H-3′′), 5.61-5.50
3
4
(m, 1H, H-2′′′), 4.98 (dd, 1H, J ) 10.5 Hz, J ) 1.2 Hz, H-3′′′),
3
4
4.64 (dd, 1H, J ) 17.3 Hz, J ) 1.2 Hz, H-3′′′), 4.50 (m, 2H,
H-1′′′), 3.78 (s, 2H, H-1′′). 13C NMR (DMSO-d6, 100 MHz): δ )
174.8 (CdS), 160.7 (CdO), 152.7 (C2′′), 151.7 (C6), 133.0 (C8′′a),
131.9 (C2′′′), 131.4 (C1′), 128.9 (C3′, C5′), 128.4 (C2′, C6′), 128.1
(C5′′), 128.0 (C4′), 127.8 (C4′′a), 127.2 (C4′′), 125.5 (C7′′), 122.5
(C8′′), 121.7 (C6′′), 117.8 (C3′′), 117.1 (C3′′′), 116.5 (C1′′a), 116.1
(C5), 52.5 (C1′′′), 22.19 (C1′′). LRMS [ES]+: m/z 423.12 [M +
Na]+ (100%). HRMS [ES]+: m/z calcd for C24H20N2O2NaS
423.1143 [M + Na]+, found 423.1142 (-0.3 ppm).

2680 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Medda et al.
122.6 (C8′′), 121.8 (C6′′), 117.8 (C3′′), 116.0 (C5), 115.9 (C1′′a),
20.1 (CH2), 18.36 (CH3). LRMS [ES]+: m/z 397.12 [M + Na]+
(100%). HRMS [ES]+: m/z calcd for C20H18N2O2NaS 397.0987 [M
+ Na]+, found 397.0987 (+0.1 ppm).
MHz, DMSO-d6): δ ) 12.55 (br s, 1H, NH), 12.21 (br s, 1H, NH),
9.38 (s, 1H, OH), 7.64 (d, 1H, J ) 8.4 Hz, H-5′′), 7.52-7.39 (m,
4H, H-4′′, H-8′′, AA′BB′ system, J ) 8.4 Hz, H-2′, H-6′),
7.28-7.15 (m, 2H, H-7′′, H-6′′), 6.86-6.83 (m, 3H, H-3′′, AA′BB′
system, J ) 8.4 Hz, H-3′, H-5′), 3.88 (s, 2H, CH2). LRMS [ES]+:
m/z 508.02 [M + Na]+ (100%).
6-(3′-Methylphenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6ii). Yielded 54 mg (0.14
mmol, 51%) as a white powder after column chromatography
(EtOAc/hexane, 1:4) and recrystallization from CHCl3. Mp 235-237
°C. IR (KBr) νmax/cm-1: 3767 and 2988 (OH), 2902 (CH3, CH2),
1646 (CdO), 1563 (NH), 1465 (CSNH), 1403, 1394, and 1233
(OH), 1211, 1198, and 1128 (CdS), 1066, 1044, and 1017 (C-O),
993 (C-H), 823 and 747 (C-HAr). 1H NMR (DMSO-d6, 300 MHz):
δ ) 12.55 (br s, 1H, NH), 12.22 (br s, 1H, NH), 9.41 (br s, 1H,
OH), 7.65 (d, 1H, J ) 7.9 Hz, H-5′′), 7.47 (d, 1H, J ) 8.8 Hz,
H-4′′), 7.36 (d, 1H, J ) 8.5 Hz, H-8′′), 7.26-7.09 (m, 4H, H-7′′,
H-6′′ + 2 × ArH), 7.00-6.96 (m, 1H, ArH), 6.93-6.85 (m, 2H,
H-3′′ + 1 × ArH), 3.91 (s, 2H, CH2), 2.18 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6): δ ) 173.8 (CdS), 162.4 (CdO), 152.7
(C2′′), 150.9 (C6), 136.9 (C3′), 133.0 (C8′′a), 131.5 (C1′), 129.9
(C2′), 128.9 (CAr), 128.3 (C4′′a), 128.0 (C4′′), 127.6 (C5′′), 127.4
(CAr), 126.2 (CAr), 125.4 (C7′′), 122.7 (C8′′), 121.8 (C6′′), 118.2
(C3′′), 116.5 (C1′′a), 115.0 (C5), 21.3 (CH2), 20.2 (CH3). LRMS
[ES]+: m/z 397.10 [M + Na]+ (100%). HRMS [ES]+: m/z calcd
for C20H18N2O2NaS 397.0987 [M + Na]+, found 397.0988 (+0.1
ppm).
6-(4′-Methylphenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6iii). Yielded 56 mg
(0.15 mmol, 50%) as a white powder after column chromatography
(EtOAc/hexane, 1:4) and recrystallization from CHCl3. Mp 220-221
°C. IR (KBr) νmax/cm-1: 3663 and 2988 (OH), 2902 (CH3, CH2),
1630 (CdO), 1540 (NH), 1515 (CSNH), 1445 (C-N), 1405, 1349,
and 1250 (OH), 1226 and 1208 (CdS), 1066 and 1028 (C-O),
818, 744, and 729 (C-HAr). 1H NMR (DMSO-d6, 300 MHz): δ )
12.52 (br s, 1H, NH), 12.26 (br s, 1H, NH), 9.46 (br s, 1H, OH),
7.65 (d, 1H, J ) 7.4 Hz, H-5′′), 7.50 (d, 1H, J ) 8.7 Hz, H-4′′),
7.35 (d, 1H, 3J ) 7.7 Hz, H-8′′), 7.22-7.08 (m, 6H, H-7′′, H-6′′ +
4 × ArH), 6.92 (d, 1H, J ) 8.7 Hz, H-3′′), 3.89 (s, 2H, CH2), 2.29
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6): δ ) 173.8 (CdS),
162.2 (CdO), 152.7 (C2′′), 150.1 (C6), 139.1 (C4′), 133.0 (C8′′a),
128.7 (C4′′a), 128.5 (CAr), 128.4 (CAr), 128.0 (C5′′), 127.4 (C4′′),
125.7 (C7′′), 122.8 (C8′′), 121.8 (C6′′), 118.4 (C3′′), 116.5 (C1′′a),
115.1 (C5), 21.1 (CH2), 20.9 (CH3). LRMS [ES]+: m/z 397.09 [M
+ Na]+ (100%). HRMS [ES]+: m/z calcd for C20H18N2O2NaS
397.0987 [M + Na]+, found 397.0989 (+0.1 ppm).
6-(4′-Chlorophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6iv). Yielded 20 mg
(0.05 mmol, 18%) as a white powder after column chromatography
(EtOAc/hexane, 1:2) and recrystallization from CHCl3. Mp 287-289
°C. IR (KBr) νmax/cm-1: 3277 and 3030 (OH), 2941 (CH2), 1702
and 1633 (CdO), 1545 (NH), 1448 (C-N), 1219 and 1204 (CdS,
CSNH), 1114, 1127, and 1090 (CdS), 1090 and 1014 (C-O), 837
and 820 (C-HAr). 1H NMR (400 MHz, DMSO-d6): δ ) 12.57 (br
s, 1H, NH), 12.23 (br s, 1H, NH), 9.38 (s, 1H, OH), 7.64 (d, 1H,
J ) 7.5 Hz, H-5′′), 7.49-7.41 (m, 2H, H-4′′, H-8′′), 7.30-7.22
(m, 1H, H-7′′), 7.21-7.14 (m, 3H, H-6′′ overlapped with AA′BB′
system, J ) 8.3 Hz, H-3′′, H-5′′), 7.04 (d, 2H, J ) 8.3 Hz, AA′BB′
system, H-2′, H-6′), 6.85 (d, 1H, J ) 8.7 Hz, H-3′′), 3.91 (s, 2H,
CH2). 13C NMR (100 MHz, DMSO-d6): δ ) 174.2 (CdS), 160.6
(CdO), 152.7 (C2′′), 151.6 (C6), 133.8 (C4′), 133.0 (C8′′a), 132.9
(C1′), 130.4 (C3′, C5′), 128.4 (C5′′), 127.9 (C2′, C6′), 127.9 (C4′′a),
127.8 (C4′′), 125.9 (C7′′), 123.1 (C8′′), 122.3 (C6′′), 118.4 (C3′′),
116.8 (C1′′a), 115.9 (C5), 20.9 (CH2). LRMS [ES]+: m/z 416.97
[M - H]+ (100%). HRMS [ES]+: m/z calcd for C21H15N2O2NaSCl
417.0440 [M + Na]+, found 417.0443 (+3.1 ppm).
6-(4′-Trifluoromethylphenyl)-5-[2′′-hydroxynaphthyl-(1′′)-
methyl]-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (6vi). Yielded
33 mg (0.077 mmol, 25%) as a white powder after column
chromatography (EtOAc/hexane, 1:2) and recrystallization from
CHCl3. Mp > 250 °C (decomposes). IR (KBr) νmax/cm-1: 2970
(OH), 2912 (CH2), 1691, 1549, and 1428 (C-N), 1635 (CdO),
1515 (CSNH), 1409 (OH), 1321, 1286 and 1167 (C-F), 1131 and
1112 (CdS), 1065 and 1017 (C-O), 860 and 702 (C-H), 821,
778, and 743 (C-HAr). 1H NMR (400 MHz, DMSO-d6): δ ) 12.67
(br s, 1H, NH), 12.28 (br s, 1H, NH), 9.40 (br s, 1H, OH), 7.65 (d,
1H, J ) 8.3, H-5′′), 7.52-7.35 (m, 4H, H-4′′, and H-8′′ overlapped
with AA′BB′ system, J ) 8.0 Hz, H-2′, H-6′), 7.17 (m, 1H, H-7′′),
7.17-7.12 (m, 3H, H-6′′ overlapped with AA′BB′ system, J ) 8.0
Hz, H-3′, H-5′), 6.80 (d, 1H, J ) 8.8 Hz, H-3′′), 3.95 (s, 2H, CH2).
13C NMR (100 MHz, DMSO-d6): δ ) 173.9 (CdS), 161.9 (CdO),
152.2 (C2′′), 148.8 (C6), 135.3 (C1′), 132.9 (C8′′a), 129.3 (C4′),
128.8 (C3′, C5′), 128.0 (C4′′), 127.9 (C5′′), 127.8 (C4′′a), 125.6
(C7′′), 124.1 (C2′, C6′), 122.6 (C6′′), 121.9 (C8′′), 119.8 (CF3),
117.6 (C3′′), 116.2 (C1′′a), 116.0 (C5), 20.54 (CH2). LRMS [ES]+:
m/z 451.05 [M + Na]+ (100%).
6-(2′-Bromophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6vii). Yielded 30 mg
(0.07 mmol, 20%) after purification by column chromatography
and recrystallization from CHCl3. Mp 224-226 °C. IR (KBr) νmax
/
cm-1: 3055 (OH), 2927 (CH2), 1627 (CdO), 1548 (N-H), 1436
(C-N), 1196 (CdS, CSNH), 1132 and 1117 (C-O, CdS), 1027
(C-O), 813 and 740 (C-HAr), 678 (C-Br). 1H NMR (acetone-d6,
300 MHz): δ ) 11.61 (br, 1H, NH), 11.23 (br, 1H, NH), 8.93 (br,
1H, OH), 7.74-7.60 (m, 1H, H-5′), 7.61-7.52 (m, 2H, H-4′′, H-8′′),
7.38-7.11 (m, 6H, H-6′′, H-7′′ + 4 × ArH), 6.88 (d, 1H, J ) 8.8
Hz, H-3′′), 4.13 (d, 1H, AB system, J ) 15.6 Hz, CH2), 3.98 (d,
1H, AB system, J ) 15.6 Hz, CH2). 13C NMR (DMSO-d6, 100
MHz): 175.3 (CdS), 164.5 (CdO), 154.6 (C2′′), 150.6 (C6), 134.4
(C1′), 133.4 (C8′′a), 133.6 (CAr), 131.9 (CAr), 132.5 (CAr), 129.9
(C4′′a), 129.1 (C4′′), 129.0 (CAr), 128.3 (C5′′), 126.7 (C7′′), 123.6
(C8′′), 123.3 (C2′), 123.1 (C6′′), 119.7 (C3′′), 117.0 (C1′′a), 117.0
(C5), 21.4 (CH2). LRMS [ES]+: m/z 460.92, 462.92 [M + Na]+.
LRMS [ES]-: m/z 437.01, 439.01 [M - H]-. HRMS [ES]+: m/z
calcd for C21H15N2O2SNa79Br 460.9935 [M + Na]+, found 460.9941
(+ 1.3 ppm); m/z calcd for C21H15N2O2SNa81Br 462.9915 [M +
Na]+, found 462.9903 (-2.6 ppm).
6-(3′-Bromophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6viii). Yielded 15 mg
(0.07 mmol, 26%) as a white powder after column chromatography
(EtOAc/hexane, 1:2) and recrystallization from CHCl3. Mp 237-239
°C. IR (KBr) νmax/cm-1: 3114 and 3081 (OH), 2935 (CH2), 1713
and 1629 (CdO), 1561 (NH), 1462 and 1439 (C-N), 1199 (CdS,
CSNH), 1119 (C-O, CdS), 820, 806, and 746 (C-HAr), 697
(C-Br). 1H NMR (400 MHz, DMSO-d6): δ )12.58 (br s, 1H, NH),
12.21 (br s, 1H, NH), 9.36 (s, 1H, OH), 7.63 (d, 1H, J ) 7.7 Hz,
H-5′′), 7.51-7.47 (m, 2H, H-4′′, H-8′′), 7.40-7.35 (m, 1H, ArH),
7.31-7.25 (m, 1H, H-7′′), 7.20-7.15 (m, 1H, H-6′′), 7.12-6.99
(m, 3H, ArH), 6.85 (d, 1H, J ) 8.8 Hz, H-3′′), 3.91 (s, 2H, CH2).
13C NMR (100 MHz, acetone-d6): δ ) 175.3 (CdS), 164.7 (CdO),
154.3 (C2′′), 150.5 (C6), 134.8 (C1′), 134.3 (C8′′a), 134.0 (CAr),
132.7 (CAr), 131.2 (CAr), 130.0 (C4′′a), 129.3 (CAr), 129.2 (C4′′),
128.8 (C5′′), 126.6 (C7′′), 123.8 (C8′′), 123.3 (C6′′), 122.7 (C3′),
120.3 (C3′′), 117.9 (C1′′a), 116.0 (C5), 22.2 (CH2). LRMS [ES]-:
m/z 436.99 [M - H]-, 439.00 [M - H]-. HRMS [ES]-: m/z calcd
for C21H14N2O2S79Br 436.9959 [M - H]-, found 436.9966 (+1.5
ppm); m/z calcd for C21H14N2O2S81Br 438.9939 [M - H]-, found
438.9948 (+2.1 ppm).
6-(4′-Iodophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-thioxo-
2,3-dihydropyrimidin-1H-4-one (6v). Yielded 13 mg (0.02 mmol,
22%) as a white powder after column chromatography (EtOAc/
hexane, 1:2) and recrystallization from CHCl3. Mp > 250 °C
(decomposes). IR (KBr) νmax/cm-1: 3090 (OH), 1628 (CdO), 1560
(NH), 1427 (C-N), 1208 (CdS, CSNH), 1154 and 1126 (C-O,
6-(4′-Chlorophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-ylmethyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6ix). Yielded 18 mg
(0.004 mmol, 32%) as a white powder after column chromatography
1
CdS), 1008 (C-O), 850, 814, and 754 (C-HAr). H NMR (400

NoVel Cambinol Analogs as Sirtuin Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2681
(EtOAc/hexane, 1:2) and recrystallization from CHCl3. Mp 229-232
°C. IR (KBr) ν max/cm-1: 3086 (OH), 2941 (CH2), 2363, 1635
(CdO), 1556 (NH), 1439 (C-N), 1257 and 1213 (CdS, CSNH),
1129 (C-O), 1041 and 1027 (C-O), 819, 750 (C-HAr). 1H NMR
(acetone-d6, 400 MHz): δ ) 11.59 (br s, 1H, NH), 11.22 (br s, 1H,
NH), 9.25 (s, 1H, OH), 7.68 (d, 1H, J ) 7.6 Hz, H-5′′), 7.57 (d,
1H, J ) 8.8 Hz, H-4′′), 7.53-7.40 (m, 4H, H-8′′ + 3 × ArH),
7.23-7.08 (m, 3H, H-7′′, H-6′′ + 1 × ArH), 7.00 (d, 1H, J ) 8.8
Hz, H-3′′), 3.92 (s, 2H, CH2). 13C NMR (acetone-d6, 100 MHz): δ
) 175.0 (CdS), 164.7 (CdO), 154.3 (C2′′), 150.6 (C6), 134.7 (C3′),
134.5 (C1′), 133.5 (C8′′a), 131.1 (CAr), 131.0 (CAr), 130.0 (C4′′a),
129.9 (CAr), 129.3 (C4′′), 129.2 (C5′′), 128.3 (CAr), 126.6 (C7′′),
123.8 (C8′′), 123.3 (C6′′), 120.1 (C3′′), 117.8 (C1′′), 116.0 (C5),
22.1 (CH2). LRMS [ES]-: m/z 393.12 [M - H]- (100%). LRMS
[ES]+: 417.10 [M + Na]+ (100%). HRMS [ES]-: m/z calcd for
C21H14N2O2SCl 393.0465 [M - H]-, found 393.0461 (-0.9 ppm).
6-(2′-Fluorophenyl)-5-[2′′-hydroxynaphthyl-(1′′)methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6x). Yielded 17 mg (0.10
mmol, 10%) as a white powder after column chromatography
(EtOAc/hexane, 1:2) and recrystallization from CHCl3. Mp 197 °C
(decomposes). IR (KBr) νmax/cm-1: 3316 and 2935 (OH), 2885
(CH2), 1630 (CdO), 1558 (NH), 1439 (C-N), 1269 and 1216
microplate. Enzyme (15 µL, 0.07 U/µL for SIRT1, 0.03 U/µL for
SIRT2) and Fluor de Lys SIRT1 or SIRT2 (15 µM) plus NAD+ (1
mM) in assay buffer were added. After incubating for 1 h at 37
°C, a developer solution (50 µL developer solution added to each
well, developer solution contains 38 µL buffer, 10 µL developer
and 2 µL nicotinamide) was added (50 µL) to each well and the
microplate was incubated for a further 45 min at room temperature.
Plates were read in a Spectra max Gemini XS fluorimeter with an
excitation wavelength of 355 nm and an emission wavelength of
460 nm.
Cell Culture and Western Blotting. Human cancer cell lines
MCF-7 were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Invitrogen, U.K.), and H1299 cells were cultured in RPMI.
Both were supplemented with 10% fetal calf serum (FCS, Hyclone,
U.K.) and gentamycin (complete medium). Cells were seeded at a
concentration of 2 × 105 (MCF-7) and 6 × 104 (H1299) in 6 well
collagen precoated plates (TPP, Helena Biosciences, U.K.) and
incubated in a humidified atmosphere containing 5% CO2-95%
air at 37 °C for 42 h (MCF-7) and 24 h (H1299). Different
concentrations of target compounds in DMSO were added to MCF-
7, and they were incubated for a further 6 h. Different concentrations
of target compounds in DMSO were also added to H1299 cells,
with 40 nM trichostatin (TSA) also added and incubated for a
further 24 h. After lysing the cells with 1 × LDS sample buffer
(100 µL per well for MCF-7 and 200 µL for H1299) (Invitrogen,
U.K.), the protein concentration was assessed with a BCA protein
assay kit (Pierce, U.K.) and the concentration of protein equalized
with 1 × LDS sample buffer (the total amount of protein loaded in
gels in Figures S14 and S15 was 2.0 µg; Figure S16, 2.3 µg).
Proteins were separated with 4-12% bis-tris gels (Invitrogen, USA)
and electrophoretically transferred to PVDF transfer membranes
(Millipore, U.K.). Membranes were blocked with Marvel nonfat
milk (45 min, 5% solution in PBS/0.1% tween) and immunoblotted
using a rabbit polyclonal antibody against p53 acetylated at K382
(Biolegend, U.K.) and DO.1 (anti total p53, in-house produced)
for the MCF-7 membranes. PC-10 mouse monoclonal antibody (in
house produced) was used to detect PCNA as a loading control.
Anti-K40 acetylated tubulin (SIGMA) and anti R-tubulin (SIGMA)
were used for the H1299 membranes. All the primary antibodies
were diluted in Marvel nonfat milk (5% solution in PBS/0.1%
tween). The secondary antibody used against antiacetylated p53
was a HRP-tagged polyclonal swine antirabbit IgG (DAKO, U.K.).
The secondary antibody used against the remaining primary
antibodies was a HRP-tagged polyclonal rabbit antimouse IgG
(DAKO, U.K.). After incubation with primary (1 h) and secondary
antibodies (45 min), bound antibody was visualized with enhanced
chemiluminescence (ECL) Western blotting developer (Amersham,
U.K.) in a darkroom.
1
(C-F), 1131 (C-O, CdS), 816 and 751 (C-HAr). H NMR (400
MHz, DMSO-d6): δ ) 12.61 (br, 1H, NH), 12.35 (br, 1H, NH),
9.27 (s, 1H, OH), 7.63 (d, 1H, J ) 7.3 Hz, H-5′′), 7.53-7.43 (m,
2H, H-4′′ + 1 × ArH), 7.30-7.22 (m, 2H, H-7′′ + 1 × ArH),
7.17-7.12 (m, 1H, H-6′′), 7.13-7.05 (m, 1H, ArH), 7.00-6.92
(m, 2H, ArH), 6.82 (d, 1H, J ) 8.8 Hz, H-3′′), 3.90 (s, 2H, CH2).
13C NMR (100 MHz, DMSO-d6): δ ) 173.3 (CdS), 161.9 (CdO),
159.9 (C2′), 157.4 (C6), 152.7 (C2′′), 132.9 (C8′′a), 131.5 (C6′),
130.3 (C4′), 128.0 (C5′′), 127.8 (C4′′a), 127.4 (C4′′), 125.4 (C7′′),
123.7 (C5′), 122.6 (C8′′), 121.8 (C6′′), 121.7 (C1′), 117.7 (C3′′),
117.2 (C5), 115.8 (C3′), 115.1 (C1′′a), 20.5 (CH2). LRMS [ES]-:
m/z 377.14 [M - H]- (100%). LRMS [ES]+: m/z 401.10 [M +
Na]+ (100%). HRMS [CI]+: m/z calcd for C21H16N2O2SF 379.0917
[M + H]+, found 379.0911 (-1.5 ppm).
6-(3′-Fluorophenyl)-5-[2′′-hydroxynaphthyl-(1′′)-methyl]-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-one (6xi). Yielded 18 mg
(0.10 mmol, 10%) as a white powder after column chromatography
(EtOAc/hexane, 1:2) and recrystallization from CHCl3. Mp 150 °C
(decomposes). IR (KBr) νmax/cm-1: 3378, 3126, 3059 (OH), 2941
(CH2), 1630 (CdO), 1554 (NH), 1439 (C-N), 1268 (C-F), 1199
(CdS, CSNH), 1158 and 1119 (C-O, CdS), 821, 792, and 746
1
(C-HAr). H NMR (400 MHz, DMSO-d6): δ ) 12.58 (br s, 1H,
NH), 12.24 (br s, 1H, NH), 9.41 (s, 1H, OH), 7.64 (d, 1H, J ) 7.5
Hz, H-5′′), 7.48-7.45 (m, 2H, H-4′′, H-8′′), 7.30-6.99 (m, 4H,
H-7′′, H-6′′ + 2 × ArH), 6.93-6.68 (m, 3H, H-3′′ + 2 × ArH),
3.92 (s, 2H, CH2). 13C NMR (100 MHz, DMSO-d6): δ ) 173.8
(CdS), 162.6 (C3′), 162.1 (CdO), 159.5 (C6), 152.7 (C2′′), 134.1
(C1′), 133.0 (C8′′a), 129.7 (CAr), 128.1 (C5′′), 128.0 (C4′′a), 127.5
(C4′′), 125.6 (C7′′), 124.4 (C6′), 122.8 (C8′′), 121.9 (C6′′), 117.9
(C3′′), 116.3 (C1′′a), 116.0 (CAr), 115.7 (C5), 115.4 (CAr), 20.8
(CH2). HRMS [ES]+: m/z calcd for C21H15N2O2NaSF 401.0736 [M
+ Na]+, found 401.0735 (-0.2 ppm).
Acknowledgment. We are grateful to support from Cancer
Research UK (funding for F.M., S.L., M.H., and J.C.) and the
Royal Society (University Research Fellowship for N.J.W.). We
would like to thank Tomas Lebl for NMR interpretation and
Chris Lawson for help in determining the purity of the analogs
using HPLC techniques.
Molecular Docking Studies. The SIRT2 structure (PDB ID:
1J8F) was used for the docking studies, which were performed using
the program GOLD.12 Hydrogens were added to the protein
structure using the program Reduce,24 and small molecule structures
were generated using the PRODRG server.25 A homology model
of SIRT1 was generated using the PHYRE server. All visualization
and analysis was performed using Pymol.26
Biology Protocols. SIRT1 and SIRT2 Inhibition Assay.
Compounds were tested for inhibition of SIRT1 and SIRT2 using
the human recombinant SIRT1 and SIRT2 enzymes provided with
the Fluor de Lys fluorescence-based assay kit (AK555, AK556,
BIOMOL, Plymouth Meeting, U.S.A.). All the other required
reagents were provided with the kit. All kit components were stored
at -78 °C for highest stability. Positive controls were provided by
the known SIRT1/T2 inhibitors tenovin-63 and cambinol.5 Fresh
dilutions of cambinol analogues were prepared in DMSO (Aldrich),
added to the assay buffer, and pipeted (10 µL) into a 96 well white
Supporting Information Available: Detailed experimental
procedures for the parallel synthesis and characterization of all
intermediates along with HPLC analysis of all analogs of 1; selected
NMR spectra; small molecule X-ray crystal structure analysis of
1. This material is available free of charge via the Internet at http://
pubs.acs.org.
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