Synthesis and biological characterisation of sirtuin inhibitors based on the tenovins

Article abstract of DOI:10.1016/j.bmc.2012.01.001

The tenovins are small molecule inhibitors of the NAD⁺-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neuro

Full text of DOI:10.1016/j.bmc.2012.01.001

Bioorganic & Medicinal Chemistry 20 (2012) 1779–1793
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological characterisation of sirtuin inhibitors
based on the tenovins
Anna R. McCarthy a,c, , Lisa Pirrie a, , Jonathan J. Hollick a, Sebastien Ronseaux b, Johanna Campbell b,
Maureen Higgins b, Oliver D. Staples b, Fanny Tran a, Alexandra M.Z. Slawin a, Sonia Lain b,c,
,
⇑
Nicholas J. Westwood a,
⇑
a School of Chemistry and Biomedical Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, UK
b Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK
c Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm SE-17177, Sweden
a r t i c l e i n f o
a b s t r a c t
The tenovins are small molecule inhibitors of the NAD+-dependent family of protein deacetylases known
as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible
applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel teno-
vin analogues, further insights into the structural requirements for activity against the sirtuins in vitro
are provided. In addition, the activity of one of the analogues in cells led to an improved understanding
of the function of SirT1 in cells.
Article history:
Received 27 September 2011
Revised 9 December 2011
Accepted 2 January 2012
Available online 12 January 2012
Keywords:
Sirtuin
Ó 2012 Elsevier Ltd. All rights reserved.
Parallel synthesis
Chemical tool
Metabolism
Neurodegenerative diseases
1. Introduction
Modifications to the unselective inhibitor cambinol 1 (Fig. 1), orig-
inally discovered by Bedalov et al.,7 have led to relatively active
The human genome contains seven sirtuins (SirT1-7).1 The ma-
jor function of these enzymes is as NAD+-dependent deacetylases
of histone and non-histone substrates although some members
of this protein family possess ADP ribosylase activity.2 Recent stud-
ies have implicated the sirtuins in a range of important cellular
processes and disease states. SirT1 has been implicated in the reg-
ulation of the transcriptional function of p533 and SirT2 has been
linked with Parkinson’s and Huntington’s disease.4 Initial interest
in small molecule modulators of sirtuin function led to the identi-
fication of activators of potential relevance to the manipulation of
cellular lifespan under conditions of calorie restriction.5 More re-
cently, there has been considerable interest in the development
of sirtuin inhibitors and excellent review articles have been pub-
lished that summarise the current situation.6 Sirtuin inhibitors
are interesting not only due to their potential use in therapeutic
applications but also as chemical tools to help dissect the function
of the various sirtuins.
compounds that show either selective inhibition of SirT1 over SirT2
or vice versa.
In addition, we have reported the discovery of the tenovins.8
A
high-throughput screen designed to detect compounds that
activate the tumour suppressor p53, led to the prioritisation of
tenovin-1 (2) (Fig. 1) for target identification studies as 2 impaired
the growth of BL2-derived tumour xenografts. However, the
planned target identification studies proved difficult with 2 due
to its limited water solubility. Here we describe the synthesis of
a series of more water soluble analogues of 2 that, in general,
retained the desired biological activity. One of these new
H
H
N
S
N
O
S
NH
O
N
H
N
H
Our contribution to date has focused on the two sirtuin inhibi-
tor classes that possess activity in preclinical cancer models.7a,8a
O
OH
⇑
2
Corresponding authors. Fax: +44 1334 462595 (N.J.W.).
E-mail address: njw3@st-andrews.ac.uk (N.J. Westwood).
1
These authors contributed equally.
Figure 1. Structure of cambinol (1) and tenovin-1 (2).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.001

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A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
analogues, tenovin-6, was subsequently used to identify the sirtu-
ins as an important target of this class of compounds.8a In addition,
we report here the parallel synthesis of a range of tenovin-6 ana-
logues enabling the determination of structure–activity relation-
ships. Insights into the potential importance of an intramolecular
hydrogen bond for the activity of this class of compounds were
gained. Preliminary tenovin-6 metabolism studies are also re-
ported. Studies with a potential metabolite of tenovin-6 led to an
improved understanding of the function of SirT1 in cells.
Tenovin-6 was tested in a cell-based p53 induction assay8a to
assess the effect of the structural changes on activity in cells.
MCF-7 cells were treated for 6 hours with tenovin-6 at concentra-
tions ranging from 2 to 10 lM after which total p53 levels were
analysed by Western blot. Tenovin-6 treatment not only led to
an increase in p53 levels (Table 1 and Fig. S19) but to a greater ex-
tent than tenovin-1 did (Table 1).8a As expected, it was shown that
tenovin-6 was significantly more water soluble than tenovin-1 (2)
(sixfold, Table 1). Subsequent studies focused on the outcome of
varying the carbon linker chain length. An analogous synthetic
route was used to prepare the 4C (8), 6C (9) and 8C (10) analogues
(Scheme 1). In addition the all carbon linker was replaced by a
polyethylene-glycol linker to give 17. Synthesis of 17 required ini-
tial preparation of the PEG-containing linker for coupling to 5
(Scheme 1B). Conversion of hexaethylene glycol (11) to the
mono-tosylate 12 was followed by oxidation of the remaining alco-
hol in 12 to give the carboxylic acid 13.10 Formation of the corre-
sponding acid chloride 14 enabled coupling with 5 to give 15 in
46% yield. The tosylate group in 15 was then substituted by
dimethylamine via bromide 16. Finally, the hydrochloride salt
was prepared giving 17.
Biological analysis showed that extension of the linker was tol-
erated with 9 and 10 demonstrating a similar activity to tenovin-6
in the p53 assay (Table 1 and Fig. S19). Shortening the linker or
incorporation of the PEG-linker led to less active analogues with
17 being inactive in cells (Table 1). Following these studies, it
was decided to carry out target identification work using teno-
vin-6 and this ultimately led to the realisation that tenovin-6
was an inhibitor of the two sirtuins SirT1 and SirT2.8a It was there-
fore decided to test the activity of the other tenovin analogues
2. Results and discussion
2.1. Synthesis of more water soluble tenovin-1 analogues
Using a series of commercially available analogues, our previous
studies showed that the N-acetyl group present in tenovin-1 could
be modified without loss of activity (see tenovins-2, -4 and -5 in
Ref. 8a) whereas replacement of the tert-butyl group in these spe-
cific cases led to a reduction in activity. It was therefore decided to
prepare analogues of 2 containing an N-acyl group modified with a
carbon linker of varying lengths and terminating in a tertiary
amine (Scheme 1). Initially a 5-carbon (5C) linker was used in com-
bination with a dimethylamino-group (tenovin-6). The synthesis of
this analogue involved conversion of 4-tert-butyl benzoic acid (3)
to the corresponding acid chloride 4 followed by reaction with so-
dium thiocyanate to generate an N-acyl-isothiocyanate which was
reacted with 1,4-phenylenediamine to give the N-acyl thiourea 5.
Subsequent N-acylation of 5 with 5-bromovaleryl chloride, substi-
tution of the bromine by dimethylamine and formation of the cor-
responding HCl salt gave the desired analogue, tenovin-6.
NH2
A
O
O
S
b
c,d,e
R
N
H
N
H
3 R = OH
4 R = Cl
5
a
6 R = (CH2)4Br
H
7 R = (CH2)4NMe2
N
R
Tenovin-1 R = CH3
O
S
Tenovin-6 R = (CH2)4NMe2.HCl
O
N
H
N
H
8
R = (CH2)3NMe2.HCl
R = (CH2)5NMe2.HCl
9
10 R = (CH2)7NMe2.HCl
B
f
g
O
O
HO
5 OH
HO
5 OTs
11
12
O
O
h
i,j,k,l
O
O
HO
5OTs
S
Cl
5OTs
13
14
H
N
O
O
O
R
4
O
N
H
N
15 R = OTs
16 R = Br
H
17 R = NMe2.HCl
Scheme 1. Reagents and conditions: (A) (a) SOCl2, DCM, rt, 16 h, 96% (0.5 g scale), 98% (50 g scale); (b) NaSCN, acetone, rt, 8 h, then 1,4-phenylene-diamine, 16 h, 58% (4 g
scale), 74% (50 g scale); (c) acid chloride, NEt3, DCM, rt, 4–8 h; (d) 40% aq HNMe2, DCM/H2O, rt, 24 h; (e) 2 M HCl in diethyl ether, yield over three steps for tenovin-6 78% (6 g
scale), 79% (35 g scale); for tenovin-7 55%; for tenovin-8 59%; for tenovin-9 62%; (B) (f) Ts-Cl, AgO, KI, DCM, rt, 1 h, 87% (see Ref. 10); (g) CrO3/H2SO4, acetone, 0 °C–rt, 16 h, 65%
(see Ref. 10); (h) SOCl2, DCM, rt, 16 h; (i) 5, NEt3, DCM, rt, 16 h, 46% over two steps; (j) LiBr, acetone, reflux, 24 h, 81%; (k) 40% aq HNMe2, DCM/H2O, rt, 24 h, 91%; (l) 2 M HCl in
diethyl ether, DCM, 73%.

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1781
Table 1
Biological data for selected tenovin-6 analogues
R
O
S
N
H
N
R1
Compound
R
R1
p53 levelsa,e
SirT1 Inhib.b
SirT2 Inhib.b
Solubilityf (
lg/mL)
2
l
M
5
l
M
10
l
M
% Inhib 60
l
M
IC50
ndd
(
lM)
% Inhib 60
lM
IC50
ndd
(lM)
c
Tenovin-1
Tenovin-6
8
9
10
17
26
NHC(O)CH3
H
H
H
H
H
H
Me
+
++c
+++c
ndd
ndd
0.12
0.74
0.57
>0.97
>1.01
>1.34
nd
NHCO(CH2)4NMe2
NHCO(CH2)3NMe2
NHCO(CH2)5NMe2
NHCO(CH2)7NMe2
NHCO(CH2OCH2)5CH2NMe2
NHCO(CH2)4NMe2
++
À
+
+++++
++
+++++
++
73.9 1.3
85.2 3.0
96.2 3.6
70.9 16.7
29.6 0.01
6.6 1.5
37.5 0.8a
35.8 9.0
16.1 2.9
24.7 9.8
>60
94.9 0.4
10.4 0.1a
12.8 4.0
21.3 4.2
41.7 17.1
>90
85.9 À0.6
77.8 5.9
59.9 11.8
19.0 0.01
46.7 4.2
+++++
++++
À
++++
+++++
À
+
À
À
À
À
nd
nd
a
p53 levels were detected in MCF-7 cells after 6 h treatment with the indicated compounds.
Enzyme inhibition as determined using the commercially available assay kit (Bioml/Enzo Life Sciences). Results were obtained in triplicate. Values are expressed as
b
means SD.
c
Value differs from previously reported due to change in peptide substrate concentration (from 7 lM (Ref. 8a) to 25 lM).
Not determined due to lack of water solubility.
The following symbols were used to represent: +, small increase; ++, moderate increase; +++, medium increase; ++++, significant increase; +++++, large increase; À, no
d
e
increase in p53 levels; nd, not determined.
f
Solubility was determined using a by UV spectrophotometric method. The extinction co-efficient for each compound was calculated and used to determine the solubility
(see Section 4.4).
against purified human recombinant SirT1 and SIRT2. Whilst rela-
tively little difference in the activity against both enzymes in vitro
was observed on varying the linker length (cf. tenovin-6 and ana-
logues 8–10), a clear difference came with 17 which was shown
to no longer inhibit SirT1 or SirT2 (Table 1). This observation is
consistent with the lack of p53 activation observed on treatment
of MCF-7 cells with 17.
2.2. Large scale synthesis of tenovin-6
As tenovin-6 retained the same biological activity as tenovin-1
and was of sufficiently high water solubility to enable the target
identification studies to be carried out,8a it was decided that a large
quantity of tenovin-6 should be prepared for use in preclinical effi-
cacy models of human tumours. Increasing the scale of the synthe-
sis of 5 proved particularly effective as on scales of over 15 g, 5
crashed out of the crude reaction mixture giving, after filtration,
a solid of sufficient purity for use in the next step. Acylation of 5
Figure 2. (A) Representation of the small molecule crystal structure of tenovin-1;
(B) schematic of tenovin-6 highlighting important regions of the molecule.
with bromovaleryl chloride initially proved more challenging on
a larger scale with the generation of an unknown by-product. How-
ever, optimised conditions were found that enabled the isolation of
a pure sample of the required bromide 6 (Scheme 1) following fil-
tration of the crude reaction mixture and washing the filter cake
with dichloromethane. On scale up, the displacement reaction to
give 7 proceeded in an analogous manner to the small scale reac-
tion and did not require further optimisation. Formation of the
HCl salt of 7 afforded a high yield of pure tenovin-6 on up to a
35 g scale.
evidence for the existence of this hydrogen bond in the solid state
(Fig. 2A).11 Interestingly, a previously reported co-crystal structure
of glycogen phosphorylase (GP) with an N-acyl-N’-aryl substituted
urea (structurally related to the N-acyl N’-aryl thiourea present in
the tenovins) showed the GP-inhibitor bound to the protein in a
conformation that contained an intramolecular hydrogen bond.12
In order to explore whether the ability to form the intramolec-
ular hydrogen bond in the tenovins correlated with biological
activity against the sirtuins, it was decided to prepare an analogue
of tenovin-6 in which the N14H was replaced by a N14Me group
(26, Scheme 2A). It was reasoned that 26 could not form the intra-
molecular hydrogen bond and would adopt an alternative pre-
ferred conformation in solution to tenovin-6. A modified version
of our initial approach to the tenovins was developed to prepare
analogue 26. In accordance with literature precedent, N-Boc pro-
tected 1,4-phenylenediamine 18 was N-trifluoroacetylated to give
19 (Scheme 2A).13 N-Methylation of 19 with concomitant removal
of the trifluoroacetate group to give 20 was followed by reprotec-
tion of the N-methylated nitrogen with a Cbz-group leading to 21.
Removal of the N-Boc-protection and coupling of the resulting
2.3. Tenovins adopt a preferred conformation in solution that
contains an intramolecular hydrogen bond
1H NMR analysis of tenovins-1, 6 and analogues 8–10 showed
the presence of an unexpectedly low field signal at higher than
12.5 ppm (see Figs. S2, S3 and Table S1). This signal was assigned
to the N14-hydrogen atom (Fig. 2A) and its chemical shift was
rationalised based on a proposal that in the preferred conformation
of the tenovins the N14H forms an intramolecular hydrogen bond
with the oxygen atom of the C11-carbonyl. Small molecule X-ray
crystallographic analysis of a crystal of tenovin-1 also provided

1782
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
A
NHBoc
NHBoc
NHBoc
O
d
a
b
R1RN
F3C
N
H
H2N
1
18
19
20
21
R = Me, R = H
R = Me, R = Cbz
c
1
H
H
N
N
R2
NMe2
O
S
g
3
O
O
R1RN
N
N
H
Me
22 R = Me, R1 = Cbz, R2 = Br
23 R = Me, R1 = Cbz, R2 = NMe2
24 R = Me, R1 = H, R2 = NMe2
25 R = R1 = H, R2 = NMe2
e
f
26
B
H
N
NH2
h
R1
j
25
O
RHN
RHN
i
1
28
29
R = Cbz, R = Br
R = Cbz, R = NMe2
27
R = Cbz
1
H
N
NMe2.HCl
R1
O
S
k,l
R2
R3
O
N
H
N
H
30a-q
Scheme 2. Reagents and conditions: (A) (a) NEt3, trifluoroacetic anhydride, DCM, 0 °C–rt, 15 min, 98%; (b) tBuOK, THF, À78 °C, then MeI, 50 °C, tBuOK, 1.5 h, 59%; (c) benzyl
chloroformate, DIPEA, DCM, 0 °C–rt, 16 h, 94%; (d) trifluoroacetic acid, DCM, rt, then 5-bromovaleryl chloride, NEt3, DCM, rt, 2 h, 32%; (e) 40% aq HNMe2, DCM/H2O, rt, 24 h,
86%; (f) 48% w/v HBr in acetic acid, rt, 1 h, 96%; (g) 4-t-butylbenzoyl chloride, acetone, NaSCN, rt, 16 h, then 24, rt, 16 h, 30%; (B) (h) 5-bromopentanoyl chloride, DIPEA, DCM,
rt, 4 h, 71%; (i) 40% aq HNMe2, DCM/H2O, rt, 16 h, 68%; (j) 48% w/v HBr in acetic acid, rt, 1 h or 10% Pd/C, H-Cube, MeOH, quantitative; (k) acid chloride, NaSCN, acetone, rt,
30 min then 25, acetone, rt, 16 h, 8–74%; (l) 2 M HCl in diethyl ether, acetone, 54–91%.
aniline to 5-bromovaleryl chloride gave the required intermediate
22. Subsequent incorporation of the water solubilising tertiary
amine led to 26 via 23 after hydrogenolysis to remove the Cbz-
group (to give 24), coupling with the N-acyl-isothiocyanate gener-
ated from 4 (Scheme 1A) and salt formation. Interestingly, 26 was
found to be a very poor inhibitor of SirT1 and a relatively weak
inhibitor of SirT2 (Table 1) and led to no increase in p53 levels in
cells (Fig. S49). This suggests that the tenovins may bind to SirT1
in a conformation that retains the intramolecular hydrogen bond
although this can only be proved by solving a co-crystal structure
of a tenovin analogue with the SirT1 enzyme.14 Comparison of the
intramolecularly hydrogen bonded conformer of the tenovins with
the structure of cambinol (1) suggests that the two compounds
may occupy the same or overlapping binding sites although the
positions in which substituents are incorporated in the fixed six-
membered ring in 1 differ from those in the tenovins. At this stage
it was decided to prepare analogues of tenovin-6 that differed in
structure in the tert-butyl-phenyl ring (Fig. 2B). Whilst modifica-
tions in this position had previously led to a reduction in activity
(see Ref. 8a), these studies were limited and further studies were
required in order to try and increase potency and selectivity.
rapid preparation of analogues of tenovin-6 that differed in the
substituents in the tert-butyl-substituted aromatic ring. This ap-
proach was inspired by our route to 26 and involved the synthesis
of the key intermediate aniline 25 (Scheme 2A and B). Compound
25 was initially prepared by HBr-mediated deprotection of the N-
Cbz protected aniline 29 (prepared as shown in Scheme 2B from
27 via 28). However, this route to 25 proved unreliable with sam-
ples of 25 decomposing possibly due to difficulties in removing ex-
cess HBr. Deprotection by hydrogenation using a palladium on
carbon catalyst proved more robust, with samples of 25 being sta-
ble over extended periods of time after using this deprotection pro-
cedure. Standard parallel synthesis protocols were then used to
prepare a range of tenovin-6 analogues using 25 with purification
of the resulting free bases taking place on short silica plugs prior to
hydrochloride salt formation (Scheme 2B and Table 2). The choice
of substituents enabled exploration of the importance of the size
and electronic properties of the 4-substituent as well as the effect
of incorporating substituents at the 2- and 3-positions. Analogues
containing two substituents were also prepared.
2.5. Biological activity of novel analogues in vitro and in cells
2.4. Parallel synthesis of tenovin-6 analogues and preliminary
SARs for SirT1 and SirT2
Assessment of the activity of the novel tenovin-6 analogues as
inhibitors of SirT1 and SirT2 in vitro was then carried out. Table 2
shows the percentage inhibition at a final concentration of inhibitor
Whilst practical for the large scale synthesis of tenovin-6, the
synthetic route described in Scheme 1A was modified to enable
of 60
l
M for all analogues and selected IC50 data. Replacement of the
4-tert-butyl substituent in tenovin-6 with
a
4-n-propyl,
a

Table 2
Biological data for selected tenovin-6 analogues
H
N
NMe2.HCl
R1
O
X
R2
R3
O
N
H
N
H
X
R1
R2
R3
SirT1 inhibition
SirT2 inhibition
p53b
Ac-tubulinc
20
Ma
IC50
(l
M)
% Inhib 60
l
Ma
IC50
(lM)
b-Gal 5
lM
b-Gal 10
l
M
b-Gal 20
lM
10
lM
l
M
30 lM
a
a
% Inhib 60
l
Tnv-6
30a
30b
30c
30d
30e
30f
30g
30h
30i
30j
30k
30l
30m
30n
30o
30p
30q
33
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
H
H
H
H
H
H
H
H
H
H
t-Bu
n-Pr
iso-Pr
n-Bu
n-Pen
H
Me
OMe
Cl
Br
H
H
H
73.9 (1.3)
50.9 (3.4)
52.9 (2.8)
56.3 (2.8)
73.2 (1.9)
3.5 (0.5)
47.3 (2.3)
48.5 (0.9)
53.4 (1.2)
13.5 (1.0)
5.2 (1.1)
25.3 (6.9)
29.2 (0.3)
70.5 (1.4)
93.5 (0.4)
8.1 (0.1)
37.5 (1.0)
60.2 (4.7)
42.7 (3.1)
46.3 (1.4)
36.7 (1.7)
>90
>90
>90
>90
>90
>90
nd
nd
39.9 (8.0)
18.3 (0.3)
70.9 (4.0)
>90
>90
21.7 (1.3)
17.9 (3.1)
94.9 (0.4)
88.9 (3.0)
78.4 (4.4)
90.1 (3.7)
85.4 (7.4)
43.7 (4.2)
40.1 (3.5)
51.1 (4.5)
61.5 (2.1)
47.7 (0.4)
20.9 (2.7)
30.5 (0.1)
57.9 (0.1)
81.1 (2.8)
91.1 (0.7)
68.5 (4.5)
48.5 (3.5)
54.3 (1.6)
35.3 (14)
84.9 (1.6)
10.4 (0.1)
7.85 (1.3)
5.71 (0.7)
8.6 (1.1)
5.6 (0.9)
>90
>90
50.6 (1.4)
>90
+ (11)
À (1.0)
+ (8.7)
+ (10.5)
+ (1.5)
+ (3.5)
+ (2.4)
+ (12)
Tox (0.28)
+ (1.2)
À (0.92)
À (1.1)
À (1.12)
À (1.01)
À (1.01)
À (0.89)
À (1.2)
À (1.0)
+ (1.2)
+ (3.0)
À (1.0)
+ (1.3)
+ (1.5)
À (1.1)
À (0.78)
À (1.1)
À (0.99)
À (1.0)
À (0.97)
À (1.0)
À (1.0)
À (0.95)
À (1.0)
À (1.0)
À (1.0)
À (1.0)
À (1.0)
+ (1.4)
À (1.2)
À (0.86)d
À (1.1)
+ (1.4)
Tox (0.66)
À (1.2)
À (0.90)
À (0.99)
À (1.0)
À (1.1)
+ (1.4)
À (1.2)
À (1.1)
À (1.2)
À (0.98)
À (1.1)
À (1.9)
À (1.1)
À (1.0)
+ (1.8)
Toxicd
+ (1.4)
+ (1.3)
Toxic
+ (11.1)
À (0.93)
À (1.1)
À (0.97)
À (1.1)
À (1.1)
À (1.2)
À (1.1)
À (0.89)
À (1.1)
À (1.1)
À (1.2)
À (1.1)
À (1.1)
À (0.98)
À (1.1)
+ (7.3)
À (0.91)
À (1.1)
À (1.1)
À (1.2)
À (1.0)
À (1.1)
À (1.0)
À (0.94)
À (1.1)
À (1.1)
À (1.1)
À (1.1)
À (1.04)
À (0.99)
À (1.2)
+ (6.9)
nd
+ (1.4)
À (0.95)
À (1.0)
À (1.0)
À (1.2)
+ (1.3)
À (1.2)
À (1.1)
À (1.0)
À (0.97)
+ (1.3)
+ (1.2)
À (1.2)
À (1.0)
+ (2.1)
+ (1.9)
H
H
55.0 (5.4)
>90
nd
Me
OMe
Cl
Br
Br
Cl
H
H
H
H
nd
H
t-Bu
Cl
H
H
21.2 (7.0)
4.6 (0.6)
31.9 (6.0)
42.5 (2.4)
>90
+ (1.4)
À (0.97)
À (0.84)
À (1.2)
+ (6.5)
9.9 (0.9)
29.3 (0.9)
93.6 (4.5)
72.5 (2.1)
t-Bu
t-Bu
88.4 (5.2)
12.4 (1.3)
39
+ (3.9)
+ (5.5)
+ (1.6)
a
Enzyme inhibition was determined using the commercially available assay kits (Biomol/Enzo Life Sciences). Results were obtained in triplicate. Values are expressed as means (SD).
p53 activation using a reporter gene assay was determined in ARN8 cells in triplicate. + Denotes active and À denotes no observed activity. Values in parentheses correspond to the ratio between compound treated cells and
b
DMSO control cells to 2 significant figures. For further details see Section 4.
c
Levels of K40 acetylated tubulin were determined in H1299 cells using western blotting in triplicate. + Denotes active and À denotes no observed activity. Values in parentheses correspond to the ratio between the intensity of
the bands in the compound treated and DMSO control treated cells to 2 significant figures.
d
Tenovin-6 was toxic to H1299 cells at higher concentrations, see Ref. 8a. For more information on the interpretation of cell data and examples of Ac-tubulin blots see Supplementary data (Figs. S8–S10).

1784
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
4-iso-propyl, a 4-n-butyl or a 4-n-pentyl substituent led to only min-
or changes in activity against SirT2 indicatingthat this enzyme is rel-
atively tolerant of the nature of the alkyl substituent at this position
(cf. Table 2, analogues 30a–d). Inhibition of SirT1 varied more with
the structure of the 4 analogues. Interestingly, whilst the analogues
with shorter alkyl chains (30a and 30b) showed the initially ex-
pected activity in cells, analogues 30c and 30d (with longer n-alkyl
chains) proved either toxic or inactive in cells. Differences in the cell
permeability of these two analogues compared to tenovin-6 and
analogues 30a and 30b may explain this observation. It should be
noted that previous studies by us7b,c,8a and others3e have looked to
correlate inhibition of SirT1 in vitro with p53 activation in cells
and in vitro SirT2 inhibition with an increase in K40-acetylated
tubulin levels in cells. Results presented in Section 2.6 below suggest
that this approach may be an oversimplification.
A decrease in activity against both SirT1 and SirT2 was ob-
served with a range of alternative 4-substituents including small
substituents such as 4-H and 4-methyl (30e and 30f), electron-
donating (4-Me, 4-OMe, 30f and 30g) and electron-withdrawing
(4-Cl and 4-Br, 30h and 30i) substituents. Some degree of selec-
tivity for inhibition of SirT2 over SirT1 was observed when a 4-
methoxy- or a 4-bromo-substituent was present (analogues 30g
and 30i). Replacement of the 4-tert-butyl group in tenovin-6
with a 3-methyl, 3-methoxy or 3-chloro group led to a loss in
activity (compounds 30j–l) against both enzymes although incor-
poration of a 3-bromo-substituent both in the absence (30m)
and the presence of the 4-tert-butyl group (30n) led to an
substituent was not mirrored by the activity in cells. Incorpora-
tion of a 3,4-dichloro-substitution pattern had a detrimental
effect on activity for both enzymes compared to tenovin-6 (com-
pound 30o). Further studies also suggested that incorporation of
an electron-donating substituent at the 2-position of this aro-
matic ring may result in tuning of activity towards SirT2 with
the 2-Me analogue showing better activity against SirT2 than
SirT1 (30p). In addition to the in vitro characterisation of these
analogues, 30a–q were all tested in cells. An alternative method
of assessing p53 activation in cells was used for these analogues
compared to compounds 8–10, 17 and 26 (see Table 2, Section 4
and Ref. 8a for details). This change was made as the p53-depen-
dent transcription assay was judged to be more robust, quantita-
tive (see values in Table 2) and cost effective for the analysis of
an increased number of analogues. As observed for the sirtuin
inhibitor cambinol (1),7b analogues that did not inhibit SirT1
activity in vitro failed to activate p53-dependent transcription
in cells. However, analogues 30c, 30d, 30m and 30n did not acti-
vate p53-dependent transcription in cells despite inhibiting SirT1
in vitro. Unexpectedly poor physicochemical properties such as
cell-permeability or cellular toxicity (see 30c) may explain these
results. The robust SirT1 inhibitors 30a and 30b did activate
p53-dependent transcription. Unlike the in vitro SirT1 inhibition,
which appears to be very sensitive to changes in compound
structure, several of the novel analogues retained activity against
SirT2 in vitro. In general, a reasonable correlation between SirT2
inhibition in vitro and increases in K40-acetylated tubulin levels
in cells was observed, with 7 of the 10 in vitro SirT2 inhibitors
leading to increased levels of K40-acetylated tubulin. All of the
analogues that were found to increase acetylated tubulin levels
in cells, inhibited SirT2 in vitro.
improvement in activity with 30n having IC50s of 18.3 0.3
and 4.6 0.6 M against SirT1 and SirT2 respectively. Unfortu-
nately, the maintenance of in vitro activity (compared to
lM
l
tenovin-6) associated with the incorporation of
a 3-bromo-
A
NHBoc
O
O
O
a,b
NH2
N
H
N
H
32
31
H
N
NMe2.HCl
O
O
O
c,d,e
N
H
N
H
33
O
B
R1
CO2H
h
N
H
RHN
RHN
36 R = Cbz, R1 = OH
37 R = Cbz, R1 = NMe2
38 R = H, R1 = NMe2
34 R = H
i,j
k
g
35
R = Cbz
O
NMe2.HCl
N
H
O
S
l,m
N
H
N
H
39
Scheme 3. Reagents and conditions: (A) (a) (COCl)2, 1,2-DCE, reflux, 24 h; (b) 14, MeCN, reflux, 3 h, 17%; (c) TFA, rt, 40 min quantitative; (d) 5-bromovaleryl chloride, NEt3,
DCM rt, 16 h (48%); (e) 40% aq HNMe2, H2O, DCM, rt, 20 h then 2 M HCl in diethyl ether, acetone 36%; (B) (g) benzyl chloroformate, satd aq NaHCO3, THF, rt, 16 h, 85%; (h)
methyl chloroformate, NEt3, DCM, 0 °C–rt, 2 h then 4-aminobutanol, rt, 6 h, 72%; (i) CBr4, PPh3, DCM, 0 °C–rt, 4 h, 54%; (j) 40% aq HNMe2, DCM/H2O, rt, 16 h, 67%; (k) 10% Pd/C,
H-Cube, MeOH, quantitative; (l) 4-t-butylbenzoyl chloride, NaSCN, acetone, rt, 30 min then 38, acetone, rt, 16 h, 52%; (m) 2 M HCl in diethyl ether, acetone, 86%.

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1785
2.6. Other issues relating to the tenovin structure
Interestingly, 39 activated p53 in cells, showing that the poten-
tial metabolism to 1,4-phenylenediamine is not essential for p53
activation by tenovin-6. However, 39 was not as potent as teno-
vin-6 in this assay. Subtle differences in the cell permeability of
the two isomeric tenovins may explain this observation.
Prior to carrying out mouse xenograft experiments,8a studies fo-
cused on possible methods by which the tenovins may be metab-
olised in vivo. This work was initially carried out in order to
determine the half-life of tenovin-6 and hence to provide informa-
tion on potential dosing regimes in xenograft experiments.8a Incu-
bation of tenovin-6 with H1299 cells for 16 h, followed by cell lysis
and extraction with methanol led to the identification of one major
degradation product (Fig. S59 retention time (rt) (tenovin-
6) = 5.61 min; rt (degradation product) = 4.46 min). Mass spectro-
metric analysis showed that degradation was accompanied by a
loss of 16 Da (data not shown), which was predicted to arise from
oxidative desulfurisation15 of the thiourea functional group to the
corresponding urea. Evidence to support this came following the
synthesis of urea compound 33 (Scheme 3A). This was achieved
in six steps from 4-tert-butylbenzamide (31) via the N-acyl-urea
32. Comparison of the retention time and fragmentation pattern
of synthetic 33 with those of the degradation product corroborates
the assignment (Figs. S6 and S79). Synthetic 33 inhibited SirT1
in vitro and also caused an increase in p53-dependent transcrip-
tion in the cellular assay (Table 2).
3. Conclusions
The development of sirtuin inhibitors continues to be of consid-
erable interest and importance. Significant advances in our under-
standing of the detailed biology of this class of deacetylases are
being made and potent and selective chemical tools can help with
this. In addition, it has become clear that modulation of sirtuin
function can be linked to a range of potential therapeutic applica-
tions and an increased range of chemical starting points for drug
discovery applications is desirable. Here, the synthesis of a series
of novel tenovin analogues is reported including a demonstration
that the chemistry is relevant to large scale work and applicable
to a medium-throughput format. During the course of the work,
1H NMR and X-ray crystallographic analysis of the tenovins
showed that this inhibitor class adopts a preferred conformation
in solution and in the solid state that contains an intramolecular
hydrogen bond. Evidence suggesting that the ability to form this
hydrogen bond is linked to the desired biological activity is pro-
vided for SirT1 inhibition. The situation for SirT2 inhibition is less
clear at present. This observation may aid future developments
in sirtuin inhibitor development with fixed ring analogues of the
tenovins being of potential interest. The results of our recent stud-
ies in this area will be reported in the near future. Finally, prior to
xenograft experiments in mice,8a it was necessary to consider po-
tential pathways by which the tenovins may degrade in cells.
The oxidative desulfurisation of the thiourea functionality was
identified as a potential issue and the synthesis of 33, the oxo-
version of tenovin-6, verified this. In addition, further experiments
with analogue 33 provide new evidence that K40-acetylated
tubulin is a substrate of SirT1 in cells. This conclusion is consistent
It therefore seems likely that once in the cell both tenovin-6 and
33 can inhibit SirT1 function. Analogue 33 was also found to in-
crease levels of K40-acetylated tubulin in cells (Table 2). Intrigued
by this unexpected result, further experiments were carried out to
investigate whether the ability of 33 to inhibit SirT1 could be
responsible for this observation.
A cytoplasmic extract from
H1299 cells treated with trichostatin A was prepared. This extract
contains relatively high levels of K40-acetylated tubulin (Fig. 3A).
The extract was then treated with purified recombinant SirT1 in
the presence/absence of NAD+. As shown in Figure 3A a decrease
in the levels of K40-acetylated tubulin was observed in the pres-
ence of SirT1 at two different concentrations and this decrease
was NAD+-dependent. In addition, overexpression of SirT1 in
H1299 cells also led to a reduction in the levels of K40-acetylated
tubulin in cells (Fig. 3B). These results are consistent with the
unexpected increase in tubulin levels on treatment of H1299 cells
with the apparently SirT1 selective inhibitor 33.
with
a previous report on tubulin deacetylation by sirtuins
although no comment was made about SirT1’s ability to deacety-
late tubulin in the earlier report.17 Synthesis of a reverse amide
analogue of tenovin-6 also confirmed that the observed activa-
tion of p53 could not be explained by formation of 1,4-
phenylenediamime.
1,4-Phenylenediamine is a known carcinogen.16 As 1,4-phenyl-
enediamine is also a potential metabolite of tenovin-6 concerns
were raised about the possibility that this could explain the ob-
served p53 activation. The fact that not all tenovin analogues pre-
pared to date activate p53 despite containing the diamine unit
argues against this. However, it was decided to prepare and assess
the biological activity of 39, an analogue in which one of the
amide bonds is reversed and which cannot be metabolised to
1,4-phenylenediamine. 39 was prepared from 4-amino-benzoic
acid (31) (Scheme 3B) and was shown to have comparable activity
in the in vitro SirT1 and SirT2 assay to tenovin-6 (Table 2).
4. Experimental section
4.1. Chemistry
Unless otherwise stated, starting materials and reagents were
obtained from commercial suppliers and were used without fur-
ther purification. 1H and 13C NMR spectra were measured on a Brü-
ker Advance 300/400 instrument. Chemical shifts are calibrated
with reference to the residual proton and carbon resonances of
the solvent (CDCl3: dH = 7.26, dC = 77.0 ppm; DMSO-d6: dH = 2.50,
dC = 39.5 ppm; CD3OD: dH = 3.31, dC = 49.0 ppm). Low and high-res-
olution mass spectral analyses were recorded using chemical ion-
isation operating in positive or negative ion mode.
4.1.1. Preparation of tenovin-6 and analogues 8–10
4.1.1.1. 1-(4-Amino-phenyl)-3-(4-tert-butyl-benzoyl)-thiourea
(5). To a solution of 4 (4.0 g, 20.4 mmol) in acetone (30 mL) was
added sodium thiocyanate (1.7 g, 20.8 mmol). The resulting pale
yellow suspension was stirred at rt for 8 h, then added to a solution
of 1,4-phenylenediamine (4.4 g, 40.8 mmol) in acetone (30 mL) at
0 °C. The brown suspension was warmed to rt and stirred for 16 h.
The solvent was concentrated in vacuo and the remaining residue
Figure 3. SirT1-mediated deacetylation of K40-acetylated
a-tubulin. (A) A cyto-
plasmic extract prepared from H1299 cells containing acetylated tubulin was
treated with purified recombinant His-tagged SirT1 in the presence/absence of
NAD+. Different concentrations of SirT1 were used; (B) comparison of K40-
acetylated tubulin levels in vector transfected H1299 cells (H1299pcDNA3) or
overexpressing SirT1 cells (H1299SirT1). Arrow indicates expression of ectopic
SirT1 isoform a.

1786
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
resuspended in CH2Cl2 and the insoluble solids filtered. The filtrate
was concentrated in vacuo to give a brown solid which was purified
by column chromatography on silica using petroleum ether/ethyl
acetate (1:1) as the eluent and subsequent recrystallisation from
ethyl acetate to give 5 (4.4 g, 13.5 mmol, 58%) as a pale yellow solid.
Mp 185–186 °C; mmax cmÀ1 (KBr) 3398, 3350, 3200, 2960, 1684,
1665, 1609, 1542, 1328, 1274, 1126, 846, 772, 720, 621; 1H NMR
(300 MHz, CDCl3) d 12.37 (1H, br s, NH), 9.02 (1H, br s, NH), 7.81
(2H, d, J = 10.8 Hz, ArH), 7.68 (2H, d, J = 8.6 Hz, ArH), 7.49 (2H, d,
J = 8.6 Hz, ArH), 6.71 (2H, d, J = 11.8 Hz, ArH), 3.75 (2H, br s, NH2),
1.36 (9H, s, (CH3)3); 13C NMR (100 MHz, CDCl3) d 178.5, 166.8,
157.7, 145.4, 128.8, 128.7, 127.4, 126.2, 125.8, 115.1, 35.0, 31.1;
LRMS (m/z): [M+H]+ 329.56; HRMS (m/z): [M]+ calcd for C18H22N3OS,
328.1484; found, 328.1486.
4.1.1.4. N-{4-[3-(4-tert-Butyl-benzoyl)-thioureido]-phenyl}-4-
dimethylamino-butyramide hydrochloride (8). Thiourea
(200 mg, 0.61 mmol) was stirred in CH2Cl2 (10 mL) under N2 atmo-
sphere. 4-Bromobutyryl chloride (169 mg, 0.92 mmol) was added,
5
followed by triethylamine (85 lL, 0.61 mmol). The orange solution
was stirred at rt for 1.5 h and diluted with CH2Cl2 (10 mL). The or-
ganic solution was washed with 1 M aqueous hydrochloric acid
(1 Â 10 mL), 10% aqueous sodium hydroxide (1 Â 50 mL), and sat-
urated brine (1 Â 10 mL). The organic phase was dried over MgSO4
and concentrated in vacuo. The resulting pale yellow foam was
purified by flash chromatography on silica using hexanes/ethyl
acetate (1:1) to give 4-bromo-N-{4-[3-(4-tert-butyl-benzoyl)-thio-
ureido]-phenyl}-butyramide (138 mg, 0.29 mmol, 48%) as a yellow
oil. 1H NMR (300 MHz, CDCl3) d 12.59 (1H, br s, NH), 9.08 (1H, br s,
NH), 7.82 (2H, d, J = 8.7 Hz, ArH), 7.60 (6H, m, ArH), 3.52 (2H, t,
J = 6.2 Hz, CH2), 2.57 (2H, t, J = 7.0 Hz, CH2), 2.26 (2H, m, CH2),
1.35 (9H, s, (CH3)3). Treatment of the prepared bromo compound
(138 mg, 0.29 mmol) with dimethylamine (3 mL) in H2O (7.5 mL)
gave a crude mixture which was dissolved in a minimal amount
of acetone and 2 M hydrochloric acid in ether solution was added
dropwise until a precipitate appeared. The solid was filtered and
the yellow solid which was recrystallised from isopropanol to give
4.1.1.2. 5-Bromo-pentanoic acid {4-[3-(4-tert-butyl-benzoyl)-
thioureido]-phenyl}-amide (6). Thiourea
5 (30.0 g, 91.5 mmol)
was stirred in CH2Cl2 (600 mL) under N2 atmosphere. 5-Bromovaleryl
chloride (18.3 g, 91.5 mmol) was added, followed by triethylamine
(12.6 mL, 91.5 mmol). The beige suspension was stirred at rt for
6 h. The suspension was filtered to afford 6 (36.0 g, 75.0 mmol,
82%) as a light yellow powder. An analogous protocol was used to
prepare 6 (1.0 g, 2.0 mmol, 65%) from 5 (1.0 g, 3.1 mmol) except
following reaction the solution was washed sequentially with 1 M
aqueous HCl (1 Â 15 mL), 1 M NaOH (1 Â 15 mL) and brine
(1 Â 15 mL). The organic layer was dried over MgSO4 and the solvent
removed in vacuo to afford an off-white gum. The residue was puri-
fied by column chromatography using hexanes/ethyl acetate (1:1) as
the eluent to afford the product. Mp 158–159 °C; mmax cmÀ1 (KBr)
3358, 3210, 2964, 2947, 1695, 1657, 1604, 1558, 1347, 1269, 1174,
1113, 952, 842, 641; 1H NMR (300 MHz, CDCl3) d 12.60 (1H, br s,
NH), 9.05 (1H, br s, NH), 7.82 (2H, m, ArH), 7.68 (2H, d, J = 8.9 Hz),
7.57 (4H, m, ArH), 7.22 (1H, br s, NH), 3.45 (1H, t, J = 6.3 Hz, CH2),
2.42 (1H, t, J = 6.9 Hz, CH2), 1.93 (4H, m,(CH2)2), 1.36 (9H, s, (CH3)3);
13C NMR (100 MHz, CDCl3) d 178.4, 170.9, 166.9, 157.9, 133.6,
128.6, 127.4, 126.3, 124.9, 119.9, 37.4, 33.6, 32.5, 31.1, 27.7, 24.6.
LRMS (m/z): [M]+ 512.06, [M+Na]+; HRMS (m/z): [M]+ calcd for
8 (75 mg, 0.16 mmol, 55%) as a yellow solid. m
max cmÀ1 (NaCl, thin
layer) 3239, 3044, 2921, 2342, 1659, 1603, 1514, 1074, 793; 1H
NMR (300 MHz, DMSO-d6) d 12.61 (1H, br s, NH), 11.44 (1H, br s,
NH), 10.17 (1H, br s, NH), 9.64 (1H, br s, NH+), 7.95 (2H, d,
J = 8.5 Hz, ArH), 7.62 (6H, m, ArH), 3.09 (2H, m, CH2), 2.79 (6H, d,
J = 4.8 Hz, N(CH3)2), 2.44 (2H, t, J = 7.3 Hz, CH2), 1.95 (2H, m, CH2)
and 1.32 (9H, s, (CH3)3); 13C NMR (75 MHz, DMSO-d6) d 179.4,
171.2, 168.3, 156.4, 137.6, 133.1, 129.5, 128.9, 125.5, 125.1,
119.1, 56.7, 42.5, 35.9, 35.3, 31.3; LRMS (m/z): [M+H]+ 441.28;
HRMS (m/z): [M]+ calcd for C24H33N4O2S, 441.2318; found,
441.2322.
4.1.1.5. 6-Dimethylamino-hexanoic acid {4-[3-(4-tert-butyl-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (9). Thio-
urea 5 (200 mg, 0.61 mmol) was stirred in CH2Cl2 (10 mL) under
N2 atmosphere. 6-Bromohexanoyl chloride (195.8 mg, 0.92 mmol)
C
23H28N3O2NaS79Br, 512.0983; found, 512.0995.
was added, followed by triethylamine (85 lL, 0.61 mmol). The
4.1.1.3. 5-Dimethylamino-pentanoic acid {4-[3-(4-tert-butyl-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (tenovin-
beige suspension was stirred at rt for 1.5 h and diluted with CH2Cl2
(10 mL). The organic solution was washed with 1 M aqueous
hydrochloric acid (1 Â 10 mL), 10% aqueous sodium hydroxide
(1 Â 50 mL), and saturated brine (1 Â 10 mL). The organic phase
was dried over MgSO4 and concentrated in vacuo. The resulting
brown foam was purified by flash chromatography on silica (hex-
anes/ethyl acetate 1:1) to give 6-bromo-hexanoic acid {4-[3-
(4-tert-butyl-benzoyl)-thioureido]-phenyl}-amide (166 mg, 0.33
mmol, 55%) as a yellow oil. 1H NMR (300 MHz, CDCl3) d 12.58
(1H, br s, NH), 9.10 (1H, br s, NH), 7.57 (7H, m, ArH, NH), 3.40
(2H, t, J = 6.7 Hz, CH2), 2.36 (2H, t, J = 7.4 Hz, CH2), 1.89 (2H, m,
CH2), 1.73 (2H, m, CH2), 1.51 (2H, m, CH2), 1.35 (9H, s, (CH3)3).
Treatment of prepared bromo compound (136 mg, 0.27 mmol)
with dimethylamine (2.5 mL) in H2O (6.25 mL) gave a crude mix-
ture containing that was dissolved in a minimal amount of CH2Cl2
and a 2 M hydrochloric acid in ether solution was added dropwise
until a cream precipitate appeared. The solid was filtered and
recrystallised from isopropanol to give 9 (80 mg, 0.16 mmol, 59%)
as a yellow solid. mmax cmÀ1 (NaCl, thin layer) 3372, 3039, 2961,
2863, 2707, 1701, 1698, 1664, 1609, 1516, 1472, 1270, 1125,
852, 772; 1H NMR (300 MHz, DMSO-d6) d 12.59 (1H, s, NH),
11.42 (1H, s, NH), 10.08 (1H, s, NH), 9.83 (1H, br s, NH+), 7.94
(2H, d, J = 8.4 Hz, ArH), 7.59 (6H, m, ArH), 3.01 (2H, m, CH2), 2.73
(6H, d, J = 4.8 Hz, N(CH3)2), 2.35 (2H, t, J = 7.1 Hz, CH2), 1.64 (4H,
m, (CH2)2) and 1.32 (9H, s, (CH3)3); 13C NMR (75 MHz, DMSO-d6)
d 179.7, 171.2, 168.0, 156.2, 137.4, 132.7, 129.2, 128.6, 125.3,
124.8, 118.9, 57.9, 43.9, 36.2, 34.8, 30.8, 26.1, 25.6, 24.8; LRMS
6). To
a stirred solution of 6 (35.0 g, 71.6 mmol) in DCM
(600 mL) was added dimethylamine (40% aqueous solution)
(240 mL) and water (600 mL) and the resulting biphasic mixture
stirred rapidly at rt for 16 h. The mixture was diluted with CH2Cl2
(200 mL) and the resulting organic layer was washed with water
(300 mL), 1 M aqueous sodium hydroxide (300 mL), saturated
brine (2 Â 300 mL), dried over MgSO4 and concentrated in vacuo
to give 7 as an off-white solid. Purification of 7 was accomplished
by conversion to the corresponding HCl salt. Compound 7 was dis-
solved in a minimal amount of acetone and 2 M hydrochloric acid
in ether solution was added dropwise until a yellow precipitate ap-
peared. The solid was filtered to give Tenovin-6 as a yellow solid
(24.1 g, 49.4 mmol, 69%). An analogous protocol was used to pre-
pare Tenovin-6 (686 mg, 1.4 mmol, 67%) from 6 (1.0 g, 2.0 mmol).
Mp 206–208 °C; mmax cmÀ1 (KBr) 3236, 3040, 2952, 2663, 2503,
1674, 1610, 1577, 1348, 1270, 1159, 1113, 966, 834, 703; 1H
NMR (300 MHz, DMSO-d6) d 12.61 (1H, br s, NH), 11.43 (1H, br s,
NH), 10.13 (1H, br s, NH), 9.77 (1H, br s, NH), 7.95 (2H, d,
J = 8.4 Hz, ArH), 7.59 (6H, m, ArH), 3.06 (2H, m, CH2), 2.75 (6H, s,
N(CH3)2), 2.40 (2H, t, J = 6.1 Hz, CH2), 1.66 (4H, m, (CH2)2), 1.33
(9H, s, (CH3)3); 13C NMR (75 MHz, DMSO-d6) d 179.4, 171.2,
168.5, 156.8, 137.9, 133.3, 129.7, 129.1, 125.8, 125.3, 119.5, 56.7,
42.5, 35.9, 35.3, 31.3, 23.8, 22.4; LRMS (m/z): [M]+ 455.24,
[(MÀHCl)+H]+; HRMS (m/z): [M]+ calcd for C25H34N4O2S,
455.2474; found, 455.2477.

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1787
(m/z): [M+Na]+ 491.38; HRMS (m/z): [M]+ calcd for C26H37N4O2S,
469.2637; found, 469.2640.
(15 mL) and the solution added dropwise at 0 °C. The mixture
was allowed to warm to rt and stirred for 16 h. The resulting green
solution was filtered through Celite and concentrated in vacuo. The
resulting filtrate was diluted with H2O and extracted with CH2Cl2
(3 Â 50 mL). The organic phase was dried over MgSO4 and concen-
trated in vacuo to give 13 (672 mg, 1.6 mmol, 65%) as a pale yellow
oil which was used without further purification. 1H NMR
(300 MHz, acetone-d6) d 7.82 (2H, d, J = 8.0 Hz, ArH), 7.49 (2H, d,
J = 8.0 Hz, ArH), 4.13 (4H, m, (CH2)2), 3.59 (18H, m, (CH2)9), 2.46
(3H, s, CH3); 13C NMR (100 MHz, acetone-d6) d 172.7, 144.9,
132.9, 129.9, 127.9, 77.4, 77.1, 76.8, 71.3, 71.2, 70.8, 70.7, 70.6,
70.6, 70.5, 70.4, 70.4, 70.3, 69.3, 69.1, 68.8, 68.6, 68.5, 21.6; LRMS
(m/z): [M+Na]+ 459.16.
4.1.1.6. 8-Dimethylamino-octanoic acid {4-[3-(4-tert-butyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (10). 8-Bromo-
octanoic acid (2 g, 8.90 mmol) was stirred in CH2Cl2 (15 mL) under
N2 atmosphere and thionyl chloride (784 lL, 10.68 mmol) added
followed by DMF (few drops). The solution was stirred for 16 h
and concentrated in vacuo to give 8-bromooctanoyl chloride
(2.04 g, 8.50 mmol, 95%) as a yellow oil which was used without
further purification. Thiourea 5 (600 mg, 1.84 mmol) was stirred
in CH2Cl2 (15 mL) under N2 atmosphere. 8-Bromooctanoyl chloride
(532 mg, 2.02 mmol) was added, followed by triethylamine
(256 lL, 1.84 mmol). The beige suspension was stirred at rt for
16 h and diluted with CH2Cl2 (20 mL). The organic solution was
washed with 1 M aqueous hydrochloric acid (1 Â 20 mL), 10%
aqueous sodium hydroxide (1 Â 20 mL), and saturated brine
(1 Â 20 mL). The organic phase was dried over MgSO4 and concen-
trated in vacuo. The resulting yellow foam was purified by flash
chromatography on silica (petroleum ether/ethyl acetate 1:1) to
give 8-bromo-octanoic acid {4-[3-(4-tert-butyl-benzoyl)-thiourei-
do]-phenyl}-amide (579 mg, 1.09 mmol, 59%) as a yellow oil. 1H
NMR (400 MHz, CDCl3) d 12.59 (1H, s, NH), 9.04 (1H, s, NH), 7.82
(2H, d, J = 8.6 Hz, ArH), 7.63 (2H, d, J = 8.9 Hz, ArH), 7.56 (6H, m,
ArH), 7.18 (1H, s, NH), 3.41 (2H, t, J = 6.8 Hz, CH2), 2.37 (2H, t,
J = 7.4 Hz, CH2), 1.86 (2H, m, CH2), 1.74 (2H, m, CH2), 1.44 (6H,
m, (CH2)3), 1.36 (9H, s, (CH3)3); 13C NMR (75 MHz, CDCl3) d
178.5, 171.6, 167.0, 157.8, 136.8, 133.4, 128.6, 127.5, 126.2,
124.8, 120.1, 37.6, 34.0, 32.7, 31.1, 29.1, 28.6, 28.0, 25.5. Treatment
of the prepared bromo compound (487 mg, 0.92 mmol) with
dimethylamine (10 mL) in H2O (25 mL) gave a crude mixture
which was dissolved in a minimal amount of CH2Cl2 and a 2 M
hydrochloric acid in ether solution was added dropwise until a
cream precipitate appeared. The solid was filtered to give a yellow
solid which was recrystallised from isopropanol to give 10
(301 mg, 0.57 mmol, 62%) as a yellow-brown solid. 1H NMR
(400 MHz, DMSO-d6) d 12.60 (1H, s, NH), 11.44 (1H, s, NH), 10.08
(1H, br s, NH+), 9.95 (1H, s, NH), 7.97 (2H, dd, J = 8.5, 16.5 Hz,
ArH), 7.62 (6H, m, ArH), 2.99 (2H, m, CH2), 2.72 (6H, br s,
N(CH3)2), 2.33 (2H, m, CH2), 1.62 (4H, m, (CH2)2), 1.32 (6H, m,
CH2)3) and 1.32 (9H, s, (CH3)3); 13C NMR (75 MHz, DMSO-d6) d
179.5, 171.3, 168.0, 156.2, 137.4, 132.7, 129.2, 128.6, 125.3,
124.7, 118.9, 58.5, 44.4, 36.2, 34.8, 30.8, 28.2, 26.1, 25.6, 25.0
24.9; LRMS (m/z): [M+H]+ 497.38; HRMS (m/z): [M]+ calcd for
4.1.2.3. Toluene-4-sulfonic acid 2-[2-(2-{2-[2-({4-[3-(4-tert-
butyl-benzoyl)-thioureido]-phenylcarbamoyl}-methoxy)-eth-
oxy]-ethoxy}-ethoxy)-ethoxy]-ethyl ester (15). Acid 13 (117 mg,
0.26 mmol) was stirred in oxalyl chloride containing a catalytic
amount of DMF. The solution was heated at reflux for 1 h then cooled
to rt and the solvent concentrated in vacuo to give the corresponding
acid chloride 14 which was used without further purification. Crude
acid chloride 14 (138 mg, 0.29 mmol, 1.3 equiv) was stirred in dry
CH2Cl2 (10 mL) under N2 atmosphere and thiourea 5 (74 mg,
0.23 mmol, 1 equiv) was added to give a bright yellow solution. Tri-
ethylamine (41 lL, 0.23 mmol, 1 equiv) was added and the mixture
stirred at rt for 16 h. The solution was diluted with CH2Cl2
(1 Â 10 mL) and washed with 1 M aqueous hydrochloric acid
(1 Â 10 mL), 1 M aqueous sodium hydroxide followed by saturated
brine (1 Â 10 mL). The organic phase was dried over MgSO4 and
the solvent concentrated in vacuo. The resulting yellow foam was
purified by flash chromatography on silica (petroleum ether:ethyl
acetate 1:1) to give 15 (79 mg, 0.10 mmol, 46%) as a yellow oil. mmax
cmÀ1 (NaCl, thin layer) 3259, 3059, 2958, 2708, 2635, 1918, 1697,
1668, 1604, 1516, 1473, 1268, 1122, 1034, 852, 734; 1H NMR
(300 MHz, CDCl3) d 12.61 (1H, s, NH), 9.15 (1H, s, NH), 8.99 (1H, s,
NH), 7.79 (4H, m, ArH), 7.67 (4H, m, ArH), 7.53 (2H, d, J = 8.6 Hz,
ArH), 7.31 (2H, d, J = 8.0 Hz, ArH), 4.10 (4H, m, (CH2)2), 3.63 (18H,
m, (CH2)9), 2.41 (3H, s, CH3), 1.34 (9H, s, (CH3)3); 13C NMR
(100 MHz, CDCl3) d 178.3, 168.4, 166.9, 157.8, 144.8, 136.1, 133.7,
129.8, 128.6, 127.9, 127.5, 126.2, 126.1, 124.6, 120.2, 71.2, 70.7,
70.6, 70.5, 70.4, 70.1, 69.3, 68.7, 35.3, 31.1, 21.7; LRMS (m/z):
[M+Na]+ 782.28; HRMS (m/z): [M]+ calcd for C37H49N3O10NaS2,
782.2757; found, 782.2756.
C
28H41N4O2S, 497.2950; found, 497.2946.
4.1.2.4. N-{4-[3-(4-tert-Butyl-benzoyl)-thioureido]-phenyl}-2-
[2-(2-{2-[2-(2-dimethylamino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-
ethoxy]-acetamide hydrochloride (17). Lithium bromide (44 mg,
0.5 mmol) was added to a stirred solution of tosylate 15 (76 mg,
0.1 mmol) in acetone (3 mL). The mixture was heated at 50 °C for
24 h. The solvent was removed in vacuo and the resulting residue
resuspended in ethyl acetate and the solid filtered. The filtrate was
concentrated in vacuo to give 16 (54 mg, 0.1 mmol, 81%) as a pale
4.1.2. Synthesis of PEG analogue 17
4.1.2.1. Benzenesulfonic acid 2-[2-(2-{2-[2-(2-hydroxy-ethoxy)-
ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethyl ester (12). Hexaethyl-
eneglycol (11) (2.82 g, 10 mmol) was dissolved in CH2Cl2
(100 mL) under
a N2 atmosphere, and silver oxide (3.48 g,
15 mmol) added followed by tosyl chloride (2.10 g, 11 mmol) and
potassium iodide (332 mg, 2 mmol). The solution was stirred at
rt for 1 h then concentrated and filtered through Celite eluting with
ethyl acetate. The filtrate was concentrated in vacuo to give a pale
yellow oil which was purified by column chromatography on silica
(ethyl acetate) to give 12 (3.79 g, 8.70 mmol, 87%) as a pale yellow
oil. 1H NMR (300 MHz, DMSO-d6) d 7.79 (2H, d, J = 8.4 Hz, ArH),
7.33 (2H, d, J = 8.0 Hz, ArH), 4.15 (2H, m, CH2), 3.64 (22H, m,
(CH2)11), 2.44 (3H, s, CH3) and 1.80 (1H, br s, OH).
yellow oil which was used without further purification. m
max cmÀ1
(NaCl, thin layer) 3396, 3054, 2963, 2921, 2873, 2599, 1694, 1668,
1610, 1514, 1475, 1266, 1115, 947, 736; 1H NMR (300 MHz, CDCl3)
d 12.61 (1H, s, NH), 9.16 (1H, br s, NH), 9.06 (1H, s, NH), 7.82 (2H,
d, J = 8.6 Hz, ArH), 7.70 (4H, q, J = 9.0 Hz, ArH), 7.55 (2H, d,
J = 8.6 Hz, ArH), 4.18 (2H, s, CH2), 3.71 (16H, m, (CH2)8), 3.45 (2H, t,
J = 6.3 Hz, CH2), 1.36 (9H, s, (CH3)3); LRMS (m/z): [M+Na]+ 692.02.
Bromide 16 (30 mg, 0.05 mmol) was converted to the dimethyl-
amine derivative by stirring in 40% aqueous dimethylamine
(0.75 mL), H2O (1.8 mL) and CH2Cl2 (2 mL) at rt for 24 h. The reaction
mixture was diluted with CH2Cl2 (20 mL) and the organic phase
washed with H2O (1 Â 10 mL), 1 M aqueous sodium hydroxide
(1 Â 10 mL) and saturated brine (1 Â 10 mL). The organic phase
was dried over MgSO4 and the solvent concentrated in vacuo to give
4.1.2.2. [2-(2-{2-[2-(2-Toluenesulfonyloxy-ethoxy)-ethoxy]-eth-
oxy}-ethoxy)-ethoxy]-acetic acid (13). A 1.5 M aqueous solution
of sulfuric acid (10 mL) was prepared at 0 °C and stirred while
chromium trioxide was added to give an orange solution. The
known alcohol 12 (1 g, 2.3 mmol) was dissolved in acetone

1788
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
the dimethylamine derivative. The pale yellow oil was taken up in a
minimal amount of CH2Cl2 and a solution of 2 M hydrochloric acid in
diethyl ether was added dropwise until a precipitate appeared. The
mixture was concentrated in vacuo to give 17 (20 mg, 0.04 mmol,
73%) as a yellow foam. mmax cmÀ1 (NaCl, thin layer) 3308, 2992,
2921, 2873, 1725, 1674, 1609, 1517, 1464, 1272, 1123, 841, 738;
1H NMR (300 MHz, DMSO-d6) d 12.62 (1H, s, NH), 11.45 (1H, s,
NH), 9.77 (1H, s, NH), 9.77 (1H, s, NH), 9.55 (1H, s, NH), 7.95 (2H, d,
J = 8.4 Hz, ArH), 7.63 (6H, m, ArH), 4.10 (4H, m, (CH2)2), 3.61 (18H,
in dry CH2Cl2 (40 mL) under N2 atmosphere and triethylamine
(4.4 mL, 31.6 mmol) was added and stirred for 20 min before addi-
tion of 5-bromovaleryl chloride (3.2 mL, 23.7 mmol) followed by
further triethylamine (6.6 mL, 47.4 mmol). The reaction mixture
was stirred at rt for 16 h then diluted with CH2Cl2 (1 Â 50 mL)
and washed with 1 M aqueous hydrochloric acid (1 Â 100 mL),
1 M aqueous sodium hydroxide (1 Â 100 mL) and saturated brine
(1 Â 100 mL). The organic phase was dried over MgSO4 and con-
centrated in vacuo to give a crude brown oil which was purified
by column chromatography on silica (petroleum ether/ethyl ace-
tate 1:1) to give 22 (2.14 g, 5.1 mmol, 32%) as a brown solid. 1H
NMR (300 MHz, CDCl3) d 7.50 (2H, d, J = 8.6 Hz, ArH), 7.39–7.29
(5H, m, ArH), 7.21 (2H, d, J = 8.6 Hz, ArH), 5.19 (2H, s, CH2), 3.47
(2H, t, J = 6.5 Hz, CH2), 3.32 (3H, s, NCH3), 2.41 (2H, d, J = 6.9 Hz,
CH2), 2.02–1.88 (4H, m, (CH2)2). 22 (500 mg, 1.30 mmol) was stir-
red in CH2Cl2 (4 mL), 40% aqueous dimethylamine (4 mL) and H2O
(10 mL) at rt for 20 h. The reaction mixture was diluted with
CH2Cl2 (20 mL) and washed with H2O (1 Â 10 mL), 10% aqueous
NaOH (1 Â 10 mL), and saturated brine (1 Â 10 mL). The organic
phase was dried over MgSO4 and concentrated in vacuo to give
23 as a yellow oil (428 mg, 4.4 mmol, 86%) which was used with-
out further purification. mmax cmÀ1 (NaCl, thin layer) 3312, 3183,
3048, 2945, 2864, 2782, 2307, 1896, 1699, 1604, 1537, 1516,
1498, 1455, 1389, 1351, 1266, 1156, 843, 734; 1H NMR
(300 MHz, CDCl3) d 8.43 (1H, s, NH), 7.40 (2H, d, J = 8.4 Hz, ArH),
7.29–7.15 (5H, m, ArH), 7.06 (2H, d, J = 8.4 Hz, ArH), 5.07 (2H, s,
CH2), 3.20 (3H, s, NCH3), 2.31–2.18 (4H, m, (CH2)2), 2.13 (6H, s,
N(CH3)2), 1.66 (2H, app. quintet, J = 7.7 Hz, CH2), 1.45 (2H, app.
quintet, J = 7.7 Hz, CH2); 13C NMR (75 MHz, CDCl3) d 171.6, 171.6,
155.6, 136.6, 136.4, 128.5, 127.9, 127.7, 126.3, 120.3, 67.3, 58.8,
45.3, 37.9, 37.1, 26.5, 23.4.
Compound 23 (550 mg, 1.45 mmol) was stirred in 48% w/v
hydrobromic acid in acetic acid (6 mL) for 1 h. Diethylether was
added until a cream precipitate appeared and the ethereal solution
was decanted. The resulting white sticky solid was suspended in
CH2Cl2 (100 mL) and 1 M aqueous sodium hydroxide (20 mL).
The aqueous layer was extracted with CH2Cl2 (1 Â 50 mL) and
the combined organic layers were washed with saturated brine
(1 Â 50 mL), dried (MgSO4) and concentrated in vacuo to give 24
as a yellow oil (153 mg, 0.61 mmol, 42%). mmax cmÀ1 (NaCl, thin
layer) 3295, 3134, 3051, 2942, 2865, 2782, 2309, 1896, 1652,
1656, 1603, 1520, 1467, 1308, 1266, 1178, 824, 736; 1H NMR
(300 MHz, CDCl3) d 7.65 (1H, s, NH), 7.23 (2H, d, J = 8.8 Hz, ArH),
6.49 (2H, d, J = 8.8 Hz, ArH), 2.74 (3H, s, NCH3), 2.26 (4H, app. quar-
tet, J = 7.3 Hz, (CH2)2), 2.16 (6H, s, N(CH3)2), 1.67 (2H, app. quintet,
J = 7.6 Hz, CH2), 1.49 (2H, app. quintet, J = 7.1 Hz, CH2); 13C NMR
(75 MHz, CDCl3) d 171.1, 147.3, 130.2, 121.9, 121.8, 112.6, 59.9,
49.6, 45.5, 37.3, 27.8, 24.2; LRMS (m/z): [M+H]+ 250.11; HRMS
calcd for C14H24N3O, 250.1919; found, 250.1922.
m, (CH2)9), 2.77 (6H, d, J = 4.0 Hz, N(CH3)2), 1.32 (9H, s, (CH3)3); 13
C
NMR (75 MHz, DMSO-d6) d 179.2, 168.2, 168.0, 156.3, 136.4,
133.3, 129.2, 128.6, 125.3, 124.7, 119.6, 70.3, 70.1, 69.8, 69.7, 69.6,
69.5, 69.3, 64.4, 63.9, 63.3, 55.6, 42.6, 34.8, 30.8; LRMS (m/z):
[(MÀHCl)+H]+ 632.82,; HRMS (m/z): [M]+ calcd for C32H49N4O7S,
633.3322; found, 633.3327.
4.1.3. Synthesis of analogue 26
4.1.3.1. [4-(2,2,2-Trifluoro-acetylamino)-phenyl]-carbamic acid
tert-butyl ester (19). A dry flask was charged with aniline 18
(1.02 g, 4.90 mmol) and CH2Cl2 (40 mL) and cooled to À78 °C. Tri-
fluoroacetic anhydride (977 lL, 6.90 mmol) was added dropwise,
followed by triethylamine (1.9 mL, 13.80 mmol). The cream slurry
was stirred for 30 min and diluted with ethyl acetate (50 mL), and
washed with 1 M aqueous hydrochloric acid (1 Â 30 mL) and satu-
rated brine (1 Â 30 mL). The organic phase was dried over MgSO4
and concentrated in vacuo to give 19 (1.43 g, 4.90 mmol, quantita-
tive) as a cream solid. 1H NMR (300 MHz, CDCl3) d 7.81 (1H, br s,
NH), 7.49 (2H, d, J = 9 Hz, ArH), 7.39 (2H, d, J = 9.0 Hz, ArH), 6.57
(1H, br s, NH) and 1.52 (9H, s, (CH3)3).
4.1.3.2. (4-Methylamino-phenyl)-carbamic acid tert-butyl ester
(20). Trifluoroacetamide 19 (695 mg, 2.36 mmol) was stirred in
dry THF (10 mL) under N2 atmosphere. To this stirring solution
was added potassium-t-butoxide (264 mg, 2.46 mmol) and methyl
iodide (147
lL, 2.83 mmol) and the reaction mixture heated at
50 °C for 5 min.
A
further portion of potassium-t-butoxide
(409 mg, 3.65 mmol) in THF (10 mL) was added dropwise slowly.
The resulting yellow suspension was stirred at rt for 3 h. The sol-
vent was concentrated in vacuo and the resulting residue resus-
pended in ethyl acetate. The undissolved solid was filtered and
the filtrate concentrated in vacuo to give a crude pale yellow oil
which was purified by column chromatography on silica (petro-
leum ether:ethyl acetate 2:1) to give 20 (235 mg, 1.09 mmol,
46%) as a brown oil. 1H NMR (300 MHz, CDCl3) d 7.13 (2H, d,
J = 8.1 Hz), 6.55 (2H, d, J = 8.7 Hz, ArH), 6.20 (1H, br s, NH), 2.79
(3H, s, NCH3) and 1.47 (9H, s, (CH3)3); LRMS (m/z): [M+Na]+ 245.11.
4.1.3.3. Benzyl (4-((tert-butoxycarbonyl)amino)phenyl)(methyl)
carbamate (21). To a solution of 20 (1.4 g, 6.4 mmol) in CH2Cl2
(20 mL) at À78 °C, under N2, was added benzylchloroformate
(1.1 mL, 7.7 mmol) followed by diisopropylethylamine (1.1 mL,
6.4 mmol) dropwise. The solution was allowed to warm to room
temperature over 2 h. The solution was washed with water
(10 mL) and brine (10 mL). The organic phase was dried over
MgSO4 and the solvent removed to afford the crude product. The
resulting residue was purified by column chromatography using
petroleum ether/ethyl acetate (2:1) to afford the product as a
white solid (2.2 g, 6.2 mmol, 97%). 1H NMR (300 MHz, CDCl3) d
7.35–7.21 (7H, m, ArH), 7.08 (2H, d, J = 7.8 Hz, ArH), 6.40 (1H, s,
NH), 5.07 (2H, s, CH2), 3.21 (6H, s, N(CH3)2), 1.45 (9H, s, (CH3)3).
4.1.3.5. 4-(tert-Butyl)-N-((4-(5-(dimethylamino)pentanamido)
phenyl)(methyl)carbamothioyl)benzamide (26). To
a stirred
solution of 4 (65 L, 0.36 mmol) in dry acetone (2 mL) was added
l
sodium thiocyanate (33 mg, 0.40 mmol) and the resulting yellow
suspension was stirred at rt for 18 h. A solution of amine 24
(90 mg, 0.36 mmol) in dry acetone (1 mL) was added and the
resulting suspension stirred at rt for a further 7 h. The solvent
was removed in vacuo and the residue re-suspended in CH2Cl2
and the undissolved salts were removed by filtration. The filtrate
was concentrated to afford a yellow solid which was purified by
column chromatography on silica (CH2Cl2/MeOH 10:0.1) to afford
26 as a bright yellow solid (50 mg, 0.10 mmol, 28%). Mp 242–
243 °C; mmax cmÀ1 (KBr) 3392, 2960, 2925, 2855, 1675, 1662,
1610, 1514, 1484, 1408, 1267, 1113, 855, 779; 1H NMR
(300 MHz, acetone-d6) d 9.50 (1H, s, NH), 7.56 (2H, d, J = 8.8 Hz,
ArH), 7.14 (6H, app. s, ArH), 3.60 (3H, s, NCH3), 3.08 (2H, app. t,
4.1.3.4.
5-(Dimethylamino)-N-(4-(methylamino)phenyl)pen-
tanamide (24). To a solution of the known compound 21 (5.7 g,
15.8 mmol) in CH2Cl2 (100 mL) was added TFA (15 mL) and the
resulting solution stirred at rt for 2 h before the solvent was re-
moved in vacuo. The resulting deprotected amine salt was stirred

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1789
J = 6.9 Hz, CH2), 2.76 (6H, s, N(CH3)2), 2.32 (2H, app. t, J = 6.9 Hz,
CH2), 1.78–1.58 (4H, m, (CH2)2), 1.08 (9H, s, (CH3)3); 13C NMR
(75 MHz, aceone-d6) d 184.6, 178.2, 172.0, 156.7, 140.5, 139.9,
133.6, 129.7, 126.8, 125.9, 120.6, 120.5, 57.8, 46.2, 42.9, 36.7,
35.5, 31.2, 24.6, 23.1; LRMS (m/z): [M+H]+ 469.31; HRMS calcd
for C26H37N4O2S, 469.2637; found, 469.2629.
4.1.5. General procedure for the synthesis of analogues 30a–q
To a solution of the acid chloride (1 equiv) in dry acetone
(10 mL per mmol) under N2, was added sodium thiocyanate
(1 equiv). The resulting suspension was stirred at room tempera-
ture for 30 min before cooling to 0 °C. A solution of amine 25
(1 equiv) in dry acetone (5 mL per mmol) was added and the
resulting suspension allowed to warm to room temperature and
stirred for 16 h. The reaction was filtered through Celite and the fil-
trate concentrated in vacuo to give the crude product. The product
was purified by column chromatography (CH2Cl2/MeOH 10:1) and
conversion to the corresponding HCl salt provided 30a–q.
4.1.4. Synthesis of analogues 30a–q
4.1.4.1.
Benzyl
4-(5-bromopentanamido)phenylcarbamate
(28). Aniline 27 (12.69 g, 52.0 mmol) was stirred in CH2Cl2
(250 mL) at 0 °C and 5-bromovaleryl chloride (8.9 mL, 52.0 mmol)
was added followed by DIPEA (9.0 mL, 52.0 mmol). The resulting
white suspension was allowed to warm to room temperature and
stirred for 3 h. The white solid was collected by filtration and recrys-
tallised from ethyl acetate to give 28 as a white solid (14.6 g,
36.9 mmol, 71%). Mp 184–186 °C; mmax cmÀ1 (KBr) 2971, 1656,
1548, 1305, 1233, 1066, 812; 1H NMR (400 MHz, CDCl3) d 7.38
(2H, d, J = 8.9 Hz, ArH), 7.36–7.25 (7H, m, ArH), 7.02 (1H, s, NH),
6.56 (1H, br s, NH), 5.12 (2H, s, CH2), 3.38 (2H, t, J = 6.4 Hz, CH2),
2.32 (2H, t, J = 6.8 Hz, CH2), 1.91–1.80 (4H, m, (CH2)2); 13C NMR
(100 MHz, CDCl3) d 174.0, 153.4, 136.0, 131.9, 131.6, 128.7, 120.8,
119.4, 67.5, 36.9, 33.7, 24.9, 24.1; LRMS (m/z): [M+Na]+ 427.20;
HRMS calcd for C19H21N2O3Na79Br, 427.0633; found, 427.0637.
4.1.5.1. 5-Dimethylamino-pentanoic acid {4-[3-(4-propyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30a). Yield
(95 mg, 0.20 mmol, 45%), light yellow solid. Mp 161–162 °C; mmax
cmÀ1 (KBr) 3391, 2960, 2871, 2717, 2084, 1668, 1608, 1530,
1408, 1338, 1265, 1149, 838, 738; 1H NMR (300 MHz, DMSO-d6):
d 12.58 (1H, s, NH), 11.40 (1H, s, NH), 10.20 (1H, s, NH), 10.10
(1H, br s, NH+), 7.92 (2H, d, J = 8.2 Hz, ArH), 7.62 (4H, AA’BB’,
J = 9.0, 20.9 Hz, ArH), 7.36 (2H, d, J = 8.3 Hz, ArH), 3.05 (2H, m,
CH2), 2.73 (6H, d, J = 4.9 Hz, N(CH3)2), 2.64 (2H, m, CH2), 2.39
(2H, t, J = 6.5 Hz, CH2), 1.64 (6H, m, (CH2)3), 0.90 (3H, t, J = 7.3 Hz,
CH3); 13C NMR (100 MHz, DMSO-d6): d 178.9, 170.8, 168.0, 148.0,
137.4, 132.9, 129.5, 128.7, 128.4, 124.7, 120.7, 56.0, 41.9, 37.0,
35.5, 33.0, 23.7, 23.1, 22.0, 13.6; LRMS (m/z): [(MÀHCl)+H]+
441.20; HRMS (m/z): [M]+ calcd for C24H33N4O2S, 441.2324; found,
441.2318.
4.1.4.2. Benzyl 4-(5-(dimethylamino)pentanamido)phenylcar-
bamate (29). Bromide 28 (5.9 g, 12.0 mmol) was stirred in CH2Cl2
(150 mL) with 40% aqueous dimethylamine (10 mL) and water
(25 mL) for 24 h. The solution was then diluted with CH2Cl2
(50 mL) and the organic phase washed with water (100 mL), 1 M
aqueous sodium hydroxide (100 mL) and brine (100 mL), dried
(MgSO4), filtered and concentrated in vacuo to give 29 as a white so-
lid (3 g, 8.1 mmol, 68%). Mp 141–144 °C; mmax cmÀ1 (KBr) 2894,
1656, 1546, 1307, 1231, 1065; 1H NMR (400 MHz, CDCl3) d 7.89
(1H, br s, NH), 7.39 (2H, d, J = 8.4 Hz, ArH), 7.35–7.22 (7H, m, ArH),
6.65 (1H, br s, NH), 5.16 (2H, s, CH2), 2.32–2.26 (4H, m, (CH2)2),
2.18 (6H, s, N(CH3)2), 1.70 (2H, app. quintet, J = 8.3 Hz, 7.3 Hz,
CH2), 1.50 (2H, app. quintet, J = 8.3 Hz, 7.3 Hz, CH2); 13C NMR
(100 MHz, CDCl3) d 171.3, 154.0, 136.2, 135.8, 133.7, 128.6,
128.3, 120.7, 67.0, 59.9, 45.4, 37.1, 26.6, 23.5; LRMS (m/z):
[M+H]+ 370.30; HRMS calcd for C21H28N3O3, 370.2131; found,
370.2131.
4.1.5.2. 5-Dimethylamino-pentanoic acid {4-[3-(4-isopropyl-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (30b). Yield
(25 mg, 0.05 mmol, 34%), light yellow oil. mmax cmÀ1 (NaCl, thin
layer) 3246, 2961, 2705, 1668, 1606, 1516, 1408, 1343, 1265,
1150, 838, 768; 1H NMR (400 MHz, DMSO-d6): d 12.59 (s, 1H,
NH), 10.21 (s, 1H, NH), 10.16 (br s, 1H, NH), 7.93 (d, J = 8.3 Hz,
2H, ArH), 7.62 (AA’BB’, J = 9.0, 27.4 Hz, 4H, ArH), 7.42 (d,
J = 8.3 Hz, 2H, ArH), 3.02 (m, 1H, CH), 2.71 (m, 6H, N(CH3)2), 1.66
(m, 4H, (CH2)2), 2.37 (m, 2H, CH2), 1.23 (d, J = 6.9 Hz, 6H, CHMe2);
13C NMR (100 MHz, CD3OD) d 180.7, 173.6, 169.6, 155.9, 138.1,
135.1, 121.1, 129.6, 128.0, 125.8, 121.9, 58.7, 43.5, 36.8, 36.7,
25.2, 24.1, 23.3; LRMS (m/z): [(MÀHCl)+H]+ 441.22; HRMS (m/z):
[M]+ calcd for C24H33N4O2S, 441.2324; found, 441.2314.
4.1.4.3. 5-Dimethylamino-pentanoic acid (4-amino-phenyl)-
amide (25). Method 1: Compound 29 (1.94 g, 5.0 mmol) was stirred
in 48% w/v hydrobromic acid in acetic acid (11 mL) for 1 h. Diethyl-
ether was added until a cream precipitate appeared and the ethereal
solution decanted. The resulting white sticky solid was suspended in
CH2Cl2 (100 mL) and 1 M aqueous sodium hydroxide (20 mL). The
aqueous layer was extracted with dichloromethane (1 Â 50 mL)
and the combined organic layers were washed with saturated brine
(1 Â 50 mL), dried (MgSO4) and concentrated in vacuo to give 25 as a
pale yellow oil (975 mg, 4.1 mmol, 82%).
4.1.5.3. 5-Dimethylamino-pentanoic acid {4-[3-(4-butyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30c). Yield
(45 mg, 0.09 mmol, 14%), yellow solid. Mp 170–171 °C; mmax
cmÀ1 (KBr) 3415, 3250, 3042, 2957, 2933, 2714, 2472, 1675,
1638, 1611, 1529, 1408, 1271, 1147, 842, 742; 1H NMR
(300 MHz, DMSO-d6): d 12.58 (1H, s, NH), 11.44 (1H, s, NH),
10.13 (1H, s, NH), 9.70 (1H, br s, NH), 7.91 (2H, d, J = 8.2 Hz,
ArH), 7.62 (4H, AA’BB’, J = 9, 20.2 Hz, ArH), 7.36 (2H, d, J = 8.3 Hz,
ArH), 3.06 (2H, m, CH2), 2.75 (6H, d, J = 4.9 Hz, N(CH3)2), 2.67
(2H, m, CH2), 2.39 (2H, t, J = 6.6 Hz, CH2), 1.60 (6H, m, (CH2)3),
1.31 (2H, q, J = 7.3 Hz, CH2), 0.91 (3H, t, J = 7.3 Hz, CH3); 13C NMR
(75 MHz, DMSO-d6) d 180.6 (C), 173.6 (C), 169.7 (C), 150.5 (C),
138.2 (C), 135.3 (C), 131.0 (C), 129.8 (CH), 129.3 (CH), 124.8 (CH),
121.0 (CH), 58.6 (CH2), 43.4 (N(CH3)2), 36.7 (CH2), 36.6 (CH2),
34.7 (CH2), 25.2 (CH2), 23.4 (CH2), 23.2 (CH2), 13.9 (CH3) LRMS
(m/z): [(MÀHCl)+H]+ 455.2; HRMS (m/z): [M]+ calcd for
Method 2: A 0.1 M solution of the protected amine 29 (1 g,
2.8 mmol) in methanol (28 mL) was passed through the H-CubeÒ,
which was equipped with a 10% Pd/C Cat-Cart, at 0 bar under the
Full H2 mode. The flow through was collected and concentrated
in vacuo to afford 25 as a colourless oil (617 mg, 2.8 mmol, quant.)
which was used without further purification. m
max cmÀ1 (NaCl, thin
layer) 3299, 3131, 3050, 2945, 2864, 2781, 2305, 1876, 1653, 1605,
1542, 1516, 1264, 1174, 831, 736; 1H NMR (300 MHz, CDCl3) d 7.68
(1H, br s, NH), 7.22 (2H, d, J = 8.7 Hz, ArH), 6.57 (2H, d, J = 8.7 Hz,
ArH), 2.30 (4H, app. quintet, J = 7.1 Hz, (CH2)2), 2.23 (6H, s,
N(CH3)2), 1.70 (2H, app. quintet, J = 7.2 Hz, CH2), 1.53 (2H, app.
quintet, J = 7.1 Hz, CH2); 13C NMR (100 MHz, CDCl3) d 168.6,
143.1, 129.7, 121.9, 115.4, 58.8, 45.2, 37.0, 26.5, 23.5; LRMS (m/
z): [M+H]+ 222.34.
C25H35N4O2S, 455.2481; found, 455.2484.
4.1.5.4. 5-Dimethylamino-pentanoic acid {4-[3-(4-pentyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30d). Yield
(178 mg, 0.35 mmol, 55%), yellow solid. Mp 173–174 °C; mmax
cmÀ1 (KBr) 3417, 3258, 3012, 2952, 2929, 2713, 2473, 1672,
1609, 1544, 1513, 1409, 1272, 1143, 840, 734; 1H NMR
(300 MHz, DMSO-d6): d 12.58 (1H, s, NH), 11.44 (1H, s, NH),

1790
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
10.18 (1H, s, NH), 9.98 (1H, br s, NH), 7.91 (2H, d, J = 8.3 Hz, ArH),
7.62 (4H, AA’BB’, J = 9.0, 20.1 Hz, ArH), 7.36 (2H, d, J = 8.4 Hz, ArH),
3.02 (2H, m, CH2), 2.73 (6H, m, N(CH3)2), 2.66 (2H, m, CH2), 1.62
(6H, m, (CH2)3), 1.29 (4H, m, (CH2)2), 0.86 (3H, t, J = 6.8 Hz, CH3);
13C NMR (100 MHz, CD3OD) d 180.6, 173.5, 169.7, 150.5, 138.2,
135.3, 131.0, 130.0, 129.4, 125.8, 121.1, 58.7, 43.5, 36.8, 36.7,
32.6, 32.1, 25.2, 23.5, 23.2, 14.4; LRMS (m/z): [(MÀHCl)+H]+
469.25; HRMS (m/z): [M]+ calcd for C26H37N4O2S, 469.2637; found,
469.2637.
4.1.5.9. 5-Dimethylamino-pentanoic acid {4-[3-(4-bromo-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30i). Yield
(36 mg, 0.07 mmol, 11%), brown oil. 1H NMR (400 MHz, DMSO-
d6): d 12.41 (1H, s, NH), 11.65 (1H, s, NH), 10.52 (1H, br s, NH),
10.30 (1H, s, NH), 7.91 (2H, m, ArH), 7.75 (2H, d, J = 8.4 Hz, ArH),
7.66 (2H, m, ArH), 7.58 (2H, m, ArH), 3.06 (2H, m, CH2), 2.73 (2H,
d, J = 4.7 Hz, N(CH3)2), 2.39 (2H, m, CH2), 1.66 (4H, m, (CH2)2);
13C NMR (100 MHz, DMSO-d6) d 178.7, 170.8, 167.3, 137.5, 132.7,
131.4, 131.4, 131.3, 129.7, 124.7, 119.2, 56.0, 41.8, 35.5, 23.2,
22.0; LRMS (m/z): [(MÀHCl)+H]+ 479.11; HRMS (m/z): [M]+ calcd
for C21H26N4O2SBr, 477.0960; found, 477.0965.
4.1.5.5. 5-Dimethylamino-pentanoic acid [4-(3-benzoyl-thio-
ureido)-phenyl]-amide hydrochloride (30e). Yield (22 mg,
0.05 mmol, 55%), brown solid. Mp 174–175 °C; mmax cmÀ1 (KBr)
3357, 2956, 2925, 2854, 1737, 1668, 1604, 1516, 1464, 1409,
1377, 1261, 1154, 802, 706; 1H NMR (400 MHz, DMSO-d6): d
12.54 (1H, s, NH), 11.55 (1H, s, NH), 10.16 (1H, s, NH), 9.92 (1H,
br s, NH+), 7.97 (2H, d, J = 7.7 Hz, ArH), 7.59 (7H, m, ArH), 3.06
(2H, m, CH2), 2.74 (6H, d, J = 4.9 Hz, N(CH3)2), 2.39 (2H, t,
J = 6.9 Hz, CH2), 1.64 (4H, m, (CH2)2); 13C NMR (100 MHz, CD3OD)
d 180.6, 174.3, 169.9, 138.1, 135.3, 134.4, 133.8, 129.9, 129.3,
126.0, 121.3, 58.8, 44.0, 36.7, 25.2, 23.1; LRMS (m/z): [(MÀHCl)+H]+
398.96; HRMS (m/z): [M]+ calcd for C21H27N4O2S, 399.1855; found,
399.1861.
4.1.5.10. 5-Dimethylamino-pentanoic acid {4-[3-(3-methyl-ben-
zoyl)-thioureido]-phenyl}-amide (30j). Yield (41 g, 0.09 mmol,
43%), white-yellow solid. Mp 175–176 °C; mmax cmÀ1 (KBr) 3250,
2921, 2677, 1665, 1651, 1604, 1521, 1406, 1314, 1271, 1184,
837; 1H NMR (400 MHz, DMSO-d6): d 12.54 (1H, s, NH), 11.50
(1H, s, NH), 10.21 (1H, s, NH), 10.17 (1H, br s, NH), 7.83 (1H, s,
ArH), 7.77 (1H, d, J = 7.3 Hz, ArH), 7.66 (1H, d, J = 9.1 Hz, ArH),
7.60 (2H, m, ArH), 7.46 (2H, m, ArH), 7.13 (2H, d, J = 7.7 Hz, ArH),
3.05 (2H, m, CH2), 2.71 (6H, m, N(CH3)2), 2.40 (3H, s, CH3), 2.39
(2H, m, CH2), 1.65 (4H, m, (CH2)2); 13C NMR (100 MHz, CD3OD) d
180.6, 174.3, 170.0, 140.0, 138.2, 135.3, 135.1, 133.7, 129.8,
129.7, 126.5, 125.8, 121.1, 59.0, 44.0, 36.8, 25.3, 23.2, 21.4; LRMS
(m/z): [(MÀHCl)+H]+ 413.19; HRMS (m/z): [M]+ calcd for
4.1.5.6. 5-Dimethylamino-pentanoic acid {4-[3-(4-methyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30f). Yield
(46 mg, 0.10 mmol, 16%), pale brown solid. Mp 202–204 °C; mmax
cmÀ1 (KBr) 3417, 3257, 3025, 2939, 2675, 2467, 1689, 1666,
1647, 1603, 1521, 1302, 1248, 1155, 839; 1H NMR (400 MHz,
DMSO-d6): d 12.70 (1H, s, NH), 11.44 (1H, s, NH), 10.19 (1H, br s,
NH), 8.00 (2H, d, J = 7.8 Hz, ArH), 7.75 (2H, d, J = 7.7 Hz, ArH),
7.45 (2H, d, J = 8.5 Hz, ArH), 7.30 (2H, d, J = 7.8 Hz, ArH), 3.16
(2H, m, CH2), 2.84 (6H, d, J = 4.6 Hz, N(CH3)2), 2.42 (2H, m, CH2),
2.15 (3H, s, CH3), 1.74 (4H, m, (CH2)2); 13C NMR (100 MHz, CD3OD)
d 180.7, 173.5, 169.7, 145.7, 138.2, 135.3, 130.8, 130.5, 129.6,
126.0, 121.3, 58.7, 43.5, 36.7, 25.2, 23.2, 21.6; LRMS (m/z):
[(MÀHCl)+H]+ 413.18; HRMS (m/z): [M]+ calcd for C22H29N4O2S,
413.2011; found, 413.2005.
C22H29N4O2S, 413.2011; found, 413.2007.
4.1.5.11. 5-Dimethylamino-pentanoic acid {4-[3-(3-methoxy-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (30k). Yield
(32 mg, 0.07 mmol, 11%), orange oil. 1H NMR (400 MHz, DMSO-d6):
d 12.53 (1H, s, NH), 11.55 (1H, s, NH), 10.17 (1H, s, NH), 9.99 (1H, br
s, NH), 7.60 (6H, m, ArH), 7.45 (1H, t, J = 7.9 Hz, ArH), 7.22 (1H, dd,
J = 2, 7.9 Hz, ArH), 3.85 (3H, s, OMe), 3.03 (2H, m, CH2), 2.73 (6H, m,
N(CH3)2), 2.38 (2H, m, CH2), 1.64 (4H, m, (CH2)2); 13C NMR
(100 MHz, DMSO-d6) d 178.8, 173.9, 170.8, 167.9, 159.0, 137.4,
133.4, 129.6, 124.7, 120.9, 119.4, 119.0, 113.2, 56.0, 55.4, 41.9,
35.5, 23.2, 22.0; LRMS (m/z): [(MÀHCl)+H]+ 429.17; HRMS (m/z):
[M]+ calcd for C22H29N4O3S, 429.1960; found, 429.1959.
4.1.5.7. 5-Dimethylamino-pentanoic acid {4-[3-(4-methoxy-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (30g). Yield
(23 mg, 0.05 mmol, 22%), pale yellow solid. Mp 190–191 °C; mmax
cmÀ1 (KBr) 3410, 2956, 2923, 2851, 1725, 1659, 1647, 1605,
1540, 1516, 1464, 1256, 1158, 1025; 1H NMR (400 MHz, DMSO-
d6): d 12.70 (s, 1H, NH), 11.44 (s, 1H, NH), 10.19 (br s, 1H, NH),
8.10 (d, J = 8.7 Hz, 2H, ArH), 7.66 (d, J = 8.7 Hz, 2H, ArH), 7.16 (d,
J = 8.7 Hz, 4H, ArH), 3.93 (s, 3H, OMe), 3.08 (m, 2H, CH2), 2.79 (d,
J = 4.7 Hz, 6H, N(CH3)2), 2.34 (t, J = 8.5 Hz, 2H, CH2), 1.72 (m, 2H,
CH2), 1.59 (m, 2H, CH2); 13C NMR (100 MHz, CD3OD) d 180.9,
174.0, 169.2, 165.5, 138.3, 135.3, 131.5, 125.8, 125.4, 121.2, 115.2,
59.6, 56.1, 44.5, 37.2, 26.7, 24.0; LRMS (m/z): [(MÀHCl)+H]+
429.21; HRMS (m/z): [M]+ calcd for C22H29N4O3S, 429.1960; found,
429.1970.
4.1.5.12. 5-Dimethylamino-pentanoic acid {4-[3-(3-chloro-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30l). Yield
(28 mg, 0.06 mmol, 23%), orange sticky oil. mmax cmÀ1 (KBr) 3410,
2920, 2671, 2347, 1669, 1647, 1604, 1539, 1514, 1472, 1407,
1335, 1267, 1141, 836, 750; 1H NMR (300 MHz, DMSO-d6): d
12.39 (1H, s, NH), 11.71 (1H, s, NH), 10.12 (1H, s, NH), 9.65 (1H,
br s, NH+), 8.03 (1H, s, ArH), 7.73 (1H, d, J = 8.2 Hz, ArH), 7.61
(5H, m, ArH), 3.06 (2H, m, CH2), 2.75 (6H, m, N(CH3)2), 2.38 (2H,
m, CH2), 1.64 (4H, m, (CH2)2); 13C NMR (100 MHz, DMSO-d6) d
178.6, 174.0, 170.8, 166.8, 137.5, 134.2, 133.0, 132.7, 130.4,
128.4, 127.4, 124.7, 119.0, 56.0, 41.9, 35.5, 23.1, 21.4; LRMS (m/
z): [(MÀHCl)+H]+ 433.13.
4.1.5.13. 5-Dimethylamino-pentanoic acid {4-[3-(3-bromo-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30m). Yield
(74 mg, 0.13 mmol, 21%), yellow solid. Mp 114–115 °C; mmax cmÀ1
(KBr) 3411, 3237, 3046, 2960, 2695, 2058, 1674, 1606, 1521, 1408,
1340, 1265, 1152, 839, 736; 1H NMR (400 MHz, DMSO-d6): d 12.55
(1H, s, NH), 12.38 (1H, s, NH), 10.14 (1H, s, NH), 9.81 (1H, br s, NH),
8.15 (1H, t, J = 1.8 Hz, ArH), 7.93 (1H, d, J = 7.9 Hz, ArH), 7.85 (1H,
d, J = 7.9 Hz, ArH), 7.64 (2H, d, J = 8.9 Hz, ArH), 7.57 (2H, d,
J = 8.9 Hz, ArH), 7.49 (1H, t, J – 7.9 Hz, ArH), 3.05 (2H, app t,
J = 7.6 Hz, CH2), 2.70 (6H, d, J = 4.9 Hz, N(CH3)2), 2.38 (2H, app. t,
J = 6.6 Hz, CH2), 1.71–1.56 (4H, m, (CH2)2); 13C NMR (100 MHz,
DMSO-d6) d 179.1, 171.2, 167.1, 137.8, 136.0, 134.8, 133.2, 131.6,
131.0, 128.1, 125.2, 121.8, 119.2, 56.5, 42.3, 35.9, 23.6, 22.3; LRMS
4.1.5.8. 5-Dimethylamino-pentanoic acid {4-[3-(4-chloro-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30h). Yield
(44 mg, 0.09 mmol, 15%), yellow sticky oil. mmax cmÀ1 (NaCl, thin
layer) 3396, 3053, 2925, 2716, 1668, 1605, 1561, 1516, 1485,
1408, 1149, 1013, 842; 1H NMR (400 MHz, DMSO-d6): d 12.54
(1H, s, NH), 11.76 (1H, s, NH), 10.34 (2H, br s, 2 Â NH), 8.08 (2H,
d, J = 7.8 Hz, ArH), 7.71 (6H, m, ArH), 3.15 (2H, m, CH2), 2.82 (6H,
d, J = 4.7 Hz, N(CH3)2), 2.49 (2H, m, CH2), 1.75 (4H, m, (CH2)2);
13C NMR (100 MHz, DMSO-d6) d 178.7, 170.8, 167.1, 137.9, 137.5,
132.8, 131.0, 130.6, 130.2, 128.6, 128.5, 124.8, 120.1, 119.0, 56.0,
41.8, 35.5, 23.2, 22.0; LRMS (m/z): [(MÀHCl)+H]+ 433.15; HRMS
(m/z): [M]+ calcd for C21H26N4O2SCl, 433.1465; found, 433.1452.

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1791
(m/z) [(MÀHCl)+H]+ 477.03; HRMS (m/z): [M]+ calcd for
was then concentrated in vacuo. After redissolving the residue in
dry acetonitrile (5 mL) aniline 18 was added. The resulting mixture
was heated at reflux for 3 h and after cooling, the resulting beige
precipitate was collected by filtration to give 32 (400 mg,
0.24 mmol, 17%) as a beige solid which was not purified further.
Mp decomp. >310 °C; mmax cmÀ1 (KBr) 3396, 3363, 3244, 2972,
2905, 2597, 1694, 1674, 1607, 1559, 1521, 1479, 1411, 1367,
1265, 1220, 1164, 1058, 828, 771; 1H NMR (300 MHz, DMSO-d6)
d 10.91 (1H, s, NH), 10.77 (1H, s, NH), 9.31 (1H, s, NH), 7.97 (2H,
d, J = 8.6 Hz, ArH), 7.55 (2H, d, J = 8.6 Hz, ArH), 7.43 (4H, m, ArH),
1.47 (9H, s, (CH3)3), 1.31 (9H, s, (CH3)3); 13C NMR (100 MHz,
DMSO-d6) d 168.4, 156.1, 152.8, 151.1, 135.5, 131.9, 129.5, 128.2,
125.4, 120.3, 118.6, 78.9, 34.8, 30.8, 28.1.
N-Acyl urea 32 (300 mg, 0.73 mmol) was stirred in trifluoroace-
tic acid (1.2 mL) for 40 min and concentrated in vacuo to give the
corresponding aniline 1-(4-amino-phenyl)-3-(4-tert-butyl-ben-
zoyl)-urea as the trifluoroacetate salt (383 mg, 0.73 mmol, quanti-
tative) as a brown solid which was not purified further. 1H NMR
(300 MHz, DMSO-d6) d 11.02 (1H, s, NH), 10.96 (1H, s, NH), 7.98
(2H, d, J = 8.6 Hz, ArH), 7.70 (2H, d, J = 8.9 Hz, ArH), 7.55 (2H, d,
J = 8.6 Hz, ArH), 7.32 (2H, d, J = 8.8 Hz, ArH), 1.30 (9H, s, (CH3)3).
The deprotected aniline (368 mg, 0.70 mmol) was then stirred in
C21H26N4O2BrS, 477.0960; found, 477.0958.
4.1.5.14. 5-Dimethylamino-pentanoic acid {4-[3-(3-bromo-4-
tert-butyl-benzoyl)-thioureido]-phenyl}-amide hydrochloride
(30n). Yield (52 mg, 0.09 mmol, 35%), yellow oil. m
max cmÀ1 (NaCl,
thin layer) 3242, 3044, 2963, 2925, 2872, 2694, 2058, 1671, 1604,
1517, 1480, 1408, 1362, 1144, 839, 731; 1H NMR (400 MHz, DMSO-
d6): d 12.59 (1H, s, NH), 12.44 (1H, s, NH), 10.14 (1H, s, NH), 9.87
(1H, br s, NH+), 8.22 (1H, d, J = 2.0 Hz, ArH), 7.92 (1H, dd, J = 2 Hz,
8.4 Hz, ArH), 7.67–7.60 (3H, m, ArH), 7.57 (2H, d, J = 8.9 Hz, ArH),
3.09–3.01 (2H, m, CH2), 2.73 (6H, d, J = 4.8 Hz, N(CH3)2), 2.38 (2H,
app. t, J = 7.1 Hz, CH2), 1.73–1.56 (4H, m, (CH2)2), 1.49 (9H, s,
(CH3)3); 13C NMR (100 MHz, CD3OD) d 180.5, 173.8, 168.0, 154.5,
138.2, 136.8, 135.2, 133.0, 130.8, 128.6, 126.0, 123.7, 121.3, 58.7,
44.0, 36.7, 30.3, 25.2, 23.2; LRMS (m/z) [(MÀHCl)+H]+ 533.16;
HRMS (m/z): [M]+ calcd for C25H34N4O2BrS, 533.1586; found,
533.1579.
4.1.5.15. 5-Dimethylamino-pentanoic acid {4-[3-(3,4-dichloro-
benzoyl)-thioureido]-phenyl}-amide hydrochloride (30o). Yield
(90 mg, 0.18 mmol, 52%), brown oil. 1H NMR (300 MHz, DMSO-d6):
d 12.29 (1H, s, NH), 12.11 (1H, s, NH), 10.26 (1H, s, NH), 9.93 (1H, br
s, NH+), 7.88 (2H, d, J = 1.93 Hz, ArH), 7.72 (5H, m, ArH), 3.16 (2H, m,
CH2), 2.84 (6H, br s, N(CH3)2), 2.48 (2H, m, CH2), 1.74 (4H, m,
(CH2)2); 13C NMR (100 Hz, CD3OD) d 180.0, 173.6, 168.4, 138.6,
138.3, 125.2, 134.3, 133.4, 131.6, 131.0, 128.7, 125.8, 121.2, 58.7,
43.5, 36.8, 25.2, 23.3; LRMS (m/z): [(MÀHCl)+H]+ 467.11
dry CH2Cl2 (10 mL) and triethylamine (98
added to give a clear brown solution. 5-Bromovaleryl chloride
(140 L, 1.05 mmol) was added followed by triethylamine (98 L,
lL, 0.70 mmol) was
l
l
0.70 mmol). The reaction mixture was stirred at rt for 16 h and
then diluted with CH2Cl2 (1 Â 20 mL) and washed with 1 M aque-
ous hydrochloric acid (1 Â 20 mL), 1 M aqueous sodium hydroxide
(1 Â 20 mL) and saturated brine (1 Â 20 mL). The organic phase
was dried (MgSO4) and concentrated in vacuo to give the crude
product which was purified by column chromatography on silica
(petroleum ether/ethyl acetate 1:1) to give N-((4-(5-bromopenta-
namido)phenyl)carbamoyl)-4-(tert-butyl)-benzamide (153 mg, 0.34
mmol, 48%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) d
10.93 (1H, s, NH), 10.82 (1H, s, NH), 9.90 (1H, s, NH), 7.98 (2H, d,
J = 8.5 Hz, ArH), 7.54 (6H, m, ArH), 3.57 (2H, t, J = 6.6 Hz, CH2),
2.33 (2H, t, J = 7.2 Hz, CH2), 1.80 (4H, m, (CH2)2), 1.31 (9H, s,
(CH3)3). The bromide (123 mg, 0.27 mmol) was then stirred in
CH2Cl2 (10 mL), 40% aqueous dimethylamine (3 mL) and H2O
(7.5 mL) at rt for 20 h. The reaction mixture was diluted with
CH2Cl2 (20 mL) and washed with H2O (1 Â 10 mL), 10% aqueous
NaOH (1 Â 10 mL), and saturated brine (1 Â 10 mL). The organic
phase was dried over MgSO4 and concentrated in vacuo to give a
cream solid which was dissolved in acetone and treated with 2 M
hydrochloric acid in diethyl ether. A white precipitate was ob-
tained and isolated by filtration to give 33 (44 mg, 0.10 mmol
36%) as a cream solid. Mp 247–249 °C; mmax cmÀ1 (KBr) 3415,
3238, 2965, 2903, 2659, 2471, 1699, 1667, 1610, 1570, 1514,
1481, 1409, 1275, 1228, 1128, 836, 770; 1H NMR (400 MHz,
DMSO-d6) d 10.93 (1H, s, NH), 10.82 (1H, s, NH), 9.98 (1H, s, NH),
9.63 (1H, br s, NH), 7.98 (2H, d, J = 8.6 Hz, ArH), 7.53 (6H, m,
ArH), 3.05 (2H, m, CH2), 2.75 (6H, s, N(CH3)2), 2.36 (2H, t,
J = 6.7 Hz, CH2), 1.64 (4H, m, CH2)2), 1.31 (9H, s, (CH3)3); 13C NMR
(100 MHz, DMSO-d6) d 171.5, 169.4, 157.1, 152.1, 136.2, 133.8,
130.5, 129.5, 126.5, 121.5, 120.5, 57.2, 43.2, 36.5, 35.8, 32.0, 24.4,
23.0; LRMS (m/z): [M]+ 439.13, [(MÀHCl)+H]+; HRMS (m/z): [M]+
calcd for C25H35N4O3, 439.2709; found, 439.2696.
4.1.5.16. 5-Dimethylamino-pentanoic acid {4-[3-(2-methyl-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30p). Yield
(28 mg, 0.06 mmol, 10%), white solid. Mp 201–202 °C; mmax cmÀ1
(KBr) 3388, 3227, 3041, 2957, 1590, 1716, 1674, 1651, 1604, 1521,
1406, 1308, 1256, 1165, 835, 746; 1H NMR (400 MHz, DMSO-d6): d
12.46 (1H, s, NH), 11.69 (1H, s, NH), 10.20 (1H, s, NH), 10.13 (1H,
br s, NH), 7.63 (4H, AA’BB’, J = 9.1, 21.5 Hz, ArH), 7.50 (1H, d,
J = 7.4 Hz, ArH), 7.43 (1H, m, ArH), 7.31 (2H, d, J = 8.0 Hz, ArH),
3.06 (2H, m, CH2), 2.73 (6H, d, J = 4.9 Hz, N(CH3)2), 2.42 (3H, s,
CH3), 2.39 (2H, m, CH2), 1.65 (4H, m, (CH2)2); 13C NMR (100 MHz,
CD3OD) 180.6, 173.7, 172.3, 138.2, 138.0, 135.4, 135.3, 132.4,
132.2, 128.7, 127.0, 125.8, 121.1, 58.7, 43.5, 36.7, 25.2, 23.2; LRMS
(m/z): [(MÀHCl)+H]+ 413.15; HRMS (m/z): [M]+ calcd for
C22H29N4O2S, 413.2011; found, 413.1998.
4.1.5.17. 5-Dimethylamino-pentanoic acid {4-[3-(2-chloro-ben-
zoyl)-thioureido]-phenyl}-amide hydrochloride (30q). Yield
(22 mg, 0.05 mmol, 8%), yellow solid. Mp 188–190 °C; mmax cmÀ1
(KBr) 3311, 3042, 2956, 2926, 2855, 2703, 1667, 1604, 1516,
1467, 1408, 1344, 1291, 1155, 839, 752; 1H NMR (300 MHz,
DMSO-d6): d 12.27 (1H, s, NH), 12.00 (1H, s, NH), 10.10 (1H, s,
NH), 9.45 (1H, br s, NH), 7.56 (8H, m, ArH), 3.07 (2H, m, CH2),
2.76 (6H, d, J = 3.7 Hz, N(CH3)2), 2.39 (2H, t, J = 6.6 Hz, 2H CH2),
1.64 (4H, m, (CH2)2); 13C NMR (100 MHz, CD3OD) d 180.2, 174.0,
168.0, 138.2, 136.7, 136.5, 135.8, 135.2, 133.4, 132.4, 130.3,
125.8, 121.9, 58.7, 43.4, 36.7, 25.2, 23.1; LRMS (m/z): [(MÀHCl)+H]+
433.11; HRMS (m/z): [M]+ calcd for C21H26N4O2SCl, 433.1465;
found, 433.1469.
4.1.6. Synthesis of 33
4.1.7. Synthesis of 39
4.1.6.1. 5-Dimethylamino-pentanoic acid {4-[3-(4-tert-butyl-
benzoyl)-ureido]-phenyl}-amide hydrochloride (33). 4-t-Butyl
benzamide (31) (250 mg, 1.41 mmol) was added to a dry flask con-
taining molecular sieves under N2. 1,2-Dichloroethane (5 mL) was
added and the mixture stirred whilst a solution of 2 M oxalyl chlo-
ride in CH2Cl2 (1.19 mL, 2.30 mmol) was added. The clear yellow
solution was heated at reflux for 24 h and cooled to rt. The reaction
4.1.7.1. Benzyl (4-((4-hydroxybutyl)carbamoyl)phenyl)carba-
mate (36). To a solution of the known acid 35 (1 g, 3.69 mmol) in
CH2Cl2 (10 mL) at 0 °C was added triethylamine (1.03 mL,
7.38 mmol) and methyl chloroformate (0.57 mL, 7.38 mmol) drop-
wise. The resulting solution was allowed to warm to rt and stirred
for 2 h before cooling to 0 °C and 4-aminobutanol (0.68 mL,
7.38 mmol) added dropwise and the mixture stirred at rt for 6 h.

1792
A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
The solvent was removed in vacuo and water added (20 mL). The
resulting white precipitate was filtered to afford 36 as a white solid
(0.9 g, 2.66 mmol, 72%). Mp 164–165 °C; mmax cmÀ1 (KBr) 3567,
3381, 3294, 3030, 2952, 2866, 2741, 1792, 1699, 1667, 1628, 1611,
1587, 1524, 1411, 1312, 1245, 1061, 736, 696; 1H NMR (400 MHz,
CDCl3) d 8.10 (1H, s, NH), 7.49 (2H, d, J = 8.8 Hz, ArH), 7.44–7.32
(7H, m, ArH), 6.85 (1H, s, NH), 5.21 (1H, s, NH), 2.58 (1H, s, OH),
2.44–2.37 (4H, m, (CH2)2), 1.79 (2H, app. quintet, J = 7.5 Hz, CH2),
1.60 (2H, app. quintet, J = 7.2 Hz, CH2); 13C NMR (75 MHz, CDCl3) d
164.3, 154.2, 143.1, 134.6, 130.4, 128.3, 128.1, 119.5, 66.1, 58.7,
38.3, 26.1, 22.9; LRMS (m/z): [M]À 341.29 [MÀH]À;
through celite and the filtrate concentrated to give the crude prod-
uct. The product was purified by column chromatography
(CH2Cl2:MeOH 10:1) and conversion to the corresponding HCl salt
provided 39 (177 mg, 0.36 mmol, 86%). Mp 184–186 °C; m
max cmÀ1
(KBr) 3253, 2921, 2641, 2478, 1709, 1662, 1609, 1564, 1365, 1261,
1126, 1014, 960, 849, 765; 1H NMR (400 MHz, d6-DMSO) d 12.80
(1H, s, NH), 11.54 (1H, s, NH), 10.30 (1H, br s, NH), 8.63 (1H, t,
J = 5.8 Hz, NH), 7.96–7.89 (4H, m, ArH), 7.82 (2H, d, J = 8.6 Hz,
ArH), 7.56 (2H, d, J = 8.6 Hz, ArH), 3.28 (2H, app. quartet,
J = 6.8 Hz, CH2), 3.08–3.02 (2H, m, CH2), 2.71 (6H, d, J = 4.9 Hz,
N(CH3)2), 1.74–1.65 (2H, m, CH2), 1.55 (2H, app. quartet,
J = 6.9 Hz, CH2), 1.31 (9H, s, (CH3)3); 13C NMR (100 MHz, DMSO-
d6) d 168.3, 165.6, 156.2, 151.0, 140.2, 129.4, 129.3, 128.6, 125.6,
125.4, 118.8, 56.2, 42.0, 34.8, 30.8, 26.1, 21.2; LRMS (m/z):
[(MÀHCl)+H]+ 455.25; HRMS (m/z): [M]+ calcd for C25H34N4O2S,
455.2474; found, 455.2474.
4.1.7.2. Benzyl (4-((4-(dimethylamino)butyl)carbamoyl)phenyl)
carbamate (37). To a stirred solution of 31 (1.2 g, 3.5 mmol) in
CH2Cl2 (40 mL) was added CBr4 (1.28 g, 3.9 mmol) and the solution
cooled to 0 °C before PPh3 (1.02 g, 3.9 mmol) was added slowly. The
resulting mixture was allowed to warm to rt and stirred for 4 h. The
solvent was removed and the residue purified by column chroma-
tography on silica (CH2Cl2:MeOH 10:0.5) to afford the bromide
(764 mg, 1.89 mmol, 54%) which was used immediately in the next
4.2. Inhibition of SirT1 and SirT2
Compounds were tested for inhibition of human recombinant
SirT1 and SirT2 using the Fluor de Lys fluorescence-based assay
kits provided by Biomol (Plymouth Meeting, USA). The assays were
carried out as described in Lain et al.8a, except the final concentra-
step. Mp 161–162 °C; m
max cmÀ1 (KBr) 3327, 3091, 2937, 1701, 1668,
1632, 1611, 1588, 1521, 1413, 1315, 1245, 1070, 738; 1H NMR
(400 MHz, CDCl3) d 7.75 (2H, d, J = 8.7 Hz, ArH), 7.49 (2H, d,
J = 8.3 Hz, ArH), 7.46–7.37 (5H, m, ArH), 6.88 (1H, s, NH), 6.14 (1H,
t, J = 5.2 Hz, NH), 5.24 (2H, s, CH2), 3.55–3.47 (4H, m, (CH2)2), 1.99
(2H, app. quintet, J = 6.7 Hz, CH2), 1.81 (2H, app. quintet, J = 7.4 Hz,
CH2); 13C NMR (75 MHz, CDCl3) d 167.0, 167.2, 153.3, 141.1, 136.1,
129.1, 128.9, 128.8, 128.5, 118.4, 67.7, 39.5, 33.8, 30.4, 28.8. To a
solution of the bromide (300 mg, 0.74 mmol) in CH2Cl2 (3 mL) was
added a 40% aqueous solution of dimethylamine (1.5 mL) and water
(12 mL) and the resulting biphasic mixture stirred at rt for 18 h. The
reaction mixture was diluted with CH2Cl2 (10 mL) and washed with
H2O (1 Â 10 mL), 10% aqueous sodium hydroxide (1 Â 10 mL), and
saturated brine (1 Â 10 mL). The organic phase was dried over
MgSO4 and concentrated in vacuo to give 37 as an off-white sticky
oil (185 mg, 0.50 mmol, 67%). 1H NMR (400 MHz, CDCl3) d 7.82
(1H, t, J = 5.4 Hz, NH), 7.74 (2H, d, J = 8.6 Hz, ArH), 7.48 (2H, d,
J = 8.6 Hz, ArH), 7.42–7.34 (5H, m, ArH), 5.20 (2H, s, CH2), 3.46–
3.39 (2H, m, CH2), 2.33 (2H, t, J = 6.4 Hz, CH2), 2.22 (6H, s,
N(CH3)2), 1.72–1.57 (4H, m, (CH2)2); 13C NMR (75 MHz, CDCl3) d
167.4, 167.0, 153.2, 141.0, 136.2, 131.3, 129.1, 128.8, 128.6, 118.2,
67.6, 59.5, 45.5, 40.2, 27.7, 25.5; LRMS (m/z): [M]+ 370.25 [M+H]+;
HRMS calcd for C21H28N3O3, 370.2131; found, 370.2131.
tion of FdL substrate used per reaction was 25 lM. Raw data was
processed using Microsoft Excel. SigmaPlot (Systat Software Inc.)
was used to fit the data to a 4-parameter equation which was used
to calculate the concentration of inhibitor resulting in 50% inhibi-
tion (IC50 value). Compounds were tested on two separate occa-
sions for % inhibition at 60 lM and the values were used to
calculate a mean weighted to the standard error. For IC50 values,
compounds were tested on three separate occasions and the IC50
values were used to calculate a mean weighted to a standard devi-
ation. These values are given in Tables 1 and 2.
4.3. Cell culture and Western Blotting
Human MCF-7 and H1299 cancer cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, UK) and
RPMI respectively. Both were supplemented with 10% foetal calf
serum (FCS, Hyclone, UK) and gentamycin (complete medium).
Cells were seeded at a concentration of 2 Â 105 (MCF-7) and
6 Â 104 (H1299) in 6 well collagen precoated plates (TPP, Helena
Biosciences, UK) and incubated in a humidified atmosphere con-
taining 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 cells which 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 16 h. After lysing the cells with
4.1.7.3. 4-Amino-N-(4-(dimethylamino)butyl)benzamide (38). A
0.1 M solution of the protected amine 32 (185 mg, 0.50 mmol) in
methanol (5.2 mL) was passed through the H-CubeÒ, which was
equipped with a 10% Pd/C Cat-Cart, at 0 bar under the Full H2 mode.
The flow through was collected and concentrated in vacuo to afford
38 as a colourless oil (118 mg, 0.50 mmol, quant.) which was used
without further purification. 1H NMR (400 MHz, CDCl3) d 7.63 (2H,
d, J = 8.6 Hz, ArH), 7.21 (1H, br s, NH), 6.67 (2H, d, J = 8.6 Hz, ArH),
3.97 (2H, br s, NH2), 3.45 (2H, app. quintet, J = 5.9 Hz, CH2), 2.36
(2H, app. t, J = 6.7 Hz, CH2), 2.25 (6H, s, NMe2), 1.73–1.59 (4H, m,
(CH2)2); 13C NMR (100 MHz, CDCl3) d 169.5, 143.1, 131.7, 120.6,
116.3, 59.8, 45.9, 38.0, 26.2, 23.7; LRMS (m/z): [M+H]+ 222.45.
1 Â LDS sample buffer (100
lL per well for MCF-7 and 200 lL for
H1299) (Invitrogen, UK), the protein concentration was assessed
with a BCA protein assay kit (Pierce, UK) and the concentration
of protein equalised with 1 Â LDS sample buffer. Proteins were
separated with 4–12% bis-tris gels (Invitrogen, USA) and electro-
phoretically transferred to PVDF transfer membranes (Millipore,
UK). Membranes were blocked with Marvel nonfat milk (45 min,
5% solution in PBS/0.1% tween) and immunoblotted using DO.1
(anti p53, in-house produced) for the MCF-7 membranes. PC-10
mouse monoclonal antibody (in-house produced) was used to de-
4.1.7.4. 4-(tert-Butyl)-N-((4-((4-(dimethylamino)butyl)carbam-
oyl)phenyl)-carbamothioyl)benzamiide hydrochloride (39). To
a solution of 4 (77
lL, 0.42 mmol) in dry acetone (4 mL) under
tect PCNA as a loading control. Anti-K40 acetylated
a-tubulin (SIG-
N2, was added sodium thiocyanate (34 mg, 0.42 mmol) The result-
ing suspension was stirred at room temperature for 30 min. before
being cooled to 0 °C. A solution of amine 38 (99 mg, 0.42 mmol) in
dry acetone (3 mL) was added and the resulting suspension al-
lowed to warm to rt and stirred for 16 h. The reaction was filtered
MA) and anti -tubulin (SIGMA) were used for the H1299
a
membranes. All the primary antibodies were diluted in Marvel
nonfat milk (5% solution in PBS/0.1% tween). The secondary anti-
body used against the primary antibodies was a HRP-tagged poly-
clonal rabbit antimouse IgG (DAKO, UK). After incubation with

A. R. McCarthy et al. / Bioorg. Med. Chem. 20 (2012) 1779–1793
1793
primary (1 h) and secondary antibodies (45 min), bound antibody
Supplementary data
was visualised with enhanced chemiluminescence (ECL) Western
blotting developer (Amersham, UK) in a darkroom. Use of the b-
galactosidase assay has been reported previously by us using T22
cells. In this study, the only change to this protocol was the use
of ARN8 cells instead of T22s.7b,c
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.001.
References and notes
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a-tubulin
H1299 cells were treated with trichostatin A (100 nM) for 6 h.
Cytoplasmic extracts were prepared following the Dignam method
and combined with dd H2O, 10 Â complete EDTA-free protease
inhibitor (Roche), 527 mM Tris pH 7.5, 5 mM NAD+, 5 mM DTT,
750 mM NaCl, 0.5 mM trichostatin A and 2 mM PMSF and incu-
bated on ice prior to the addition of recombinant SirT1 (0.23 and
4. (a) Outeiro, T. F.; Kontopoulos, E.; Altmann, S. M.; Kufareva, I.; Strathearn, K. E.;
Amore, A. M.; Volk, C. B.; Maxwell, M. M.; Rochet, J.; McLean, P. J.; Young, A. B.;
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0.36
carried out for 90 min at 37 °C. Levels of K40-acetylated
total -tubulin and SirT1 were detected by Western Blot analysis.
Antibodies against K40-acetylated tubulin and tubulin (as de-
scribed above) and SirT1 (# 05-1243, Millipore) were used.
l
M final concentrations, Enzo Life Sciences). Reactions were
a
-tubulin,
a
4.5. SirT1 overexpression
H1299 cells were transfected with pcDNA3 or pcDNA3-SirT1,
which was prepared by cloning SirT1 from pCMV-SirT1 (Origene)
into pcDNA3. K40-acetylated tubulin, tubulin and SirT1 levels were
detected by western blotting as described.
4.6. Protocol for solubility analysis
Relative solubilities of compounds were determined using a UV
spectroscopy based method. Generation of the extinction coeffi-
cient for each compound was achieved by dissolving 1 mg of com-
pound in spectroscopic grade acetonitrile (100 mL) (some
compounds required dissolving in a minimal amount of DMSO
first). A series of 6 Â 2 fold dilutions were then made, giving a total
of 7 solutions. These solutions were analysed by UV spectroscopy
and their absorbance at 273 nM was plotted against molar concen-
tration. The extinction coefficient was calculated from the gradient
of the line of best fit. The extinction coefficient was then used to
calculate the actual concentration of a 100
of compound.
lM aqueous solution
The actual concentration of the 100
l
M aqueous solution was
L) of a 40 mM DMSO solu-
L) of this solution was
9. See ESI for Supplementary figures and tables.
determined by diluting an aliquot (4.2
l
10. Loiseau, F. A.; Hii, K. K.; Hill, A. M. J. Org. Chem. 2004, 69, 639.
11. Crystallographic data (excluding structure factors) for 4iv has been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 857728. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax:
+44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
12. Klabunde, T.; Wendt, K. U.; Kadereit, D.; Brachvogel, V.; Burger, H.; Herling, A.
W.; Oikonomakos, N. G.; Kosmopoulou, M. N.; Schmoll, D.; Sarubbi, E.; Von
Roedern, E.; Schönafinger, K.; Defossa, E. J. Med. Chem. 2005, 48, 6178.
13. Klotz, P.; Chatrenet, B.; Coppo, M.; Rousseau, B.; Goeldner, M.; Hirth, C. J.
Labelled Compd. Radiopharm. 1991, 29, 149.
tion to 1 mL with water. An aliquot (60
l
added to water (40 lL) and any undissolved compound was pel-
leted by centrifugation. The supernatant (80 lL) was diluted to
4 mL with acetonitrile to generate a theoretical 2 Â 10À6 M solu-
tion. This solution was then analysed by UV spectroscopy.
Acknowledgements
14. For a recent publication on the challenges of X-ray crystallographic analysis of
SirT1 see: Holbourn, K. P.; Lloyd, M. D.; Thompson, A. S.; Threadgill, M. D.;
Acharya, K. R. Acta Crystallogr., Sect. F 2011, 67, 461.
15. Katori, E.; Nagano, T.; Kunieda, T.; Hirobe, M. Chem. Pharm. Bull. 1981, 29, 3075.
16. Chung, K.; Murdock, C. A.; Stevens, S. E., Jr.; Li, Y.; Wei, C.; Huang, T.; Chou, M.
W. Toxicol. Lett. 1995, 81, 23.
N.J.W. was a Royal Society University Research Fellow at the
time this work was done. We would like to thank Tenovus, Scottish
Enterprise, SULSA and Cancer Research UK for funding. We thank
Professor Sir David Lane for his continuous support of this work
and Professor Roland Wolf for allowing access to the required mass
spectrometer.
17. North, B. J.; Marshall, B. L.; Borra, M. T.; Denu, J. M.; Verdin, E. Mol. Cell 2003, 11,
437.