Set-up of a new series of HDAC inhibitors: The 5,11-dihydrodibenzo[b,e] azepin-6-ones as privileged structures

Article abstract of DOI:10.1016/j.bmcl.2012.07.067

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of 5,11-dihydrodibenzo[b,e] azepine-6-ones alkylated on the amide nitrogen with an alkyl chain bearing an hydroxamic acids moiety at the end, has been designed (based upon the general motif for HDAC inhibitors), synthesized and tested. This allowed us to identify a new series of submicromolar HDAC inhibitors, which showed antiproliferative activity on HCT-116 colon carcinoma cells.

Full text of DOI:10.1016/j.bmcl.2012.07.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5360–5362
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Set-up of a new series of HDAC inhibitors: the
5,11-dihydrodibenzo[b,e]azepin-6-ones as privileged structures
Mario Bigioni a, Alessandro Ettorre a, Patrizia Felicetti a, Sandro Mauro a, Cristina Rossi a,
Carlo Alberto Maggi b, Elena Marastoni a, Monica Binaschi a, Massimo Parlani a, Daniela Fattori a,
⇑
a Menarini Ricerche Pomezia, via Tito Speri 10, 00040 Pomezia, Rome, Italy
b Menarini Ricerche Firenze, via Rismondo 12A, 50131 Florence, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC)
inhibitors.
A series of 5,11-dihydrodibenzo[b,e]azepine-6-ones alkylated on the amide nitrogen with an alkyl
chain bearing an hydroxamic acids moiety at the end, has been designed (based upon the general motif
for HDAC inhibitors), synthesized and tested.
Received 17 April 2012
Revised 16 July 2012
Accepted 18 July 2012
Available online 26 July 2012
This allowed us to identify a new series of submicromolar HDAC inhibitors, which showed antiprolif-
erative activity on HCT-116 colon carcinoma cells.
Keywords:
HDAc inhibitors
Privileged structures
Tricycles
Ó 2012 Elsevier Ltd. All rights reserved.
It well established that the acetylation of lysine residues on the
tails of histone proteins plays an important role in the regulation
of gene expression in eukaryotic cells.1 The control of lysine acetyla-
tion relies upon the opposing actions of histone acetyl transferases
(HATs) and histone deacetylases (HDACs). In the last 10 years a large
amount of work has been done, patented and published on HDAC
inhibitors. Two compounds (vorinostat2 and romidespin,3 Fig. 1)
are already on the market, while several others are in different stage
of clinical trials. HDAC inhibition has been shown to be a successful
strategy to treat certain forms of cancer; in addition they are also un-
derextensivestudytotreata widerangeofdisorders, includingHun-
tington’s disease,4 fibrosis,5 cardiac hypertrophy,6 multiple
sclerosis,7 spinal muscle atrophy8 and as antiinfective agents.9
A number of structurally diverse HDAC inhibitors has been re-
ported,10 and most of them belongs to the class of hydroxamic
acids. This moiety is known to chelate the zinc ion in the active site
in a bidentate fashion through its CO and OH groups. Three differ-
ent regions can be recognized in these molecules: (a) metal binding
moiety; (b) a linker; (c) a surface recognition (Fig. 1).
ments are frequently found in biologically active compounds.
Rich suggests that these molecules are templates that are resistant
to the hydrophobic collapse in aqueous medium, while retaining
the ability to form productive hydrophobic and aromatic interac-
tions. These two modes of binding usually account for most of
the binding energy in protein–ligand interactions.11
To begin with, we focussed on a tryciclic template.12
We used as a starting material the commercially available tricy-
cle core 1. The endocyclic ketone was reduced to methylene and
the core thus obtained was evaluated with three linkers of differ-
ent length (Scheme 1): three (4a), four (4b) and five (4c) methylene
linker
surface recognition motif
Zn chelating moiety
O
As a part of an internal project aimed at the identification of
new and proprietary classes of HDAC inhibitors, we decided to
use the classical Zn chelating moiety and linker, and explore a
new surface recognition motif, a privileged structure belonging
to the diphenylmethyl family.
H
O
N
NH
S
O
O
H
N
HN
S
OH
O
NH
N
H
O
Diphenylmethyl motives are well known in medicinal chemis-
try and often referred as privileged structures (Fig. 2). These frag-
O
vorinostat
O
romidespin
⇑
Corresponding author. Tel.: +39 06 911 84522; fax: +39 06 000 0000.
E-mail address: dfattori@menarini-ricerche.it (D. Fattori).
Figure 1. General structure of hydroxamate based HDACs inhibitors, and the
formulae of vorinostat and romidespin.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.067

M. Bigioni et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5360–5362
5361
Table 1
Pharmacological activity of SAHA and tricycle hydroxamic acids
Cmpnd (%) of inhibition
IC50 HDAC
M)
IC50 HCT-116
(lM)
(l
diphenylmethyl
0.01
lM
0.1
55
<20
<20
67
lM
1.0
81
lM
A
SAHA
4a
4b
4c
<20
<20
<20
21
0.079
nd
nd
0.042
0.153
0.060
0.053
nd
0.60
nd
nd
0.71
>1
2.00
1.6
E
D
A
B
B
D
<20
29
84
77
81
84
42
78
A
C
X
B C
Y
spirocyclics
benzodiazepines
7
11
47
trycycles
9
21
58
11
13
15
<20
<20
<20
62
<20
50
Figure 2. General formulas of privileged structures inspired at the diphenylmethyl
motif.
nd
0.60
0.111
units.13 As indicated above, a terminal hydroxamate group was
introduced as Zn chelating moiety.
The reduction of the ketone was obtained by treatment with tri-
ethyl silane (TES) in acid medium (TFA) and produced 2. This was
NH
(CH2)5CO2Et
O
(CH2)5CONHOH
N
N
O
a
O
O
alkylated with the appropriate
x-bromoalkyl esters in standard
c, d
O
O
conditions (NaH, DMF) to obtain 3a–3c. The hydroxamic acids
4a–4c were produced by treatment of the ethyl esters 3 with
NH2OH in a water/MeOH mixture.
7
5
1
b
Compounds 4a–4c14 were screened at three concentrations
(CH2)5CONHOH
(1.0, 0.1 and 0.01 lm) using an enzymatic assay which measured
N
H
the total HDAC activity in HeLa cell extracts (Table 1).15 The results
showed clearly that n = 5 was the best of the linkers, seen since 4c
had an activity comparable with that of the reference compound
SAHA (IC50 42 nM vs 79 nM ‘respectively’).
CO2H
O
6
In order to improve the pharmacological properties of this hit,
we decided to explore the three regions of the molecule: the ring
X, ring Y and the methylene between the rings (Fig. 2).
We report here the work done on the methylene position, while
the functionalization of the rings A and B will be described
elsewhere.
First of all we restored the endocyclic ketone to see whether we
could gain additional binding affinity via a polar interaction. The
derivative 5 was prepared by alkylation of lactam 1 with ethyl 6-
bromo hexanoate and NaH in DMF at room temperature. In this
case the synthesis of the hydroxamic acid by direct treatment of
5 with aqueous hydroxylamine was not possible, since these con-
ditions caused the opening of the lactam ring to give compound
6. We were forced to hydrolyze the ester under very mild and con-
trolled conditions and perform a classical coupling reaction with
hydroxylamine using EDAC and HOAt to give compound 7 (Scheme
2).
Scheme 2. (a) NaH, DMF, 0 °C for 30 min, then Br(CH2)5CO2Et; (b) NH2OH, MeOH/
THF/H2O; (c) 0.01 N NaOH in MeOH/THF/H2O; (d) EDAC, HOAt, NH2OH-HCl.
(CH2)5COR
(CH2)5CO2Et
(CH2)5COR
O
N
N
N
O
a
O
e
MeO
MeO
HO
O
5
8 R = OEt
b
9
R = NHOH
12
R = OEt
d
b
13 R = NHOH
c
(CH2)5COR
O
N
(CH ) COR
6
2 5
F
N
Compound 5 was used as the starting point for further modifi-
cations (Scheme 3). Treatment with NaBH4 in MeOH gave 8 as a
racemic mixture, which was then transformed into the corre-
sponding hydroxamic acid 9 by treatment with hydroxylamine in
O
F
F
14
R = OEt
b
15 R = NHOH
10
R = OEt
b
11 R = NHOH
Scheme 3. (a) NaBH4, MeOH; (b). NH2OH, MeOH/water; (c) DAST, CH2Cl2, rt; (d)
BAST, 80 °C; (e) Me2SO4, HCl, MeOH.
(CH2)nCO2Et
N
NH
NH
O
b
O
O
a
O
aqueous solution. Reaction of alcohol 8 with DAST in dichloro-
methane solution at room temperature provided the clean mono-
fluoro derivative 10.16 The stability of the bis aromatic ketone 5
meant that a higher eaction temperature was required for the fluo-
rination reaction compared to compound 8, and this temperature
was not compatible with DAST (decomposed). BAST is reported
to have a higher thermal stability than DAST17 and thus difluoro
derivative 14 was obtained smoothly and in high yield by heating
a solution of BAST, 5 and a catalytic amount of ethanol at 80 °C.
Dimethoxyketale 12 was obtained by stirring ketone 5 in MeOH
saturated with HCl and in the presence of Me2SO4. Treatment of
3
2
1
(CH2)nCONHOH
O
N
c
4a
4b
n = 3
n = 4
4c n = 5
Scheme 1. (a) TES, TFA, rt; (b) Br(CH2)nCO2Et, NaH, DMF; (c) NH2OH, MeOH/water.

5362
M. Bigioni et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5360–5362
7. Gray, S. G.; Dangond, F. Epigenetics 2006, 1, 67.
ethyl esters 8, 10, 12 and 14 with hydroxylamine in MeOH/water,
gave the corresponding hydroxamic acids 9, 11, 13 and 15 respec-
tively (Scheme 3). Again the compounds were tested in a three
points experiment for HDAC inhibition and those showing an inhi-
8. Minamiyama, M.; Katsuno, M.; Adachi, H.; Waza, M.; Sang, C.; Kobayashi, Y.;
Tanaka, F.; Doyu, M.; Inukai, A.; Sobue, G. Hum. Mol. Genet. 2004, 13, 1183.
9. Meinke, P. T.; Liberator, P. Curr. Med. Chem. 2001, 8, 211.
10. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
11. Wiley, R. A.; Rich, D. Med. Res. Rev. 1993, 3, 327.
bition >40% at 0.1 lM were evaluated for cytotoxicity on tumor
12. Deziel, R.; leit, S.; Beaulieu, P.; Chantigny, I. A.; Mancuso, J.; Tessier, P.; Shapiro,
P.; Chesworth, R.; Smil, D. US 2,008/020,759,0 A1.
HCT-116 tumor cell lines.18
13. Longer linker chains had already been introduced on analogous series without
any significant improvement over the five methylenes one. Unpublished
results.
The endocyclic ketone was tolerated, but was not beneficial to
the binding affinity, since it caused a three-fold drop in the IC50
.
The effect on cell activity was more dramatic probably due to de-
creased permeability. More or less the same effect was seen with
alcohol 9, while the dimethoxyketal 13 was clearly detrimental.
One (11) or two (15) fluorine atoms were tolerated, but no major
improvement of the biological activity was seen compared to 4c.
In conclusion we have been able to exploit the diphenyl methyl
privileged fragment as recognition motif in the identification of a
new series of HDAC inhibitors.
14. All final compounds were characterized by 1H NMR and LC–MS analyses, and
showed a purity >97%. When necessary they were purified either by flash
chromatography on silica gel or by reverse phase preparative HPLC. NMR
experiments were recorded on
a Brucker Avance 400 MHz spectrometer
equipped with a 5 mm inverse probe and processed using Xwin-NMR version
3.5. Mass spectra were recorded using a WATERS Alliance 2795 HPLC system
fitted with a UV-PDA 996 diode array detector, a ZMD mass spectrometer and a
GL Science Inertsil ODS-3 column (50 Â 3 mm 3
lm). Generally, the gradient
used was 20–80% B in 8 min. at a flow rate of 0.8 mL/min (the eluents used
were A: H20 + 0.1% TFA and B MeCN + 0.1% TFA), the sample concentrations
were 0.1 mg/mL and the injection volume 10 lL. Part of the eluent (20 lL/min)
was diverted to the mass spectrometer and subjected to ESI+ ionisation (cone
voltages 20 V and 50 V, source temperature 105 °C).
Additional work aimed at improving the pharmacological activ-
ity has been carried out on this scaffold and will be the subject of
future reports.
15. The compounds were dissolved in DMSO and stored at À80 °C. HDAC
enzymatic activity was tested using
a commercially available assay kit
(HDAC Fluorescent Activity Assay/Drug Discovery Kit AK-500, Biomol,
International LP, Plymouth Meeting PA) based on the Fluor de LysTM
Acknowledgments
substrate and developer combination. Enzymatic reactions (50 lL) were run
in 96 well plates. The release of the fluorophore was monitored with a Victor
1420 fluorescent plate reader set at excitation/emission wavelength of 335/
460 nm. The activity of compounds was expressed as IC50 (drug concentration
causing a 50% inhibition of enzymatic activity) and calculated with Easy-fit
software application. All the experiment were carried out in triplicate.
The authors wish to thank Alessandro Sisto for the assistance in
the preparation of the compounds and Christopher Fincham for
manuscript revision.
16. When the reaction was performed on the non alkylated tricyle it went on
smoothly, but the subsequent alkylation produced a mixture of products.
17. Vu, N. Q.; Gree, R.; Brown, E.; Dujardin, G. Tetrahedron Lett. 2003, 44, 6425.
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l
l
l
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