Benzofused hydroxamic acids: Useful fragments for the preparation of histone deacetylase inhibitors. Part 2: 7-Fluorobenzothiophenes and benzofurans

Article abstract of DOI:10.1016/j.bmcl.2015.02.007

In the search for a new class of histone deacetylase inhibitors, we prepared a series of very simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular weight: they showed very good ligand efficiencies. Following these findings, classical fragment growing work was performed to increase binding energy and selective cytotoxicity. In the second phase of the work, information from the SARs of the benzothiophene series and data available in literature, we explored the in vitro pharmacological properties of the 6-substituted-7-fluoro-benzothiophene hydroxamates and the 5-susbtituted-benzofuran hydroxamates.

Full text of DOI:10.1016/j.bmcl.2015.02.007

Accepted Manuscript
Benzofused hydroxamic acids: Useful fragments for the preparation of histone
deacetylase inhibitors. Part 2: 7-Fluorobenzothiophenes and benzofurans
Elena Marastoni, Sandra Bartoli, Marco Berettoni, Amalia Cipollone,
Alessandro Ettorre, Christopher I. Fincham, Sandro Mauro, Marielle Paris,
Marina Porcelloni, Mario Bigioni, Monica Binaschi, Federica Nardelli,
Massimo Parlani, Carlo A. Maggi, Paola Paoli, Patrizia Rossi, Daniela Fattori
PII:
S0960-894X(15)00112-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.02.007
BMCL 22419
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 October 2014
30 January 2015
2 February 2015
Please cite this article as: Marastoni, E., Bartoli, S., Berettoni, M., Cipollone, A., Ettorre, A., Fincham, C.I., Mauro,
S., Paris, M., Porcelloni, M., Bigioni, M., Binaschi, M., Nardelli, F., Parlani, M., Maggi, C.A., Paoli, P., Rossi, P.,
Fattori, D., Benzofused hydroxamic acids: Useful fragments for the preparation of histone deacetylase inhibitors.
Part 2: 7-Fluorobenzothiophenes and benzofurans, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.02.007
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Benzofused Hydroxamic Acids: Useful
Fragments for the Preparation of Histone
Deacetylase Inhibitors. Part 2: 7-Fluoro
benzothiophenes and Benzofurans
Leave this area blank for abstract info.
Elena Marastoni, Sandra Bartoli, Marco Berettoni, Amalia Cipollone, Alessandro Ettorre, Christopher I.
Fincham, Sandro Mauro, Marielle Paris, Marina Porcelloni, Mario Bigioni, Monica Binaschi, Federica Nardelli,
Massimo Parlani, Carlo A. Maggi, Paola Paoli, Patrizia Rossi, Daniela Fattori

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Benzofused Hydroxamic Acids: Useful Fragments for the Preparation of Histone
Deacetylase Inhibitors. Part 2: 7-Fluorobenzothiophenes and Benzofurans
Elena Marastonia, Sandra Bartolia, Marco Berettonia, Amalia Cipollonea, Alessandro Ettorrea, Christopher I.
Finchama, Sandro Mauroa, Marielle Parisa, Marina Porcellonia, Mario Bigionia, Monica Binaschia, Federica
Nardellia, Massimo Parlania, Carlo A. Maggib, Paola Paolic, Patrizia Rossic, and Daniela Fattoria∗
aMenarini Ricerche, Via Tito Speri 10, 00040 Pomezia (Rome), Italy
bMenarini Ricerche, Via Rismondo12A, 50131 Florence, Italy
cDip. di Ingegneria Industriale, Università degli Studi di Firenze, Via Santa Marta 3, 50139 Florence (Italy)
ARTICLE INFO
ABSTRACT
Article history:
Received
In the search for a new class of histone deacetylase inhibitors, we prepared a series of very
simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular
weight: they showed very good ligand efficiencies. Following these findings, classical fragment
growing work was performed to increase binding energy and selective cytotoxicity.
In the second phase of the work, information from the SARs of the benzothiophene series and
data available in literature, we explored the in vitro pharmacological properties of the 6-
substituted-7-fluoro-benzothiophene hydroxamates and the 5-susbtituted-benzofuran
hydroxamates.
Revised
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
HDAC
Hydroxamic acids
Fragment approach
Histone deacetylases (HDACs) are zinc dependent hydrolases
that play a key role in the epigenetic modulation of gene
expression by removal of acetyl groups from N-acetyl lysine
residues of histones and other cellular proteins.1 Histone
hyperacetylation, induced by HDAC inhibitors, correlates with
gene expression, cell-cycle arrest, differentiation and apoptosis in
tumor cells. Based on their homology to the yeast HDACs,
mammalian. HDACs can be categorized into four classes.
Mechanistically, Class I, II and IV are distinct from Class III in
co-factor requirement, that is, the former require an active site
zinc to mediate deacetylation catalysis, while the latter are
dependent on NAD+2. Virtually all the known inhibitors fall into
distinct structural classes: hydroxamic acids, aminobenzamides,
short chain fatty acids, and cyclic peptides. They generally target
the Zn+2 containing catalytic domain within Class I and Class II
HDACs and are non-selective or slightly selective. Currently
there are several HDAC inhibitors in clinical trials in a variety of
haematological and solid tumors,2 and two (Vorinostat3 and
Romidepsin) have been approved by the Food and Drug
Administration (FDA) for the treatment of cutaneous T-cell
lymphoma (CTCL).
As a part of an internal project aimed at the identification of
new and proprietary classes of HDAC inhibitors, we tested a
series of fragments characterized by a hydroxamic acid moiety
linked to a benzofused thiophene derivative for their ability to
inhibit HDAC activity in vitro. The benzothiophene and the 7-
fluoro-benzothiophenes showed quite good ligand efficiencies
and were selected for further exploration. Next we expanded our
SARs of the benzothiophene scaffold and obtained then
preliminary in vivo data for compound 1a (Figure 1). 4
Figure 1. Examples of compounds from ref [4]
The object of the present paper is the study of the
pharmacological properties of the 7-fluoro derivatives, and of the
———
∗ Corresponding author. Tel.: +39-06-91184522; e-mail: dfattori@menarini-ricerche.it

benzofuran hydroxamates, expected, from previous experience
with similar structures, to be more water soluble.
Taking advantage of the SARs from the benzothiophene series
and the data available in literature, we prepared the 7-fluoro-
benzothiophene hydroxamates listed in Table 1. This set of
compounds was obtained by radical bromination of the 6-methyl
derivative 2, followed by a nucleophilic substitution with a
secondary amine (4) or azide ion. In the latter case a catalytic
reduction gave the aminomethyl derivative 5 (Scheme 1).
Coupling of this amine with an appropriate carboxylic acid
moiety, in standard conditions, resulted in amides 6. Upon
treatment with hydroxylamine, amines 4 and amides 6 afforded
the hydroxamic acids 7.
Figure 3. Figure 1. Ortep-3 view of compound 1b (ellipsoid probability =
25%)
With this information we began introducing the best
substituents identified for the benzothiophene series into the 5-
position of the benzofuran scaffold.
The 5-amino derivatives of benzofuran (13) were prepared by
taking advantage of solid phase methodologies. The free amino
group of the commercially available 5-aminobenzofurane-2-
carboxylic acid was protected as Fmoc derivative by treatment
with Fmoc-N-hydroxysuccinimide to produce acid 9, which was
then loaded onto hydroxylamino chlorotrityl resin using standard
conditions (Scheme 2). Fmoc deprotection with 20% piperidine
in DMF gave amine 11, which was coupled with selected
carboxylic acids. The acyl hydroxamates 13 were released from
the solid support upon treatment with a mixture of trifluoroacetic
acid and triethylsilane in dichloromethane.
Scheme 1. (a) NBS, AIBN, CCl4; (b) NHR1R2, EDAC, HOBt, DIPEA,
DMF; (c) NaN3, DMF and then H2, Pd/C, THF; (d) R3CO2H and coupling
agent; (e) KOH, NH2OH-HCl, MeOH/THF; (f) NaOH, H2O/THF and then
NH2OH-HCl, EDAC, HOAt, DMF.
As stated at the beginning of this paper, we also prepared
some benzofurane derivatives (Table 2). From previous work we
knew that, while for 2-benzothiophene carboxamides the
preferred conformation is the one where the carbonyl group is
syn with the sulfur, for 2-benzofuran carboxamides the
preference is the opposite (Figure 2) with the heterocyclic oxygen
facing the amide nitrogen.5 In addition, concerning the amidic
group, it is well known that the sp conformation for the torsion
angle R-N-C-O is the preferred one.
Scheme 2. (a) Fmoc N-hydroxysuccinimide; (b) DIPC, HOAt, pyridine,
hydroxylamine chlorotrityl resin; (c) 20% piperidine in DMF; (d) DIPC,
HOAt, pyridine; (e) TES, TFA, DCM
For the preparation of the 5-aminomethyl derivatives 18, the
starting point was commercially available 5-methylbenzofuran-2-
carboxylic acid 14, which was converted to the methyl ester 15 in
acidic methanol, submitted to radical bromination and then to
nucleophilic substitution with NaN3. Catalytic hydrogenation of
the azido derivative, in the presence of Pd/C, afforded amine 17,
which was then coupled with selected carboxylic acids,
submitted to basic hydrolysis and then coupled with
hydroxylamine (Scheme 3).
Figure 2. Possible planar conformations for benzothiophene- and
benzofuran-2-carboxamides.
No information concerning hydroxamic acids was available in
the Cambridge Structural Database (v 5.35); in any case the solid
state structure of compound 1b (Figure 1), determined by means
of X-ray diffraction analysis, seems to suggest that, at least for
benzothiophenes, the conformational preference is the same as
that of carboxamides, i.e. the carbonyl oxygen syn coplanar with
the sulphur facing the amide nitrogen (Figure 3).

Finally, to test our conformational hypothesis, we also
prepared the 6-aminomethyl derivative 24, isomer of 18b.
The 6-methylbenzofuran-2-carboxylic acid was obtained in
the form of the t-butyl ester by the condensation of hydroxyl
aldehyde 19 and t-butylbromoacetate, in the presence of a base.
Radical bromination, azide displacement, catalytic hydrogenation
and coupling with a carboxylic acid followed. The acid was
deprotected with TFA in DCM. The corresponding hydroxamate
was again obtained by coupling with hydroxylamine.
Scheme 3. (a) HCl 4M/ dioxane / MeOH; (b) NBS, AIBN, CCl4; (c)
NaN3, DMF; (d) H2, Pd/C, AcOH, THF; (e) 2-furane carboxylic acid, EDAC,
HOBt or acylchloride, TEA, DCM/CH3CN /DMF (1/1/1) 0°C; (f) LiOH 2M,
THF/MeOH; (g) hydroxylamine, EDAC, HOBt.
CHO
OH
OtBu
O
b
a
O
Me
Br
Me
Table 1. HDAC inhibition of compounds 7, and cytotoxicity on HCT-116
19
20
cells
OtBu
O
c,d
OtBu
O
O
H2N
O
21
22
R
e
H
N
O
O
O
O
% Inh at
0.1 M
IC50
( M)
23 (R = tBu)
24 (R = NHOH)
0.01
<20
M
Compd
R
HCT-116
f,g
55
SAHA
0.60
Scheme 4. (a) t-butyl bromoacetate, Cs2CO3, DMF, 110°C; (b) NBS, AIBN,
CCl4; (c) NaN3, DMF; (d) H2, Pd/C, THF; (e) 2-furan carboxylic acid, EDAC,
HOBt; (f) TFA, DCM; (g) hydroxylamine, EDAC, HOBt.
OH
<20
<20
48
45
0.80
3
7a
7b
All the compounds were evaluated for their ability to inhibit
HDAC in a two point experiment, 0.1 and 0.01 µM.6 Those
having a % inhibition >40% at a 0.1 µM were also tested for their
toxicity on HCT-116 cells.7
N
N
HN
O
The fluoro derivatives generally showed a higher binding
affinity than the des-fluoro counterparts, but this was not
paralleled by the cytotoxycity, where the activity remained in the
same range as the des-fluoro analogues with the exception of 7f,
7k and 7i.
7c
7d
7e
41
23
20
80
67
61
0.12
0.31
0.21
S
Me
HN
MeO
O
O
Table 2. HDAC inhibition of compounds 13, 16 and 24, and cytotoxicity on
Me
HCT-116 cells
HN
O
N
5
HN OH
R
O
O
O
6
HN
7f
39
21
78
59
0.09
0.47
O
O
O
Me
HN
7g
O
Me
HN
7h
7i
45
24
76
64
0.3
N
H
O
HN
0.98
O
HN
7j
<20
56
1.2
O
OMe
HN
O
MeO
78
61
7k
7l
39
0.05
0.06
HN
Me2N
<20
O

5. Fattori, D.; Porcelloni, M.; D’Andrea, P.; Catalioto, R.-M.; Ettorre, A.;
Giuliani, A.; Marastoni, E.; Mauro, S.; Meini, S.; Rossi, C.; Altamura, M.;
Maggi, C.A. J. Med. Chem. 2010, 53, 4148-4165.
6. The compounds were evaluated for their ability to inhibit isolated HDACs
in a commercially available assay (Fluor de Lys Assay System, Biomol
International LP, Plymouth Meeting PA), employing SAHA and LAQ-xx as
positive controls. The reaction mixture, 50 µL run in a 96 well plates,
contains a HeLa cell nuclear extract and a commercial substrate containing
acetylated lysine side chains. The substrate and extracts are incubated in the
presence of the appropriate concentration of the inhibitor. Deacetylation of
the substrate followed by mixing with the provided developer generates a
fluorophore, 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 and reported
values are the average of those determinations.
7. Colon cancer HCT-116 cells were plated in a 96-well tissue culture plates
containing 200 µL of complete medium. After 24 h HDAC inhibitors were
added at different concentrations, ranging from 0.1 to 100 µM in
quadruplicate. After 5 days, 20 µL of Alamar blue were added to each well
and the plates were further incubated for 4 h. The chemical reduction of
Alamar Blue in the growth medium is a fluorometric/colorimetric indicator of
cellular growth based on the detection of metabolic activity. Fluorescence
was monitored in a multilabel counter Victor 1420 at 530 nm excitation and
590 nm emission wavelength. All results were expressed as IC50 (drug
concentration causing a 50% inhibition of cellular proliferation) calculated
using the Easy-fit software.
For the benzofuran hydroxamic acids, the potency on the
isolated enzyme was generally lower than that of the
corresponding benzothiophene. Nevertheless, we identified some
compounds with cell potency in the submicromolar range (18a,
18b). To test, we synthesised a single compound with a
substituent in position 6 (24), which, as expected, was less potent
than the 5-substituted analogue (18b).
In summary, starting from a selection of small rigid fragments
derived from the benzothiophene scaffold, we have been able to
identify two additional chemical series, the 6-substituted-7-
fluoro-benzothiophene hydroxamic acids and the 5-substituted-
benzofuran hydroxamic acids, which showed significant potency
on the isolate enzymes and IC50s on the HCT-116 cells in the
nanomolar range.
Supplementary Material
A brief description of the solid state structure of 1b, as well as
experimental details of the X-ray data collection are available.
CIF file for 1b has been deposited in the Cambridge Structural
Database (CCDC 1044933)
References and notes
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Bruno, R.;Laurence, D.; Ron, G.; Ian, H.; Veronique, V.; Eugene, C.; Kees,
B.; Willem, T.; Luc, A.; Marc, D. J.; Michel, J.; Martin, P.; Kristof, V. E.;
Patrick, A. Clin. Cancer Res. 2009, 15, 6841.
3. Richton, V. Brit. J. Cancer. 2006, 95, S2-S6.
4. Marastoni, E.; Bartoli, S.; Berettoni, M.; Cipollone, A.; Ettorre, A.;
Fincham, C.I.; Mauro, S.; Paris, M.; Porcelloni, M.; Bigioni, M.; Binaschi,
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