Alkyl piperidine and piperazine hydroxamic acids as HDAC inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.085

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of N-substituted 4-alkylpiperazine and 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of HDAC inhibitors (zinc binding moiety-linker-capping group) has been designed, prepared, and tested for HDAC inhibition. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells.

Full text of DOI:10.1016/j.bmcl.2011.02.085

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2305–2308
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Alkyl piperidine and piperazine hydroxamic acids as HDAC inhibitors
Cristina Rossi a, Marina Porcelloni a, Piero D’Andrea a, Christopher I. Fincham a, Alessandro Ettorre a,
Sandro Mauro a, Antonella Squarcia a, Mario Bigioni a, Massimo Parlani a, Federica Nardelli a,
Monica Binaschi a, Carlo A. Maggi b, 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 N-substituted 4-alkylpiperazine and 4-alkylpiperidine hydroxamic acids, corresponding to
the basic structure of HDAC inhibitors (zinc binding moiety-linker-capping group) has been designed,
prepared, and tested for HDAC inhibition.
Received 19 January 2011
Revised 22 February 2011
Accepted 23 February 2011
Available online 26 February 2011
Linker length and aromatic capping group connection were systematically varied to find the optimal
geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed anti-
proliferative activity on HCT-116 colon carcinoma cells.
Keywords:
HDAC
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Lead finding
The last decade has seen histone deacetylase (HDAC) inhibition
emerge as an attractive target for the development of new antican-
cer agents.1 In addition, more it has been recently demonstrated that
HDAC inhibitors may also be used for novel therapeutic applications
such as inflammation,2 neurodegenerative diseases3 and malaria.4
HDACs are a family of enzymes (classes I–IV) that remove the
– use a hydroxamic acid as Zn binding moiety (Fig. 2, B);
– test alkyl chains spanning from n = 0 to n = 5;
– follow a minimalist approach, that is, functionalize the terminal
nitrogen with simple phenyl rings positioned in different
regions of space through the different geometry of the connect-
ing bonds (amide, sulfonamide, urea, Fig. 2, C). This is because
the aromatic ring is able to establish a large variety of molecular
interactions: aromatic-aromatic interactions, hydrogen bonding,
van der Waals, etc. In this first round, functionalization of the
aromatic rings was avoided on purpose.
acetyl group from the e-amino groups of the lysine residues of
the core histones. The enzymes of the classes I, II, and IV share
many common features in their active sites, which are composed
of a long, narrow hydrophobic tunnel leading to a cavity that con-
tains a catalytic Zn2+ ion, complexed to two Asp and one His resi-
dues. Inhibitors of these HDACs also have a common general
structure, consisting of a Zn chelating moiety (often a hydroxamic
acid), a linker and a so-called ‘surface recognition motif’1 (Fig. 1).
A number of HDAC inhibitors are at present undergoing clinical
trials and two, SAHA and depsipeptide (Fig. 1), are already on the
market.
linker
Here we describe the design, the synthesis and the primary
in vitro pharmacological characterization of a novel series of HDAC
inhibitors.
Capitalizing on the above information, we selected two possible
linkers for the design of new classes of HDAC inhibitors, 4-alkyl
piperidine and 4-alkyl piperazine (Fig. 2, A). With both these link-
ers the free heterocyclic nitrogen allows rapid chemical functional-
ization, useful for explore the space at the entrance of the active
site. We also decided to:
surfacer ecognition motif
Zn chelating moiety
O
H
O
N
NH
S
O
H
N
HN
S
OH
O
O
O
NH
N
H
O
SAHA
O
Depsipeptide
⇑
Corresponding author. Tel.: +39 06 91184522; fax: +39 06 9106137.
E-mail address: dfattori@menarini-ricerche.it (D. Fattori).
Figure 1. General structure of inhibitors for class I, II and IV HDACs, and the
formulae of SAHA and Depsipeptide
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.085

2306
C. Rossi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2305–2308
X
HN
RXCl
R
N
+
N
X
(CH2)n-
a
OH
OH
A
11
12
O
O
X
X
R
N
b
c
R
N
N
N
X
X
(CH2)n-CONHOH
H
N
Cl
OH
B
13
O
1-4 a
O
(CH2)n-CONHOH
R = Ph, PhCH2
X = CO; SO2
C
Scheme 1. Reagents: (a) BSA, DIPEA, THF; (b) (COCl)2, DMF, CH2Cl2; (c) NH2OH–
HCl, NaHCO3, CH2Cl2/H2O.
Figure 2. Scheme of the strategy followed for the design of new HDAC inhibitors.
The synthetic sequence followed for the synthesis of piperidines
with n = 1, 3, 4, and 5 is shown in Scheme 2.
Following this plan, compounds of general formulas shown in
Figure 3 were prepared. Representatives with n = 0–5 were not
synthesized for all the series, since for some chains the length
was clearly not appropriate from the very beginning, and one of
the two selected scaffolds demonstrated a clear superiority over
the other.
For the n = 0 compounds, only piperidine derivatives were pre-
pared, using the sequence shown in Scheme 1. Commercially avail-
able acid 11 was coupled with the desired acyl or sulfonyl chloride
by treatment with bis-trimethylsilylacetamide (BSA) in tetrahy-
drofuran (THF), in the presence of diisopropylethylamine (DIPEA),
to obtain, after workup, the free acids 12. These were converted
into the corresponding acyl chlorides using oxalyl chloride with a
catalytic amount of dimethylformamide (DMF); subsequent reac-
tion with hydroxylamine hydrochloride afforded the desired
hydroxamic acids 1a–4a.
Commercially available piperidines 4-alkylcarboxylic acids
were converted into the methyl esters 15 in acidic methanol, sub-
mitted to base catalyzed reaction with the opportune acyl chloride,
sulfonyl chloride or isocyanate (16) and then treated with hydrox-
ylamine in basic media for the preparation of the hydroxamates 1–
4b, d and 5, 6d, e, f.
Compounds 1c–4c were prepared according to the synthetic
pathway of Scheme 3.
Boc protected piperidinyl alcohol 17 was oxidized to the corre-
sponding aldehyde (18) and then submitted to a Wittig reaction
with (ethoxycarbonylmethylene)triphenylphosphorane (19), fol-
lowed by catalytic hydrogenation of the double bond, Boc depro-
tection afforded the free amine (20), which was reacted with
selected acyl and sulfonyl chlorides. Aminolysis of the ethyl ester
with hydroxylamine in basic media afforded the desired hydroxa-
mic acids 1c–4c.
O
O
S
O
N
H
N
O
n
HN
O
n
N
O
HN
O
n
b or c
a
OH
OH
N
H
N
H
OMe
OH
6
n
1
15
14
O
O
O
O
O
S
S
N
O
X
X
N
O
R
N
O
n
d
R
N
O
n
N
OH
OH
OH
N
H
n
N
H
OMe
NHOH
n
7
16
2
1-4 b,d; 5, 6 d,e,f
O
O
N
O
S
X = CO; SO2, NHCO
R = Ph, PhCH2
N
O
N
H
N
N
OH
OH
Scheme 2. Reagents: (a) SOCl2, MeOH; (b) RXCl, DIPEA, THF; (c) RNCO; (d) NH2OH–
HCl, KOH, MeOH.
n
N
H
n
3
8
O
O
O
N
O
BOC
N
N
O
BOC
N
b
N
a
OH
OH
N
H
O
N
H
OH
n
n
18
HN
9
4
17
19
H
O
e, f
c, d
BOC
1c-4c
N
N
O
HN
O
CO2Et
N
OH
CO2Et
N
H
N
H
20
n
n
5
10
Scheme 3. Reagents: (a) (COCl)2, DMSO, CH2Cl2; (b) Ph3P@CHCO2Et, THF; (c) H2,
Pd/C 10%, MeOH; (d) 4 N HCl in dioxane; (e) RXCl (R = Ph, CH2Ph; X = CO, SO2),
DIPEA, CH2Cl2; (f) NH2OH–HCl, NaOH, MeOH.
Figure 3. For n = 0: a; n = 1: b; n = 2: c; n = 3: d; n = 4: e; n = 5: f.

C. Rossi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2305–2308
2307
Table 1
BOC
a
c
Pharmacological activity of the HDAC inhibitors 1–10a
N
O
n
O
BOC
+
N
N
Br
Compound
% of inhibition
IC50
M)
IC50 HCT-116
M)
OEt
NH
OEt
n
(
l
(l
23
0.1
l
M
1
l
M
10
l
M
50 lM
22
21
n = 1-4
SAHA
1a
2a
3a
4a
1b
2b
3b
4b
7b
8b
10b
1c
2c
3c
4c
7c
8c
9c
55
—
—
—
—
—
—
—
—
—
—
—
8
9
11
2
—
—
81
—
—
—
—
—
—
—
—
—
—
—
33
33
30
24
—
86
1.0
29
0
33
21
41
7
39
16
3.0
6.0
—
—
—
—
1.0
14
—
0.079
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
33
50
16
65
22
68
41
61
51
10
19
—
—
—
—
38
44
—
X
HN
O
n
R
N
O
b
N
N
OEt
OEt
n
24
25
X
d
R
N
O
n
R = Ph, PhCH2
X = CO; SO2
N
NHOH
7-10b-e
Scheme 4. Reagents: (a) DIPEA or Et3N, THF; (b) 4 N HCl/dioxane; (c) RXCl, DIPEA,
CH2Cl2; (d) NH2OH–HCl, NaOH, MeOH.
—
3
—
—
—
—
3
10c
1d
2d
3d
4d
5d
6d
7d
8d
9d
10d
2e
3e
4e
5e
6e
7e
8e
9e
10e
5f
—
—
0
2
—
—
12
23
11
36
53
59
—
—
—
—
13
12
11
23
18
4
9
3
19
16
20
58
63
—
72
81
69
—
—
—
—
45
44
54
63
49
9
12
9
40
54
62
71
84
69
80
86
86
<10
30
13
28
—
77
87
77
85
—
0.84
0.54
1.39
0.26
0.10
0.09
—
—
—
—
—
—
—
—
—
—
—
—
—
3.2
2.7
17.2
5.1
0.7
0.3
—
—
—
—
—
—
—
—
—
—
—
—
—
For the piperazine derivatives 7–10 the synthesis is shown in
Scheme 4.
Boc-protected piperazine 21 was alkylated with the
x-bro-
moalkyl esters 22 to obtain the ethyl -piperazinecarboxylates
x
—
23. Boc deprotection in acidic media was followed by acylation
or sulfonylation (25), and finally ethanol aminolysis gave the de-
sired hydroxamic acids 1–4 b–d.
19
50
30
52
—
—
—
—
—
—
—
—
—
—
—
All the final compounds were characterized by 1H NMR and LC–
MS analyses, showing a purity >97%.5
—
—
—
Compounds were initially screened at two or three concentra-
tions, selected from 50, 10, 1.0, and 0.1 lM, using an enzymatic as-
say measuring total HDAC activity in HeLa cell extracts (Table 1).6,7
From the results obtained it was clear that the optimal linker
length was n = 3, and this was true for all the ‘external recognition
motives’ (1d–8d). Another observation was that the binding of the
compounds with the piperazine linker was very poor. This is prob-
ably due to its basic character, which requires a desolvation energy
gap to be overcome prior to entering the hydrophobic tunnel. From
the inhibitory activity data six molecules (1d–6d), all belonging to
the piperidine class with n = 3, were selected and the IC50 for HDAC
inhibition evaluated. Both the benzyl (1d) and phenyl (3d) sulfon-
amides had comparable submicromolar activity, while differences
were seen between the phenyl (3d) and the benzyl (4d) amides,
with the former being the least active of the four analyses (IC50
—
—
—
—
—
—
—
—
—
—
6f
a
For details see the References and notes section.
Acknowledgment
The authors wish to thank Sandra Bartoli for useful discussions
and manuscript revision.
1.39 lM). However, the most active compounds in the series were
the urea analogs, with the phenyl (5d) and the benzyl (6d) com-
pounds showing inhibition values comparable to that of SAHA.
To determine whether HDAC inhibition in vitro was paralleled
by similar effects in cultured cells, the antiproliferative activity of
the selected compounds was determined in HCT-116 colon carci-
noma cells.8,9 Again the urea derivatives (5d, 6d) showed the best
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.085.
values (IC50 0.7 and 0.3 lM, respectively), comparable with the val-
ues for SAHA obtained in parallel.
References and notes
1. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
2. Glauben, R.; Sonnenberg, E.; Zeitz, M.; Siegmund, B. Cancer Lett. 2009, 280, 154.
3. Kazantsev, A. G.; Thompson, L. M. Nat. Rev. Drug Disc. 2008, 7, 854.
4. Wells, T. N. C.; Alonso, P. L.; Gutteridge, W. E. Nat. Rev. Drug Disc. 2009, 8, 879.
5. NMR experiments were recorded on a Bruker 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
In conclusion, applying a minimalist and systematic approach
we were able to find a new series of HDAC inhibitors with a molec-
ular weight around 300 and activity comparable to that of SAHA.
This series is characterized by having a 4-propylpiperidine as the
linker, a hydroxamic acid as the zinc binding moiety and can toler-
ate a phenyl ring linked through a sulfonamide, an urea and an
amide with or without a methylene as an additional spacer. We be-
lieve that all the three classes of compounds (sulfonamide, urea,
and amide) deserve further exploration to improve inhibitory
activity and PK properties with the goal of producing an orally ac-
tive clinical candidate. These studies and the subsequent findings,
will be the subject of future communications.
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 and 50 V, source temperature 105 °C).
6. Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. Chem. Biol. 2003, 10, 61.

2308
C. Rossi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2305–2308
7. 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 substrate and developer combination.
8. Page, B.; Page, M.; Noel, C. Int. J. Oncol. 1993, 3, 473.
9. HTT-116 cells were plated in a 96-well tissue culture plates containing 200 lL of
complete medium. After 24 h HFAC inhibitors were added at different
concentrations, ranging from 10 M to 0.1 M in quadruplicate. After 5 days,
20 L of Alamar blue were added to each well and the plates were further
l
l
Enzymatic reactions (50
l
L) were run in 96 well plates. The release of the
l
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 experiments
were carried out in triplicate.
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 the results
were expressed as IC50 calculated using the Easy-fit software.