4-N-Hydroxy-4-[1-(sulfonyl)piperidin-4-yl]-butyramides as HDAC inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.042

A series of N-substituted 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of histone deacetylase (HDAC) inhibitors (zinc binding moiety-linker-capping group) has been previously reported by our group. Linker length and aromatic ca

Full text of DOI:10.1016/j.bmcl.2011.09.042

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6767–6769
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Bioorganic & Medicinal Chemistry Letters
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4-N-Hydroxy-4-[1-(sulfonyl)piperidin-4-yl]-butyramides as HDAC inhibitors
Cristina Rossi a, Christopher I. Fincham a, Piero D’Andrea a, Marina Porcelloni a, Alessandro Ettorre a,
Sandro Mauro a, Mario Bigioni a, Monica Binaschi a, Carlo A. Maggi b, Federica Nardelli 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:
Received 18 July 2011
Revised 12 September 2011
Accepted 13 September 2011
Available online 18 September 2011
A series of N-substituted 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of his-
tone deacetylase (HDAC) inhibitors (zinc binding moiety-linker-capping group) has been previously
reported by our group. 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.
We report here the second part of the strategy used in our research group to find a new class of HDAC
inhibitors, namely the SAR study for the compounds bearing a sulfonyl group on the piperidine nitrogen.
In the present work, we have considered both sulfonamides and sulfonyl ureas.
Keywords:
HDAC
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Lead optimization
There are two enzyme families that play a crucial role in con-
trolling the acetylation status of histones, histone acetyl transfer-
ases (HATs) and histone deacetylases (HDACs).1,2 Their activity
alters the chromatin structure and has consequences on the tran-
scription process. In particular HDACs remove an acetyl group from
specific located lysine residues. The inhibition of HDACs results in
an increase in the acetylation status of chromatin which has been
linked to many pharmacological activities and in particular to the
down regulation of genes involved in tumor progression and to
the re-expression of aberrantly silenced tumor suppressor genes,3
although which genes are activated or down-regulated by HDAC
inhibitors remain unclear.
and the hydroxamic acid, since in our previous work we demon-
strated, after a systematic study, this to be the optimal length.
The most common aromatic substituents were systematically
positioned in the ortho, meta, and para positions (for compounds
3–17, we were, in some cases limited, by the commercial availabil-
ity of the precursors). Examples of di-substituted (18–20) and
linker
A
surface recognition motif
Zn chelating moiety
We have recently reported how, starting from the basic HDAC
inhibitor structure (Fig. 1A) and following a minimalist approach,
we were able to identify
a new class of HDAC inhibitors
N
(CH2)n-CONHOH
characterized by a hydroxamic acid as the Zn binding moiety, a
4-propylpiperidine as the linker and variously connected aromatic
rings as the capping groups (Fig. 1B).4
Molecules 1 and 2, containing an aromatic ring (phenyl and
benzyl, respectively) linked to the piperidine nitrogen, were found
to be high nanomolar in vitro inhibitors of HDAC and also showed
antiproliferative activity on HCT-116 colon carcinoma cells. Thus,
we decided that this motif deserved further SAR examination.
We prepared the series of compounds shown in Figure 2. The
propyl chain was maintained as linker between the piperidine ring
B
O O
S
R
N
O
OH
N
H
1
R = Ph
2 R = CH2Ph
IC50 (HDAC)
IC50 (HCT-116)
µM
µM
1
0.54
0.84
2.7
3.2
2
⇑
Corresponding author.
E-mail address: dfattori@menarini-ricerche.it (D. Fattori).
Figure 1. General strategy that has produced the 4-N-hydroxy-4-[1-(sulfonyl)
piperidin-4-yl]-butyramides as HDACs inhibitors.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.042

6768
C. Rossi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6767–6769
O O
S
X
R =
Me
R
N
O
Me
O
OH
Me
X
X
N
H
MeO
OMe
3 X = Me
14 X = Me
10 X = Me
Me
19
4
15
X = OH
X = tBu
11
X = OMe
18
5 X = F
16 X = Cl
12 X = Cl
13 X = CF
MeO
MeO
6 X= Cl
17 X = CF3
3
Me
N
7
X = CF3
H
N
O
Me
O
8 X = Ipr
21
9
X = Ph
20
N
Me
O
29
33
32
26
O
24
S
30
N
N
N
H
N
O
27
34
23
22
Me
Me
N
Me
35
O
25
28
Me
31
O O
S
N
O
OH
N
H
36
Figure 2. General structure for sulfonylpiperazine HDAC inhibitors, substitutions at R and the structure of compound 36.
BOC
b
b, c
OEt
a
a
N
N
HN
O
HN
Ph
HN
O
OH
O
OH
O
CHO
OMe
43
O O
42
38
37
O O
O O
S
c
BOC
S
S
d
N
O
R
N
R
N
O
OH
OEt
OMe
N
H
39
3-31
44
45
O O
S
Scheme 1. (a) SOCl2, MeOH; (b) RSO2Cl, DIPEA, THF; (c) NH2OH–HCl, KOH, MeOH.
e, f
Ph
N
O
OH
N
H
O
O
36
b, c
S
a
N
N
O
38
Scheme 3. (a) (COCl)2, DMSO, CH2Cl2, À80 °C; (b) Ph3P = CHCO2Et, THF; (c) 4 N HCl
in dioxane; (d) PhSO2Cl; (e) LiOH 8 equiv, THF/MeOH/H2O 4:1:0.1; (f) EDAC, HOAt,
DMF and then NH2OHÁHCl DIPEA.
N
O
OMe
40
O O
O O
S
the reaction with the appropriate sulfonyl chloride, in the presence
of ethyldiisopropylamine (DIPEA) in tetrahydrofuran (THF). The es-
ter 39 was converted into the hydroxamic acid by aminolysis with
hydroxylamine in the presence of a base.5
The preparation of sulfonyl ureas is outlined in Scheme 2.
Amine 38 was treated with sulfonyldiimidazole to obtain the sta-
ble intermediate 40, which was activated by treatment with meth-
yltriflate and reacted with an amine, aniline, N-methyl aniline,
benzylamine or N-benzylamine to obtain the corresponding sulfo-
nyl ureas.6 The conversion of the esters 41 to the corresponding
hydroxamic acids was done according to Ref. 5.
Compound 36, was synthesized starting from alcohol 32
(Scheme 3). A Swern oxidation gave aldehyde 33, which was than
submitted to a Wittig reaction with (ethoxycarbonylmethylene)
triphenylphosphorane. Boc deprotection of the amine with 4 N
HCl in dioxane and reaction with phenylsulfonyl chloride gave
the desired compound. In this case it was not possible to perform
the direct aminolysis with hydroxylamine in basic media, because
this led to a degradation of the product. Instead, it was necessary to
carefully hydrolyze the ester and the form the hydroxamic acid
using a classical coupling method (Scheme 3).7
d
S
R'RN
N
R'RN
N
O
OH
OMe
N
H
41
32-35
R = Ph, Bn
R' = H, Me
Scheme 2. (a) Sulfonyldiimidazole; (b) methyl triflate; (c) RR’NH.
trisubstituted (21) analogs were also prepared. Compounds 22 and
23 are examples of meta and para hetero-biphenyls, while 24–31
represent saturated and unsaturated benzofused molecules. Com-
pounds 32–35 were prepared to examine the effect of phenyl
and benzyl sulfonylureas, (both methylated and not). Finally, we
prepared 36, where a double bond was inserted into the linker to
limit its flexibility.
The synthesis of the simple sulfonamides followed the sequence
shown in Scheme 1. The commercially available aminoacid 37 was
converted into the methyl ester by classical treatment with SOCl2/
methanol; the free piperidino nitrogen was then sulfonylated via

C. Rossi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6767–6769
6769
Table 1
To complete the evaluation of this series of compounds the anti-
proliferative activity of a selected panel of sulfonamide derivatives
was determined in HCT-116 colon carcinoma cells.10
With the exception of 28, the IC50s were all found to be in the
low micromolar range.
Pharmacological activity of SAHA and compounds 1–36
a
a
Cmpnd % Of inhibition
IC50 HDAC
GI50 HCT-116
M)
(
l
M)
(l
0.1
lM
1.0
lM
10 lM
SAHA
1
2
3
4
5
6
7
8
55
23
17
28
9
21
25
15
18
20
24
28
4
13
29
37
42
30
22
22
31
32
35
38
36
30
29
26
48
32
53
40
40
19
29
29
19
81
63
58
71
51
60
62
55
66
44
61
67
40
57
69
75
77
69
65
62
71
75
74
75
72
75
68
69
82
71
68
80
70
48
65
56
48
86
84
71
nt
nt
nt
nt
nt
84
80
83
83
nt
nt
nt
84
86
nt
86
83
84
nt
nt
85
nt
86
nt
84
86
nt
86
74
nt
nt
nt
nt
78
0.079
0.54
0.84
nt
0.6
2.7
3.2
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
2.8
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
Some in vitro PK parameters were evaluated for compounds 30
and 31. In protein binding experiments they were found to be only
2% and 3% free, respectively, while after incubation with the S9
fraction of rat, only 2% of 30 and 14% of 31 remained.
In conclusion, we have prepared a series of sulfonamide and
sulfonyl urea analogs starting from compounds 1 and 2 published
previously.(Ref. 4) A slight improvement was seen in the IC50 val-
ues for HDAC inhibition for compounds 16, 23, 28, 30, and 31,
although these did not always translate into an improvement in
the HCT-116 activity.
The work also brought to light problems with the solubility of
the compounds in the test medium, and the metabolic stability
of certain analogs in the presence of rat liver S9 fraction homoge-
nates. Further studies are planned, with a view to improving the
enzyme inhibition activity, and the physicochemical and PK prop-
erties of this class of compounds.
nt
nt
nt
nt
0.537
nt
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
nt
nt
nt
nt
nt
nt
0.173
nt
nt
nt
0.317
nt
Acknowledgments
nt
0.213
nt
The authors wish to thank Valerio Caciagli for assistance in the
synthesis of sulfonyl ureas, and Debora Tagliacozzi for the evalua-
tion of protein binding and in vitro metabolic stability.
nt
nt
nt
nt
0.124
nt
0.128
0.181
nt
17.3
nt
1.9
1.04
2.0
nt
References and notes
1. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
2. Manzo, F.; Tambaro, F. P.; Mai, A.; Altucci, L. Exp. Opin. Ther. Patents 2009, 19, 761.
3. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Discov. 2006, 5, 769.
4. Rossi, C.; Porcelloni, M.; D’Andrea, P.; Fincham, C. I.; Ettorre, A.; Mauro, S.;
Squarcia, A.; Bigioni, M.; Parlani, M.; Nardelli, F.; Binaschi, M.; Maggi, C. A.;
Fattori, D. Bioorg. Med. Chem. Lett. 2011, 2, 2305.
nt
nt
nt
nt
nt
nt
nt
5. Hauser, C. R.; Renfrow, W. B., Jr. Org. Synth. Coll. 1943, 2, 67.
6. Lee, H. K.; Bang, M.; Pak, C. S. Tetrahedron Lett. 2005, 46, 7139.
nt, not tested.
a
7. General procedure for the synthesis of hydroxamic acids from carboxylic acids.
A solution of carboxylic acid, EDAC (1.2 equiv) and HOAt (1.2 equiv) in DMF
(5 V) was stirred at room temperature for 30 min, then hydroxylamine
hydrochloride (1.2 equiv) and DIPEA (3 equiv) were added and stirring was
continued at room temperature for 12 h. Five percent aqueous KHSO4 was
added to have an acidic solution, followed by Et2O and the mixture was
transferred in a separatory funnel. The organic layer was washed with H2O,
brine and Na2SO4 then concentrated under reduced pressure. The crude
compound obtained in this way was purified by preparative HPLC to afford the
corresponding hydroxamic acid derivative.
Variability is maintained within 10.
All final compounds were characterized by 1H NMR and LC–MS
analyses, and showed a purity >97%.8 When necessary they were
purified either by flash chromatography on silica gel or by reverse
phase preparative HPLC.
Compounds were initially screened at two concentrations (1.0
8. 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
and 0.1 lM), using an enzymatic assay measuring total HDAC
activity in HeLa cell extracts (Table 1).9
On the basis of these results a group of compounds was selected
for the evaluation of their IC50s. However, for some of them the sol-
ubility in the test medium, at high concentrations, was found to be
so low (often in the order of a few micrograms/mL) that the result-
ing data were unreliable.
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: H2O + 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+ ionization (cone
voltages 20 and 50 V, source temperature 105 °C).
A first observation is that rigidifying the chain with a double
bond, (36 vs 1), gave no advantage in terms of inhibition; the same
was true for the para-substituted phenyl sulfonamides 3–9, which,
with the exception of 8, were all worse than the unsubstituted
phenyl compound 1. The best results, in terms of inhibition, were
obtained with the naphthyl derivatives 28, 30, and 31.
Benzyl and phenyl sulfonylureas, both simple (34 and 32) and
methylated (35 and 33) gave disappointing results: methylation
was very detrimental to inhibition, while the simple ones were al-
most equiactive with the corresponding sulfonamides, in spite of
the different geometry of this functional group. Very likely all the
modifications we introduced, when not detrimental, gave no addi-
tional productive binding interactions with the enzyme, and lim-
ited their influence essentially to the physicochemical properties
of the molecules.
9. 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. 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 experiments were carried out in triplicate.
10. HTT-116 cells were plated in a 96-well tissue culture plates containing 200
of complete medium. After 24 h HFAC inhibitors were added at different
concentrations, ranging from 10 to 0.1 M in quadruplicate. After 5 days, 20 lL
lL
l
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 wave length. All the results
were expressed as IC50 calculated using the Easy-fit software.