Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.11.072

A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, w

Full text of DOI:10.1016/j.bmcl.2013.11.072

Bioorganic & Medicinal Chemistry Letters 24 (2014) 61–64
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lactam based 7-amino suberoylamide hydroxamic acids as potent
HDAC inhibitors
Maurizio Taddei , Elena Cini , Luca Giannotti , Giuseppe Giannini , Gianfranco Battistuzzi b,
a,
⇑
a
a
b,
⇑
b
b
b,
Davide Vignola , Loredana Vesci , Walter Cabri
a
Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040 Pomezia, Roma, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7
of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data
compared with the corresponding linear products, without substituent in position 7. In general, this mod-
ification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH
group orientation did not strongly influence the inhibition, the contemporary modification of the sub-
eroylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that
showed IC50 values between 2 and 10 nM against all Class I HDAC isoforms, demonstrating it to be a large
Received 22 October 2013
Revised 25 November 2013
Accepted 29 November 2013
Available online 4 December 2013
Keywords:
HDAC
Anticancer
Pan-inhibitor
Hydroxamic acid
Heterocycles
spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC50 = 0.5 lM
against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far.
Ó 2013 Elsevier Ltd. All rights reserved.
Epigenetic regulation of gene expression is a mutable dynamic
process that can contribute to human diseases, driven by epige-
netic protein families that include the histone deacetylases
specific cancers.3b Despite such intense efforts, there is still a large
medical need for pan inhibitors since it has been demonstrated
that the various cancer diseases do not involve the same HDAC iso-
HDACs).1 HDACs are a group of enzymes found in several organ-
forms. The availability of a new generation of HDAC inhibitors,
5
(
isms as bacteria, fungi, plants, and animals. They belong to the
huge class of so-called ‘lysine-deacetylase’, a class of enzymes that
more powerful than those available today, and nontoxic such as
Trichostatin A, could represent an opportunity for a clinical use
as single agent, unlike what happens now where the HDAC inhib-
itors are mainly used in combination with other cytotoxic agents.
Looking to SAHA (suberoylanilide hydroxamic acid, the active prin-
ciple of Vorinostat) with medicinal chemists eyes, the molecule is
characterized by a very simple chemical structure, represented by
a pharmacophoric model that consists of three parts, a zinc-bind-
ing group—ZBG (hydroxamic acid), a linker (poly-methylene) and
a cap-group (phenyl ring). The introduction of substituents on
the methylene chain has been extensively studied leading to more
work removing acetyl groups from
e-amino-lysine residues on
many different substrates (histone, nonhistone nuclear and cyto-
2
plasmic proteins), the acetylation/deacetylation ratio working as
a regulatory signaling network in cells. Over the last years, great ef-
forts have been spent on the HDAC inhibitors in the oncology
3
field: so far two drugs (Vorinostat and Romidepsin) have emerged
for new promising anticancer treatments approved by the US Food
and Drug Administration (FDA). Vorinostat was the first-in-class
FDA approved HDAC-inhibitor to treat cutaneous T-cell lymphoma,
4
6
in patients who have received at least one prior systemic therapy.
powerful and/or more selective new derivatives. Over the years
It works as a pan inhibitor, active against the eleven HDAC human
isoforms. Recently many efforts have been dedicated to find power
selective HDAC inhibitors, although researchers are divided with
regard to the assessment of particular HDAC isoforms bound to
several authors, such as Breslow and coworkers, have reported a
series of 7-substituted ethers, amines and amide derivatives of
7
,8
SAHA with excellent inhibitory activities.
Other researchers,
through a molecular dynamic simulations of HDAC/inhibitor com-
plexes have demonstrated that interaction between the protein
9
surface and inhibitor play an important role. Efforts in the same
general direction were made, also in our laboratory, to probe
specifically the influence on enzyme binding, studying different
⇑
Corresponding authors. Tel.: +39 0577234275; fax: +39 0577234333 (M.T.);
tel.: +39 0691393640; fax: +39 0691393638 (G.G.).
E-mail addresses: maurizio.taddei@unisi.it (M. Taddei), giuseppe.giannini@
sigma-tau.it (G. Giannini).
10
11
7
-alkoxy derivatives alone or inserted in a macrocyclic fraction
with the goal to highlight the role of different substituents in this
position.
Present address: Indena, SpA, viale Ortles 12, I-20139 Milano, Italy.
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.072

6
2
M. Taddei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 61–64
We report here the results relative to the introduction of new
with (2S)-6-oxo-2-piperidinecarboxylic or (2S)-5-oxo-2-pyrroli-
amido lactams in position 7 of the SAHA skeleton. The insertion
of these ‘peptide resembling fragments’ gave a strong increase of
inhibition of different HDAC isoforms together with a general high
value of cytotoxicity against selected tumor cell lines (see
Scheme 1).
dine-carboxylic acids was instead followed by direct transforma-
tion of the
aqueous NH
x
-methylcarboxylate into hydroxamic acids with
2
OH to yield compounds 15–17 (Scheme 3).
As an alternative, commercially available (S)-2-tert-butoxycar-
bonylamino-hept-6-enoic acid 18, was coupled with the proper
aromatic amines to obtain compounds 19–22. (Scheme 4), Then
chain elongation was achieved by olefin metathesis with methyl
acrylate (Grubbs 2 Rh complex) followed by reduction of the
double bond (Scheme 4). The obtained methyl esters (23–26) were
hydrolyzed with NaOH in MeOH and the resulting carboxylic acids
were coupled with O-benzyl hydroxylamine using PyBOP, DIPEA.
Boc removal with TFA, gave the 7-amino suberoyl derivatives that
were reacted with the proper lactam–carboxylic acids. The final
benzyl removal by hydrogenolysis generated hydroxamic acids
27–30.
The molecules described here were prepared following two dif-
ferent synthetic approaches in order to optimize the introduction
of the hydroxamic acid function in the presence of polar lactam/
amide structures. The aldehyde 2, derived from (S)-glutamic acid
1
2
1,
was transformed into alkene 3 via Horner–Wadsworth–
Emmons reaction (Scheme 2). Treatment with H and Pd/C gave
2
contemporary double bond reduction and deprotection to yield
the free amino acid that was protected again at position 2 with
2
Boc O to produce compound 4. Coupling with aniline in the
1
3
presence of DMTMM followed by carboxymethyl hydrolysis
and coupling with O-benzyl hydroxylamine gave compound 5 that
was finally submitted to Boc removal to yield amido amino
hydroxamate 6. Alternatively intermediate 4 was coupled with
cyclopentylamine or phenethyl amine (using always DMTMM as
the coupling agent) followed by Boc removal to give amido amino
esters 7 and 8 (Scheme 2).
The amino lactam substituted suberoylamide hydroxamic acids
9–17 and 27–30 were evaluated for their activity against Class I
(1–3, 8), IIb (6, 10) and IV (11) HDAC isoforms. Class IIa HDACs
were avoided due to their lower sensitivity in this assay. HDAC
profiling was performed in the presence of a 50
lM solution of
the fluorogenic tetrapeptide RHKK(Ac) substrate (from p53
On scaffold 6, the introduction of different lactam carboxylic
acids was carried out using DMTMM as the coupling agent,
followed by hydrogenolysis of the benzyl protection to form the
anilide lactam hydroxamic acids 9–14. The coupling of 7 and 8
residues 379–382) or in the presence of a 50
diacetylated analogue RHK(Ac)K(Ac) for HDAC8.
lM solution of its
1
4
Upon its deacetylation, the fluorophore was released giving rise
to fluorescence emission which was detected by a fluorimeter, and
the IC50 values of the compounds were calculated from the result-
ing sigmoidal dose–response inhibition slopes.
A first exploration was done keeping intact the original SAHA
structure and introducing different lactam amides on the
stereogenic center in position 7 (S configuration). Six, five and four
R
H
N
O
H
N
X
O
OH
OH
N
H
7
N
H
O
O
SAHA
X = O, S, NH, NHCO
R = H, alkyl, aryl, arylalkyl
Ref. 7-8, 11
O
(
)
n
N H
O
H
H
N
N
O
O
H
N
O
O
OH
OH
7
N
H
7
N
H
O
O
Macrocyclic ether based SAHA derivatives 7-Lactam-carboxyamide-based SAHA derivatives
Ref. 12
This work
Scheme 1. SAHA like HDAC inhibitor carrying substituents at position 7.
Scheme 2. (a) Ref. 11. (b) (MeO)
Boc O, H (6 bar), Pd(OH) /C (10% mol), MeOH, rt, 12 h, 62%. (d) (i) PhNH
NMM, THF, rt, 12 h, 96%; (ii) NaOH aq 1 M, MeOH, rt, 12 h; (iii) BnONH
2
OPCH
2
COOMe, LiCl, DIPEA, CHCl
3
, rt, 12 h, 79%. (c)
, DMTMM,
, DMTMM,
2
2
2
2
2
NMM, THF, rt, 12 h, 75%. (e) TFA, DCM, rt, 1 h, 79%. (f) (i) Cyclohexyl amine or
phenethylamine, DMTMM, NMM, THF, rt, 12 h, 86% and 90%, respectively; (ii) TFA,
DCM, rt, 1 h, 80%.
Scheme 3. (a) (i) Lactam carboxylic acids DMTMM, NMM, THF, rt, 12 h, 76–82%; (ii)
H
2
(1 bar) Pd/C, MeOH, rt, 12 h, 52%. (b) (i) Lactam carboxylic acids DMTMM, NMM,
THF, rt, 12 h 72–89%; (ii) NH OH, NaOH, MeOH/H O, rt, 12 h, 52–77%.
2
2

M. Taddei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 61–64
63
Scheme 5. Reference linear compound. Products 31–35 were prepared following
the same synthetic procedure already described for SAHA (see Ref. 13).
stereocenters, this substituted variant was not further investi-
gated. Then, different selected amines were introduced at the
amide position of the Cap group in order to assess a possible
additive effect on the inhibition activity that was always compared
with the 7-unsubstituted SAHA like analogous compounds 31–35
that were prepared following
a reported general procedure
1
5
(Scheme 5).
Scheme 4. (a) ArNH
Grubbs Cat. 2nd generation, DCM, rt, 12 h; (ii) H
NaOH 1 M in MeOH, rt then BnONH EDC, Et N, DCM rt; (ii) TFA, DCM, 0 °C to rt; (iii)
2
, PyBOB, DIPEA, DMF/DCM, rt 12 h. (b) (i) Methyl acrylate
The molecule that included a m-CF aniline and a c-lactam
3
2
, (1 bar) Pd/C MeOH, rt. (c) (i)
showed high activity with one digit nanomolar inhibition on all
Class I HDACs tested, proving to be one of the most potent
molecule described so far. In fact, moving from SAHA to the linear
m-trifluoromethylanilide 32, the introduction of the lactam
improves the activity of 28 on Class I HDACs and HDAC6 to very
interesting IC50 values, with maximum of 2 nM. An analogous
trend was observed with the phenyl-ethylamide derivative, where
the introduction of the lactam resulted in an increase of activity
(16 and 17 vs the linear analogue 35). Finally, introduction in the
amide position of an aliphatic amine instead of an aromatic one,
gave less active compounds if compared with the previous tested
arylamides (31 and 15).
2
3
lactam carboxylic acids, PyBOP, DIPEA, DMF/DMF rt; (iv) H
2 2
(1 bar) Pd(OH) /C
EtOH/EtOAc rt.
membered lactams were included in the structure resulting in a
general ten times increase of the inhibition potency on HDACs.
The d and b-lactams 9 and 27 showed a promising activity in the
range 5–100 nM on Class I enzymes, while on Class IIb and IV they
resulted less potent (although more active than SAHA, see Table 1).
The
c lactams 10 and 11 also inhibited all HDAC isoforms much
better than SAHA although with more modest IC50 values with re-
spect to 9 and 27. The configuration of the stereocentre present in
the lactam was irrelevant for the activity as the diastereomers 7S/
These products were also studied for their effects on tumor cell
lines and again the introduction of an amido-lactam in position 7
of the SAHA structure resulted in more active molecules compared
with the unsubstituted analogues. The meta- and para-substituted
0
0
2
R (10) and 7S/2 S (11) showed a comparable inhibition on all the
investigated isoforms. The relative position of the C@O and NH
parts of the lactam has some influence on the activity as demon-
strated by the reduced inhibition recorded with compound 12
and 13 (compared with 9 and 10). In this inverted lactam series,
the introduction of a bulky substituent close to the lactam connec-
tion point gave a product (14) with the inhibition potency aligned
with the first more active variants. However, due to the more
elaborated structure, also complicated by the presence of two
anilides with the
c-lactam in position 7 were highly cytotoxic
under sub-micromolar concentrations with again a peak of activity
for compound 28 (also 29 and 30) that showed a IC50 = 0.5 lM
against the H460 cell lines.
In conclusion, SAHA variants containing (S)-7-amino carboxy-
lactams exhibited nanomolar IC50 values against different HDAC
Table 1
Different isoform inhibitory activities and in vitro cytotoxicity of lactam based 7-aminosuberoylamide hydroxamic acids
Entry
Product
HDAC (IC50 nM)
HDAC6
(IC50 lM)
HDAC1
HDAC2
HDAC3
HDAC8
HDAC10
HDAC11
H460
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
SAHA
9
258
25
43
59
131
72
33
34
1790
1450
63
2
197
7
200
32
1140
102
77
921
89
350
20
67
89
116
74
29
5
8
6
19
4
4
3
75
31
6
2
22
3
12
4
25
8
243
97
456
51
93
125
244
147
88
113
4890
6830
374
10.6
503
30.3
649
163
1350
227
210
362
47
75
3.4
2.5
4.0
5.4
5.1
4.0
2.4
2.2
21
2.4
2.5
0.5
0.8
0.5
4.1
0.6
18.2
8.2
4.9
10
11
12
13
14
27
31
15
32
28
33
29
34
30
35
16
17
142
143
689
238
110
89
4390
5040
165
8
239
27
589
93
5160
956
180
161
137
251
118
123
93
1410
2130
113
32.9
523
52.0
336
156
376
131
31.6
37
255
150
70
14
62
135
2630
1440
75.1
55.5
1600
15.5
78.6
127
1130
262
47.8
3330
5660
185
2
216
11
452
66
3610
539
144
1
1
1
1
1
1
1
1
1
1
6

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M. Taddei et al. / Bioorg. Med. Chem. Lett. 24 (2014) 61–64
isoforms, pointing out that the presence of a cyclic amide on the
solvent exposed capping group area has a particular influence on
the affinity with the enzyme. Combined with some suberoyla-
mide fragments, the lactam introduction yielded extremely
active compounds on Class I and Class IIb isoforms, corroborated
with a very promising antitumor activity on selected cancer cell
lines. Compound 28 (ST8078AA1) can be considered an interest-
ing lead compound as well, suitable for simple modifications in
order to develop new potent antitumor molecules as HDAC
inhibition.
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Acknowledgment
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
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