N-Hydroxy-(4-oxime)-cinnamide: A versatile scaffold for the synthesis of novel histone deacetilase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.02.029

With the aim to discover novel HDAC inhibitors with high potency and good safety profiles, we have designed a small library based on a N-hydroxy-(4-oxime)-cinnamide scaffold. We describe the synthesis of these novel compounds and some preliminary in vitro

Full text of DOI:10.1016/j.bmcl.2009.02.029

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2346–2349
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Hydroxy-(4-oxime)-cinnamide: A versatile scaffold for the synthesis
of novel histone deacetilase (HDAC) inhibitors
*
Giuseppe Giannini , Mauro Marzi, Riccardo Pezzi, Tiziana Brunetti, Gianfranco Battistuzzi,
Maria Di Marzo, Walter Cabri, Loredana Vesci, Claudio Pisano
R&D Sigma-Tau S.p.A., Via Pontina, km 30, 400 I-00040 Pomezia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to discover novel HDAC inhibitors with high potency and good safety profiles, we have
designed a small library based on a N-hydroxy-(4-oxime)-cinnamide scaffold. We describe the synthesis
of these novel compounds and some preliminary in vitro cytotoxic activity on three tumor cell lines, NB4,
H460 and HCT116, as well as their inhibitory activity against class I, II and IV HDAC. Several 4-oxime
derivatives demonstrated a promising inhibitory activity on HDAC6 and HDAC8 coupled to a good selec-
tivity profile.
Received 12 January 2009
Revised 3 February 2009
Accepted 4 February 2009
Available online 12 February 2009
Keywords:
HDAC inhibitors
Cinnamide
Ó 2009 Elsevier Ltd. All rights reserved.
Hydroxamic acids
Antitumor
Histone deacetylases (HDACs) are enzymes involved in the
remodeling of chromatin and play a key role in the epigenetic reg-
ulation of gene expression. HDACs catalyze the removal of acetyl
groups from the lysine residues of core histones and other proteins
that control cellular functions such as proliferation, migration, dif-
ferentiation and cell death. Therefore, inhibition of HDACs repre-
sents an actual therapeutic approach to discover new targeted
anticancer agents.1
The 18 HDACs identified in humans can be subdivided into four
classes based on their homology with the ones from yeast, their
subcellular localization and their enzymatic activities.
Class I, II and IV HDACs contain a zinc atom in their catalytic
site, whereas class III HDACs (sirtuins) have a unique enzymatic
mechanism of deacetylation dependent on the cofactor NAD+ and
are virtually unaffected by any of the HDAC inhibitors (HDACi) cur-
rently in development.2,3
HDACi entered in clinical studies can be divided into several
structural classes including hydroxamates, cyclic peptides, ali-
phatic acids and benzamides.4 ZolinzaÒ (SAHA, vorinostat), belong-
ing to the hydroxamic acid class, represents the first-in-class drug
as pan-HDACi approved by the FDA in October 2006.5,6
Actually, there is a great interest in identifying compounds with
improved HDAC isoform specificity7 and many studies are in pro-
gress to assess which HDAC isotype(s) should be targeted for anti-
cancer activity.8
A number of molecules containing a cinnamoyl hydroxamic
acid residue are or have been under clinical investigations as non-
selective inhibitors. Those include LAQ-824 (Phase II, retired)9 1,
LBH-589 (Phase II, Panobinostat)10 2, PDX-101 (Phase II, Belino-
stat)11 3, PCI-24781 (Phase I)12 4 and SB-939 (Phase I)13 5. Com-
pound 614 is an example of N-(2-aminophenyl)-acrylamide with
interesting antitumor activity in vivo (Fig. 1).
It is evident that many of these pharmacophores share a N-
hydroxycinnamide moiety as a common scaffold. The structure fits
with the standard modular construction of HDACi (Fig. 2) where
O
OH
O
OH
OH
H
N
N
H
N
H
N
Me
2 LBH-589 (Panobinostat)
N
H
N
H
1 LAQ-824
N
O
O
O
O
OH
OH
S
H
N
N
N
H
N
H
H
O
O
O
3 PXD-101 (Belinostat)
4 PCI-24781
O
O
OH
N
H
H
N
N
N
H
MeO
MeO
NH2
N
N
6
5 SB-939
OMe
* Corresponding author. Tel.: +39 0691393640; fax: +39 0691393638.
E-mail address: giuseppe.giannini@sigma-tau.it (G. Giannini).
Figure 1. Structure of molecules currently undergoing clinical trials.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.029

G. Giannini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2346–2349
2347
The 4-oxime-cinnamoyl hydroxamate derivatives (7–14) and 4-
oxime-cinnamic benzamide (15–16) (Scheme 1) were synthesized
through condensation of p-formyl cinnamic acid with various O-
substituted hydroxylamines. Intermediates 23 were converted to
hydroxamic acids (7-14) by coupling with O-protected hydroxyl-
amine via ethyl chloroformate and subsequent deprotection by
treatment with HCl in dioxane. Alternatively, intermediates 23
were directly converted to cinnamic benzamide (15–16) by addi-
tion of benzene-1,2-diamine in the presence of PyBOP coupling
reagent.
The synthesis of 17–18 were similar to the synthetic approach
used to achieve 4-oxime-cinnamoyl hydroxamate derivatives syn-
thesis. Compound 17 was obtained via a Heck reaction starting
from 4-bromo-benzaldehyde (24) and 2,3-diethoxypropene.17
After basic hydrolysis and condensation with 4-nitro-benzylhydr-
oxylamine hydrochloride, intermediate 26 was obtained. Coupling
of 26 with O-protected hydroxylamine using HATU and subse-
quent TFA-mediated deprotection yielded the desired compound
17 (Scheme 2).
Hydrophobic
spacer
Zn binding
Zn
CAP group
CU
group
Connection
Unit
Figure 2. Standard structure of HDACi.
the cinnamide moiety corresponds to the hydrophobic spacer and
the hydroxamic acid is used as the Zn binding group (ZBG).15
Keeping in mind the structural requirements for this commonly
accepted pharmacophore, we designed a small library of com-
pounds (7–21) bearing few structural modifications of the CAP
group, the connecting unit (CU) and the ZBG, starting from the ver-
satile scaffold N-hydroxy-(4-oxime)-cinnamide.16
4-Oxime-cinnamoyl hydroxamate derivatives 7–14 (Fig. 3)
were obtained through modification of the capping group starting
from various O-substituted hydroxylamine. Compounds 15–16
(Fig. 3) are cinnamic benzamide derivates, synthesized to evaluate
alternative ZBG’s.
With the aim to explore the influence of the double bond of the
cinnamic structure on biological activity, we have also synthesized
compounds 17–18 (Fig. 4) as analogues of 12.
We have also explored the influence of the position of the oxime
moiety on the aromatic ring (i.e., compound 19 (Fig. 5) vs com-
pound 14).
Compound 18 was obtained starting from 4-iodo-benzaldehyde
(28) under Sonogashira reaction conditions.18
Intermediate 29 was converted to compound 30 through con-
densation with 4-nitro-benzylhydroxylamine hydrocholoride.
Coupling with O-protected hydroxylamine and subsequent hydro-
lysis using Amberlyst 15(R) yielded the desired compound 18
(Scheme 3).
The 3-oxime analogue of compound 14 (compound 19) was
synthesized, starting from m-formyl cinnamic acid according to
the synthetic approach cited above (Scheme 1).
Oxazolidine 20 and oxazoline 21 groups were synthesized as
described precedently19,20 using allyl alcohol and propargyl alco-
hol, respectively (Scheme 4).
For all these compounds, we evaluated the anti-proliferative
activity in vitro using the human cell lines NB4 (promyelocytic leu-
kemia), NCI-H460 (non-small cell lung carcinoma) and HCT116
(colon carcinoma) (Table 1).
Compounds 11–14 and 21 were selected for further studies.
Compounds 11–13 were chosen for their significant anti-prolifera-
tive activity, compound 14 for its good solubility owing to the mor-
pholine structure and compound 21 to evaluate the effect of
replacement of the oxime moiety with an oxazoline group.
For these selected compounds, we evaluated the in vitro inhib-
itory activity against 11 HDACs isoforms.
Finally, the oxime moiety was incorporated into a ring to form
the oxazolidine 20 and oxazoline 21 adducts (Fig. 6).
7
8
9
R = H
R = allyl
R = phenyl
X = NHOH
X = NHOH
O
X = NHOH
10 R = benzyl
X = NHOH
X
11 R = pentafluorobenzyl
12 R = 4-nitrobenzyl
X = NHOH
H
X = NHOH
N
13 R = 4-methoxycarbonylbenzyl
14 R = 2-morpholinylethyl
15 R = 4-nitrobenzyl
X = NHOH
O
R
X = NHOH
X = N-(2-Amino-phenyl)
X = N-(2-Amino-phenyl)
16 R = 2-morpholinylethyl
Figure 3. Structure of new 4-oxime-cinnamoyl hydroxamate.
O
O
NHOH
NHOH
H
O
H
O
N
N
In Table 2 we show the inhibition profile using human HDAC
enzymes and a fluorogenic peptide, from p53 residues 379–382
O2N
O2N
(RHKKAc) at 50 l
M, as the substrate.21
17
18
In summary, a versatile scaffold for the synthesis of novel HDAC
inhibitors has been investigated.
Figure 4. Structure of two analogues of compound 12 obtained through modifi-
cation of its unsaturation grade.
O
N
N
O
O
O
O
O
O
O
NHOH
H
N
H
OH
OH
b
a
H
O
H
H
O
HCl
R
N
O
R
N
19
23
22
24
Figure 5. Structure of a meta-substituted cinnamoyl hydroxamate derivative.
c
d
O
O
O
O
NHOH
NHOH
NHOH
N
H
H
NH2
R
N
N
R
N
N
O
O
O
O
HCl
N
O
HCl
N
O
7-14
15-16
Scheme 1. Reagents and conditions: (a) RO–NH2ÁHCl (1.2 equiv), DMF, 18 h, 40–
50 °C; (b) (i) ethyl chloroformate (1.2 equiv), TEA (2 equiv), THFa; (ii) O-tetrahydro-
2H-pyran-2-yl-hydroxylamine (1.2 equiv), 4 h, rt; (c) 4 M HCl in dioxane, 3-18 h, rt;
(d) benzene-1,2-diamine (5 equiv), PyBOP (1.1 equiv), DIPEA (3 equiv), DMF, rt.
21
20
Figure 6. Structure of oxazolidine and oxazoline groups.

2348
G. Giannini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2346–2349
O
O
NHOH
OH
O
O
a
b
H
c
H
H
O
H
O
Br
N
N
O
OEt
O2N
O2N
24
17
25
26
Scheme 2. Reagents and conditions: (a) 3,3-diethoxy-propene (3 equiv), TBACl (1 equiv), n-Bu4 N (2 equiv), Pd(OAc)2 (0.03 equiv), DMF, 90 °C, overnight (84%); (b) (i) LiOH
sol. THF/H2O 1:1, 50 °C, 3 h (100%); (ii) 4-nitrobenzylhydroxylamine HCl (1.1 equiv), DMF, 50 °C; (c) (i) HATU (1.1 equiv), TEA (2 equiv), (Ph)3CONH2 (1.1 equiv), DMF, rt
(78%); (ii) TFA, DCM (95%).
O
OMe
O
O
I
OMe
NHOH
a
b
c
H
O
H
O
N
H
O
N
O
H
O2N
O2N
28
29
18
30
Scheme 3. Reagents and conditions: (a) methylpropiolate (4 equiv), K2CO3 (2 equiv), CuI (0.04 equiv), PdCl2(PPh3)2 (0.02 equiv), THF, 70 °C, 12 h (80%); (b) 4-
nitrobenzylhydroxylamine HCl, DMF, 50 °C, 18 h (96%); (c) (i) O-THP-hydroxylamine (1 equiv), sodium HMDS 1 M THF solution (1 equiv); THF, À78 °C, 2 h (35%); (ii)
Amberlyst 15(R), MeOH; 50 °C, 2 h (60%).
O
O
O
O
O
I
I
NHOH
O
N
N
N
d
e
HO
f
N
O
N
N
N
O
O
O
c
20
33
34
35
I
I
I
a
b
H
Cl
H
N
N
g
OH
31
O
OH
32
O
28
O
O
O
I
O
I
O
NHOH
N
N
l
h
d
N
HO
N
N
O
O
N
N
O
O
21
37
36
38
Scheme 4. Reagents and conditions: (a) NH2OHÁHCl (1 equiv), NaOH (2.5 equiv) sol. 50% w/w H2O/EtOH = 2/1, rt, 1 h; (b) NCS (1 equiv), DMF, 50 °C, 1 h; (c) allyl alcohol
(1.5 equiv), TEA (1.5 equiv), DCM, rt; (d) (i) MsCl (1.1), TEA (1.3), DCM, 1 h, 0 °C to rt; (ii) morpholine (3 equiv), THF, 60 °C, 4 h; (e) tert-butyl acrylate (4 equiv), TEA (3 equiv),
PdCl2(PPh3)2 (0.10 equiv), DMF, N2, 50 °C, overnight; (f) (i) 4 M HCl in dioxane, THF, 50 °C, 2d; (ii) ethyl chloroformate (1.2 equiv), TEA (10 equiv), NH2O–THP (1.2 equiv), THF,
0.3 h, 0 °C to rt; (iii) HCl sol. 4 M dioxane, overnight, rt; (g) propargyl alcohol (1.5 equiv), TEA (1.5 equiv), DCM, rt; (h) methyl acrylate (5 equiv), TBAOAc (2 equiv), K2CO3
(1.5 equiv), Pd(OAc)2 (0.03 equiv), DMF, Ar, 70 °C, 3 h (91%); (l) (i) MeOH, NaOH 1 M, 3 h, rt (100%); (ii) ethyl chloroformate (1.2 equiv), TEA (10 equiv), NH2O–THP (1.2 equiv),
THF, 0.3 h, 0 °C to rt; (iii) 4 M HCl in dioxane, overnight, rt.
A structure–activity-relationship (SAR) analysis was performed
allowing to gather more information about this scaffold in the de-
sign of selective HDAC6 and HDAC8 inhibitors.
Table 1
In vitro cytotoxic activity of novel N-hydroxy-(4-oxime)-cinnamide derivatives for
compounds 7–21
While selective HDAC8 inhibitors (e.g., CRA-A, PCI-34051, SB-
Compounds
Cell growth inhibitiona (IC50
NCI-H460
, l
Mb)
379278A) as well as selective HDAC6 inhibitors (e.g., tubacin, some
coumarin and thiolate derivatives) were already known,7 this
study has given a surprising result: the highlighted compounds
represent, to the best of our knowledge, a new class of inhibitors
active on HDAC6 as well as on HDAC8.
NB4
HCT116
7
8
9
2.9
1.4
2.7
0.62
0.7
0.76
0.82
1.7
>20
8.9
0.95
>20
1.9
6.3
3.2
5.1
1.6
1.6
2.6
1.7
2.9
>20
>20
7.7
>20
6.6
9.9
7.0
3.5
3.5
3.7
2.8
2.4
1.2
1.7
1.8
>20
>20
3.1
>20
4.0
3.0
4.0
10
11
12
13
14
15
16
17
18
19
20
21
Many other details emerged from our SAR analysis. The require-
ment of a significant steric hindrance for the CAP groups was not
confirmed (7 vs 8 and 9) Hydrophilic groups, such as the water-sol-
uble morpholine, confer an interesting biological profile (14). The
oxime can be enclosed in a cycle (oxazolidine or oxazoline) with
scarce influence on cytotoxic and HDACi activities (14 vs 20 and
21). In the cinnamic system, substitution in the para position is
preferred to the meta position (14 vs 19). Regarding the ZBG, data
confirm that the moiety including the hydroxamic acid and the
double bond is more potent than the benzamide moiety (12 and
14 vs 15 and 16, respectively). Furthermore, the double bond
shows to confer more potency than the single or triple bond (12
vs 17 and 18, respectively).
nd
nd
a
Growth inhibition was measured by SRB (sulphorodamine B) assay after 24 h of
treatment and 48 h of recovery.
b
Values are means of three experiments (nd = not determined).

G. Giannini et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2346–2349
2349
Table 2
Compounds 11, 12, 13, 14, 21 HDAC isoform selectivity profile
Compoundsa
IC50 (lM)
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HADAC9
HADC10
HDAC11
11
12
13
14
21
TSA
5.480
0.790
1.820
3.060
6.260
0.007
11.00
2.67
15.80
27.70
13.2
2.800
0.160
0.659
4.080
3.140
0.010
38.80
2.040
12.90
16.10
11.90
0.012
7.20
1.16
3.67
7.36
10.80
0.016
0.661
0.124
0.094
0.303
0.175
0.0004
20.00
2.30
36.30
33.70
22.30
0.022
1.290
0.070
0.603
0.235
0.127
0.090
12.00
2.470
6.430
13.10
11.20
0.038
7.360
1.580
5.430
7.840
6.100
0.020
5.48
0.790
10.80
3.060
6.260
0.015
0.023
a
Assay condition was started with a 50 lM solution, 10 doses with 1:3 dilution. Trichostatin A (TSA) was used as reference compound.
11. Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.; Romero, M. R.; Watkins,
C. J.; La Thangue, N. B.; Brown, R. Mol. Cancer Ther. 2003, 2, 721.
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Among the selected derivatives, compound 12 (ST2987) turned
out to be particularly active versus three isoforms: HDAC8, HDAC3
and HDAC6.
13. Chen, D.; Deng, W.; Lee, K. C. L.; Lye, P. L.; Sun, E. T.; Wang, H.; Yu, N.
WO2007030080 A1.
Supplementary data
14. Moradei, O.; Leit, S.; Zhou, N.; Fréchette, S.; Paquin, I.; Raeppel, S.; Gaudette, F.;
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.02.029.
15. Mai, A.; Massa, S.; Rotili, D.; Simeoni, S.; Ragno, R.; Botta, G.; Nebbioso, A.;
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