Influence of the adamantyl moiety on the activity of biphenylacrylohydroxamic acid-based HDAC inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.021

To investigate the influence of the adamantyl group on the biological properties of known HDAC inhibitors with a 4-phenylcinnamic skeleton, a series of compounds having the adamantyl moiety in the cap structure were synthesized and compared to the corresponding hydroxamic acids lacking this group. An unexpected finding was the substantial reduction of inhibitory activity toward the tested enzymes, in particular HDAC6, following the introduction of the adamantyl group. In spite of the reduced ability to function as HDAC inhibitors, the compounds containing the adamantyl moiety still retained a good efficacy as antiproliferative and proapoptotic agents. A selected compound (2c; ST3056) of this series exhibited an appreciable antitumor activity against the colon carcinoma xenograft HCT116.

Full text of DOI:10.1016/j.ejmech.2014.04.021

European Journal of Medicinal Chemistry 79 (2014) 251e259
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Influence of the adamantyl moiety on the activity of
biphenylacrylohydroxamic acid-based HDAC inhibitors
Raffaella Cincinelli a, Loana Musso a, Giuseppe Giannini b, Valentina Zuco c,
,
Michelandrea De Cesare c, Franco Zunino c, Sabrina Dallavalle a
*
a Department of Food, Environmental and Nutritional Sciences, Division of Chemistry and Molecular Biology, Università di Milano, Via Celoria 2,
20133 Milano, Italy
b R&D Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30,400, I-00040 Pomezia, RM, Italy
c Molecular Pharmacology Unit, Dept. Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Via Amadeo 42,
I-20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
To investigate the influence of the adamantyl group on the biological properties of known HDAC in-
hibitors with a 4-phenylcinnamic skeleton, a series of compounds having the adamantyl moiety in the
cap structure were synthesized and compared to the corresponding hydroxamic acids lacking this group.
An unexpected finding was the substantial reduction of inhibitory activity toward the tested enzymes, in
particular HDAC6, following the introduction of the adamantyl group. In spite of the reduced ability to
function as HDAC inhibitors, the compounds containing the adamantyl moiety still retained a good ef-
ficacy as antiproliferative and proapoptotic agents. A selected compound (2c; ST3056) of this series
exhibited an appreciable antitumor activity against the colon carcinoma xenograft HCT116.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 26 November 2013
Received in revised form
21 March 2014
Accepted 5 April 2014
Available online 8 April 2014
Keywords:
Adamantyl moiety
Hydroxamic acids
HDAC inhibitors
Antiproliferative activity
1. Introduction
modifying the cap portion of the HDAC pharmacophore, i.e. the
distal phenyl ring of 1.
In a recent study [1] we have reported novel structurally simple
histone deacetylase (HDAC) inhibitors with a hydroxamic acid as a
zinc chelating head group, a cinnamic linker domain and an aro-
matic ring as a cap structure (1, Chart 1). The compounds were
designed on the basis of a model of the HDAC2 binding site, based
on the homology model derived and validated by Wang et al. [2].
Our modeling studies indicated that the proximal phenyl ring of
Among various substituents a growing interest in adamantyl
derivatives is gaining prominence, since the incorporation of this
moiety into various molecules resulted in compounds with modi-
fied and/or improved biological availability [3].
The advantages of introducing this bulky group mainly reside in
an enhancement of lipophilicity and a protection from metabolic
cleavage of functional groups in its proximity (e.g. a phenolic OH),
thus enhancing the duration of action. On the other hand, the
adamantyl group being “biocompatible”, since metabolism can take
place in the liver, toxic effects by accumulation upon chronic
treatment are not expected [4]. Most adamantyl-based drugs are, in
fact, excreted largely unmodified, presenting therefore the added
benefit that potential side effects arising from bioactive metabolites
are intrinsically improbable [5].
prototype compound 1a (R ¼ H) exhibited a
pep stacking inter-
action with the benzyl side chain of Phe151, Phe206 and Tyr304 of
HDAC2. The distal phenyl ring of 1 (cap structure) appeared to
accommodate in a large cavity, without any significant steric clash.
In vitro assay with the isoenzyme HDAC2 validated the model,
confirming that a variety of substitutions in the distal ring were
well tolerated [1].
Therefore, in order to produce highly potent and/or receptor-
selective HDAC inhibitors, we mainly concentrated our efforts at
Seven adamantyl-derived drugs are available in the market:
Amantadine for use both as antiviral and antiparkinsonian drug [6];
Memantine, a NMDA-type glutamate receptors inhibitor [7], active
in Alzheimer’s disease; Rimantadine and Tromantadine as antiviral
drugs [8]; Vildagliptin and Saxagliptin, as oral anti-diabetic drugs
[9]; and Adapalene, a retinoid for topic use against acne [10]
(Chart 2).
* Corresponding author.
E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
http://dx.doi.org/10.1016/j.ejmech.2014.04.021
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
cinnamic spacer (2iel) (Scheme 3) were synthesized following
standard coupling procedures.
2.2. Biological assays
To understand the role of the adamantyl group as a deter-
minant of the antitumor activity, we performed a comparative
study of compounds containing or lacking this group (i.e. 1aed vs
2aed).
Chart 1. Structure of biphenyl-4-yl-acrylohydroxamic acids.
The compounds were tested for their inhibitory activity towards
HDAC2 isoform and for antiproliferative activities against a panel of
tumor cell lines from different tissue origin: H460 (human lung
carcinoma cell line), HCT116 (human colon cancer cell line), IGROV-
1 and its subline resistant to cis-platinum IGROV-1/Pt1 (human
ovarian carcinoma cell lines).
Derivatives 2aed showed inhibitory activities in the HDAC2
assay lower than the corresponding compounds lacking the ada-
mantyl group. However, compounds 2aed exhibited significant
antiproliferative profile against H460 cell line, 2bed showing
higher activity than the corresponding compounds 1bed. A com-
parable effect between the two series was found against HCT116
and IGROV-1 cell lines. The growth-inhibitory activities were
apparently p53-independent, because the cellular effects were
comparable in cells with functional (IGROV-1) or p53 defective
(IGROV-1/Pt1). SAHA, used as reference compound, exhibited
similar antiproliferative effects under the same treatment condi-
tions (72 h exposure) (Table 1).
A series of derivatives with different substituents on the
phenolic oxygen (2eeh) and with a naphthalene instead of a cin-
namic spacer (2iel) (Scheme 3) were also synthesized and tested
for antiproliferative activity (Table 2).
Interestingly, these compounds also exhibited a significant
antiproliferative profile on all the cell lines, in spite of a low
inhibitory activity in the HDAC2 assay (IC50 > 5 mM).
Chart 2. Adamantyl-derived drugs in clinical practice.
In order to unveil whether compounds 2 were active on other
HDAC isoforms, a comparative study on the different HDAC iso-
forms was also carried out (Table 3).
Compounds lacking the adamantyl group, e.g. 1b and 1c,
exhibited preferential inhibition of HDAC6 (IC50 ca. 40 nM), an ef-
fect consistent with tubulin acetylation. The introduction of the
adamantyl moiety reduced the inhibitory activity against all the
isoforms, including HDAC6 and HDAC8.
These findings confirmed the detrimental effect of the
adamantly group on the HDAC inhibitory activity for this class of
compounds.
In light of the promising results obtained from the anti-
proliferative activity evaluation, further studies on selected com-
pounds were carried out.
The ability of compounds 1c,d vs 2c,d to induce apoptosis was
investigated in IGROV-1 cells following 72 h exposure to equitoxic
concentrations, i.e. drug concentrations (deduced from dosee
response curve) which produced 80% cell growth inhibition (IC80).
As shown in Fig. 1, all compounds were able to induce high levels of
apoptosis (i.e. superior to that obtained with SAHA). Compounds
lacking the adamantyl group (i.e. 1c and 1d) were more effective as
apoptosis inducers than the analogues containing the adamantly
moiety. The former compounds induced apoptotic cell death in 90%
of treated cells.
As shown in Fig. 2, compound 1c was found to be a potent
inducer of acetylation of tubulin, an effect consistent with a pref-
erential inhibition of HDAC6 isoform [15], even if to a lesser extent
with expect of SAHA. A specific feature of these compounds was
their ability to induce an early acetylation of p53. Conversely,
compounds 2c and 2d exhibited a complete loss of ability to induce
acetylation of tubulin and histone H4 at equitoxic doses, IC80
After the discovery of amantadine as an antiviral drug [11], the
exploitation of the properties of the bulky, apolar adamantyl group
has been pioneered in a series of papers from Gerzon et al. [12],
followed by a large number of studies on compounds for diverse
medicinal applications [3,4].
Interestingly, in a recent paper from Gopalan et al. [13],
adamantyl-based HDAC inhibitors were found to exhibit cytotox-
icity in the nanomolar range.
On the basis of these considerations, we selected some of the
most active compounds among our previously synthesized HDAC
inhibitors [1] (compounds 1aed), and prepared the corresponding
analogues 2aed, having the adamantyl moiety in the cap structure
(Scheme 1).
2. Results and discussion
2.1. Chemistry
Compounds 1aed were synthesized according to the procedures
described previously [1]. Compounds 2aed were prepared by
reacting the corresponding carboxylic acids [14] with hydroxylamine
using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride (WSC) and 1-hydroxybenzotriazole hydrate (HOBt) as
coupling agents (2aec) or from the corresponding esters via reaction
with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (THPONH2), fol-
lowed by hydrolysis with p-toluenesulfonic acid monohydrate
(PTSA) (2d) (Scheme 2).
Successively, a series of derivatives with different substituents
on the phenolic oxygen (2eeh) and with a naphthalene instead of a

R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
253
Scheme 1. Structures of biphenylacrylohydroxamic acids lacking (1) or containing (2) the adamantyl moiety in the cap structure.
Scheme 2. Synthesis of compounds 2aed. Reagents and conditions: a) TfO2, Py, 3 h, rt, 87%; b) Et3SiH, dppp, Pd(OAc)2, DMF, 24 h, 60 ꢀC, 42%; c) tri (o-tolyl)phosphine, Pd(OAc)2,
TEA, methyl acrylate, 3 h, reflux, 67%; d) LiOH.H2O, THF/H2O, rt, overnight, 63%; e) WSC, HOBt, DME, rt, 1.5e24 h, then NH2OH.HCl, 1e4 h, Et3N, 40e75%; f) Pd tetrakis, 4-
formylbenzeneboronic acid, 2 M Na2CO3, toluene, 2 h, reflux, 72%; g) Ph3PCH]COOMe, chloroform, 3 h, reflux, 86%; h) NaOH, methanol, 7 h, reflux, 70%; i) Bis(pinacolato)
diboron, PdCl2(dppf), KOAc, dioxane, 100 ꢀC, 13 h, then 8, 2 M Na2CO3, PdCl2(dppf), reflux, 4 h, 86%; j) PTSA, MeOH, rt, 4 h, 44%.
(Fig. 2). This observation is consistent with the reduced potency of
compounds 2 as HDAC inhibitors.
xenografts [1]. Therefore, we evaluated the efficacy of 2c against
the human colon carcinoma model HCT116 under similar treatment
conditions. A daily oral administration (5 day/week) for five weeks
produced a significant tumor growth inhibition (60e70%) at the
end of treatment without manifestation of toxicity. The antitumor
activity of 2c was comparable to that of the analogue lacking the
adamantly group (1c) (Fig. 5). The good tolerability profile allowed
prolonged treatment in order to achieve optimal antitumor
response.
Since compound 2c is structurally related to the atypical reti-
noid containing the adamantly group 7 [16], which is known to
induce DNA damage as detected by the cellular response (i.e. acti-
vation of p53 and induction of phosphorylation of H2AX histone),
we examined the markers of DNA damage response in IGROV-
1 cells (Fig. 3).
In contrast to the atypical retinoid, which induces an early
phosphorylation of g-H2Ax, RPA-2 cleavage and p53 phosphory-
lation (at Ser15) [16], 2c did not exhibit the same profile, phos-
phorylation of p53 being detected only.
3. Conclusions
In spite of the different pattern on protein acetylation, all
compounds exhibited a comparable cell-cycle perturbation with
evidence of a marked sub-G1 peak and cell accumulation in G1
(Fig. 4).
On the basis of the above results, the therapeutic potential of a
representative derivative as an anticancer agent was analyzed.
In a previous study we have reported the good antitumor ac-
tivity of 1c following oral administration in a panel of human tumor
The present study was designed to investigate the influence of
the adamantyl group on the biological properties of known HDAC
inhibitors with a 4-phenylcinnamic skeleton [1].
Indeed, hydrophobic bulky groups in the cap region of HDAC
inhibitors have been reported to confer a lipophilic nature and
some isoform selectivity to the parent compounds. An unexpected
finding of our study was the substantial reduction of inhibitory
activity toward the tested enzymes, in particular HDAC6, following

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R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
COOH
a
CONHOH
R2
R2
R
R
R1
R1
R, R = OCH CH O R = H
,
2e
2f
R, R1 = -OCH2CH2O, R2= H
R = OH, R1= H, R2 = Cl
10a
10b
1
2
2
2
R = OH, R = H, R2 = Cl
1
COOCH3
CONHOH
b
O
CH CH N
O
CH2CH2N
CH2CN
11a R =
11b R =
2g R =
2h R =
2
2
RO
HO
R
CH CN
2
CONH
O
Br
CONH O
Br
13
14
2i
12
HO
c
d
CONHOH
CONHOH
HO
COOH
e
2l
MeO
15
MeO
Scheme 3. Synthesis of compounds 2eel. Reagents and conditions: a) 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU),
DIPEA, NH2OH$HCl, DMF, rt, 4 h, 65% for 2e; 1.5 h, 26% for 2f; b) for 2g: THPONH2, LiHMDS, THF, ꢁ78 ꢀC, 2 h, 100%, then PTSA, MeOH, rt, 24 h, 63%; for 2h: LiOH$H2O, THF/H2O, rt,
overnight, 93%, then WSC, HOBt, DMF, NH2OH$HCl, TEA, rt, overnight, 33%; c) Bis(pinacolato)diboron, PdCl2(dppf), KOAc, reflux, 2 h, then 6-bromonaphthalene-2-carboxylic acid
benzyloxyamide (13), 2 M Na2CO3, 2 h, 28%; d) H2/Pd/C, MeOH, 36 h, rt, 70%; e) HATU, DIPEA, NH2OH$HCl, DMF, rt, overnight, 30%.
carcinoma xenograft HCT116. Compound 2c was found to induce a
dose-dependent phosphorylation of histone H2AX but this effect,
observed after 24 h exposure, could reflect the onset of apoptosis. It
should be emphasized that zinc-dependent proteins other than
HDACs may be targeted by hydroxamic-acid-based compounds, but
the biological relevance of off-target effects remains to be explored.
Notwithstanding the fact that the molecular mechanism of ac-
tion of the reported compounds remains to be defined, it has been
confirmed that the presence of the adamantyl moiety sustains the
antiproliferative effect found for the unsubstituted series of
Table 1
Inhibition of HDAC2 (IC50, mM) and antiproliferative activity (IC50, mM) of compounds
1aed and 2aed and of reference HDAC inhibitor (SAHA) against a panel of tumor cell
lines.
Cpd.
HDAC-2 assay H460
HCT-116
IGROV-1
IGROV-1/Pt1
SAHA 0.1 ꢂ 0.02
3.4 ꢂ 0.8
5.4 ꢂ 0.1
>10
0.31 ꢂ 0.02
1.58 ꢂ 0.3
10.3 ꢂ 1.2
0.33 ꢂ 0.05
2.2 ꢂ 0.3
3.5 ꢂ 0.6
1.23 ꢂ 0.3
7.6 ꢂ 3.5
1.1 ꢂ 0.6
2.2 ꢂ 0.2
4.3 ꢂ 0.7
5.7 ꢂ 0.9
6.5 ꢂ 2.0
1.3 ꢂ 0.6
2.3 ꢂ 0.3
1.8 ꢂ 0.07
1a
2a
1b
2b
1c
2c
1d
2d
0.82 ꢂ 0.14
>10
0.59 ꢂ 0.2
2.64 ꢂ 0.5
1.16 ꢂ 0.27
2.3 ꢂ 0.6
>5
6.0 ꢂ 0.9
0.83 ꢂ 0.28 0.58 ꢂ 0.01
3.7 ꢂ 0.1
1.42 ꢂ 0.23
>20
0.22 ꢂ 0.01 0.75 ꢂ 0.4
1.3 ꢂ 0.1
2.4 ꢂ 0.1
1.8 ꢂ 0.4
0.96 ꢂ 0.35
0.57 ꢂ 0.09 2.01 ꢂ 0.91
1.3 ꢂ 0.1 2.03 ꢂ 0.4
>10
1.4 ꢂ 0.02
Table 2
Antiproliferative activity (IC50, mM) of compounds 2eel and of reference HDAC in-
hibitor (SAHA) against a panel of various tumor cell lines.
the introduction of the adamantyl group. This was consistent with a
marginal (if any) ability of the modified compounds to induce
acetylation of tubulin (Fig. 2). In spite of the reduced ability to
function as HDAC inhibitors, the compounds containing the ada-
mantyl moiety still retain a good efficacy as antiproliferative and
proapoptotic agents.
Cpd.
H460
HCT-116
IGROV-1
IGROV-1/Pt1
SAHA
2e
2f
2g
2h
2i
3.4 ꢂ 0.8
1.0 ꢂ 0.04
5.4 ꢂ 0.3
2.4 ꢂ 0.2
2.7 ꢂ 0.02
1.0 ꢂ 0.06
1.3 ꢂ 0.1
0.31 ꢂ 0.02
2.55 ꢂ 0.03
5.4 ꢂ 0.9
2.5 ꢂ 0.3
2.5 ꢂ 0.01
0.7
2.2 ꢂ 0.3
1.2 ꢂ 0.04
1.34 ꢂ 0.04
0.38 ꢂ 0.03
0.9 ꢂ 0.2
2.2 ꢂ 0.2
0.8 ꢂ 0.2
1.6 ꢂ 0.16
1.36 ꢂ 0.16
1.6 ꢂ 0.1
1.1 ꢂ 0.3
4.16
0.65 ꢂ 0.11
1.5 ꢂ 0.5
Indeed, a selected compound of this series (2c; ST3056)
exhibited an appreciable antitumor activity against colon
2l
1.4 ꢂ 0.1

R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
255
Table 3
Inhibition of HDAC isoforms by selected compounds.a
HDAC-1
HDAC-3
HDAC-4
HDAC-5
HDAC-6
HDAC-7
HDAC-8
HDAC-9
HDAC-10
HDAC-11
IC50 (M)
Ab
7.12 ꢃ 10ꢁ9
2.58 ꢃ 10ꢁ7
5.51 ꢃ 10ꢁ6
>10ꢁ4
1.03 ꢃ 10ꢁ8
3.50 ꢃ 10ꢁ7
4.57 ꢃ 10ꢁ6
5.56 ꢃ 10ꢁ5
8.45 ꢃ 10ꢁ5
e
1.21 ꢃ 10ꢁ8
4.93 ꢃ 10ꢁ7
1.71 ꢃ 10ꢁ5
6.14 ꢃ 10ꢁ5
9.94 ꢃ 10ꢁ5
e
1.65 ꢃ 10ꢁ8
3.78 ꢃ 10ꢁ7
1.12 ꢃ 10ꢁ5
7.84 ꢃ 10ꢁ5
2.96 ꢃ 10ꢁ5
e
4.19 ꢃ 10ꢁ10
2.86 ꢃ 10ꢁ8
4.18 ꢃ 10ꢁ8
2.80 ꢃ 10ꢁ6
3.68 ꢃ 10ꢁ8
e
3.26 ꢃ 10ꢁ6
1.67 ꢃ 10ꢁ5
4.08 ꢃ 10ꢁ7
2.25 ꢃ 10ꢁ8
3.44 ꢃ 10ꢁ8
1.56 ꢃ 10ꢁ5
7.51 ꢃ 10ꢁ5
2.13 ꢃ 10ꢁ4
e
8.95 ꢃ 10ꢁ8
2.43 ꢃ 10ꢁ7
1.62 ꢃ 10ꢁ7
6.96 ꢃ 10ꢁ6
6.98 ꢃ 10ꢁ8
e
1.56 ꢃ 10ꢁ6
3.06 ꢃ 10ꢁ5
2.91 ꢃ 10ꢁ6
3.81 ꢃ 10ꢁ8
3.16 ꢃ 10ꢁ7
8.92 ꢃ 10ꢁ6
4.14 ꢃ 10ꢁ5
3.93 ꢃ 10ꢁ5
e
2.01 ꢃ 10ꢁ8
4.56 ꢃ 10ꢁ7
1.10 ꢃ 10ꢁ5
>10ꢁ4
1.52 ꢃ 10ꢁ8
3.62 ꢃ 10ꢁ7
5.01 ꢃ 10ꢁ6
5.72 ꢃ 10ꢁ5
7.30 ꢃ 10ꢁ5
7.70 ꢃ 10ꢁ5
>10ꢁ4
Bc
1b
2b
1c
8.55 ꢃ 10ꢁ6
9.10 ꢃ 10ꢁ5
2cd
1d
e
e
>10ꢁ4
>10ꢁ4
>10ꢁ4
>10ꢁ4
>10ꢁ4
>10ꢁ4
>10ꢁ4
2d
2h
6.01 ꢃ 10ꢁ5
>10ꢁ4
>10ꢁ4
>10ꢁ4
7.72 ꢃ 10ꢁ5
>10ꢁ4
>10ꢁ4
3.81 ꢃ 10ꢁ5
4.85 ꢃ 10ꢁ5
3.40 ꢃ 10ꢁ4
4.01 ꢃ 10ꢁ5
1.24 ꢃ 10ꢁ4
4.92 ꢃ 10ꢁ5
4.23 ꢃ 10ꢁ5
6.39 ꢃ 10ꢁ5
6.34 ꢃ 10ꢁ5
a
Assay condition for compounds is to start with 50
Trichostatin A (TSA): Assay condition is to start with 5
SAHA.
m
M, 1:3 dilution, 10 doses.
M, 1:3 dilution, 10 doses.
b
c
m
d
Compound 2c has a fluorescent background at 50 mM.
Fig. 1. Apoptosis induced in ovarian carcinoma cells IGROV-1 by selected compounds at equitoxic concentrations (concentrations causing comparable cell growth inhibition, IC80
after 72 h exposure. SAHA is shown as a reference HDAC inhibitor. Apoptosis was determined after 72 h exposure by TUNEL assay and flow cytometry analysis.
)
4. Experimental section
4.1. Chemistry
All reagents and solvents were of reagent grade or were purified
by standard methods before use. Melting points were determined
in open capillaries on a Büchi melting point apparatus and are
uncorrected. NMR spectra were recorded at 300 MHz with a Bruker
instrument. Chemical shifts (d values) and coupling constants (J
values) are given in ppm and Hz, respectively. Solvents were
routinely distilled prior to use; anhydrous tetrahydrofuran (THF)
and diethyl ether (Et2O) were obtained by distillation from sodium-
benzophenone ketyl; dry methylene chloride was obtained by
distillation from phosphorus pentoxide. All reactions requiring
anhydrous conditions were performed under a positive nitrogen
flow, and all glassware were oven dried and/or flame dried. Isola-
tion and purification of the compounds were performed by flash
column chromatography on silica gel 60 (230e400 mesh) or RP-18
silica gel. Analytical thin-layer chromatography (TLC) was con-
ducted on Fluka TLC plates (silica gel 60 F254, aluminum foil).
Fig. 2. Effects of selected compounds on acetylation of tubulin, histone H4 and p53 in
IGROV-1 cells, where each compound was treated at IC80 dosage, for the indicated
exposure times.
hydroxamic acids. While a better understanding of the mechanism
of action will provide a basis for rational design of antitumor
agents, structure-activity information inferred from our study
could be the starting point for their optimization.
Fig. 3. Effect of compound 2c on the modulation of proteins implicated in cellular response to DNA damage. Compound 7 is an atypical retinoid containing the adamantly group.

256
R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
Fig. 4. Time course of cell cycle perturbation in ovarian carcinoma cells, IGROV-1, following exposure to equitoxic concentrations (IC80) of each compound. The cell cycle was
analyzed by FACScanÔ analysis of PI-stained cells.
4.2.2. 3-(30-Adamantan-1-yl-40-hydroxybiphenyl-4-yl)-N-
hydroxyacrylamide (2b)
Stirred for 3 h, then 4 h after the addition of hydroxylamine. The
crude precipitate was purified by flash chromatography with
CH3OH/H2O 75:25 v/v on RP-18 silica gel. Yield: 54%. M.p. 184 ꢀC.
1H NMR (DMSO-d6)
d: 10.70 (1H, brs); 9.50 (1H, s); 9.01 (1H,
brs); 7.65e7.50 (4H, m); 7.45 (1H, d, J ¼ 16.2 Hz); 7.35e7.25 (2H, m);
6.82 (1H, d, J ¼ 8.8 Hz); 6.45 (1H, d, J ¼ 16.2 Hz); 2.12 (6H, s); 2.04
(3H, s); 1.72 (6H, s). 13C NMR (DMSO-d6)
d: 162.9, 156.4, 141.9, 138.1,
136.0, 132.7, 129.9, 128.1, 126.4, 124.9, 124.7, 118.3, 117.0, 38.7, 36.7,
36.3, 28.4. Anal. Calcd for C25H27NO3: C, 77.09; H, 6.99; N, 3.60.
Found: C, 77.20; H, 6.93; N, 3.62.
4.2.3. 3-(30-Adamantan-1-yl-40-methoxybiphenyl-4-yl)-N-
hydroxyacrylamide (2c)
Stirred for 24 h, then 4 h after the addition of hydroxylamine.
The solid obtained was filtered and washed with acetone. Yield:
75%. 1H NMR (DMSO-d6)
d
: 10.72 (1 h, brs); 9.03 (1H, brs); 7.70e
7.39 (7H, m); 7.07 (1H, d, J ¼ 8.6 Hz); 6.47 (1H, d, J ¼ 16.0 Hz); 3.84
(3H, s); 2.10 (6H, s); 2.05 (3H, s); 1.74 (6H, s). 13C NMR (DMSO-d6)
d
:
Fig. 5. Antitumor effect of compound 2c against the human colon carcinoma HCT116.
Animals were treated by oral route with 50 mg/kg (Once-daily administration for 4e5
days a week for 5 weeks, for a total of 24 doses).
162.8, 158.5, 141.5, 137.9, 133.1, 131.4, 128.1, 126.6, 125.2, 124.6, 118.6,
112.7, 55.4, 36.6, 28.4. Anal. Calcd for C26H29NO3: C, 77.39; H, 7.24;
N, 3.47. Found: C, 77.51; H, 7.27; N, 3.41.
4.3. 5-(30-Adamantan-1-yl-40-methoxybiphenyl-4-yl)penta-2,4-
dienoic acid (tetrahydropyran-2-yloxy)amide (9)
Compounds 4 [16], 5 [14], 7 [16], 10a [16], 10b [14], 11aeb [17],
and 15 [18] were obtained following the procedures reported in the
literature.
To a solution of 6 (68 mg, 0.21 mmol) in dioxane (1.5 mL) bis(pi-
nacolato)diboron (59 mg, 0.23 mmol), KOAc (62 mg, 0.63 mmol) and
PdCl2(dppf) (5 mg, 0.006 mmol) were added. The mixture was
refluxed for 1.45 h under nitrogen. The solution was cooled at room
temperature, then compound 8 (144 mg, 0.41 mmol), 2 M Na2CO3
4.2. General procedure for the synthesis of compounds 2aec
(270 mL, 0.53 mmol) and PdCl2(dppf) (5 mg, 0.006 mmol) were added.
To a suspension of the appropriate acid (0.53 mmol) in dry DMF
(8.3 mL) HOBt (86 mg, 0.64 mmol) and WSC (132 mg, 0.69 mmol)
were added. The mixture was stirred at room temperature under
nitrogen for 1.5e24 h, then NH2OH$HCl (185 mg, 2.66 mmol) and
The resulting mixture was refluxed for 4 h. After addition of ethyl
acetate, the organic phase was washed with water, brine, dried over
Na2SO4 and filtered. The solvent was removed under reduced pressure
to give a crude which was purified by flash chromatography (hexane/
ethyl acetate 60:40) to obtain 91 mg of 9. Yield: 86%. M.p.190e192 ꢀC.
TEA (368 mL, 2.66 mmol) were added. The reaction was stirred at
room temperature for 1e4 h. After addition of water, a precipitate
formed, which was filtered and dried to obtain the desired
hydroxamic acid.
1H NMR (DMSO-d6)
d: 11.20 (1H, brs); 7.69e7.60 (4H, m); 7.55
(1H, m); 7.43 (1H, d, J ¼ 1.9 Hz); 7.30 (1H, m); 7.12e7.00 (3H, m); 6.05
(1H, d, J ¼ 16.1 Hz); 4.90 (1H, m); 3.92 (3H, s); 3.55 (2H, m); 2.12 (6H,
s); 2.05 (3H, s); 1.75 (6H, s); 1.72 (2H, m); 1.58 (4H, m). Anal. Calcd for
4.2.1. 3-(30-Adamantan-1-yl-biphenyl-4-yl)-N-hydroxyacrylamide
(2a)
C33H39NO4: C, 77.16; H, 7.65; N, 2.73. Found: C, 77.01; H, 7.69; N, 2.78.
Stirred for 1.5 h, then 1 h after the addition of hydroxylamine.
4.4. 5-(30-Adamantan-1-yl-40-methoxybiphenyl-4-yl)penta-2,4-
dienoic acid hydroxyamide (2d)
Yield: 33%. M.p. 132e134 ꢀC. 1H NMR (DMSO-d6)
d: 9.10 (1H, brs);
7.75e7.35 (9H, m); 6.51 (1H, d, J ¼ 16.1 Hz); 2.10 (3H, s); 1.95 (6H, s);
1.74 (6H, s). Anal. Calcd for C25H27NO2: C, 80.40; H, 7.29; N, 3.75.
Found: C, 80.62; H, 7.32; N, 3.80.
To a solution of compound 9 (50 mg, 0.1 mmol) in methanol
(1.5 mL) p-toluenesulfonic acid monohydrate (PTSA) (6 mg,

R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
257
0.03 mmol) was added and the mixture was stirred at room tem-
4.8. 3-(30-Adamantan-1-yl-40-cyanomethoxybiphenyl-4-yl)-N-
hydroxyacrylamide (2h)
perature for 4 h. The solid formed was filtered and dried to give
19 mg of compound 2d. Yield: 44%. M.p. 242e244 ꢀC (dec). 1H NMR
(DMSO-d6)
d
: 10.71 (1H, s); 8.95 (1H, s); 7.65e7.59 (4H, m); 7.55
A solution of compound 11b (240 mg, 0.56 mmol) and LiOH$H2O
(117 mg, 2.8 mmol) in THF:H2O 1:1 v/v (24 mL) was stirred over-
night at room temperature. THF was evaporated and the aqueous
phase was acidified with 1 N HCl to pH 1. The precipitate was
filtered to give 3-(30-adamantan-1-yl-40-cyanomethoxybiphenyl-4-
yl)acrylic acid as a light brown solid. Yield 93%. M.p. 333e334 ꢀC.
The above compound was coupled with hydroxylamine hydro-
chloride following the general procedure described for the syn-
thesis of compounds 2aec to give compound 2h as a white solid.
(1H, dd, J ¼ 8.8, 1.9 Hz); 7.42 (1H, d, J ¼ 1.9 Hz); 7.25 (1H, m); 7.15e
6.90 (3H, m); 6.02 (1H, d, J ¼ 16 Hz); 3.85 (3H, s); 2.12 (6H, s); 2.05
(3H, s); 1.75 (6H, s). 13C NMR (DMSO-d6)
d: 162.9,158.4,140.5,139.0,
137.9, 137.7, 134.6, 131.5, 127.6, 126.7, 126.5, 125.1, 124.5, 122.1, 112.7,
55.3, 36.6, 28.4. Anal. Calcd for C28H31NO3: C, 78.29; H, 7.27; N, 3.26.
Found: C, 78.11; H, 7.23; N, 3.22.
Yield 33%. M.p. 172e173 ꢀC. 1H NMR (DMSO-d6)
d: 9.35 (1H, s);
4.5. 3-[4-(8-Adamantan-1-yl-2,3-dihydrobenzo[1,4]dioxin-6-yl)-
phenyl]-N-hydroxyacrylamide (2e)
7.70e7.40 (6H, m); 7.32 (1H, d, J ¼ 1.9 Hz); 6.98 (1H, d, J ¼ 8.8 Hz);
6.49 (1H, d, J ¼ 16.1 Hz); 4.52 (2H, s); 2.10 (6H, s), 2.05 (3H, s); 1.74
(6H, s). 13C NMR (DMSO-d6)
d: 169.7, 162.8, 157.1, 141.3, 138.3, 137.9,
A solution of HATU (55 mg, 0.14 mmol), acid 10a (60 mg,
0.14 mmol) and DIPEA (0.05 mL, 0.23 mmol) was stirred for 2 min
under nitrogen. NH2OH$HCl (40 mg, 0.58 mmol) was added and the
resulting solution was stirred at room temperature overnight. The
solvent was evaporated, water was added and the suspension was
stirred 1 h at room temperature. The white solid formed was
133.2, 132.1, 128.1, 126.7, 125.2, 124.8, 118.6, 113.8, 67.4, 36.6, 36.5,
28.4. Anal. Calcd for C27H28N2O3: C, 75.68; H, 6.59; N, 6.54. Found:
C, 75.75; H, 6.54; N, 6.57.
4.9. 6-(3-Adamantan-1-yl-4-hydroxyphenyl)-naphthalene-2-
carboxylic acid benzyloxyamide (13)
filtered. Yield: 65% M.p. 211e213 ꢀC. 1H NMR (DMSO-d6)
d: 10.75
(1H, s); 9.04 (1H, s); 7.68e7.52 (4H, m); 7.47 (1H, d, J ¼ 16.0 Hz);
7.03 (1H, d, J ¼ 2.1 Hz); 7.00 (1H, d, J ¼ 2.1 Hz); 6.47 (1H, d,
To
a solution of 6-bromo-naphthalene-2-carboxylic acid
J ¼ 16.0 Hz); 4.27 (4H, s); 2.10 (6H, s); 2.05 (3H, s); 1.74 (6H, s). 13
C
(154 mg, 0.61 mmol) in anhydrous THF (3 mL) at 0 ꢀC ethyl chlor-
oformate (0.88 mL, 0.92 mmol) and triethylamine (0.138 mL,
0.99 mmol) were added. The resulting mixture was stirred for
10 min then a solution of O-benzyl-hydroxylamine in CH3OH (ob-
tained from O-benzyl-hydroxylamine hydrochloride (147 mg,
0.92 mmol), KOH (52 mg, 0.92 mmol) in 0.8 mL of CH3OH) was
added. The reaction was stirred for 1 h, the solvent was evaporated
and the residue was washed with ethyl acetate and water. The
crude was washed with diethyl ether to obtain 150 mg of 6-
bromonaphthalene-2-carboxylic acid benzyloxyamide (13). Yield:
NMR (DMSO-d6) d: 162.8, 144.0, 142.7, 141.2, 138.7, 138.0, 133.3,
131.6, 128.0, 126.7, 118.6, 116.6, 113.2, 63.7, 63.5, 36.7, 36.5, 28.4.
Anal. Calcd for C27H29NO4: C, 75.15; H, 6.77; N, 3.25. Found: C,
75.25; H, 6.73; N, 3.21.
4.6. 3-(30-Adamantan-1-yl-2-chloro-40-hydroxybiphenyl-4-yl)-N-
hydroxyacrylamide (2f)
Prepared from 10b following the procedure described for 2e.
69%. M.p. 171 ꢀC. 1H NMR (DMSO-d6)
d: 11.80 (1H, bs); 8.32 (1H, d,
Yield 26%. M.p.160 ꢀC. 1H NMR (DMSO-d6)
d: 10.77 (1H, s); 9.58 (1H,
J ¼ 1.9 Hz); 8.25 (1H, d, J ¼ 1.9 Hz); 8.02e7.87 (2H, m); 7.38 (1H, d,
s); 9.10 (1H, s); 7.71 (1H, d, J ¼ 1.8 Hz); 7.56 (1H, d, J ¼ 8.2 Hz); 7.51e
7.36 (2H, m); 7.20e7.13 (2H, m); 6.84 (1H, d, J ¼ 8.2 Hz); 6.51 (1H, d,
J ¼ 16.0 Hz); 2.09 (6H, s); 2.02 (3H, s), 1.72 (6H, s). 13C NMR (DMSO-
J ¼ 8.2 Hz); 7.67 (1H, m); 7.52e7.25 (5H, m); 4.92 (2H, s).
To a solution of 12 (123 mg, 0.40 mmol) in dioxane (2.4 mL) were
added bis(pinacolato)diboron (112 mg, 0.44 mmol), PdCl2(dppf)
(8.8 mg, 0.012 mmol), KOAc (118 mg, 1.2 mmol) and the resulting
solutionwas refluxed for 2 h. After addition of 13 (285 mg, 0.8 mmol)
and 2 M Na2CO3 (0.5 mL), the solution was refluxed for further 2 h.
Ethyl acetate (2 mL) was added and the organic phase was washed
with water, 1 M HCl (4 mL) and brine, then dried and evaporated.
Purification by flash column chromatography (hexane: ethyl acetate
2:1) afforded compound 14 as a white solid. Yield 28%. M.p. 135e
d6) d: 167.7, 156.2, 141.8, 141.5, 135.1, 134.5, 131.8, 129.5, 128.4, 127.5,
126.8, 120.8, 116.1, 36.6, 36.3, 28.4. Anal. Calcd for C25H26ClNO3: C,
70.83; H, 6.18; N, 3.30. Found: C, 70.66; H, 6.13; N, 3.33.
4.7. 4-{2-[3-Adamantan-1-yl-40-(2-hydroxycarbamoylvinyl)-
biphenyl-4-yloxy]ethyl}-morpholin-4-ium toluene-4-sulfonate (2g)
A solution of 11a (153 mg, 0.3 mmol), THP-ONH2 (36 mg,
0.3 mmol), 1.06 M LiHMDS (0.6 mL) in dry THF (4 mL) was stirred
at ꢁ78 ꢀC for 2 h. After warming to room temperature, water was
added and the aqueous phase was extracted with ethyl acetate
(3 ꢃ 5 mL). The collected organic layers were dried and evaporated to
give 3-[30-adamantan-1-yl-40-(2-morpholin-4-yl-ethoxy)biphenyl-
4-yl]-N-(tetrahydropyran-2-yloxy)acrylamide as a white solid. Yield
98%. M.p. 139e141 ꢀC.
138 ꢀC. 1H NMR (DMSO-d6)
d: 11.90 (1H, bs); 9.57 (1H, bs); 8.33 (1H,
d, J ¼ 1.9 Hz); 8.14 (1H, d, J ¼ 1.9 Hz); 8.12e7.97 (2H, m); 7.80e7.72
(2H, m); 7.63e7.28 (7H, m); 6.93 (1H, d, J ¼ 8.19 Hz); 4.97 (2H, s);
2.46e1.98 (9H, m); 1.90e1.59 (6H, m). Anal. Calcd for C34H33NO3: C,
81.08; H, 6.60; N, 2.78. Found: C, 81.29; H, 6.64; N, 2.76.
4.10. 6-(3-Adamantan-1-yl-4-hydroxyphenyl)-naphthalene-2-
carboxylic acid hydroxyamide (2i)
A suspension of the above compound (176 mg, 0.3 mmol) and
PTSA (56 mg, 0.3 mmol) in MeOH (25 mL) was stirred at room
temperature for 24 h. The solvent was evaporated and the residue
was washed with ethyl acetate and water to give the title com-
Compound 14 (24 mg, 0.05 mmol) was hydrogenated for 36 h at
room temperature in MeOH (5 mL) using 20% Pd/C as a catalyst
(5 mg). The catalyst was filtered and the solvent evaporated. The
crude was purified by flash chromatography (hexane:ethyl acetate
1:2 v/v, then MeOH) to give the title compound as a white solid.
pound as a white solid. Yield 63%. 1H NMR (DMSO-d6)
d: 10.77 (1H,
s); 10.16 (1H, s); 9.05 (1H, s); 7.74e7.40 (9H, m); 7.21e7.06 (3H, m);
6.48 (1H, d, J ¼ 16.0 Hz); 4.52e4.32 (2H, m); 4.17e3.89 (2H, m);
3.86e3.49 (4H, m), 2.28 (3H, s); 2.08 (9H, s), 1.76 (6H, s). 13C NMR
(DMSO-d6) d: 162.8, 157.0, 145.6, 141.2, 138.2, 138.0, 137.7, 133.2,
132.2, 128.1, 126.7, 125.5, 125.2, 124.9, 118.7, 114.0, 63.9, 55.4, 52.2,
36.6, 36.5, 28.4, 20.8.
Yield 70%. M.p. 204 ꢀC. 1H NMR (DMSO-d6)
d: 9.65 (1H, brs); 8.35
(1H, d, J ¼ 1.9 Hz); 8.12 (1H, d, J ¼ 1.9 Hz); 8.10e7.95 (2H, m); 7.88e
7.76 (2H, m); 7.52e7.45 (2H, m); 6.95 (1H, d, J ¼ 8.6 Hz); 2.19 (6H, s);
2.07 (3H, s); 1.75 (6H, s). 13C NMR (DMSO-d6)
d: 164.3, 156.4, 139.8,
136.1, 134.7, 130.8, 130.1, 129.5, 129.3, 128.1, 126.9, 125.8, 125.4,

258
R. Cincinelli et al. / European Journal of Medicinal Chemistry 79 (2014) 251e259
125.2, 124.0, 123.6, 117.0, 36.7, 36.4, 28.4. Anal. Calcd for C27H27NO3:
C, 78.42; H, 6.58; N, 3.39. Found: C, 78.56; H, 6.53; N, 3.34.
4.15. Cell cycle analysis
The cell cycle distribution was analyzed in propidium iodide-
4.11. 6-(3-Adamantan-1-yl-4-methoxyphenyl)naphthalene-2-
stained cells by FACScan flow cytometry, as described [19].
carboxylic acid hydroxyamide (2l)
4.16. TUNEL assay
The title compound was obtained from 15 following the same
procedure described for the synthesis of 2e. The crudewas purified by
RP-18 silica gel chromatography (MeOH:H2O 85:15 v/v). Yield 30%.
Apoptosis was determined in ovarian carcinoma IGROV-1 cells
by TUNEL assay following 72 h-exposure to the drug and fixed in 4%
paraformaldehyde for 45 min, at room temperature. The in situ cell
death detection kit fluorescein (Roche, Mannheim, Germany) was
used according to manufacturers instructions. Samples were
analyzed by flow cytometry (Becton Dickinson).
M.p. 222 ꢀC.1H NMR (DMSO-d6)
d
: 9.10 (1H, s); 8.35 (1H, d, J ¼ 1.9 Hz);
8.19 (1H, d, J ¼ 1.9 Hz); 8.10e8.00 (2H, m); 7.88 (1H, dd, J ¼ 8.6,1.9 Hz);
7.83 (1H, dd, J ¼ 8.6, 1.9 Hz); 7.65 (1H, dd, J ¼ 8.9, 1.9 Hz); 7.57 (1H, d,
J ¼ 1.9 Hz); 7.12 (1H, d, J ¼ 8.6 Hz); 3.86 (3H, s); 2.14 (6H, s); 2.07 (3H,
s); 1.76 (6H, s). 13C NMR (DMSO-d6)
d: 164.2,158.5,139.5,138.0,134.6,
131.6, 130.9, 129.7, 129.4, 128.2, 126.9, 125.9, 125.7, 125.0, 124.1, 123.6,
112.7, 55.4, 36.63, 36.59, 28.4. Anal. Calcd for C28H29NO3: C, 78.66; H,
6.84; N, 3.28. Found: C, 78.81; H, 6.81; N, 3.25.
4.17. Western blotting
Cells treated with various concentrations of HDAC inhibitors for
4 h were collected and lysed as previously described [19].
4.12. HDAC2 assay
4.18. Tumor models and evaluation of antitumor activity
HDAC2 was immunoprecipitated from HeLa cells. Whole cell
lysates were obtained by lysing the cells in a buffer containing
20 mM TriseHCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol,
0.5% NP-40, 1 mM PMSF, protease inhibitors cocktail (Roche). Ly-
sates were clarified by centrifugation (12,000 ꢃ g) for 10 min at 4 ꢀC
and were diluted with TBST (20 mM TriseHCl pH 7.5, 150 mM NaCl
and 0.1% Tween 20) containing 1 mM PMSF. Purified IgG from
rabbit antisera to HDAC2 (Santa Cruz Biotechnology sc-7899) were
then added and immune complexes allowed to form for 1 h at 4 ꢀC.
The experiments were performed using female athymic Swiss
nude mice. Mice were maintained in laminar flow rooms keeping
temperature and humidity constant. Mice had free access to food
and water. Experiments were approved by the Ethics Committee for
Animal Experimentation of the Istituto Nazionale Tumori of Milan
according to institutional guidelines.
Exponentially growing tumor cells (107 cells/mouse) were s.c.
injected into the right flank of athymic nude mice. Tumor lines
were achieved by serial s.c. passages of fragments (about
2 ꢃ 2 ꢃ 6 mm) from growing tumors into healthy mice. Animals
were treated 3 days after tumor implantation. Compounds were
dissolved in DMSO and diluted in PBS containing 5% Cremophor to
a final concentration of 10% DMSO.
Protein A-Sepharose (10 ml of settled beads) were added and the
components mixed on a rotor overnight at 4 ꢀC. Immune complexes
were collected by centrifugation and washed with cold TBST.
HDAC2 activity was assayed with a pan-HDAC substrate (KI-104;
Biomol Research Laboratories Inc., PA, USA) and the reaction was
carried out in half-volume white 96-well plates. The assay com-
ponents were incubated at 37 ꢀC for 40 min. The reaction was
Acknowledgments
quenched with the addition of 50
ml HDAC-FDL Developer (KI-105,
Biomol) 20ꢃ stock diluted in KI-143 buffer with 2
m
M TSA. The
We are indebted to MIUR (PRIN 2009 project, grant number:
2009KFMP7Z_005) for financial support.
plates were incubated 30 min at room temperature to allow the
fluorescent signal to develop. The fluorescence generated was
monitored at 355/460 nm (excitation/emission) wavelength.
Appendix A. Supplementary data
4.13. Histone deacetylase profiling (HDAC1, HDAC3-11)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.021. These data include the
experimental procedures for the synthesis of compounds 4, 5, 7,
10aeb, 11aeb, 15 and NMR spectra of the new compounds.
HDAC profiling was performed by Reaction Biology Corp. (Mal-
vern, PA) against 10 isolated isoforms of human HDAC (HDAC1,
HDAC3-11) in the presence of the fluorogenic tetrapeptide RHKKAc
(p53 residues 379e382) as the substrate (50 mM). Isolated human
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sequentially diluted solutions were used for testing. IC50 values
were calculated from the resulting sigmoidal doseeresponse inhi-
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