Novel spiroindoline HDAC inhibitors: Synthesis, molecular modelling and biological studies

Article abstract of DOI:10.1016/j.ejmech.2018.07.069

This paper describes the rational development of a series of novel spiroindoline derivatives endowed with selective inhibitory activity on the HDAC6 isoform. A convenient multicomponent one-pot protocol was applied for the assembly of the desired N1-substituted spiroindoline core which allowed a straightforward analoging. Computational studies and in vitro determination of inhibitory potency for the developed compounds against HDAC6 and HDAC1 isoforms were flanked by cell-based studies on histone H3 and α-tubulin acetylation. The effects on cancer cell cycle and apoptosis of the best performing derivatives were assessed on cancer cell lines highlighting a promising antitumor potential. In view of cell-based data and calculated drug-like properties, the selective HDAC6 inhibitor 5b, with a spiroindoline-based hydroxamate bearing a tert-butyl carbamate functionality, was selected to be further investigated for its potential in inhibiting tumor cells migration. It was able to potently inhibit cell migration in SH-SY5Y neuroblastoma cells and did not display toxicity in NIH3T3 mouse fibroblasts. Taken together, these data foster further investigation and optimization for this class of compounds as novel anticancer agents.

Full text of DOI:10.1016/j.ejmech.2018.07.069

Accepted Manuscript
Novel spiroindoline HDAC inhibitors: Synthesis, molecular modelling and biological
studies
Margherita Brindisi, Johanna Senger, Caterina Cavella, Alessandro Grillo, Giulia
Chemi, Sandra Gemma, Dora Mariagrazia Cucinella, Stefania Lamponi, Federica
Sarno, Concetta Iside, Angela Nebbioso, Ettore Novellino, Tajith Baba Shaik,
Christophe Romier, Daniel Herp, Manfred Jung, Stefania Butini, Giuseppe Campiani,
Lucia Altucci, Simone Brogi
PII:
S0223-5234(18)30634-2
10.1016/j.ejmech.2018.07.069
EJMECH 10604
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 March 2018
Revised Date: 27 July 2018
Accepted Date: 29 July 2018
Please cite this article as: M. Brindisi, J. Senger, C. Cavella, A. Grillo, G. Chemi, S. Gemma, D.M.
Cucinella, S. Lamponi, F. Sarno, C. Iside, A. Nebbioso, E. Novellino, T.B. Shaik, C. Romier, D. Herp,
M. Jung, S. Butini, G. Campiani, L. Altucci, S. Brogi, Novel spiroindoline HDAC inhibitors: Synthesis,
molecular modelling and biological studies, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.07.069.
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ACCEPTED MANUSCRIPT
Graphical abstract
Novel Spiroindoline HDAC inhibitors: Synthesis, Molecular Modelling and
Biological Studies

ACCEPTED MANUSCRIPT
Novel Spiroindoline HDAC inhibitors: Synthesis, Molecular
Modelling and Biological Studies
a
b
a
a
a
Margherita Brindisi , Johanna Senger , Caterina Cavella , Alessandro Grillo , Giulia Chemi ,
a
a
a
c,d
Sandra Gemma , Dora Mariagrazia Cucinella , Stefania Lamponi , Federica Sarno , Concetta
c,d
c,d
e
f
f
Iside , Angela Nebbioso , Ettore Novellino , Tajith Baba Shaik , Christophe Romier , Daniel
b
b
a,
a,
c,d
a
Herp , Manfred Jung , Stefania Butini *, Giuseppe Campiani **, Lucia Altucci , Simone Brogi
a
European Research Centre for Drug Discovery and Development (NatSynDrugs) and Department
of Biotechnology, Chemistry, and Pharmacy, Department of Excellence 2018-2022, University of
Siena, via Aldo Moro 2, 53100 Siena, Italy.
b
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstraße 25, 79104
Freiburg, Germany.
c
Department of Biochemistry, Biophysics & General Pathology, Second University of Napoli, Vico
Luigi De Crecchio 7, 80138 Napoli, Italy.
d
Institute of Genetics and Biophysics Adriano Buzzati-Traverso, IGB-CNR, Via Pietro Castellino
1
11, 80131 Napoli, Italy.
e
Department of Pharmacy, University of Napoli Federico II, Via D. Montesano 49, 80131 Napoli,
Italy.
f
Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et
Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 67404 Illkirch Cedex,
France
Keywords
Antitumor agents, bioinformatics, drug design, enzyme inhibitors, epigenetics, HDAC
Corresponding Authors:
*Stefania Butini, Tel. +390577234161, email: butini3@unisi.it
**Giuseppe Campiani, Tel. +390577234172, email: campiani@unisi.it

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Abstract
This paper describes the rational development of a series of novel spiroindoline derivatives
endowed with selective inhibitory activity on the HDAC6 isoform. A convenient multicomponent
one-pot protocol was applied for the assembly of the desired N1-substituted spiroindoline core
which allowed a straightforward analoging. Computational studies and in vitro determination of
inhibitory potency for the developed compounds against HDAC6 and HDAC1 isoforms were
flanked by cell-based studies on histone H3 and α-tubulin acetylation. The effects on cancer cell
cycle and apoptosis of the best performing derivatives were assessed on cancer cell lines
highlighting a promising antitumor potential. In view of cell-based data and calculated drug-like
properties, the selective HDAC6 inhibitor 5b, with a spiroindoline-based hydroxamate bearing a
tert-butyl carbamate functionality, was selected to be further investigated for its potential in
inhibiting tumor cells migration. It was able to potently inhibit cell migration in SH-SY5Y
neuroblastoma cells and did not display toxicity in NIH3T3 mouse fibroblasts. Taken together,
these data foster further investigation and optimization for this class of compounds as novel
anticancer agents.

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1. Introduction
In the past decades, histone deacetylases (HDACs) have emerged as versatile therapeutic targets.
Histones deacetylation process causes a variation of the chromatin structure thus enabling the
accessibility of transcription factors to their target genes, including tumor suppressor genes or
oncogenes [1, 2]. The abnormal modulation of this process can influence tumor progression [3] and
can also be involved in a variety of neurological disease states, tissue fibrosis, autoimmune and
inflammatory diseases, and metabolic disorders [4, 5]. Based on these evidences, HDAC inhibitors
(HDACi) gained increasing interest as promising agents for treating several diseases. Regarding
their anticancer potential, HDACi were demonstrated to induce cell cycle arrest, apoptosis and
tumor growth decrease [6]. For this purpose, the Food and Drug Administration (FDA) has recently
approved a series of HDACi for the treatment of different types of cancers. In particular, Vorinostat
(
1, Figure 1) was licensed in 2006 for the treatment of refractory cutaneous T-cell lymphoma
CTCL) [7], Romidepsin (2, Figure 1) was approved in 2009 for the treatment of CTCL and in 2011
(
for treating peripheral T-cell lymphoma (PTCL) [8], Belinostat (3, Figure 1) licensed in 2014 for
the treatment of PTCL [9], and in 2015 Panobinostat (4, Figure 1) was approved for the treatment of
recurrent multiple myeloma [10]. Other HDACi such as Mocetinostat (MGCD0103) [11],
Abexinostat (PCI-24781) [12], Entinostat (MS-275) [13], Givinostat (ITF2357) [14] and
Ricolinostat (ACY-1215) [15] are currently undergoing clinical trials for various types of cancers
[
[
16]. Moreover, Tucidinostat (Chidamide) was recently approved by Chinese FDA to treat PTCL
17].

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Figure 1. Approved histone deacetylase pan inhibitors 1-4.
Notably, these drugs can show several side-effects since they are mainly pan-HDACi or targeting
multiple HDAC isoforms [18]. Isoform-selective HDACi could display therapeutic benefits due to
minimal toxicity and limited off-target effects [19]. Therefore, in the last years, isoform- or class-
specific inhibition have caught the attention in HDACi research [20].
In our quest for the search of novel cancer therapeutics we decided to focus on HDAC6 isoform for
a series of reasons. HDAC6 is crucially involved in several cellular processes including cell growth,
survival and migration, but also in protein degradation and intracellular trafficking. Consequently,
HDAC6 has been considered a potential target for the treatment of different types of malignancies
inhibiting cancer cell migration and metastasis [21, 22]. HDAC6 displays a unique cytoplasmic
localization [21, 23, 24], and it is active on a variety of non-histone protein substrates such as α-
tubulin, peroxiredoxin, cortactin, and Hsp90 [25, 26]. On the contrary, among class I HDAC
enzymes, HDAC 1, 2, and 3 are found primarily in the nucleus, whereas HDAC8 is found in both
the nucleus and the cytoplasm. Additionally, the closest homolog of HDAC6, namely the cytosolic
isozyme HDAC10, also belonging to class IIb HDACs, is not effective as lysine deacetylase. This is
mainly due to the presence of a glutamate gatekeeper (replacing the lysine gatekeeper of
HDAC6) and to a sterically constricted active site, which confer specificity for acetylpolyamine

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substrates while disfavoring acetyllysine hydrolysis. Moreover, HDAC10 mainly promotes
autophagy-mediated effects [27]. Class IIa HDAC enzymes (HDAC 4, 5, 7, and 9) are usually
inefficient enzymes on standard substrates [28], although they lately displayed some utility in
enhancing metabolic health in chronic diseases driven by physical inactivity [29].
In this scenario, selective HDAC6i have been developed as potential anticancer agents [23, 30-32].
However, design of structurally innovative and more selective inhibitors still represents an
important task in drug discovery. In particular, although a variety of molecules advanced to clinic
are considered selective HDAC6 inhibitors (eg. Ricolinostat and Citarinostat), their selectivity for
HDAC6 isoform is only about 10-fold over HDAC1/2/3 isoforms (class I HDACs) [33]. On the
contrary, preclinical candidates displaying higher HDAC6 selectivity (SI around 60), such as
Tubastatin A, show severe liabilities due to its intrinsic toxicity to normal cells. As a continuation
of our efforts in this field [34] the present study describes a thorough investigation of the active
catalytic sites of HDAC1/6 and the development of a series of spiroindoline derivatives with
preferred binding at HDAC6 isoform (Figure 2). Selectivity was investigated over HDAC1
(nuclear) and HDAC8 isoforms (nuclear/cytoplasmic) (Tables 1,2).
Figure 2. Retrosynthetic one-pot approach to the spiroindoline scaffold of title compounds 5a-g
structures are defined in Table 1).
(

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For the rational design of the new class of inhibitors, we took into account the general features for
the HDACs inhibition, namely: i) a cap group interacting with the surface of the enzyme, ii) a linker
and iii) a metal-binding group interacting with the catalytic zinc ion (zinc-binding group [ZBG] [35,
36]). In order to efficiently shape the newly conceived chemical entities to achieve selective
HDAC6 inhibition, we envisaged the combination of a bulky spiroindoline cap group with a linker
moiety containing an aromatic ring at appropriate distances from the ZBG (Figure 2). In particular,
keeping in mind the notion that HDAC6, differently from other isoforms, can accommodate bulky
rigid cap groups [37, 38], we aimed at building a cap group able to nicely accommodate in the
HDAC binding site while also targeting a specific lipophilic pocket identified in HDAC6 and
shaped by three residues, namely L749, F679 and F680. In the HDAC1 homolog the F679 is
replaced by Y204 residue, and this could represent a further handhold for gaining potency and
selectivity on HDAC6 over HDAC1 isoform. The spirocyclic indoline scaffold was preferred over
an indoline substituted at C-3 by a flat aromatic system to favor interaction with the targeted
lipophilic HDAC6 pocket. Beyond their increasing appearance in literature as useful medicinal
chemistry and drug discovery templates [39-42], spiro-fused scaffolds may display a number of
additional potential benefits. First, the insertion of a spiro-fused ring could allow the development
of achiral molecules. Moreover, recent literature supports the use of spiro-compounds since the
conformational restriction by spiro ring formation could potentially improve bioavailability and
metabolic stability [43], reduce off-target activities and impart more favorable physical properties
with respect to compounds containing many flat rings [44-48]. Spiro-ring fusion could also
represent a useful method of increasing molecular complexity and may offer greater benefit than
introduction of flat rings [46, 47]. Docking studies finally helped identifying a spiroindolyl
cyclohexane and a spiroindolyl 4-piperidine templates as the preferential cap groups to be explored.
Regarding the linker and ZBG portions, our primary choice was a benzyl linker anchoring a para-
hydroxamic functionality as the ZBG. This group already conferred strong HDAC6 selectivity to
inhibitors such as TubA and its selection is also supported by recent studies highlighting a

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monodentate interaction for phenylhydroxamate inhibitors as a signature of selectivity for HDAC6
versus class I HDACs [49, 50]. We also herein explored a thiophene-containing isosteric linker.
To generate the new molecules a straightforward multicomponent one-pot protocol was applied for
the assembly of the desired N1-substituted spiroindoline core which allowed to rapidly perform a
small series of focused structural variations at the spiro-fused piperidine terminal ring level in order
to explore their effects on HDAC6 activity and selectivity (Figure 2). We then compared the
outcome in HDAC1 and HDAC6 isoforms, in order to obtain unambiguous suggestions on general
inhibitors’ potency and selectivity, since HDAC1 is a representative member of a different
deacetylase class displaying different phylogeny. Specifically, HDAC1 is representative of the
HDAC enzymes with nuclear localization, while HDAC6 represents a cytoplasmic isoform. To
further confirm the inhibitor selectivity profile in cell-based settings, the effects of selected
compounds on histone H3 and α-tubulin acetylation levels (taken as markers of class I HDACs and
HDAC6 inhibition, respectively) were determined. Moreover, the most promising inhibitors, in
terms of both inhibitory profile and calculated drug-like properties, were evaluated for their effects
on cell cycle and apoptosis in several tumor cell lines. The effects on the inhibition of cell migration
and cell toxicity on mouse fibroblasts were also evaluated.
2. Chemistry
Compounds 5a-g were synthesized exploiting a practical one-pot procedure for the simultaneous
construction of the spiroindoline template bearing the linker moiety already in place. This chemical
path was conceived for allowing the simple functionalization of all the portions of the indoline
skeleton, with subsequent rapid analoging potential. In more details, the proposed scheme for the
one-pot synthesis of indolines from arylhydrazines envisaged three key steps: i) a Fischer indole
synthesis, starting from the suitable arylhydrazines and α,α-disubstituted aldehydes, providing the
corresponding 3,3-disubstituted indolenines; b) a C=N bond reduction of the intermediate
indolenines and c) reductive amination for N1 alkylation [51-53]. Accordingly, compound 5a was

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obtained following the procedure reported in Scheme 1. Cyclohexanecarboxaldehyde 6a was
reacted with phenyl hydrazine 7 in acetic acid at 80 °C. The indolenine formation was generally
completed in 2-3 h and easily monitored by TLC and LC–MS analyses. Dilution with 1,2-
dichloroethane was then followed by sequential addition of methyl 4-formylbenzoate 8a and
NaBH(OAc) . This procedure led to easily achieve the desired N1 functionalized spiroindoline
3
template. Methyl ester functionality of compound 9 was then subjected to the final reaction with
hydroxylamine and sodium hydroxide providing final compound 5a.
Scheme 1. Reagents and conditions: a) AcOH, 80 °C, 2 h then 0 °C, 1,2-DCE, NaBH(OAc) , 25
3
°
C, 12 h; b) NH OH (50 wt. % in H O), NaOH, DCM/MeOH from 0 °C to 25 °C, 12 h.
2 2
The synthesis of compounds 5b-g is described in Scheme 2. All the final compounds were obtained
starting from two key intermediates, namely compounds 12 and 14, deriving from the one-pot
scaffold construction, thus demonstrating the versatility of the procedure.
For the synthesis of final compounds 5b-5e the one-pot protocol previously described was
employed starting from 1-Boc-piperidine-4-carboxaldehyde 6b, in turn prepared from 1-Boc-4-
piperidone 10. This latter was homologated by means of a Wittig reaction. Treatment with
(methoxymethyl)triphenylphosphonium chloride provided the enol ether 11 which was
subsequently hydrolyzed with cerium(III) chloride in acetonitrile to the 1-Boc-piperidine-4-
carboxaldehyde 6b in good overall yield. For the preparation of the target compound 5b, compound
6b was subjected to a one-pot reaction with phenylhydrazine (7), methyl-4-formyl benzoate (8a),

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and NaBH(OAc) to obtain ester intermediate 12. Reaction of this latter with hydroxylamine
3
solution and sodium hydroxide led to final compound 5b. Intermediate 12 was also Boc-deprotected
and reacted with formaldehyde in the presence of NaBH CN thus affording the corresponding N-
3
methyl derivative, finally reacted with hydroxylamine, providing compound 5c.
Intermediate 12 was also employed for the preparation of the two amide derivatives 5d,e. After Boc
deprotection, the resulting free amine was reacted with acetyl or pivaloyl chloride, respectively.
Reaction with aqueous hydroxylamine and sodium hydroxide led to target compounds 5d,e.
For the preparation of thiophene-based compounds 5f and 5g, aldehyde 8b was obtained from
bromoderivative 13 [34] which underwent reaction with 4-methylmorpholine N-oxide in
acetonitrile. One pot reaction of 6b and phenylhydrazine followed by addition of aldehyde 8b led to
the key intermediate methyl ester 14 employed for the synthesis of final compounds 5f,g, following
the procedure described for compound 5b,c.
Scheme 2. Reagents and conditions: a) (Ph) P(Cl)CH OMe, NaHMDS, dry THF, 0 °C to 25 °C,
3
2
.
3
0 min; b) CeCl 7H O, NaI, 40 °C, MeCN, 16 h; c) NMO, MeCN, 25 °C, 12 h; d) 7, AcOH then
3 2
8
a, NaBH(OAc) , 1,2-DCE, 80 °C, 3 h, then 25 °C, 14 h; e) NH OH (50 wt. in % H O), NaOH,
3
2
2
DCM/MeOH from 0 °C to 25 °C, 12 h; f) AcCl, MeOH, 25 °C, 18 min; g) CH O (37% aqueous
2

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sol.), 1,2-DCE, 25 °C, 1 h, NaBH CN, 25 °C, 12 h; h) AcCl, TEA, DCM, 25 °C, 12 h; i) pivaloyl
3
chloride, TEA, DCM, 25 °C, 12 h; j) 7, AcOH then 8b, NaBH(OAc) , 1,2-DCE, 80 °C, 3 h, then 25
3
°
C, 14 h.
3
3
. Results and discussion
.1. Structure–activity relationship and molecular modelling studies
The inhibitory potency of the newly conceived compounds was evaluated in vitro against HDAC1
and HDAC6 enzymes, using a fluorescence-based activity assay [54]. The inhibition potency of
compounds 5a-g on HDAC1 and HDAC6 enzymes is reported in Table 1. Structure–activity
relationship (SAR) analysis was performed coupling in vitro data with computational studies based
on molecular docking calculations using Glide employing Standard Precision (SP) as scoring
function. The calculation outcome provides a general trend that highlights the preferred binding of
spiroindoline compounds at HDAC6 isoform.
Table 1. Inhibitory activity as IC50 (nM) ± standard error and Glide docking score (standard
precision, SP; kcal/mol) against HDAC1 and HDAC6 enzymes and HDAC1/HDAC6 selectivity
ratio.
IC HDAC1 IC HDAC6
HDAC1/ Glide SP HDAC1 Glide SP HDAC6
5
0
50
Cpd
Structure
(
µM)
(nM)
HDAC6
(kcal/mol)
(kcal/mol)
5
a
6.79 ± 1.02
40.9 ± 6.9
166
-8.367
-9.967
5
b
4.00 ± 0.78
41.9 ± 7.9
95
57
-8.701
-8.550
-10.194
5
c
11.71 ± 0.71 203.8 ± 28.4
-9.435

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5
d
7.29 ± 0.79
62.5 ± 14.5
116
-8.707
-9.838
5
e
4.47 ± 0.50
60.1 ± 7.9
74
-8.373
-8.004
-9.539
-9.124
5
f
11.44 ± 3.29
1780 ± 350
6
5
g
10.87 ± 5.67 718.6 ± 73.5
15
63
-8.138
-8.277
-9.013
-9.878
Tubastatin
A
-
1.91 ± 0.42
30.4 ± 2.1
Using docking calculations, we observed a conserved trend in the binding modes of this series of
developed compounds into HDAC1 and 6 isoforms’ binding sites. In particular, 5b could be able to
form a metal coordination bond into both isoforms (Figure 3), although we noticed a different
pattern of interaction. Compound 5b into HDAC1 binding site is able to form only one H-bond with
the backbone of G149 and limited hydrophobic contacts with F150 (especially π-π stacking
interaction) and L271. The bulky piperidyl-N-tert-butyl carbamate group appears to be largely
solvent exposed with few hydrophobic interactions with Y204. This binding mode could support its
micromolar inhibitory potency against HDAC1. On the contrary, the binding mode of compound 5b
into HDAC6 shows an increased number of contacts. In detail, besides the potential metal
2
+
coordination bond with the Zn ion, 5b forms a series of H-bonds with H610, G619 (backbone),

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and Y782. The benzene ring of the linker moiety is able to establish a double π-π stacking with
F620 and F680. Gratifyingly, in full agreement to our design strategy, the spiropiperidyl moiety
nicely fits within the targeted hydrophobic pocket (L749, F679 and F680). Interestingly, the
optimum accommodation of the spiro-fused portion also allowed the terminal t-butylcarbamate
functionality to establish strong hydrophobic interactions with the side chains of T678 and F679
(Figure 3), contrarily to what observed for HDAC1 isoform. The major number of established
contacts is in agreement with the registered higher inhibitory potency of compound 5b against
HDAC6 vs HDAC1 with 100-fold selectivity (IC HDAC1 = 4.00 µM; IC HDAC6 = 41.9 nM,
50
50
Table 1).
Figure 3. Docked poses of 5b into HDAC1 (panel A) and HDAC6 (panel B). Compound 5b is
represented as sticks while the residues in the active sites are represented as line and the protein is
2
+
represented as cartoon. Zn is represented as a gray sphere. H-bonds are represented as black
dotted lines, while red and blue dotted lines represent the metal coordination bonds.
Similarly to 5b, 5a showed a comparable behavior into HDAC1 and HDAC6 enzymes (Figure S1).
The absence of the t-butylcarbamate functionality allows the indoline moiety to improve the
hydrophobic contacts with F150 (HDAC1) and F620 (HDAC6), maintaining the other interactions
already described for 5b. Accordingly, the experimental IC s against HDAC1/6 are comparable to
5
0

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those found for 5b. The introduction of an N-Me group replacing the t-butylcarbamate led to
compound 5c displaying a slight decrease of affinity towards both HDAC isoforms with an
inhibitory potency of IC HDAC1 = 11.71 µM; IC HDAC6 = 203.8 nM. Although the docking
5
0
50
outputs depicted in Figure S2 are very similar to those found for 5a, the introduction of a
protonatable group such as N-Me on the spiro-fused terminal ring is not well tolerated by both
enzymes. This event could be due to the presence, in both binding sites of the two isoforms, of
positively charged amino-acids such as K200 in HDAC1 and R673 in HDAC6 and to the not
favorable desolvation energy for this compound. The insertion on the terminal spiro-fused ring of
an N-acetyl or a N-trimethylacetyl group led to compounds 5d and 5e, respectively. These
substitutions are well tolerated from HDAC6 and allow keeping significant selectivity over
HDAC1. The docking outputs of 5d (Figure S3) and 5e (Figure S4) into both enzyme subtypes are
comparable to those obtained for 5b, as supported by experimentally determined inhibitory
potencies (Table 1). The replacement of the benzene ring in compound 5c with thiophene led to
compound 5f. In this latter, the presence of a protonatable group (N-Me) combined with a smaller
aromatic moiety in the linker caused a dramatic decrease in both affinity and isoform selectivity.
Beyond the already discussed unfavorable effects due to the N-Me insertion, we registered also a
reduction of π-π stacking in both enzymes. As reported in Figure 4, 5f was not able to establish a
double π-π stacking into HDAC6 but is able to stack only with F620. Regarding HDAC1 a different
orientation of the thiophene was encountered with respect to the benzene ring of the previously
described compounds, resulting in an unfavorable conformational energy of the poses as detected
by docking calculation, and in the lower inhibitory potency against HDAC1 and HDAC6 (IC50
HDAC1 = 11.44 µM; IC HDAC6 = 1780 nM).
5
0

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Figure 4. Docked poses of 5f into HDAC1 (panel A) and HDAC6 (panel B). Compound 5f is
represented as sticks while the residues in the active sites are represented as line and the protein is
2
+
represented as cartoon. Zn is represented by a gray sphere. H-bonds are represented as black
dotted lines, while red and blue dotted lines represent the metal coordination bonds.
The replacement of the basic tertiary amine of 5f by a t-butylcarbamate led to compound 5g. As
expected, this compound showed a slight improvement in inhibitory potency against HDAC6,
although lower than its benzene counterpart 5b. 5g (Figure S5) demonstrates a similarity to 5f in the
binding mode. These findings support the in vitro and computational data reported in Table 1.
As a further indication on the selectivity of compounds, we also assessed the potency of three
representative inhibitors (compounds 5a,b,g, Table 2) on HDAC8 isoform, which is unique in class
I HDACs, since it recognizes both histone and non-histone substrates. Moreover, HDAC8 is
ubiquitously expressed and can localize to either the nucleus (primary site) or the cytoplasm [55].
Compound 5b displayed the lower activity of HDAC8 (IC = 1.602 µM), thus confirming its very
5
0
selective interaction with HDAC6 enzyme. Compounds 5a and 5g displayed moderate selectivity
over HDAC8 isoform.
Table 2. Inhibitory activity as IC (µM) ± standard error against HDAC8 enzyme.
5
0
Cpd
5a
5b
5g
TubA
IC (µM) 0.253 ± 0.018 1.602 ± 0.166 1.344 ± 0.157 0.695
5
0

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We also predicted the in silico drug-like features of the novel chemical entities in order to support
the selection process for the compounds to be advanced in cell-based studies. In particular, we used
different tools such as QikProp, FaFDrugs [56] and an in-house 3D-QSAR model for predicting
+
hERG K channel affinity (3D-chERGi) [57]. The outputs of these calculations are reported in
Table S1. Additionally, the compounds were evaluated for their potential ability to behave as Pan
Assay Interference Compounds (PAINS). This calculation was performed by means of FAFDrugs.
Remarkably, the results showed that none of the compounds contain sub-structural features that
would label them as “frequent hitters” in high-throughput screens. The molecular properties of the
compounds showed satisfactory results, displaying suitable predicted aqueous solubility (cLogS),
and cLogP. The membrane permeability was considered acceptable as well as the predicted human
oral adsorption. No violation of Lipinski’s rules was found. Compounds 5a, 5b, 5d, 5e and 5g were
+
predicted to possess limited affinity for the hERG K channel, while 5c and 5f could potentially
interact with it. Although the molecular properties of the compounds showed satisfactory results,
compounds 5d and 5f were not furthered in cell-based assays, since globally possessing the less
satisfactory drug-like profile.
4. Cellular studies
Based on in vitro determined HDAC affinity and HDAC6 isoform selectivity and on the calculated
drug-like properties, a selection of four derivatives (5a-c and 5e) were engaged in Western blotting
analyses to evaluate the levels of acetylation of α-tubulin (AcTub) and of histone H3 in lysines 9
and 14 (H3K9,14ac). Acute promyelocytic leukemia NB4 and glioblastoma U87 cell lines were
stimulated with compounds 5a, 5b, 5c at 1 and 5 µM for 30 hours, in comparison with Vorinostat
(1, SAHA) and Tubastatin, used as positive controls (Figure 5A). In NB4 cells all the assayed
compounds were able to increase the levels of AcTub at 5 µM, while only compounds 5a and 5c
increased the levels of H3K9,14ac (Figure 5A, top). Compound 5b, in particular, showed a HDAC6-

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selective mode of action. In U87 cell line all compounds were able to affect the levels of histone
acetylation but 5c and 5a induced a dose-dependent increase of AcTub (Figure 5A, bottom).
In addition, due to the superior profile of 5b in terms of activity and selectivity, its outcome on
histone and tubulin acetylation was evaluated in neuroblastoma SH-SY5Y cells (Figure 5B).
Interestingly, 5b induced a dose-dependent increase in acetylation levels of tubulin, but not of
histone H3, compared to the positive controls. The levels of tubulin acetylation were also higher
than those measured with Tubastatin A (a selective HDAC6 inhibitor) and SAHA (1, a pan-HDAC
inhibitor). All these data highlight compound 5b as an extremely selective HDAC6 inhibitor also in
cell-based settings.
Finally, in order to directly compare their HDAC6 selectivity in cell-based assays, compounds 5b
and 5e (one of the best performing compounds in terms of calculated physico-chemical properties)
were tested in NB4 and U87 cell lines (Figure 5C) by Western blotting analyses. Both compounds
showed improved selectivity for HDAC6 compared to 1.

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Figure 5. Western blot analyses of acetylation levels of the α-tubulin (AcTub) and histone H3 in
lysines 9 and 14 (H3K9,14ac) after 30 hours of the indicated treatments in NB4 and U87 (A), SH-
SY5Y (B), and NB4 and U87 (C) cell lines. ERKs, total (Tot) histone H4 and Ponceau Red signals
were used as loading controls.
By FACS analysis in NB4 cells, compound 5e, when tested at 5 µM, induced a G1-phase arrest of
cell cycle progression, similar to 1 (Figure 6A). Notably, both 5b and 5e were able to induce cell
death, as revealed by the percentage of DNA fragmentation. Compound 5b, at 5 µM, displayed a

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higher cytotoxic activity than 1 and 5e (Figure 6B). Collectively, these data suggest a promising
antitumor activity for these two compounds.
Moreover, since HDAC6 displays unique features with respect to other HDAC isoforms, being
mainly devoted to the deacetylation of non-histone proteins strongly involved in cell-cell
interaction, motility and apoptosis, selective HDAC6 inhibitors display crucial importance for
investigating and dissecting the specific roles of different HDAC enzymes in cancer. In particular,
due to the role of HDAC6 in the regulation of microtubule- and actin-dependent cell mobility [58],
inhibitor 5b was evaluated in real-time migration assay (see Experimental Section for description).
Interestingly, 5b significantly inhibited migration of SH-SY5Y neuroblastoma cells (Figure 6C).
The migration inhibitory effect of 5b evaluated by slope analysis showed a strong decrease of
migration rate after about 4 days of treatment, suggesting a strong anti-invasion effect of the drug.
These data strongly imply wide-ranging anti-proliferative, anti-invasiveness and apoptotic effects of
5b in low micromolar range.

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Figure 6. (A, B) Cell cycle and pre-G1 analyses in NB4 cell lines treated for 30 h with indicated
compounds. (C) Migration inhibitory action of 5b measured in SH-SY5Y as Slope (1/h) by
xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche).
To further characterize the profile of compound 5b, we also performed a cytotoxicity assay to
establish the effect on mouse fibroblasts NIH3T3 cells. Cell viability was measured by the Neutral
Red Uptake test and data normalized as % control. We observed a TC on NIH3T3 cells of 23 ꢀM,
50
thus indicating a low toxicity for normal cells and a potentially favourable safety margin.
5. Conclusion

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We herein described a series of spiroindoline derivatives rationally designed to preferentially bind
the HDAC6 isoform. This goal was reached by combining medicinal chemistry and biological
efforts. Our focused design strategy ultimately led to compounds displaying up to 170-fold
selectivity for HDAC6 over HDAC1 isoform. A practical one-pot synthetic path was developed to
build a focused set of N1-substituted spiroindoline cores. The versatility of the strategy will allow
future scaffold manipulation and rapid analoging. The developed inhibitors were submitted to
enzyme assays and the best performing compounds were engaged in cell-based assays to establish
selectivity on cells and anticancer potential. Their influence on tubulin and histone acetylation
status was evaluated to further confirm their HDAC6 selectivity. Moreover, the best performing
derivatives were assessed for their effects on cell cycle and apoptosis, showing a promising
antitumor potential in tumor cell lines. A selected derivative, 5b, characterized by a piperidyl-N-
tert-butyl carbamate, was further investigated revealing a significant potential in inhibiting tumor
cells migration. Additionally, this compound did not display toxicity in NIH3T3 mouse fibroblasts
and was found to possess satisfactory predicted drug-like properties. Regarding the tert-butyl
carbamate moiety of our hit 5b, it is interesting to mention that carbamates are present in many
approved drugs [59] and in particular, tert-butyl carbamates of piperidine derivatives have been
reported to accomplish drug-like properties [60]. Taken together, these data highlight a promising
antitumor potential for the developed compounds. Further studies and compound optimization are
ongoing in our laboratories.
6
6
. Experimental Section
.1. Chemistry
Unless otherwise specified, materials were purchased from commercial suppliers and used without
further purification. Reaction progress was monitored by TLC using silica gel 60 F254 (0.040–
0.063 mm) with detection by UV. Silica gel 60 (0.040–0.063 mm) was used for column
chromatography. 1H NMR and 13C NMR spectra were recorded on a Varian 300 MHz
spectrometer or a Bruker 400 MHz spectrometer by using the residual signal of the deuterated

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solvent as internal standard. Splitting patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q) and broad (br); the values of chemical shifts (δ) are given in ppm and coupling constants
(J) in Hertz (Hz). Microwave reactions were performed by a CEM Discovery apparatus. ESIMS
spectra were performed by an Agilent 1100 Series LC/MSD spectrometer. The yields are referred to
purified products and are not optimized. All moisture-sensitive reactions were performed under
argon atmosphere using oven-dried glassware and anhydrous solvents. Experimental procedures
and characterization for the novel compounds are reported in Supplementary Material. Final
compounds were analyzed by combustion analysis (CHN) to confirm purity > 95% (Table S2).
6.2. Computational details
All calculations were performed using two workstations (Intel Core i5–2400 CPU @3.10 GHz
Quad; Intel Core i5–2500 CPU @ 3.30 GHz Quad) with Ubuntu 17.04 OS, running Maestro 10.1
(Schrödinger, LLC, NY, USA, 2015). Figures depicting docking outputs were prepared using
PyMOL (The PyMOL Molecular Graphics System, v1.8.4.0, Schrödinger LLC, New York, 2015).
6
6
.2.1. Molecular Docking simulations
.2.1.1. Proteins and ligands preparation
Crystal structures of human HDAC1 (PDB ID 4BKX [61]) and HDAC6 (PDB ID 5EDU [37]) were
taken from PDB and submitted to Protein Preparation Wizard (PPW) protocol implemented in
Maestro suite 2015 in order to obtain suitable protein structures for molecular docking calculations
as previously reported [62-64]. Ligands, water molecules and compounds used in the crystallization
2
+
process were removed maintaining the Zn ion [34]. By following PPW protocol we performed a
series of computational steps to: (1) generate metal binding state for the enzymes (2) add
hydrogens, (3) optimize the orientation of hydroxyl groups, Asn, and Gln, and the protonation state
of His, and (4) perform a constrained minimization refinement with the impref utility. The refined
proteins were used in molecular docking calculation as reported in the next paragraph.
The 3D structure of ligands reported in Table 1 and those of reference compounds (Trichostatin A,
Tubastatin A and 1) were built in Maestro 10.1 (Schrödinger, LLC, New York, NY, 2015).

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MacroModel software was used for the energy minimizations employing the OPLS-2005 force
field. The solvent effects were simulated using the analytical Generalized-Born/Surface-Area
(GB/SA) model [65], and no cutoff for nonbonded interactions was selected. Polak-Ribiere
conjugate gradient (PRCG) method with 1000 maximum iterations and 0.001 gradient convergence
threshold was employed. All compounds reported in this paper were treated by LigPrep application
(
LigPrep version 3.3, Schrödinger, LLC, New York, NY, 2015) in order to generate the most
probable ionization state at cellular pH (7.4 ± 0.2) as reported by us [66, 67]. Moreover, according
to the evidences reported in literature [68-73], we used a neutral hydroxamic acid moiety of the
spiroindoline compounds, since the hydroxamic acid proton should not be transferred in HDAC
isoforms containing histidines in the binding site close to the reactive metal center, as in the case of
HDAC1 and HDAC6.
6.2.1.2. Molecular Docking
Glide software (Glide, version 6.6, Schrödinger, LLC, New York, NY, 2015) has been employed to
perform the docking studies presented in this paper, using the ligands and proteins prepared as
above-mentioned, applying Glide standard precision (SP) scoring function. Energy grids were
prepared using default value of protein atom scaling factor (1.0 Å) within a cubic box centered on
the zinc ion which roughly represents the center of the active sites [34]. After grid generation with
the introduction of metal constrains, the ligands were docked into the enzymes. The number of
poses entered to post-docking minimization was set to 50. Glide SP score was evaluated. In order to
assess the validity of docking protocol, 1 and Trichostatin A were used as reference compounds for
a re-docking procedure. The docking results revealed a similar accommodation for the above
mentioned reference compounds with respect to the previously published results (data not shown)
[
37, 74].
.2.2. Molecular properties prediction
6
Molecular properties were evaluated by means of QikProp (QikProp, version 4.3, Schrödinger,
LLC, New York, NY, 2015), while the assessment for potential Pan Assay Interference Compounds

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(PAINS) within the set of selected molecules was conducted by means of FAFDrugs4.0
+
(http://fafdrugs4.mti.univ-paris-diderot.fr/ access date December 2017). The evaluation of hERG K
channel affinity was performed using our inclusive in-house 3D-QSAR model (3D-chERGi) [57].
.3. In vitro testing of HDAC1, HDAC6 and HDAC8
6
OptiPlate-96 black microplates (Perkin–Elmer) were employed with an assay volume of 60 ꢀL.
Human recombinant HDAC1 (BPS Bioscience, Catalog #: 50051) or human recombinant HDAC6
(BPS Bioscience, Catalog #: 50006) were diluted in incubation buffer (50 mM Tris-HCl, pH 8.0,
1
37 mM NaCl, 2.7 mM KCl, 1 mM MgCl and 1 mg/ml BSA). A total of 52 ꢀL of this dilution
2
were incubated with 3 ꢀL of different concentrations of inhibitors in DMSO and 5 ꢀL of the
fluorogenic substrate ZMAL (Z-(Ac)Lys-AMC) [75, 76] (126 ꢀM) at 37 °C. After 90 min
incubation time, 60 ꢀL of the stop solution (33 ꢀM Trichostatin A and 6 mg/mL trypsin in trypsin
buffer [Tris-HCl 50 mM, pH 8.0, NaCl 100 mM]), were added. After a following incubation at 37
°
C for 30 min, the fluorescence was measured on a BMG LABTECH POLARstar OPTIMA plate
reader (BMG Labtechnologies, Germany) with an excitation wavelength of 390 nm and an emission
wavelength of 460 nm [54, 76].
Recombinant hHDAC8 was produced as described before [77]. The HDAC8 activity assay was
performed using a commercial HDAC8 fluorimetric drug discovery kit [Fluor de Lys(R)-HDAC8,
BMLKI178] according to the manufacturer’s instructions as described earlier [78]. The enzyme was
incubated for 90 min at 37 °C, with a substrate concentration of 50 ꢀM and increasing
concentrations of inhibitors. Measurement was performed as described for HDAC1/6.
6.4. Biological section
6.4.1. Cell lines
Glioblastoma, U87 (ATCC) and neuroblastoma cell lines, SH-SY5Y (ATCC) were propagated in
DMEM medium (Euroclone, Milan, Italy) with 10% fetal bovine serum (FBS) (Euroclone), 2 mM
L-glutamine (Euroclone) and antibiotics (100 U/ml penicillin, 100 l g/ml streptomycin)
(Euroclone). Human Acute Promyelocytic Leukemia, NB4 cell line, was propagated in RPMI 1640

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medium with 10% FBS (Euroclone), 100 U/mL penicillin-streptomycin (Euroclone) and 2 mM L-
glutamine (Euroclone). Cells were stimulated with drugs for 30 h at 1µM and 5µM.
6.4.2. Western blot analysis
To quantify the acetylation levels, we performed the total protein and the histone extraction by
evaluation respectively of Ac-tubulin and histone H3K9,14ac or H3Ac.
6.4.2.1. Total protein extraction
The cells were harvested, washed with PBS (Euroclone), and lysed for 15 min at 4 °C in lysis
extraction buffer with protease and phosphatase inhibitors: 50 mM Tris-HCl pH 8.0, 150 mM NaCl,
1
% NP40, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 40 mg/mL
phenylmethylsulfonyl fluoride (PMSF), 20 g/mL aprotinin, 20 mg/ml leupeptin, 2 mg/mL antipain,
0 mM p-nitrophenyl phosphate, 10 mg/mL pepstatin A and 20 nM okadaic acid. Cells were then
1
centrifuged at 13000 rpm for 30 minutes at 4 °C, and protein content of supernatant was used to
determine the protein concentration by colorimetric assay (Biorad, Italy). Cell extracts were diluted
1:1 in sample buffer 2X Laemmli (0.217 M Tris-HCl pH 8.0, 52.17% SDS, 17.4% glycerol, 0.026%
bromo-phenol blue, 8.7% beta-mercapto-ethanol), and then boiled for 3 minutes. Equal amounts of
protein (50 ꢀg) were run and separated by SDS-PAGE gel (acrylamide gel). Primary antibodies
used were acetyl-tubulin (Sigma) and anti-ERKs antibody (Santa Cruz Biotechnology) was used for
normalization.
6
.4.2.2. Histone extraction
Cells were harvested and washed twice with cold 1X PBS and lysed in Triton extraction buffer
TEB; PBS containing 0.5% Triton X 100 (v/v), 2 mM PMSF, 0.02% (w/v) NaN ) at a cell density
(
3
of 107 cells/mL for 10 min on ice, with gentle stirring. After centrifugation (2,000 rpm at 4 °C for
0 min), the supernatant was removed, and the pellet was washed in half the volume of TEB and
1
centrifuged as before. The pellet was suspended in 0.2 N HCl at a cell density of 4 × 107 cells/mL
overnight at 4 °C on a rolling table. The samples were then centrifuged at 2,000 rpm for 10 min at 4
°
C, the supernatant was removed, and the protein content was determined using a Bradford assay

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(Bio-Rad, CA, USA). For the detection of histone H3 acetylation, we used H3Ac (Abcam) or
H3K9,14ac (diagenode). Histone H4 (Abcam) antibodies and Pounceau Red (Sigma) were used to
normalize for equal loading. Semi-quantitative analysis was performed using Fiji software [79].
6.4.3. Cell cycle analysis
Cells after stimulation were collected. The cells were then centrifuged (1,200 rpm for 5 min) and
suspended in 500 µL a solution containing 1 × PBS, 0.1% sodium citrate, 0.1% NP40 and 50
mg/mL propidium iodide (Sigma Aldrich). The samples were incubated for 30 min at room
temperature in the dark. Cell cycle was evaluated by FACS-Calibur (Becton Dickinson) and
analyzed with ModFit v3 software (Verity Software House). Apoptosis was measured as Pre-G1
analyzed by FACS with Cell Quest software (BD Biosciences) [80]. Experiments were performed
in duplicate.
6.4.4. Cell migration assay
Cell migration was analysed in real time without exogenous labels using the Roche xCELLigence
Real-Time Cell Analyzer (RTCA) DP instrument. The RTCA DP instrument uses the CIM (cellular
invasion/migration)-Plate 16 featuring microelectronic sensors integrated into the underside of the
micro-porous polyethylene terephthalate (PET) membrane of a Boyden-like chamber. In response to
the chemo-attractant (FBS) the cells migrate from the upper chamber through the membrane into
the bottom chamber thus contacting and adhering to the electronic sensors on the underside of the
membrane, resulting in an increase in impedance that is proportional to increasing numbers of
migrated cells on the underside of the membrane. CI values reflecting impedance changes are
registered by the RTCA DP instrument. The membrane was pre-incubated for one hour in the CO2
5% at 37 °C with serum-free medium before obtaining a background measurement. After the cell
index measure, the SH-SY5Y cells were added to the top chamber CIM-Plate at the desired
concentration. The migration was monitored every two minutes for several hours. The cells were
analysed in absence or presence of 10% FBS in the bottom chamber. Cell migration was detected

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by real-time monitoring and low and high seeding densities were quantitatively monitored and
reflected by the CI values [81].
Acknowledgement
The authors thank COST Action CM1406. This study was supported by grants from MIUR (Rome,
grants PRIN-2010M2JARJ to G. Campiani) and by the Deutsche Forschungsgemeinschaft (DFG, Ju
295/13-1) to J. S. and M. J. T. B. S. and C. R. are supported by institutional funds from the Centre
National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche
Médicale (INSERM) and the Université de Strasbourg.
Supplementary data
Supplementary data associated to this article can be found at….
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