Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4

Article abstract of DOI:10.1021/acs.jmedchem.8b00050

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tub

Full text of DOI:10.1021/acs.jmedchem.8b00050

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Article
Design and Synthesis of Tubulin and Histone
Deacetylase Inhibitor Based on isocombretastatin A-4
Diana Lamaa, Hsin-Ping Lin, Lena Zig, Cyril Bauvais, Guillaume Bollot, Jérôme Bignon,
Helene Levaique, Olivier Pamlard, Joëlle Dubois, Mehdi Ouaissi, Martin Souce, Athena
KASSELOURI, François Saller, Delphine Borgel, Chantal Jayat-Vignoles, Hazar Al-
Mouhammad, Jean Feuillard, Karim Benihoud, Mouad Alami, and Abdallah Hamze
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00050 • Publication Date (Web): 13 Jul 2018
Downloaded from http://pubs.acs.org on July 13, 2018
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Design and Synthesis of Tubulin and Histone
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Deacetylase Inhibitor Based on isocombretastatin Aꢀ
4
a,‡
a,‡
b
c
c
d
Diana Lamaa, Hsin-Ping Lin, Lena Zig, Cyril Bauvais, Guillaume Bollot, Jérôme Bignon,
d
d
d
e
f
Helene Levaique, Olivier Pamlard, Joelle Dubois, Mehdi Ouaissi, Martin Souce, Athena
f
g
g
h
Kasselouri, François Saller, Delphine Borgel, Chantal Jayat-Vignoles, Hazar Al-
h
h,i
b
,a
,a
Mouhammad, Jean Feuillard, Karim Benihoud, Mouad Alami,* Abdallah Hamze*
a
BioCIS, Univ. ParisꢀSud, CNRS, équipe labellisée Ligue Contre le Cancer, Université ParisꢀSaclay, 92290,
ChâtenayꢀMalabry, France
b
Vectorologie et thérapeutiques anticancéreuses, UMR 8203 CNRS, Univ. ParisꢀSud, Institut Gustave Roussy,
Université ParisꢀSaclay, Villejuif, Franceꢀ94805
c
SYNSIGHT, 86 rue de Paris Orsay 91400, France
d
CIBI platform, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS avenue de la terrasse, Fꢀ91198 Gif
sur Yvette, France
e
CHRU Hôpital de Tours Trousseau, Service de chirurgie digestive, oncologique, endocrinienne et de
transplantation hépatique, avenue de la République, 37170 ChambrayꢀlèsꢀTours
f
Lip(Sys)², Chimie Analytique Pharmaceutique, Univ ParisꢀSud, Université ParisꢀSaclay, Fꢀ92290 Châtenayꢀ
Malabry, France.
g
INSERM, UMRꢀS1176, University ParisꢀSaclay, Fꢀ94276 Le KremlinꢀBicêtre, France
h
Univ Limoges, Faculté de Médecine, CNRS UMR 7276, Laboratoire CRIBL, Fꢀ87025 Limoges, France
i
CHU Limoges, Hôpital Dupuytren, Service d'hématologie, Fꢀ87025 Limoges, France
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‡
D.L. and L. HꢀS contributed equally to this work.
ABSTRACT
Designing multiꢀtarget drugs have raised considerable interest due to their advantages in the
treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor
drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC
activity. On the basis of 1,1ꢀdiarylethylenes (isoCAꢀ4) and belinostat, a series of hybrid
molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were
proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent
antiproliferative activity.
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Keywords: histone deacetylase, tubulin, antiproliferative activity, multiꢀtarget drugs, dual
inhibitor
INTRODUCTION
In the last decade, multiꢀtarget drugs have gained considerable interest due to their advantages
in the treatment of complex diseases. In cancer, because of the affected network complexity and
the involvement of different cell types, a selective single target drug can rarely achieve its
desired effect, and most curative cancer treatments are based on the combination of multiple
1
effective drugs. However, the polypharmacology approach is often linked with side effects.
Interestingly, when rationally designed drugs with multiple targets could have a superior
2
therapeutic effect and display low side effect profile. Based on these challenges the paradigm of
hybrid molecules with dual activity was developed. Hybridization of drugs is growing and
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undergoing development in both industry and academia, and their design is a niche in the large
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field of drug discovery. Hybrid molecules are defined as new chemical entities which combine
two distinct biologically active molecules, which could act as two distinct pharmacophores at
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different targets. The aim of this hybrid drug is to modulate, amplify, or exert dualꢀdrug action.
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The main challenge in the development of dual molecules is the need to design a drug that
modulates multiple disease targets simultaneously.
Cancer treatment protocols are mostly designed to interfere with one or more mechanisms
involved in cell proliferation. Thus, this heterogeneity open up a research opportunities for the
multiꢀtarget drugs’ design and development. Histone deacetylases (HDACs), a class of enzyme
which ensures the removal of acetyl groups from εꢀNꢀacetyl lysine residues of histones,
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consequently play an important role in epigenetic regulations. They have essential role in many
biological processes, largely through their repressive influence on transcription, which is
responsible for regulation of proliferation, angiogenesis, migration, differentiation, and
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metastasis. Given the biological function of HDACs, it is not surprising that they play a highly
important role in different diseases, and are the target of many drugs. Overexpression of HDACs
is observed in various types of human tumors, including colorectal, gastric, liver breast, and lung
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cancers. Therefore, inhibition of HDACs has been approved as a successful strategy treating
many cancer forms, and multiple products have reached the market as antitumor drugs, such as
romidepsin (2004), vorinostat (2006), belinostat (2014), panobinostat (2015), and chidamide
(2015). Over the past several years, a large number of clinical trials have been carried out, and
some are onꢀgoing involving HDACi as a single therapy or in combination with other anticancer
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agents to improve their antitumor activities. Many studies have shown that HDACi could
synergistically enhance the inhibitory effects of other antitumor agents, such as HSP90,
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topoisomerase, and tubulin inhibitors.
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The tubulin/microtubule system plays an essential role in several primordial eukaryotic cellular
processes such as cell division, motility, intracellular trafficking. It constitutes an important
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target for anticancer therapy, hence in recent years, new tubulinꢀbinding agents have been
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under preclinical or clinical development. The tumor vascular disrupting agent combretastatin Aꢀ
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(CAꢀ4, Figure 1) is one of these classes of molecules and its association with carboplatin,
paclitaxel, and bevacizumab, is currently under investigation in phase II of clinical trials in
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advanced nonꢀsquamous nonꢀsmallꢀcell lung cancer. Its combination with pazopanib is in the
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phase 1b for the treatment of advanced ovarian cancer. The SARs study has shown that the
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optimal biological activity of CAꢀ4 is linked to its cisꢀolefinic bond configuration.
Unfortunately, the Zꢀconfiguration of CAꢀ4 is associated with in vivo stability problems, due to
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its ZꢀE isomerization which currently remains the main obstacle to the broad clinical
application of CAꢀ4. From a chemical point of view, the pharmaceutical process of scalingꢀup of
this molecule to produce the pure Zꢀenantiomer represents another weak point. Therefore, several
attempts to modify this Zꢀolefinic bridge have been reported with the aim of preventing the
isomerization problem. This mostly included the synthesis of restricted CAꢀ4 analogs in which
1
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the Z double bond was incorporated into a heterocyclic system. In our ongoing effort to modify
this linkꢀbridge, we have recently reported a series of combretastatin Aꢀ4 analogs where isoCAꢀ4
(Figure 1) emerged as the most potent agents with a comparable antiproliferative activity to CAꢀ
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without the isomerization problem. IsoCAꢀ4 showed an excellent antitumor activity against a
1
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variety of cancer cell lines. Moreover, indolylꢀbased isocombretastatins also displayed potent
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cytotoxic activity (Figure 1). Recently, we have demonstrated that the nanocomposites formed
between squaleneꢀgemcitabine/isoCAꢀ4 induced complete regression of tumors xenografted on
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nude mice.
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A recent study has shown a synergic effect of the combination of microtubuleꢀdestabilizing
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agent vincristine and vorinostat (HDACi) in leukemia in vitro and in vivo, suggesting that
vorinostat may alter microtubule dynamics through HDAC inhibition. Consequently, the
discovery of a single molecule targeting both tubulin and HDAC proteins efficiently constitutes a
significant challenge. Based on these results, isocombretastatins derivatives (3a or
indoleisocombretastatin) and HDACi (vorinostat or belinostat) could be used as a good template
to design dual tubulin/HDAC inhibitors.
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In this study, we have rationally designed and synthesized a series of novel isocombretastatinꢀ
vorinostat or belinostat hybrids as dual antitumor agents. IsoCAꢀ4 and vorinostat were first
merged into new hybrid molecule 5aꢀb (Figure 1). Since structureꢀactivity relationship studies on
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isoCAꢀ4 derivatives have shown that the substitution at the hydroxyl group of isoCAꢀ4 was
tolerable, the linker bearing the hydroxamic acid group was attached to this position. Secondly,
to probe the impact of the length and the nature of the linker on the activity, an olefinic linker
derived from belinostat was used (5eꢀg), as well as an acetylenic linker 5cꢀd (Figure 1), and we
finally tested the direct connection (no linker) between isoCAꢀ4 and the zincꢀbinding group
(
ZBG). Third, a benzamide motif 5i was introduced replacing the hydroxamic acid ZBG. Lastly,
in order to validate the importance of the ring B of isoCAꢀ4 in the newly designed molecules, a
series of indoleisocombretastatin were also designed and synthesized. These modifications are
very interesting in terms of ligandꢀprotein interactions, assessing new binding possibilities, as
well as activity modulation.
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Figure 1. Chemical structures of CAꢀ4, isoCAꢀ4, and design of chimeric tubulin and HDAC
inhibitors.
RESULTS AND DISCUSSION
Chemistry
Nꢀtosylhydrazones were used as a source for the in situ generation of diazo substrates in the
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presence of a base. Recently, Nꢀtosylhydrazones have emerged as a new type of versatile
coupling partners for transition metalꢀcatalyzed crossꢀcoupling reactions, making this type of
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crossꢀcoupling reactions practical in organic synthesis. NꢀTosylhydrazones are easily derived
from the corresponding ketones or aldehydes. Therefore, we found this methodology was
perfectly suited for the preparation of the 1,1ꢀdiaryl ethylene scaffold of isoCAꢀ4, as it results in
the formation of the requisite double bond in a oneꢀstep procedure, in comparison to the classical
Wittig reaction which requires many steps for synthesis.
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As depicted in Scheme 1, the key intermediate isoCAꢀ4 was prepared in two steps. First, the
coupling of the corresponding Nꢀtosylhydrazone derivative 1a and aryl halide 2a under our
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previously reported conditions, using PdCl (MeCN) /dppf system. Secondly, the deprotection
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of OTBS group in the presence of K CO in MeOH led to the formation of compound 3a, in
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good overall yield of 71%. The reaction of the phenol group of 3a with alkyl halides gave the
corresponding alkyl aryl ethers 4a-c, in an 82, 97 and 85% yields respectively, which were
converted quantitatively to the desired hydroxamic acids 5a, 5b, and 5c.
Scheme 1. Synthesis of esters derivatives 4a-c and compounds 5a-c.
The synthesis of compounds 5d−j was carried out as shown in Scheme 2. Key intermediates
3bꢀd were prepared as above using Nꢀtosylhydrazone partner 1a and diꢀhalogenated aryl or
heteroaryl derivatives 2bꢀd. In the case of electrophilic pyridine partner, the dppf ligand was
found to be more efficient than Sphos.
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Compounds 4dꢀe containing alkyne function in metaꢀposition of isoCAꢀ4 were obtained by
using standard Sonogashira coupling of intermediate 3b with the corresponding alkynes in
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excellent yield. Treatment of ester 4dꢀe with hydroxylamine gave the desired hydroxamic acid
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derivatives 5dꢀe.
The acrylates derivatives 4f−h were obtained in good yield by reacting the corresponding aryl
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halides 3bꢀd with methyl or ethyl acrylate, using MizorokiꢀHeck crossꢀcoupling. Those
compounds were converted into compounds 5f−h by treatment with NH OH in MeOH. To attach
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the hydroxamic acid directly on the aromatic ring of the isoCA4, we initially prepared, ester 4i
by reaction between aryl bromide 3b and ethyl chloroformate. This key intermediate 4i was used
subsequently for the preparation of hydroxamic derivative 5i, but also for the formation of
benzamide derivative 5j by reaction with oꢀphenylenediamine in the presence of EDCI/HOBt.
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MeO
MeO
Br
I
I
Br
OMe
OMe
OMe
OMe
Br
Method A
3b, 70%
NNHTs
+
2b
PdCl (MeCN) 5%, dppf 10%,
2
2
MeO
MeO
LiO-t-Bu 2,2 éq, dioxane 100°C,
MeO
MeO
N
Br
N
sealed tube
from 2c,
method A
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OMe
OMe
Method B
Pd(OAc)2 (5 mol %),
SPhos (10 mol %),
OMe
1a
2c
3c, 74%
Br
Br
I
LiO-t-Bu 2,2 éq, dioxane 100°C
MeO
MeO
OMe
2d
OMe
O
OMe 3d, 78%
O
n
O
MeO
MeO
n
MeO
MeO
n
OEt
NHOH
OEt
NH OH/MeOH
3
b
2
OMe
OMe
PdCl (PPh ) /CuI
2
3 2
OMe
Et N/THF
OMe
n = 2; 5d, 85%
n = 3; 5e, 82%
3
n = 2; 4d, 95%
n = 3; 4e, 73%
O
O
O
MeO
OMe
MeO
MeO
OMe
NH OH/MeOH
NHOH
2
3b
MeO
OMe
Pd(OAc) /P(o-Tol)
2
3
OMe
OMe
Et N/THF, sealed tube
OMe
f, 82%
3
OMe
5f, 70%
4
O
O
O
MeO
MeO
N
OMe 1) NaOH/EtOH
MeO
MeO
N
OMe
NHOH
3c
OMe
OEt
Pd(OAc) /P(o-Tol)
2) EDCI, HOBt
DMF/TEA
NH2OH
2
3
Et N/THF, sealed tube
OMe
3
a
OMe
4g
5g, 58%
O
O
NHOH
O
MeO
MeO
MeO
MeO
OEt
NH OH/MeOH
2
3d
Pd(OAc) /P(o-Tol)
OMe
OMe
2
3
Et N/THF, sealed tube
OMe
3
OMe
a
4
h
5h, 69%
O
O
O
MeO
MeO
OEt
OMe
MeO
MeO
Cl
OEt
NH2OH/MeOH
NHOH
OMe
3b
tBuLi/THF
OMe
4
i, 36%
OMe
OMe
5
i, 77%
H N
O
2
1
) KOH
MeO
MeO
2
) o-Phenylenediamine
EDCI, HOBt, DMF/TEA
N
H
OMe
5j, 60%
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5
5
5
5
5
5
5
6
a
Scheme 2. Synthesis of compounds 4d-i and 5d-j. The compound was used for the next step
without any purification.
Next, we used our standard conditions to perform the coupling of Nꢀtosylhydrazone 1a and
indole derivatives. The reaction is carried out under simple conditions and quickly leads to
coupling products in less than an hour. The electrophilic partner is changed each time in order to
vary the nature and the position of the substituent on the indole ring B, with the aim of studying
the influence of substituents on biological activity.
0
1
2
3
4
5
6
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9
0
1
2
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of indolesꢀesters derivatives 4k-o. The compound was used for the next
step without any purification.
Hydroxamic derivatives of indoles 5kꢀo were synthesized as illustrated in Scheme 4. Ester 4kꢀn
were converted into acids under basic conditions. Then, the coupling with hydroxylamine in the
presence of EDC/HOBt gives the desired products in moderate yield after HPLC purification.
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0
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0
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4
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8
9
0
1
2
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4
5
6
7
8
9
0
Scheme 4. Synthesis of indoles derivatives 5k-o
Biological results
In vitro antiproliferative activity
The synthesized compounds were assayed by MTS method for growth inhibition of human
colon carcinoma cells (HCT116). With the aim of determining IC values, a treatment at eight
5
0
different concentrations was used to determine the IC values for each compound in comparison
5
0
to the control compound isoCAꢀ4. The summarized results in Table 1 show that most of the
tested compounds had a cytotoxic activity at micromolar concentrations or lower. Compounds
evaluated in their ester form (cf, 4aꢀe), except for the compound 4f which displayed low antiꢀ
proliferative activity on this cell line. An overall decrease in activity has been identified as the
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5
6
chain length of the linker reaches over five carbons seen with the compounds (cf, 4a vs 4b; 4b vs
4
c; 4d vs 4e). The optimal activity was obtained with 3 carbons (compound 4f).
However, potency was gained by switching from ester to hydroxamic acid group (cf, 5a vs 4a;
b vs 4b; 5c vs 4c; 5d vs 4d; 5e vs 4e and 5f vs 4f).The compound bearing oꢀphenylenediamineꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
based zinc binding group 5j was turned out to be less active than the hydroxamic acid derivative
cf, 5j vs 5i). Above all 1,1ꢀdiarylethylene derivatives (4aꢀ5j), compounds 5d, 5f, and 5h
(
exhibited the best antiꢀproliferative activities. Compound 5f with an Nꢀhydroxycinnamamide
linker, in meta position, displayed more potent activity than its counterpart 5h. The introduction
of a pyridine ring was probed systematically as a bioisostere of the phenyl ring. However, in our
case, this modification led to a drop in the activity (cf, 5f vs 5g). Also, phenyl ring can be often
replaced by a heteroaromatic ring. Accordingly, some new analogs of indoleꢀisocombretastatin
were also synthesized and evaluated. Again, the length and the position of the linker on the
indole ring was studied, the results have shown that the best activity was obtained once more
with an Nꢀhydroxycinnamamide linker (cf, 5m, and 5n vs 5kꢀl, 5o). In addition, positioning this
linker on C3 of the indole was more favorable for the activity than C2 position (cf, 5n vs 5m).
Inhibition of tubulin polymerization.
Compounds 4 and 5 were next evaluated to examine whether these derivatives were interacting
with microtubules (Table 1). The results have clearly indicated that tubulin is the intracellular
target of dual 1,1ꢀdiarylethylenes and indoleꢀisocombretastatin analogs. Indeed, all efficient antiꢀ
proliferative compounds were potent inhibitors of tubulin assembly with micromolar IC values.
5
0
It is important to note that indoleꢀisocombretastatin analogs (5kꢀo) exhibited less effective IC50
values against tubulin than their 1,1ꢀdiarylethylenes analogs (5d, 5f, and 5h).
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a
Table 1. Cytotoxic activity of compounds 4aꢀf and 5aꢀo against HCT116 cells.
compd
GI50
HCT116
IC50
ITP
(µM)
compd
GI50
HCT116
(nM)
IC50
ITP
(µM)
b
c
b
c
(
nM)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
a
a
150 ± 20
22 ± 0.58
5 ± 0.5
2.4 ± 0.2
d
5f
1.5 ± 0.3
1.6 ± 0.3
7.5 ± 1.2
5g
37.2 ± 0.6
4
b
1000 ± 200 NA
5h
8 ± 0.02
2.1 ± 0.4
20 ± 1.2
d
5
4
b
c
500 ± 52
NA
5i
5j
233 ± 22
340 ±2
d
d
3000 ± 150 NA
NA
d
d
5
c
NA
5k
5l
36 ± 4
81 ± 15
2
500 ± 100
4d
5d
1000 ± 100 NA
30 ± 1.5
7.3 ± 3.3
8 ± 2
1.7 ± 0.3
5m
5n
20 ± 2.5
14 ± 0.3
5.4 ± 1.5
3.7 ± 0.5
d
4e
5e
3000 ± 100 NA
372 ± 5
85 ± 4
18 ± 1.2
5o
393 ± 4.1
2 ± 0.1
22 ± 2
4
f
4 ± 0.4
isoCAꢀ4
2.0 ± 0.3
a
b
HCT116 human colon carcinoma cells. Compound concentration required to decrease cell
c
growth by 50%; values represent the average ± sd of three experiments. IC50 dis the
concentration of a compound that inhibits 50% of the rate of tubulin polymerization. NA=
not active at 200 µM.
Among all tested analogs, compounds 5f and 5h were as active as isoCAꢀ4 against tubulin with
an IC value of 1.5 and 2 ꢁM respectively. This reinforces our idea that the phenyl ring is
5
0
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5
6
important and more tolerable than other heterocycles for potent antiꢀcancer activity in relation to
a significant involvement of βꢀtubulin.
Inhibition of HDAC.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
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9
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0
1
2
3
4
5
6
7
8
9
0
These preliminary cytotoxicity and inhibition of tubulin assembly results have shown that
derivatives 5d, 5f, 5h, 5m and 5n (IC ≤ 20 nM) are promising drugs in this novel series. In this
50
context, we next investigated their effects on the inhibition of HDAC. Profiling of these
compounds was performed by Eurofins Cerep S.A. Compounds showing higher inhibition (or
stimulation for assays run in basal conditions) than 50% are considered to represent significant
effects of the test compounds. For example, in the enzyme assays, compound 5f have shown an
inhibition above 50% on HDAC8 (95% at 1Eꢀ05 M), and HDAC11 (60% at 1Eꢀ05 M) (Figure
2
). At the highest tested concentration, 5f caused light (< 50%) or no inhibition of other studied
HDAC activities. On this basis, we have determined the IC for compound 5f on HDAC8 and
5
0
HDAC11.
At this point, testing the effect of our reference isoCAꢀ4 on the inhibition of HDAC's seemed
necessary to rule out the possibility of the effect being inherent in the tested structure. As
expected, isoCAꢀ4, not bearing a hydroxamic acid function (zinc chelator) does not inhibit the
ꢀ5
different HDACs, even at a high concentration of 10 M (Figure 2).
Accordingly, we continued our in vitro HDAC screening by examining the IC50 values of
compounds 5d, 5f, 5h, 5m and 5n against recombinant human HDACs 6, 8, and 11. The results
27
of these studies are summarized in Table 2. The most studied HDACi trichostatin A (TSA),
Scriptaid and specific HDAC8i (PCIꢀ34051) were used as a control. Overall, all tested
compounds proved to be selective for HDAC8. Compounds 5f and 5n showed a close activity to
TSA, and compound 5h possess a better HDACi activity compared to TSA. The SAR studies
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revealed that the conjugate bearing a long linker (C5, compound 5d) have a weak inhibition
activity while the Nꢀhydroxycinnamamide linker seems to have the optimal length. Also, we can
notice the importance of the position of the linker on the aromatic ring (cf, compound 5f vs 5h
and 5m vs 5n). These results are very encouraging, taking in account that the isozyme HDAC8 is
involved in the pathogenesis of neuroblastoma and that its expression significantly correlates
1
1
1
1
1
1
1
1
1
1
2
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2
2
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2
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3
3
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4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
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8
9
0
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4
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0
1
2
3
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8
9
0
1
2
3
4
5
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9
0
1
2
3
4
5
6
7
8
9
0
2
8
with advanced tumor stage.
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9
0
1
2
3
4
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8
9
0
Figure 2. Inhibition profile of selected compounds against HDAC isoforms 1ꢀ11. Selected
ꢀ5
compounds were used at 1.0 M.
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Table 2. In vitro inhibition of recombinant HDACs 6, 8 and 11 for selected compounds.
IC50 (nM)
Compds
HDAC6
HDAC8
HDAC11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
5d
5f
Nt
Nt
11000 ± 520
15000 ± 1300
5000 ± 45
3800 ± 30
6900 ± 120
15 ± 1.4
340 ± 26
60 ± 10
10000 ± 850
8000 ± 100
5
h
a
5
m
6200 ± 100
240 ± 20
280 ± 20
12 ± 2.2
Nt
5
n
5000 ± 100
a
TSA
Nt
b
a
PCIꢀ34051
Scriptaid
3100 ± 30
Nt
a
a
Nt
Nt
22000 ± 1000
c
c
c
isoCAꢀ4
Na
Na
Na
a
b
c
Nt = not tested. PCIꢀ34051 (CAS number = 950762ꢀ95ꢀ5). Na = not active.
Cytotoxic effect of compounds 5f and 5h on different tumor cell lines.
To further determine the potential of these compounds, the most cytotoxic and HDAC8i
derivatives 5f and 5h were evaluated on nine different cancer cell lines, which include human
lung epithelial cells (A549), chronic myeloid leukemia cells (K562), doxorubicinꢀresistant K562
cells (K562R), human prostate cancer cells (PC3), human glioblastoma (U87ꢀMG), breast cancer
cells (MCF7), human primary pancreatic adenocarcinoma (BXPC3), human pancreatic
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2
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2
2
2
2
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5
5
5
5
5
5
5
5
6
carcinoma (MiaPaca2), and colon carcinoma cell lines (HT29). IsoCAꢀ4, TSA and PCIꢀ34051
were included as reference compounds for comparison. As depicted in Table 3, all of these
compounds displayed good antiꢀproliferative potency with GI values ranging from 0.4 to 80
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nM. Hybrid compounds 5f and 5h showed a higher antiꢀproliferative activity on all studied cells
lines compared to the HDACi (TSA) and to the representative HDAC8 selective inhibitor PCIꢀ
3
4051. The compound 5h was less active in term of cytotoxicity in comparison to the reference
isoCAꢀ4. On the other hand, compound 5f displayed more antiꢀproliferative efficiency compared
to the controls compounds isoCAꢀ4, TSA or PCIꢀ34051. Interesting activity was obtained with 5f
against doxorubicinꢀresistant K562R cells (60ꢀ and 2ꢀfold higher than TSA and isoCAꢀ4,
respectively). The difference is even more important in the case of human glioblastoma U87ꢀ
29
MG (650ꢀ and 5ꢀfold higher than TSA and isoCAꢀ4, respectively). The screening results also
revealed that compound 5f has the most potent inhibitory activity against human prostate cancer cells
(PC3) cells (IC = 0.4 nM). This could be explained by the presence of an Nꢀhydroxycinnamamide
50
linker on meta position of the 1,1ꢀdiarylethylene which has an optimal inhibition of microtubule,
and HDAC, resulting in high antiproliferative properties. In comparison to selective HDAC8i PCIꢀ
3
405, hybrid molecule 5f has a much higher antiproliferative activity on all tested tumor cell
lines. This proves that the combination of the two effect: inhibition of tubulin polymerization and
HDACi is beneficial for the antiproliferative effect.
To highlight the efficiency of these dual compounds, we investigated their potential in CAꢀ4
30
refractory human colon adenocarcinoma cell line (HTꢀ29, IC = 9137 nM). HTꢀ29 cells are
5
0
3
1
resistant to CAꢀ4 due to the overexpression of the multidrugꢀresistance protein (MRPꢀ1). As
expected in Table 3, HTꢀ29 cells were less sensitive to the combretastatin analogue isoCAꢀ4 with
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an IC of 276 nM. TSA showed also a moderate activity. To our delight, compound 5f showed a
5
0
marked activity and was l35ꢀfold more active than isoCAꢀ4 and 17ꢀfold than TSA in the HTꢀ29
cells suggesting a clear and definite advantage of this dual molecule (Figure 3). One can note
that TSA (IC = 34 nM) seems to be less potent than 5f (IC = 2 nM), but more effective (%
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
cell viability <20%). Also, to confirm the beneficial effect of this dual compound (5f), we carried
out a control study (Figure 3), in which, we did a comparison between the antiproliferative effect
on HTꢀ29 cells of isoCAꢀ4, TSA, 5f, 5h, and a combination of isoCAꢀ4 + TSA. As shown in
figure 3, reference compound (isoCAꢀ4) was nearly inefficient against this cell line (IC = 276
5
0
nM). Compound 5h and TSA led to a moderate antiproliferative activity with an IC = 63 nM
5
0
and 34 nM respectively. The combination of isoCAꢀ4 + TSA led to a significant improvement of
the antiproliferative activity in comparison to isoCAꢀ4 and TSA used separately, with an IC =
5
0
3
.8 nM. To our delight, the effect of dual compound 5f was as active as the combination of
isoCAꢀ4 + TSA with an IC = 2.04 nM. These results are in favor of a synergetic effect of the
5
0
hybrid compound 5f. The advantage of this hybrid molecule permit to avoid administrating the
combination of TSA and isoCAꢀ4.
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9
1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Table 3. Cytotoxic activity of selected derivatives against human tumor cell lines
GI (nM)
50
Compds
HCT116
A549
K562
K562R
PC3
U87
MCF7
BXPC3
MiaPaca2
HT-29
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
f
1.5 ± 0.3
1.7 ± 0.5
1.6
±
1.4 ± 0.1
0.4 ± 0.1
28 ± 0.2
1.6
±
2 ± 0.1
2.9 ± 0.08
5.1 ± 0.2
2.04 ± 0.25
0.04
0.02
5
h
8 ± 0.02
2 ± 0.2
22 ± 1.1
10 ± 0.1
20 ± 0.5
80 ± 0.5
2.2 ± 0.8
11 ± 1.4
8 ± 0.3
17 ± 0.1
8 ± 0.4
25 ± 0.3
20 ± 1.2
18 ± 0.4
10 ± 1.2
63.6 ± 6.28
276 ± 56
isoCAꢀ4
5.2 ± 0.2
3.0
±
0
.05
PCI-
2640 ±
1130
> 20000
80 ± 10
2010 ±
850
2200
±
2660
1100
±
> 15000
2970
2700
45 ± 5
±
> 20000
100 ± 36
> 20000
200 ± 60
> 10000
b
3
4051
1270
TSA
35 ± 0.7
262 ± 7.4
88 ± 0.14
387 ± 88
1038 ± 2
34.1 ± 2.96
a
GI = Compound concentration required to decrease cell growth by 50%; values represent the
5
0
b
average ± sd of three experiments. PCIꢀ34051 (CAS number = 950762ꢀ95ꢀ5).
1
0 0
Iso C A 4 + T S A
TSA
8
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0
0
0
0
0
Iso C A 4
5
5
f
h
-
1 1
-1 0
-9
-8
-7
-6
-5
lo g c o n c e n tra tio n (M )
Figure 3. Effect of TSA, isoCAꢀ4, TSA + isoCAꢀ4, 5f and 5h on the HTꢀ29 cells.
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HDACi are wellꢀknown for their ability to trigger H2AX phosphorylation (γH2AX),
a
hallmark of DNA doubleꢀstrand breaks. Therefore, we examine whether compounds 5f and 5h,
which are able to inhibit HDAC activities, modulate γH2AX level. Compared to mockꢀtreated
cells, HT29 cells treated with compound 5f or 5h led to γH2AX induction (Figure 4).
Interestingly, increase in drug concentration led to a doseꢀdependent increase in γH2AX.
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Figure 4. γH2AX induction after treatment of colon carcinoma cells with compound 5f and 5h.
HT29 cells were mockꢀtreated or treated with compounds at the indicated doses. After 3 days,
total H2AX, phosphorylated of H2AX (γH2AX) and actin were measured by western blot. The
numbers indicate the level of γH2AX relative to mockꢀtreated cells
In addition to in vitro studies, HT29 colon carcinoma cell line was treated with compounds 5f
and 5h as well as the reference compound PCIꢀ34051, an HDAC8 specific inhibitor and the cell
lysates were analyzed by western blot. Compared to mockꢀtreated cells, cells treated with 5f and
5h displayed a strong increase in acetylation of SMC3, a wellꢀknown HDAC8 substrate, the
increase being more prominent after 24h of treatment (Figure 5A). In contrast, no significant
modification of α tubulin (a substrate of HDAC6) acetylation was found (Figure 5B).
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Altogether these data demonstrate that compounds 5f and 5h are able to inhibit selectively
HDAC8 while avoiding inhibition of HDAC6.
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Figure 5. Compounds 5f and 5h are both able to inhibit HDAC8 activity on SMC3 protein.
HT29 colon carcinoma cells were mockꢀtreated or treated with compounds 5f, 5h or PCIꢀ34051
(25 µM) and cell lysates obtained after 6 or 24h were analyzed by western blot. (A) acetylated
SMC3 and total SMC3; (B) acetylated α tubulin and total α tubulin. The numbers indicate the
level of the acetylated form of a given protein relative to total level of the same protein.
Molecular modelling
Compounds 5f and 5h displaying the best antiproliferativeꢀactivity and effective inhibitions of
both the tubulin polymerization and HDAC8 were selected for molecular modelling study.
To elucidate the potential interactions of these compounds with the tubulinꢀmicrotubule
system, docking experiments have been achieved in the colchicine binding site of tubulin (Figure
33
6
). For compound 5f, classical hydrogen bond interaction between the Cysβ241 and the
3
4
trimethoxyphenyl motif has been identified. Three additional hydrogen bonds were also
observed between the hydroxamic chain of 5f, and the side chains of Tyrα224, Glnα11, and the
carbonyl of the backbone of Glnβ247. The side chain of Leuβ248 also established two CH/π
interactions with the two aromatic rings of 5f.
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Switching the linker from meta to orthoꢀposition, in the molecule 5h led to a new orientation
of the molecule in the colchicine binding site due to the steric hindrance near the double bond. A
hydrogen bond between the methoxy group of the Bꢀring of 5h and the side chain of Thrα179
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was observed. The carbonyl group of the hydroxamic acid interacts with NH function of the
backbone of Serα178, while the hydroxyl group of the hydroxamic acid establishes two
hydrogens bonds with the Thrβ353. Two CH/π interactions were also observed: one with the side
chain of Leuβ248 and the aromatic Bꢀring of 5h, and the other between the OMe group of the Aꢀ
ring and the phenyl ring of Tyrα224.
Figure 6. Putative binding mode of compounds 5f, shown in orange sticks (left), and 5h, shown
in blue sticks (right) in the colchicine binding site of tubulin (code PDB: 3HKC). Protein
residues involved in the interaction are shown in yellow sticks (β chain) and pink sticks (⍺
chain), Hꢀbond as blue dashed lines, apolar interactions as yellow dashedꢀlines.
In HDAC8, as expected, the hydroxamic acid group of compounds 5f and 5h coordinates the
zinc atom in the catalytic site of HDAC8 (Figure 7). Compound 5f establishes four hydrogen
bonds to the side chain of His142, His143, Asp178, and His180. Moreover, a CH/π interaction
was detected between the side chain of Met274 and the aromatic Aꢀring of 5f. Similarly,
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compound 5h establishes three hydrogen bonds with His142, His143, and His180, and an
additional hydrogen bond was also detected between the OMe group of Bꢀring and NH function
of the backbone of Gly151.
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Figure 7. Putative binding mode of compounds 5f (left) and 5h (right) in the binding site of
HDAC8 (code PDB: 1T69). Protein residues involved in the interaction are shown in green
sticks, zincꢀion as gray spheres, Hꢀbond as blue dashed lines, CH/ꢀ interactions as yellow
dashedꢀlines. For clarity purposes, ꢀ ꢀ ꢀ stacking between compounds and Phe152 and Phe208
are not depicted here.
Effect of compound 5f on the cell cycle
Since compound 5f displayed the best antiproliferative activity, we have selected it for the cell
cycle studies. As shown in Figure 8, compound 5f had a dramatic effect on the cell cycle,
inducing cell cycle arrest at the G /M phase in both HCTꢀ116 and K562 cells. The arrest of the
2
cell cycle was observed even at low concentration (5 nM).
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Figure 8. Effects of compound 5f (blue) on cell cycle distribution in a) HCT116 and b) K562
cells at 5 and 10 nM, as determined by flow cytometry analysis (DMSO control in red). DNA
content was assessed via propidium iodide staining.
Cell death type by compound 5f
Antiproliferative activity and cell cycle arrest at the G /M phase was also observed for Burkittꢀ
2
lymphoma (BL2) cells after treatment by compound 5f. As expected, again compound 5f showed
an interesting antiproliferative activity against this cell line with an IC = 10 nM. To further
5
0
characterize cell death type we analyzed phosphatidylserine externalization, an early event of
apoptosis. Compound 5f behaves like a potent inducer of apoptosis, (Figure 9), a type of death
that avoids inflammatory phenomena during antiꢀtumor treatments. Compound 5f seems to have
a much more important cytotoxic effect on BL2 cells than the HDACi vorinostat and it was as
toxic as the mitotic spindle poison isoCAꢀ4.
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Figure 9. Burkittꢀlymphoma BL2 cells were incubated with vorinostat, isoCAꢀ4 or compound 5f
at 10 nM for 48h. Percentage of apoptotic cells was evaluated by flow cytometry after labelling
of external phophatidyl serine residues (AnnexinꢀVꢀpositive cells). Plasma membrane integrity is
maintained during early apoptosis (propidium iodide negative cells).
Evaluation of cytotoxicity in human non-cancer cells
To demonstrate the safety profile of compound 5f, and to obtain a preliminary indication of the
cytotoxic potential of this compound for normal human cells, we evaluated its activity on
quiescent peripheral blood lymphocytes (PBL) isolated from two different healthy donors, and
compared to isoCAꢀ4 and vorinostat. In our hands, doseꢀresponse curves yielded an IC value of
5
0
1
.7 µM for isoCAꢀ4, 3.4 µM for vorinostat and 7.0 µM for compound 5f. These results indicate
clearly the safety profile of 5f and confirm that this compound is not more cytotoxic than isoCAꢀ
or vorinostat in quiescent PBLs.
4
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Immunofluorescence studies
Considering the importance of the tubulinꢀmicrotubule system in the maintenance of cellular
morphology and basic cellular functions, an assay involving the disruption of microtubule
network was performed in order to reveal whether compound 5f could affect microtubule
morphology in living cells. As shown in Figure 10, in the control experiment (DMSO), the
microtubule network in the K562 cells exhibited normal organization, with microtubules
extending from the central regions of the cell to the cell periphery. In contrast, after the exposure
to 1 nM of compound 5f for 24h, the spindle formation showed distinct abnormalities and was
heavily disrupted. The changes of the mitotic spindles were more clearly observed by increasing
the concentration to 10 nM. In this case, the microtubule spindle has significantly shrunk, and a
multinucleation phenomenon was also observed. These results have confirmed that compound 5f
has dramatically disrupted the microtubule organization, and has interfered with the mitosis of
K562 cells at low concentrations.
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Figure 10. Effect of 5f on microtubule network organization. K562 cells were treated with 5f for
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4 h. After incubation, the cells were fixed and stained with monoclonal βꢀtubulin antibody
(red), cell nuclei were stained with DAPI (blue).
Physicochemical properties
In order to ascertain the drugꢀlikeness of our products, physicochemical properties of compounds
5
f and 5h were assessed in parallel with the reference compound isoCAꢀ4. Compounds 5f and 5h
have shown to conform to Lipinski’s rule of five (Table 4). Additionally, 5f and 5h have shown
an improved aqueous solubility compared to isoCAꢀ4 indicating that the presence of the
hydroxamic acid moiety fosters water solubility. As predicted, the experimental logP values were
in the desirable druglike range (1 < logP < 3) assuring a good lipophilicꢀhydrophilic balance.
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Therefore, our findings showed that the presence of hydroxamic group could improve aqueous
solubility with a comparative antiꢀproliferative activity of the isoCAꢀ4.
Table 4. Water solubility and physicochemical properties of compounds 5f, 5h and isoCA-4
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a
b
c
d
e
f
g
Cmpd.
MW
HBA
HBD
cLogP
logP
tPSA
Water solubility (ꢀg/ml)
isoCA-4
316.35
385.42
385.42
<450
5
1
2.86
2.16
2.93
2.13
<5
57.16
86.26
86.26
11.62
5
f
7
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2
2
2.84
2.84
<5
69.28
88.33
5
h
h
RO5
<10
<5
<90
a
b
c
MW: molecular weight; HBA: hydrogenꢀbond acceptor atoms. HBD: hydrogenꢀbond donor
d
e
atoms. clogP: calculated logarithm of the octanol− water partition coefficient. tPSA:
topological polar surface area, calculated using http://www.molinspiration.com/cgiꢀ
f
g
h
bin/properties. Water solubility. Experimental values of logP. RO5: Lipinski’s rule of five.
CONCLUSION
To conclude, we reported the discovery of a new series of dualꢀtargeting inhibitors as novel
antiꢀtumoral agents. IsoCAꢀ4 and belinostat were firstly merged into a new hybrid molecule. A
series of a grafted linker bearing a zinc binding group onto the 1,1ꢀdiarylethylene scaffold were
designed, synthesized and evaluated as multiꢀtarget inhibitors of tubulin and HDAC. Among all
synthesized compounds, 5f and 5h exhibited the most potent antiꢀproliferative activity with an
IC50 in the nanomolar range. Moreover, they showed a potent inhibitory activity against the
tubulin assembly and HDAC8. The most active compound 5f induced the cell cycle arrest of
cancer cells at the G /M phase by dramatically disrupting the microtubule organization.
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