Design and synthesis of novel dual-target agents for HDAC1 and CK2 inhibition

Article abstract of DOI:10.1039/c6ra09717k

Drug entities able to address multiple targets can be more effective than those directed to just one biological target. We disclose herein a series of novel dual inhibitors to target histone deacetylase 1 (HDAC 1) and protein kinase CK2. Our bifunctional compounds combine two complementary chemo-active prototypical scaffolds: a hydroxamate essential for the chelation of the zinc ion present in the active site of HDAC (Zinc Binding Group), and a 4,5,6,7-tetrabromobenzotriazole (TBB) moiety introduced to interact with the ATP binding site in CK2 and to act simultaneously as the cap group in the interaction with HDAC1. The synthesized dual-acting agents exhibited promising inhibitory activities towards HDAC1 and CK2. The best result was obtained for 5c with an IC₅₀ of 5 μM for both enzymes. However, its N-2 substituted isomer 5e presented the best profile in cell-based assays, with cytotoxic activity in the low micromolar LC₅₀ in two mammalian cancer cell lines and 4-fold less activity towards a pseudonormal mammalian cell line. Furthermore, this hybrid molecule induced apoptosis in leukemia cells in a concentration-dependent manner. All together this makes 5e a promising lead compound for future in vivo assays in animal tumor models.

Full text of DOI:10.1039/c6ra09717k

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Design and synthesis of novel dual-target agents
for HDAC1 and CK2 inhibition†
Cite this: RSC Adv., 2016, 6, 66595
M. Purwin,ab J. Hernandez-Toribio,a C. Coderch,a R. Panchuk,c N. Skorokhyd,c
´
a
a
a
*
*
K. Filipiak, B. de Pascual-Teresa and A. Ramos
Drug entities able to address multiple targets can be more effective than those directed to just one
biological target. We disclose herein a series of novel dual inhibitors to target histone deacetylase 1
(HDAC 1) and protein kinase CK2. Our bifunctional compounds combine two complementary chemo-
active prototypical scaffolds: a hydroxamate essential for the chelation of the zinc ion present in the
active site of HDAC (Zinc Binding Group), and
a 4,5,6,7-tetrabromobenzotriazole (TBB) moiety
introduced to interact with the ATP binding site in CK2 and to act simultaneously as the cap group in the
interaction with HDAC1. The synthesized dual-acting agents exhibited promising inhibitory activities
towards HDAC1 and CK2. The best result was obtained for 5c with an IC50 of 5 mM for both enzymes.
However, its N-2 substituted isomer 5e presented the best profile in cell-based assays, with cytotoxic
activity in the low micromolar LC50 in two mammalian cancer cell lines and 4-fold less activity towards
a pseudonormal mammalian cell line. Furthermore, this hybrid molecule induced apoptosis in leukemia
cells in a concentration-dependent manner. All together this makes 5e a promising lead compound for
future in vivo assays in animal tumor models.
Received 14th April 2016
Accepted 1st July 2016
DOI: 10.1039/c6ra09717k
www.rsc.org/advances
of action.1–3 However, the employment of these multi-
component drug cocktails can also introduce adverse effects
Introduction
related to pharmacokinetics, unpredictable drug–drug interac-
tions, toxicity and patient incompliance.4,5 To address these
limitations, an attractive and more elegant strategy in modern
cancer medicine is to design a single drug with efficient inhib-
itory activity for more than one biological target, while main-
taining lower side effects.6–8 In addition, from an efficacy
perspective, as well as in terms of cost reduction, the design of
multitarget drugs instead of discrete inhibitors is also of
increasing interest and prominence.9,10 In recent years, the
development of dual inhibitors has attracted a great deal of
attention among several research groups and successful exam-
ples have been published for the treatment of cancer, psychiatric
pathologies, inammation, diabetes and neurodegenerative
diseases.11,12
Histone deacetylases (HDACs) are a class of epigenetic
enzymes that have generated much interest in cancer therapy,
since their aberrant activity has been linked to cell proliferation
in a variety of human diseases, most notably myeloid neoplasia
and solid tumors.13–18 These enzymes catalyze the hydrolysis of
acetylated lysine side chains in histone and non-histone
proteins for transcriptional regulation, cell cycle progression,
and apoptosis.19,20 There are 18 human HDAC isoforms sub-
divided into four different classes (I–IV), among which classes I
(1, 2, 3 and 8), II (4–7, 9 and 10), and IV (11) are Zn2+-dependent
for enzymatic activity. Class I HDACs are ubiquitously expressed
and play essential roles in proliferation, while classes II and IV
Owing to the wide-range importance of chemotherapy as the
primary treatment of choice for most cancer cases, the devel-
opment of new efficient drug entities represents a challenge. To
date, signicant progress has been made in this area, although
most traditional cytotoxic agents are associated with severe
toxicities and other undesirable side effects. More recently,
combination cancer therapies have expanded the treatment
options, including the use of multiple drugs with different
pharmacological targets, which can directly achieve the desir-
able therapeutic outcome concertedly via different mechanisms
a
´
´
Departamento de Quımica y Bioquımica, Facultad de Farmacia, Universidad CEU San
´
Pablo, Urbanizacion Monteprincipe, 28668, Boadilla del Monte, Madrid, Spain.
E-mail: bpaster@ceu.es; aramgon@ceu.es; Tel: +34 913724777; +34 913724796
bDepartment of Organic Chemistry, The Faculty of Pharmacy with the Division of
Laboratory Medicine, Medical University of Bialystok, A. Mickiewicza 2A, 15-222,
Bialystok, Poland
cInstitute of Cell Biology, NAS of Ukraine, Drahomanov Str. 14/16, 79005 Lviv, Ukraine
† Electronic supplementary information (ESI) available: PyMOL representation of
the binding of a substrate-like molecule to HDAC-8 as found in 2V5W; the PyMOL
representation of the binding of an ATP analog and two bromobenzimiazole
derivatives to CK2; the PyMOL representation of the best docking poses of
compounds 5a to 5h bound to HDAC1 and CK2; , the graphical representation
of the number of choline atoms that interact with the vicinity of the Mg2+ atom
in the CK2 complexes; and the PyMOL superimpositions of the HDAC1
structure obtained with SWISSMODEL and the crystal structure deposited under
PDB code: 4BKX. 1H and 13C NMR spectra of compounds 5a–5h. See DOI:
10.1039/c6ra09717k
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have tissue-specic functions.21,22 The maintenance of equilib- a potential target for cancer treatment.46 Up to date many
rium between acetylation and deacetylation of histones and compounds have been reported as potent CK2 inhibitors.47,48
nonhistone substrates is essential for normal cell growth. As Among the CK2 inhibitors reported so far, ATP-competitive
a result, the use of HDAC inhibitors (HDACi) as cancer thera- compounds such as coumarin derivatives, ellagic acid, DMAT
peutics has emerged as an area of active investigation.23,24 By or TBB stand out for their high efficiency and availability.49–52
inducing histone hyperacetylation, these compounds alter gene More recently, the benzonaphthyridine derivative CX-4945 has
transcription and can exert antitumor effects through growth been reported to be a rst-in-class, orally bioavailable selective
arrest, differentiation and/or apoptosis.25,26 HDAC inhibition CK2 inhibitor in clinical trials (Fig. 1B).53,54 Hence, since both
has been consolidated as a strategy in epigenetic drug discovery, CK2 and HDAC are closely related relevant proteins in cancer
with 12 inhibitors currently in clinical trials and the FDA therapy, we envisioned that concurrent inhibition of these two
approvals of vorinostat (SAHA)27 and romidepsin (FK228)28 for enzymes, by the design of a novel potential multi-target single
treatment of cutaneous T-cell lymphoma (Fig. 1A).
molecule, might enhance drug efficacy and overcome the current
The use of HDACi in combination with other anticancer pharmacokinetic limitations, as well as likely provide benecial
agents has become a practical method to increase the efficien- additive or synergistic biological effects. Herein we describe our
cies of these agents. There are a few examples of this subtype of efforts toward this goal by reporting a new family of dual
bifunctional HDACi derived compounds as therapeutically inhibitors of CK2 and HDAC1. These chimeric compounds
viable anticancer agents.29–35 One particularly promising contain a TBB moiety as a capping group and a hydroxamic acid
approach is the modulation of receptor tyrosine kinase (RTK) as a zinc-binding group (ZBG), which have been linked using
pathways by the action of HDACs inhibitors, leading to potential a click chemistry approach (Fig. 1D).
synergistic effects.36,37 In this context, an attractive starting point
for a secondary target is protein kinase CK2, which is not only
responsible for the regulation of HDACs by post-translational
modication (particularly phosphorylation), but also appears
to be involved in HDAC activation in hypoxia-associated cancer
HDACis can be broadly described by a common pharmacophore,
processes, thus contributing to tumor angiogenesis.38–40
CK2 is a ubiquitously expressed, constitutively active and
highly conserved protein serine/threonine kinase essential for
Results and discussion
Design rationale
consisting of a cap group, an appropriate linker and a metal-
binding moiety (ZBG), being the latter the primary affinity
determinant that coordinates to the active site Zn2+ ion (Fig. 2A).
cell viability, which is composed of tetrameric complexes con-
Among the HDACis, hydroxamic acids exhibit the most potent
sisting of two catalytic subunits (CK2a, CK2a0) and a dimer of
efficacy since, aer ionization, an exceptionally stable 5-
regulatory b subunits.41,42 CK2 is a pleiotropic protein kinase
membered ring chelate with the active site Zn2+ ion is created.
with more than 300 substrates, which are localized in different
We postulated that appropriate conjugation of the surface
cell compartments and are implicated in various important
functions, including gene expression, cell growth and differen-
tiation, signal transduction and apoptosis.43,44 It has been
recently demonstrated that CK2 plays an important role in many
pathologies and abnormal high levels of its enzymatic activity
and also maintain its activity against the CK2 protein (Fig. 2B).
have been found in a large variety of cancer cells.45 Its prolifer-
recognition group (cap) of a prototypical HDACi to other
hydrophobic antitumor pharmacophore (e.g., TBB) could
furnish the desired bifunctional agents. Presumably, the TBB
moiety would act as a surrogate of the cap group in the HDACi
This assumption is supported by our previous results,55 where
we described some “clicked” TBB derivatives in which the
terminal group is an amino group instead of the hydroxamate
ative and anti-apoptotic properties create a favourable cellular
environment for tumor maintenance and progression, making it
Fig. 1 Representative examples of (A) HDAC, (B) CK2 inhibitors, (C)
“clicked” CK2 inhibitors, and (D) chemical structure of one of the Fig. 2 (A) Prototypical structure of HDACi, as exemplified by SAHA. (B)
designed dual inhibitors.
Our click chemistry approach toward bifunctional inhibitors.
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present in these new dual-targeting compounds. Those
compounds (see Fig. 1C) showed Ki values in the low mM range
and high degree of selectivity against a panel of 24 kinases.
Chemistry
A series of HDAC-CK2 dual inhibitors has been synthesized. Our
approach involves a Cu-catalyzed azide–alkyne 1,3-dipolar
cycloaddition (CuAAC)56 as the crucial step to couple both CK2
and HDAC inhibition scaffolds. As shown in Fig. 2B, the
designed inhibitor has a triazole-alkyl linker, resulting of the
click connection of the azide bearing the ZBG and the corre-
sponding TBB-alkyne moiety.
Scheme 2 Reagents: (a) NaN3, DMF, 60 ꢁC, 12 h; (b) NH2OTHP, HOBt,
EDCI, NMM, DMF, rt, 12 h.
For any synthetic method to be useful, the substrates must
be readily accessible. As depicted in Scheme 1, alkyne reagents 1
and 2 were prepared by alkylation of TBB with the corre-
sponding alkynyl bromide in the presence of K2CO3.57,58 While
derivative 1 was obtained as a single product aer the reaction
with propargyl bromide, the presence of an extra carbon in the
bromide reagent resulted in the formation of both N-1 (2a) and
N-2 (2b) alkylated isomers. Fortunately, they were successfully
isolated by column chromatography and further tested sepa-
rately to evaluate the inuence of the N-substitution pattern in
the inhibitor structure.
Likewise, azides with the hydroxamate function were easily
obtained in two steps from the corresponding bromo-derived
carboxylic acids (Scheme 2). Thus, the synthesis of the inter-
mediates 3a–d was accomplished by standard azide formation
starting from the corresponding bromide. Further reaction of
the acid group with O-THP hydroxylamine by EDC coupling led
to the desired aliphatic and aromatic azides 4a–d.
Scheme 3 Reagents: (a) CuSO4 (10 mol%), Na-ascorbate (30 mol%),
DMF, rt, 24 h; (b) 4 N HCl in dioxane, MeOH, rt, 2 h.
To examine the effect of the linker length on both sides of
the triazole ring, TBB derivatives 1 and 2a–b as well as azides
4a–c were selected for the click reaction.
The CuAAC reaction is a regioselective process, forming
exclusively the 1,4-substituted adduct.59 Accordingly, we found
that only one isomer was obtained, thus achieving the triazole-
based conjugates in excellent yields (Scheme 3). We identied
the use of CuSO4/Na-ascorbate catalytic system and DMF as the
optimized conditions for the cycloaddition step. Subsequent
removal of the THP protecting group furnished the nal
hydroxamic acid-based dual inhibitors 5a–f.
Compounds 5g–h were selected to study the inuence of the
introduction of an aromatic ring attached to the hydroxamate
group on the inhibition of the target proteins.
Biological evaluation
The inhibitory activities of the synthesized compounds were
determined against both enzymes CK2 and HDAC1. The
percentage of inhibition at 50 mM concentration of the tested
compound is collected in Table 1. The IC50 for those
compounds showing an inhibition higher than 75% was
calculated, and the values are depicted also in Table 1.
The activity towards human recombinant HDAC1 was tested
using a uorimetric substrate. Compounds 5c, 5e and 5f pre-
sented the highest potency within the compounds with
aliphatic linkers, with IC50 of 5.0 ꢀ 1.8 mM, 2.2 ꢀ 0.2 mM and 5.1
ꢀ 2.4 mM respectively (inhibitory activities between 88 and 97%
at 50 mM) while 5d inhibited the deacetylase activity at a lower
extent (65.2% at 50 mM) and weak or no inhibition was observed
for compounds 5a and 5b in the same conditions.
The inuence of the position of the attachment (N-1 or N-2)
of the linker to the TBB moiety was analyzed by preparing
a representative set of N-1 and N-2 alkyl derivatives (5a–f).
The introduction of an aromatic ring in the linker was
benecial for the inhibition of HDAC1, and 5g displayed the
highest HDAC1 inhibition of all tested compounds with an IC50
of 1.6 ꢀ 1.1 mM.
Scheme 1 Reagents: (a) K2CO3, acetone, MW, 150 ꢁC, 1 min; (b)
K2CO3, CH3CN, RT, 24 h.
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Table 1 Percentage of inhibition of 5a–h for CK2a and HDAC1 at 50
mM. (IC50 for compounds with an inhibition higher than 75% at 50 mM)
express the effective inhibition. Both compounds exhibited
cytotoxic activity in the micromolar range towards several
mammalian tumor cell lines (Fig. 3).
% inhibition CK2a,a
50 mM (IC50, mM)
% inhibition HDAC1,a
50 mM (IC50, mM)
Compound
5a
22.7
32.2
5b
42.0
3.5
5c
5d
79.0 (5.4 ꢀ 1.2)
90.0 (5.0 ꢀ 1.8)
61.2
65.2
5e
5f
5g
5h
TBB
SAHA
61.6
47.7
47.2
22.2
97.0 (2.2 ꢀ 0.2)
88.3 (5.1 ꢀ 2.4)
99.0 (1.6 ꢀ 0.4)
86.0 (2.9 ꢀ 1.1)
—
99.8 (0.8 ꢀ 0.1)
—
25.8 (at 0.1 mM)
a
Enzymatic data are mean values from at least two independent
experiments ꢀ SD.
The inhibitory activities of these compounds for CK2 were
measured using a radiometric method with a short peptide
(RRRADDSDDDDD) as a substrate. Recombinant human CK2a
was used as a source of CK2. The best potency against CKa was
found for compound 5c (79% of inhibition at 50 mM, IC50 of 5.4
ꢀ 1.2 mM), which bears the linker attached to the N-1 position of
TBB and two CH2 groups connecting the triazole ring to both
TBB and the ZBG. The distance between TBB and the triazole
ring provided by a 2(CH2) linker seems to be crucial for activity.
Thus, compounds 5a and 5b, where only one methylene group is
present at this position are weaker inhibitors (22.7% of inhibi-
tion for 5a; 42% for 5b). However, more than 60% of inhibition at
50 mM was maintained for compound 5d, which differ from 5b in
the presence of one additional CH2 group at this position.
The effect of changing the position of attachment of the alkyl
linkers to the N-2 position of TBB was examined through
compounds 5e–f. Again the best activity was obtained for the
compound bearing 2(CH2) linkers at both sides of the con-
necting triazole (compound 5e, 61.6% of inhibition at 50 mM),
while an increase in the number of CH2 groups, such as in 5f
brought about a loss of activity.
To study the inuence of the introduction of an aromatic
ring in the linker, compounds 5g–h were synthesized. Both
compounds showed a signicant loss of activity (47.2% of
inhibition for 5g and 22.2% of inhibition for 5h) when
compared to its non-aromatic analogues 5d and 5f of similar
chain length. This loss of activity is probably due to an increase
on the rigidity of the molecule.
In summary, the described compounds have a potential
interest as dual target inhibitors, and compound 5c presents
the best prole, with identical IC50 values in the low micromolar
range for CK2 and HDAC1. Therefore, it was selected to evaluate
its antiproliferative effect on several cancer cell lines, and pro-
apoptotic activity towards human Jurkat T-leukemia and MCF-7
breast adenocarcinoma. We selected also compound 5e as
representative of the N-2 substituted derivatives.
Fig. 3 Cytotoxic effect of dual CK2/HDAC inhibitors 5c and 5e
towards human T-leukemia cells of Jurkat line, human breast
adenocarcinoma cells of MCF-7 line, murine leukemia cells of L1210
line, and human immortalized keratinocytes of HaCat line, after 24 h of
incubation.
Cytotoxic activity of the selected inhibitors was evaluated in
vitro and the LC50 index, calculated as concentration of drug
which kills 50% cells in comparison to control, was used to
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Interestingly, 5e showed 1.6–4.7 fold higher activities
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compared to its isomer 5c. Human leukemia cells of Jurkat line
(LC50 ¼ 2.9 mM) and human breast adenocarcinoma cells of
MCF-7 line (LC50 ¼ 4.26 mM) were the most sensitive to 5e
action, while human immortalized keratinocytes of HaCat cells
were found to be 3-fold more resistant to this compound (LC50
¼ 13.66 mM), indicating a selectivity of action of this dual CK2/
HDAC inhibitor towards tumor cells. Its isomeric compound 5c
demonstrated weaker cytotoxic activity towards all studied cell
lines (LC50 in the range of 13.59–27.81 mM), and lack of selec-
tivity for tumor against pseudonormal mammalian cell lines
(Table 2).
Fig. 4 Proapoptotic activity of dual CK2/HDAC inhibitors (Jurkat T-
leukemia cells, combined annexin V/PI staining, 24 h). (A) Control, (B)
5c (15 mM), (C) 5c (30 mM), (D) 5f (3 mM), (E) 5f (10 mM). Green color –
FITC-conjugated annexin V, orange color – propidium iodide.
Three main cell death mechanisms have been identied –
apoptosis, autophagy, and necrosis.60 Cell necrosis is an unde-
sirable phenomenon during chemotherapy treatment, since it
results in the development of massive inammatory processes
in the organism of cancer patients which can signicantly
complicate the treatment process. By contrast, the induction of
apoptosis by anticancer drugs leads to quick phagocytosis of
remnants of tumor cells by the macrophages which consider-
ably diminish the development of side effects of chemotherapy.
Cytomorphologic studies were performed to identify by
which way – apoptotic or necrotic – our dual CK2-HDAC
inhibitors induced the death of target cells. Phosphatydylser-
ine translocation to the external layer of the cell membrane of
dying cells, measured by the Annexin V test, is considered one of
the earliest hallmarks of apoptosis.61 Propidium iodide is a DNA
targeting dye which detects only necrotic cells, since alive cells
use membrane pumps to move it out to the extracellular
medium. Supravital double staining with FITC-conjugated
Annexin V and propidium iodide of Jurkat T-leukemia cells
treated with 5c and 5e in LC50 and LC75 doses, revealed that
both compounds induced apoptosis in leukemia cells (Fig. 4). It
should be stressed that the rate and intensity of apoptosis under
the action of these novel dual CK2/HDAC inhibitors increased
in a concentration-dependent manner. In particular, the treat-
ment at LC75 concentration led to a proportional increase in the
number of annexin V positive, PI-negative (early apoptotic cells)
compared to their action at LC50 (Fig. 4). On the other hand,
inhibitor 5e led to induction of apoptosis at lower concentration
(3 mM for LC50 and 10 mM for LC75) than 5c, which was able to
induce appearance of comparable percent of annexin V+/PIꢂ
cells only at signicantly higher concentrations (15 mM for LC50
and 30 mM for LC75, respectively). This result is in accordance
with the higher cytotoxic activity displayed by 5e in this tumor
cell line. Cytotoxic and proapoptotic activities of 5c and 5e were
compared to the ones of TBB and SAHA in human Jurkat T-
leukemia cells (ESI Fig. S1†). Interestingly 5e possesses signi-
cantly higher activity (LC50 ¼ 2.5 mM) than TBB (LC50 ¼ 20 mM)
and, although LC50 of 5e is identical to that of SAHA, its pro-
apoptotic activity is signicantly higher, as revealed by quanti-
tative annexin V/PI assay by ow cytometry. Thus, dual CK2-
HDA1C inhibitor 5e demonstrated an improved behavior in
cell-based antitumor assays compared to its predecessors TBB
and SAHA, demonstrating the interest of this type of dual tar-
geting inhibitors.
Quantitative annexin V/PI assay by ow cytometry showed
that both 5c and 5e in sub-LC75 doses (15–25 mM for 5c and 10
mM for 5e) led to signicant and almost identical increase (24–
25%) in population of late apoptotic cells (AnV(+)/PI(+)).
However, cell treatment with 5c was accompanied by the
appearance of a higher number of necrotic cells (12–18%
AnV(ꢂ)/PI(+)) compared to 5e (9.5%), and the same dependency
was also observed in previous experiment (see Fig. 4). Thus, 5e
induces mostly late apoptosis in Jurkat T-leukemia cells, while
5c, besides having weaker cytotoxic activity, is inducing both
necrosis and late apoptosis in these cells, which is considered
harmful on the physiological level. Surprisingly, both 5c and 5e
demonstrated much higher proapoptotic activity compared to
TBB (8.5% late apoptotic cells) and SAHA (18% late apoptotic
cells), also taken in sub-LC75 concentrations (Fig. 5). These
results clearly demonstrate better proapoptotic activities of
novel hybrid CK2-HDAC1 inhibitors compared to their parental
compounds.
Table 2 Comparison of LC50 values of dual CK2/HDAC inhibitors 5c
and 5e towards several mammalian cell lines
Cell line Origin
5c, LC50, mM 5e, LC50, mM 5c : 5e
Nuclear chromatine hypercondensation is considered to be
another typical hallmark of apoptosis. It can be easily measured
by using DNA-intercalating dyes, such as DAPI (40,6-diamidino-
2-phenylindole). Both 5c and 5e led to chromatine hyper-
condensation, vacuolization of cytosol and formation of
membrane blebs at 25 mM and 10 mM concentrations (Fig. 6),
which correspond to apoptosis induction. These results fully
conrm the annexin V/PI data.
Jurkat
L1210
MCF-7
Human leukemia
Murine leukemia
Human breast
adenocarcinoma
Human embryonic
kidney cells, transfected
by SV40 virus
Human immortalized
keratinocytes
13.59 ꢀ 1.04 2.87 ꢀ 1.14 4.74
20.90 ꢀ 1.03 13.09 ꢀ 1.07 1.60
17.27 ꢀ 1.07 4.26 ꢀ 1.09 4.05
293T
27.81 ꢀ 1.05 14.55 ꢀ 1.11 1.91
HaCat
22.06 ꢀ 1.05 13.66 ꢀ 1.08 1.61
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hydroxamic acid-bearing inhibitors establish
a hydrogen
bonding network between conserved His residues in the active
site and coordinate Zn2+ in a bidentate way, replacing the active-
site water molecule with one of the oxygen atoms.62 Moreover,
computational studies suggested that one of the His residues in
the binding site could be protonated as part of the deacetylation
reaction mechanism.65 In this same line, the binding poses ob-
tained for the set of our newly synthesized inhibitors buried the
hydroxamic acid moiety deep into the active site, coordinating
the two oxygen atoms with the catalytic Zn2+ ion. Apart from
chelating the Zn2+, the hydroxamic moiety established several
hydrogen bond interactions, and we observed that the terminal
OH could be easily deprotonated by the imidaloze side chain of
His140. Therefore the dockings and further calculations were
carried out with the deprotonated hydroxamates and a proton-
ated side chain of His140. In addition to the hydrogen bond
resulting from this deprotonation, the NH group of the hydrox-
amate interacts with the N3 of the imidaloze side chain of
His141, and the carbonyl oxygen with the OH group on the side
chain of Tyr303. The linker between the hydroxamate and the
triazole can establish van der Waals interactions with Phe150
and Phe205, making the TBB cap protrude out of the binding
tunnel. A linker of two or three methylene groups between the
hydroxamate and the triazole keep the latter inside the tunnel
and at van der Waals interaction reach of the side chains of
Phe150 and Phe205 as it’s the case for compounds 5a, 5c and 5e–
f, whereas 5b and 5d that present four methylene groups make
the triazole protrude out of the binding tunnel.
Fig. 5 Comparison of proapoptotic activity of compounds 5c, 5e, TBB
and SAHA towards human Jurkat T-leukemia cells. After a 24 h
exposure cells were stained with FITC-Annexin V and PI for further
analysis by flow cytometry. ***p < 0.001 relative to control, unpaired t-
test, **p < 0.01 relative to 5e, 10 mM, unpaired t-test, **p < 0.001
relative to 5e, 10 mM, unpaired t-test.
The introduction of an aromatic moiety to the linker between
the hydroxamate and the triazole in compounds 5g–h increases
the interaction with the side chains of Phe150 and Phe205 due
to the establishment of p–p interactions between the aromatic
groups. On the other hand, a shorter linker with only one
methylene group between the TBB moiety and the triazole
present in compounds 5a and 5b reduces the exibility at the
end of the ligand and, therefore, the ability to further accom-
modate the TBB cap to the surface of the protein outside the
tunnel (ESI Fig. S2†).
Given their interesting IC50, the complexes with compounds
5c and 5e were subsequently used for MD simulations to further
study their binding-stability. In both complexes (Fig. 7) the two
methylene groups attached to the hydroxamate and the triazole
interact with the side chains of Phe150 and Phe205 that make
up the walls of the tunnel, while the second two-carbon linker
and the TBB moiety protrude out of the binding site and
interact with the surface of the protein. During the 10 ns
simulation the binding modes of both compounds 5c and 5e
remained stable and the interactions with the residues in the
active site where maintained, while the triazole accommodated
itself into the tunnel optimizing the van der Waals contacts with
the side chains of Phe150 and Phe205. The different attachment
(N1 or N2) of the linker to the TBB moiety does not change
much the binding pose outside the catalytic cle: compound 5c
sits mainly on the side chain of Leu271, whereas compound 5e
sways a little more so it interacts, not only with the side chain of
Leu271, but also with Pro29 and with Phe150 in an edge to face
interaction with the latter.
Fig. 6 Cytomorphological studies of chromatin structure of MCF-7
cells under treatment with dual CK2/HDAC inhibitors. DAPI staining, 24
h. (A) Control, (B) 5c (25 mM), (C) 5e (10 mM).
Computational studies
In order to give a rationale for the results shown in Table 1, the
predicted binding-mode of the described compounds to both
enzymes was studied by means of molecular modeling
techniques.
HDAC1 binding. The catalytic site of HDAC1 is located at the
centre of the enzyme and is accessed through a small hydro-
phobic tunnel. The side chains of Phe150 and Phe205 leave a gap
˚
of approximately 8 A between them making up this tunnel, at the
bottom of which lays the catalytic Zn2+ ion. In normal condi-
tions, the acetylated Lys of the substrate plunges into the tunnel
reaching the Zn2+ and becomes part of the coordination sphere
through the carbonyl oxygen, which catalyses the deacetylation
reaction with the participation of a water molecule, as can be
seen in the crystal structure of HDAC8 in complex with
a substrate-like molecule (ESI Fig. S1†).62 HDAC inhibitors
carrying a ZBG target the enzyme by chelating the Zn2+ and
becoming part of its coordination sphere, while the linker
interacts with the walls of the tunnel and the capping moiety
protrudes out of it and interacts with the surface of the
protein.63,64 Crystallographic studies have shown that
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burying it deep inside the hydrophobic pocket of CK2 in such an
orientation that, in some cases, a halogen bond could be
established with the carbonyl oxygen of Val116, similar to the
one established by tetrabromobenzoimidazole in PDB code
2OXY. Moreover, in all of these dockings strong van der Waals
contacts are established with the side chains of Val45, Val53,
Ile66, Val95, Phe113, Val116, Met163 and Ile174, analogue to the
ones in the crystallized complexes. Depending on the length of
the ligands and the position of the substitution on the TBB
moiety they adopt different orientations within the binding site
and display different orientations of their substituents.
Compounds 5a–5f orient the hydroxamic acid moiety towards
the side chains of Asp156, Lys158, Asn161 and Asp175 that make
up one of the Mg2+-binging sites in CK2. The presence of the
additional aromatic ring of compounds 5g–h does not give good
Fig. 7 PyMOL stick and semi-transparent cartoon representation of
the bound conformation of compounds 5c and 5e to HDAC1 after the
10 ns MD simulation. The main amino acids involved in the binding are
represented as sticks and the catalytic Zn2+ is represented as a grey
sphere. Hydrogen bonding and coordinating interactions are shown as
dashed lines and the residues involved are labelled in bold. For the sake
of clarity only polar hydrogens are shown.
CK2 binding. The catalytic subunit of CK2 has the common
characteristic structure of all kinases, which is two distinct
domains with the ATP binding site between them. This binding
site is mainly a hydrophobic at pocket made up of the side
chains of Val53 and Ile66 at the ceiling, and Met163 and Ile174
at the bottom. The natural substrate ATP binds by inserting the
adenine into the binding site, thus establishing van der Waals
interactions with the amino acids at the bottom and ceiling of
the pocket. The position is further stabilized by hydrogen
bonding interactions between N1 and N7 of the base and the
backbone NH of Val116 and carbonyl of Glu114, respectively;
whilst the triphosphate moiety and the two Mg2+ ions interact
with Asp156, Lys158, Asn161 and Asp175 (ESI Fig. S3†).66 Albeit
having only halogen bond acceptors, TBB is a selective
competitive inhibitor of the ATP-binding to CK2. It occupies the
ATP-binding pocket establishing strong van der Waals interac-
tions with the side chains of Val53, Ile66, Met163 and Ile174,
which due to the shape and size of the pocket they make up,
they are responsible for the marked selectivity of TBB for CK2.67
Moreover, a hydrogen bond between N2 and Lys68 is estab-
lished, as TBB is deprotonated and negatively charged at
physiological pH, as it can be seen in the PDB structure 1J91
(ESI Fig. S4†).67 In the case of the TBB analogue tetra-
bromobenzimidazole, the lack of the N2 changes the overall
charge of the ligand and thus the ionic interaction with Lys68 is
not established. This change modies the orientation of the
binding that promotes the formation of a halogen bond
between one of the Br atoms and the backbone carbonyl of
Val116 (ESI Fig. S5†).68 These modications not only do not
impair the binding to CK2, but also make it possible to intro-
duce different substitutions at position 2.
Fig. 8 PyMOL stick and semi-transparent cartoon representation of the
bound conformation of compounds 5c and 5e to CK2 after the 10 ns MD
simulation. The main amino acids involved in the binding are represented
as sticks. For the sake of clarity only polar hydrogens are shown.
The docking of the majority of the designed inhibitors lodged
the TBB moiety in a similar way as in the crystallized complex,
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binding poses as they are not able to t within the binding site apparatus at the Microanalyses Service of the Universidad
due to the increase in length derived from the extra aromatic Complutense de Madrid.
ring and the linker (ESI Fig. S6†). They are not able to interact
4,5,6,7-Tetrabromo-1H-benzotriazole (TBB),69 N-alkyl deriva-
with the side chains of Asp156, Lys158, Asn161 and Asp175, and tives 1 and 2a–b58 and azides 3a–e70,71 were synthesized
due to this increased length these compounds interact with according to literature procedures. Melting points, 1H and/or
Val116 through the hydroxamic acid in a bidentate interaction 13C NMR data were in agreement with those published.
while the TBB moiety sits on top of the side chain of Lys158,
4,5,6,7-Tetrabromo-1-(prop-2-yn-1-yl)-1H-benzo[d][1,2,3]triazole
probably stabilized by a p-cation interaction. These results (1). To a suspension of TBB (500 mg, 1.1 mmol) and K2CO3 (1.4
suggest that equilibrium between length and ligand exibility g, 10.15 mmol) in acetone (20 mL) was added 3-bromo-1-
has to be met in order to have an efficient binding of TBB propyne (0.3 mL, 3.4 mmol). This reaction was carried out by
derivatives to the ATP-binding site in CK2. The complexes of CK2 microwave assistance at 150 ꢁC for 1 min. Aer cooling to room
with compound 5c and its direct analogue 5e, that presented the temperature, the solvent was removed under vacuum and the
highest inhibitory activity, were subjected to MD simulations to residue was dissolved in 20 mL of AcOEt and washed with brine
further study their binding stability and try to understand the (2 ꢃ 20 mL). The organic layer was dried (MgSO4), ltered,
slight differences brought about by distinct TBB substitution.
evaporated to dryness and puried by ash chromatography
1
The initial good van der Waals interactions between the TBB (Hex/DCM 4 : 1) to afford 1 as a white solid (382 mg, 72%), H
moiety of both compounds and the side chains of Val45, Val53, NMR (400 MHz, DMSO) d 5.81 (d, J ¼ 2.3 Hz, 2H), 3.68 (t, J ¼ 2.3
Ile66, Phe113, Met163 and Ile174 (as can be seen in the corre- Hz, 1H). 13C NMR (101 MHz, DMSO) d 144.8, 131.4, 129.1, 124.1,
sponding images of ESI Fig. S6†) were maintained during the MD 115.8, 106.6, 78.3, 77.4, 40.2.
simulation, in addition to the interaction of the triazole moiety
4,5,6,7-Tetrabromo-1-(but-3-yn-1-yl)-1H-benzo[d][1,2,3]triazole
with the protonated side chain of His160. Both complexes (2a) and 4,5,6,7-tetrabromo-2-(but-3-yn-1-yl)-2H-benzo[d][1,2,3]
remained quite stable due to the interaction of the hydroxamic triazole (2b). To a suspension of TBB (200 mg, 0.46 mmol) and
acid moiety with the side chains of Asp156, Lys158 and Asp175 K2CO3 (565 mg, 4.1 mmol) in acetonitrile (10 mL) was added 4-
and the establishment of a halogen bond between the carbonyl bromo-1-butyne (0.12 mL, 1.38 mmol) and the mixture was
oxygen of the backbone of Val116 and the Br at position 4 and 5 stirred at RT under argon for 24 hours. Aer cooling to room
of the TBB moieties of compounds 5e and 5c, respectively (Fig. 8). temperature, the solvent was removed under vacuum and the
residue was dissolved in 20 mL of AcOEt and washed with brine
(2 ꢃ 20 ml). The organic layer was dried (MgSO4), ltered,
Experimental
Materials and methods
evaporated to dryness and puried by ash chromatography
(Hex/DCM 4 : 1) to afford 2a (47 mg, 21%) and 2b (114 mg,
Melting points (uncorrected) were taken in open-end capillary 51%). 2a: 1H NMR (400 MHz, DMSO) d 5.07 (t, J ¼ 6.8 Hz, 2H),
tubes and were determined on a Stuart Scientic SMP3 appa- 2.96–2.86 (m, 3H). 13C NMR (101 MHz, DMSO) d 144.8, 131.8,
ratus. Thin-layer chromatography (TLC) was run on Merck silica 128.7, 123.6, 115.8, 106.9, 79.7, 74.0, 48.1, 20.5. 2b: 1H NMR (400
gel 60 F254 plates. Flash column chromatography was per- MHz, DMSO) d 4.94 (t, J ¼ 6.7 Hz, 2H), 3.03 (td, J ¼ 6.7, 2.6 Hz,
formed using silica gel Merk-60 (230–400 mesh). Unless stated 2H), 2.87 (t, J ¼ 2.6 Hz, 1H). 13C NMR (101 MHz, DMSO) d 142.5,
otherwise, purchased starting materials used were high-grade 125.8, 113.6, 79.8, 73.6, 55.5, 19.1.
commercial products. NMR spectra were recorded at 400 MHz
(1H) and 101 MHz (13C) on a Bruker 400-AC magnetic resonance
spectrometer at room temperature in CDCl3 [calibrated at 7.26
General procedure for the synthesis of azides 3a–d
ppm (1H) and 77.0 ppm (13C)] and in DMSO-d6 [calibrated at To a solution of the corresponding alkyl bromide (1.0 mmol) in
2.50 ppm (1H) and 39.5 ppm (13C)]. Data are presented as DMF (5 mL) under argon was added NaN3 (65 mg, 1.0 mmol)
follows: chemical shi (ppm), multiplicity (s singlet, bs broad and the mixture was stirred at 60 ꢁC overnight. Aer cooling to
singlet, d doublet, t triplet, q quartet, m multiplet), coupling room temperature, AcOEt (20 mL) and aqueous HCl 0.1 M (20
constant J (Hz) and integration. Data for 13C NMR are reported mL) were successively added, and the water phase was extracted
in terms of chemical shis (ppm). HPLC-MS was performed at with AcOEt (2 ꢃ 20 mL). The combined organic extracts were
Synthelia Organics SL., using an Agilent 1100 HPLC system and washed with brine, dried over MgSO4, ltered and concentrated
Xterra C18-5 mm or ACE C18-3 mm reverse phase columns. The under vacuum to afford the desired azides, which were used in
following conditions were used: method A, Xterra C18-5 mm the next step without further purication.
column, a ow rate of 2 mL minꢂ1, eluting with solvent A (20
3-Azidopropanoic acid (3a). Following the general proce-
mM NH4HCO3/water) and solvent B (ACN) at an isocratic run of dure, the reaction of 3-bromopropionic acid (1.0 g, 6.5 mmol)
40% of solvent A and 60% of solvent B over 15 min with with NaN3 (423 mg, 6.5 mmol) gave 3a as a white solid (501 mg,
detection at 254 nm; method B, a ow rate of 0.75 mL minꢂ1
,
67%). 1H NMR (400 MHz, CDCl3) d 3.58 (t, J ¼ 6.4 Hz, 2H), 2.62
eluting with solvent A (20 mM NH4HCO3/water) and solvent B (t, J ¼ 6.4 Hz, 2H).
(ACN) at a gradient run from 20–95% of solvent B over 6 min
4-Azidobutanoic acid (3b). The reaction of 4-bromobutanoic
with detection at 254 nm. For reporting HPLC-MS data, m/z acid (835 mg, 5 mmol) with NaN3 (325 mg, 5 mmol) gave 3b as
values and retention time for each method are given. Elemental a white solid (561 mg, 87%). 1H NMR (400 MHz, CDCl3) d 3.38 (t,
analyses (C, H, N, S) were performed on a LECO CHNS-932 J ¼ 7.2 Hz, 2H), 2.26 (t, J ¼ 7.2 Hz, 2H), 1.97–1.88 (m, 2H).
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5-Azidopentanoic acid (3c). The reaction of 5-bromopenta-
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chromatography (hexane/AcOEt 3 : 2), 4c as a white solid (295
mg, 61%). 1H NMR (400 MHz, CDCl3) d 8.41 (s, 1H), 4.93 (t, J ¼
6.8 Hz, 1H), 3.66–3.58 (m, 2H), 2.39 (t, J ¼ 6.9 Hz, 2H), 1.97–1.55
(m, 12H).
noic acid (1.0 g, 5.5 mmol) with NaN3 (358 mg, 5.5 mmol) gave
1
3c as a white solid (464 mg, 59%). H NMR (CDCl3, 400 MHz)
d 3.26–3.33 (m, 2H), 2.36–2.42 (m, 2H), 1.60–1.75 (m, 4H).
4-(Azidomethyl)benzoic acid (3d). The reaction of 4-(bro-
momethyl)benzoic acid (1.0 g, 4.7 mmol) with NaN3 (306 mg,
4.7 mmol) gave 3d as a white solid (752 mg, 91%). 1H NMR (400
MHz, CDCl3) d 8.12 (s, 1H), 7.50–7.38 (m, 4H), 2.48 (s, 2H).
4-Azidobenzoic acid (3e). To a solution of methyl 4-amino-
benzoate (1.0 g, 6.6 mmol) in anhydrous CH3CN (60 mL) under
argon were successively added t-BuONO (1.3 mL, 9.9 mmol) and
TMSN3 (1.0 mL, 7.9 mmol) dropwise at 0 ꢁC. The reaction
mixture was stirred at the same temperature for 30 min and at
room temperature for 3 h. The solvent was removed under
vacuum and the residue was puried by ash chromatography
(Hexane/AcOEt 95 : 5) to afford 640 mg of a white solid, which
was subsequently dissolved in methanol (3 mL). To this solu-
tion, aqueous NaOH 1 M (3 mL) was added and the mixture was
stirred for 2 h at room temperature. Aer addition of AcOEt (10
mL) the solution was neutralized with aqueous HCl 1 M (pH z
1). The two phases were separated and the water phase was
extracted with AcOEt (2 ꢃ 10 mL). The combined organic
extracts were washed with brine, dried over MgSO4 and
concentrated under vacuum to give 3e as a white solid (570 mg,
53% over two steps). 1H NMR (400 MHz, CDCl3) d 8.10 (d, J ¼ 8.5
Hz, 2H), 7.11 (d, J ¼ 8.5 Hz, 2H).
4-(Azidomethyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide
(4d). Following the general procedure, the reaction of 4-(azi-
domethyl)benzoic acid 3d (354 mg, 2.0 mmol) gave, aer ash
chromatography (hexane/AcOEt 7 : 3), 4d as a white solid (309
mg, 56%). 1H NMR (400 MHz, CDCl3) d 8.98 (s, 1H), 7.77 (d, J ¼
8.1 Hz, 2H), 7.38 (d, J ¼ 8.1 Hz, 2H), 5.07 (t, J ¼ 6.8 Hz, 1H), 4.39
(s, 2H), 4.05–3.93 (m, 1H), 3.72–3.52 (m, 1H), 1.85–1.52 (m, 6H).
4-Azido-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide
(4e).
Following the general procedure, the reaction of 4-azidobenzoic
acid 3e (326 mg, 2.0 mmol) gave, aer ash chromatography
(DCM/MeOH 9 : 1), 4e as a white solid (309 mg, 59%). 1H NMR
(400 MHz, DMSO) d 9.02 (s, 1H), 7.82 (d, J ¼ 7.6 Hz, 2H), 7.40 (d,
J ¼ 7.6 Hz, 2H), 4.98 (t, J ¼ 7.2 Hz, 1H), 3.52 (t, J ¼ 7.1 Hz, 2H),
1.86–1.48 (m, J ¼ 7.1 Hz, 6H).
General procedure for the click reaction: synthesis of triazole-
based dual inhibitors 5a–h
To a mixture of the azide (0.1 mmol) and the corresponding
alkyne (1.1 equiv, 0.11 mmol) in DMF (1 mL) under argon, were
successively added sodium ascorbate (30 mol%, 0.03 mmol, 30
mL of freshly prepared 1 M solution in water) and copper(II)
sulfate pentahydrate (10 mol%, 0.01 mmol, 33 mL of freshly
prepared 0.3 M solution in water). The heterogeneous mixture
was stirred vigorously overnight at room temperature. Aer
removing the solvent under vacuum, AcOEt (10 mL) was added
General procedure for the synthesis of hydroxamate
derivatives 4a–e
To a solution of the corresponding acid (3a–e) (2.0 mmol) in and the resulting solution was washed with NH4Cl (10 mL) and
DMF (10 mL) were successively added HOBt (1.2 equiv, 324 mg, brine and dried over (MgSO4). Filtration and evaporation of the
2.4 mmol), NMM (3 equiv, 0.66 mL, 6.0 mmol), NH2OTHP (2 solvent gave a residue which was puried by ash chromatog-
equiv, 468 mg, 4.0 mmol) and EDCI (1.4 equiv, 538 mg, 2.8 raphy (the eluent is indicated in each case). Final removal of the
mmol) and the mixture was stirred at room temperature over- THP protecting group was carried out as follows: to a solution of
night. The solution was then diluted with AcOEt (40 mL) and the protected hydroxamic acid in MeOH (4 mL) was added 4 M
subsequently washed with saturated aqueous NH4Cl (40 mL) and HCl in dioxane (0.4 mL, 0.4 mmol) and the mixture was stirred
brine, dried over MgSO4. The drying agent was ltered off, the at room temperature for 2 h. Solvent co-evaporation with MeOH
solvent was removed in vacuo and the residue was puried by (ꢃ3) afforded the corresponding adducts 5a–h.
ash chromatography on silica gel (the eluent is indicated in
N-Hydroxy-3-(4-((perbromo-1H-benzo[d][1,2,3]triazol-1-yl)
each case) to afford the desired hydroxamic acid derivatives 4a–e. methyl)-1H-1,2,3-triazol-1-yl) propanamide (5a). Following
3-Azido-N-((tetrahydro-2H-pyran-2-yl)oxy)propanamide (4a). the general procedure, the reaction of 4a (30 mg, 0.14 mmol)
Following the general procedure, the reaction of 3-azidopropa- with 1 (71 mg, 0.15 mmol) afforded, aer ash chromatography
noic acid 3a (230 mg, 2.0 mmol) gave, aer ash chromatog- (Hex/AcOEt 1 : 3) and further hydroxamate deprotection, 5a as
raphy (DCM/MeOH 95 : 5), 4a as a white solid (205 mg, 48%). 1H a white solid (59 mg, 71%), mp 130–132 ꢁC. 1H NMR (400 MHz,
NMR (400 MHz, CDCl3) d 8.42 (s, 1H), 4.93 (t, J ¼ 6.8 Hz, 1H), DMSO) d 10.12 (br s, 1H), 9.93 (br s, 1H), 8.09 (s, 1H), 6.25 (s,
3.68–3.52 (m, 2H), 2.33 (t, J ¼ 6.9 Hz, 2H), 1.77 (t, J ¼ 6.8 Hz, 2H), 2H), 4.54 (t, J ¼ 6.7 Hz, 2H), 2.94 (t, J ¼ 6.7 Hz, 2H). 13C NMR
1.69–1.47 (m, 6H).
(101 MHz, CDCl3) d 170.8, 145.6, 131.6, 129.80, 124.7, 123.9,
4-Azido-N-((tetrahydro-2H-pyran-2-yl)oxy)butanamide (4b). 116.8, 114.1, 106.0, 52.3, 46.1, 34.4. HPLC-MS: m/z 603 [M ꢂ
Following the general procedure, the reaction of 4-azidobuta- 2H]+, tR: 11.12 min, method A. Anal. calcd for C12H11Br4N7O2
noic acid 3b (258 mg, 2.0 mmol) gave, aer ash chromatog- requires C, 23.83; H, 1.83; Br, 52.84; N, 16.21; O, 5.29; found: C,
raphy (hexane/AcOEt 3 : 2), 4b as a white solid (333 mg, 73%). 23.72; H, 1.92; N, 16.31.
1H NMR (400 MHz, CDCl3) d 8.36 (s, 1H), 4.96 (t, J ¼ 7.0 Hz, 1H),
N-Hydroxy-5-(4-((perbromo-1H-benzo[d][1,2,3]triazol-1-yl)
3.69 (t, J ¼ 7.1 Hz, 2H), 2.31–2.24 (m, 2H), 1.99–1.52 (m, 10H). methyl)-1H-1,2,3-triazol-1-yl)pentanamide (5b). The reaction
5-Azido-N-((tetrahydro-2H-pyran-2-yl)oxy)pentanamide (4c). of 4c (30 mg, 0.12 mmol) with 1 (64 mg, 0.14 mmol) afforded,
Following the general procedure, the reaction of 5-azidopenta- aer ash chromatography (Hex/AcOEt 1 : 5) and further
noic acid 3c (286 mg, 2.0 mmol) gave, aer ash hydroxamate deprotection, 5b as a white solid (48 mg, 64%), mp
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167–169 ꢁC. 1H NMR (400 MHz, DMSO) d 10.04 (br s, 1H), 9.85 calcd for C14H13Br4N7O2 requires C, 26.65; H, 2.08; Br, 50.66; N,
(br s, 1H), 8.09 (s, 1H), 6.26 (s, 2H), 4.31 (t, J ¼ 7.0 Hz, 2H), 2.30 15.54; O,5.07; found: C, 26.84; H, 1.75; N, 15.48.
(t, J ¼ 7.4 Hz, 2H), 1.84–1.68 (m, 2H), 1.51–1.37 (m, 2H). 13C
N-Hydroxy-4-((4-(2-(perbromo-1H-benzo[d][1,2,3]triazol-1-yl)
NMR (101 MHz, DMSO) d 172.9, 144.9, 142.0, 131.8, 128.8, ethyl)-1H-1,2,3-triazol-1-yl)methyl)benzamide (5g). The reac-
123.7, 123.3, 115.7, 106.8, 51.2, 45.6, 32.4, 28.9, 21.2. HPLC-MS: tion of 4d (50 mg, 0.18 mmol) with 2a (96 mg, 0.20 mmol)
m/z 631 [M]+, tR: 11.67 min, method A. Anal. calcd for afforded, aer ash chromatography (DCM/MeOH 95 : 5) and
C
14H13Br4N7O2 requires C, 26.65; H, 2.08; Br, 50.66; N, 15.54; O, further hydroxamate deprotection, 5g as a white solid (87 mg,
5.07; found: C, 26.49; H, 2.07; N, 15.63.
71%), mp 139–141 ꢁC. 1H NMR (400 MHz, DMSO) d 11.24 (br s,
N-Hydroxy-3-(4-(2-(perbromo-1H-benzo[d][1,2,3]triazol-1-yl) 1H), 7.98 (s, 1H), 7.74 (d, J ¼ 8.2 Hz, 2H), 7.26 (d, J ¼ 8.2 Hz, 2H),
ethyl)-1H-1,2,3-triazol-1-yl)propanamide (5c). The reaction of 5.59 (s, 2H), 5.18 (t, J ¼ 7.1 Hz, 2H), 3.34 (t, J ¼ 7.0 Hz, 2H). 13
C
4a (30 mg, 0.14 mmol) with 2a (75 mg, 0.15 mmol) afforded, NMR (101 MHz, DMSO) d 164.2, 145.4, 143.1, 139.6, 133.0,
aer ash chromatography (Hex/AcOEt 1 : 3) and further 132.3, 129.1, 128.1, 127.8, 124.1, 116.3, 107.3, 52.7, 50.0, 27.5.
hydroxamate deprotection, 5c as a white solid (63 mg, 73%), mp HPLC-MS: m/z 680 [M + H]+, tR: 3.22 min, method A. Anal. calcd
130–132 ꢁC. 1H NMR (400 MHz, DMSO) d 10.50 (br s, 1H), 8.83 for C18H13Br4N7O2 requires C, 31.84; H, 1.93; Br, 47.07; N, 14.44;
(br s, 1H), 7.88 (s, 1H), 5.17 (t, J ¼ 7.2 Hz, 2H), 4.51 (t, J ¼ 5.3 Hz, O, 4.71; found: C, 31.71; H, 1.73; N, 14.63.
2H), 3.30 (t, J ¼ 7.2 Hz, 2H), 2.56 (t, J ¼ 5.2 Hz, 2H). 13C NMR
N-Hydroxy-4-((4-(2-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)
(101 MHz, DMSO) d 165.8, 144.9, 142.0, 131.8, 128.5, 123.5, ethyl)-1H-1,2,3-triazol-1-yl) methyl)benzamide (5h). The reac-
123.2, 115.7, 106.8, 49.4, 45.5, 32.7, 27.0. HPLC-MS: m/z 618 [M + tion of 4d (50 mg, 0.18 mmol) with 2b (96 mg, 0.20 mmol)
H]+, tR: 3.01 min, method B. Anal. calcd for C13H11Br4N7O2 afforded, aer ash chromatography (Hex/AcOEt 1 : 5) and
requires C, 25.31; H, 1.80; Br, 51.81; N, 15.89; O, 5.19; found: C, further hydroxamate deprotection, 5h as a white solid (79 mg,
25.43; H, 1.75; N, 15.96.
65%), mp 166–168 ꢁC. 1H NMR (400 MHz, DMSO) d 11.21 (br s,
N-Hydroxy-5-(4-(2-(perbromo-1H-benzo[d][1,2,3]triazol-1-yl) 1H), 7.89 (s, 1H), 7.71 (d, J ¼ 8.3 Hz, 2H), 7.26 (t, J ¼ 8.3 Hz, 2H),
ethyl)-1H-1,2,3-triazol-1-yl)pentanamide (5d). The reaction of 5.57 (s, 2H), 5.07 (t, J ¼ 7.0 Hz, 2H), 3.45 (t, J ¼ 7.0 Hz, 2H). 13
C
4c (25 mg, 0.1 mmol) with 2a (56 mg, 0.12 mmol) afforded, aer NMR (101 MHz, DMSO) d 163.6, 142.7, 142.5, 139.0, 132.4,
ash chromatography (Hex/AcOEt 1 : 6) and further hydrox- 127.6, 127.3, 125.6, 123.3, 113.6, 56.4, 52.2, 25.7. HPLC-MS tR:
amate deprotection, 5d as a white solid (45 mg, 66%), mp 141– 10.33 min, non-ionizable, method A. Anal. calcd for
1
ꢁ
142 C. H NMR (300 MHz, DMSO) d 10.19 (br s, 1H), 7.85 (s,
C18H13Br4N7O2 requires C, 31.84; H, 1.93; Br, 47.07; N, 14.44; O,
1H), 5.18 (t, J ¼ 7.1 Hz, 2H), 4.28 (t, J ¼ 6.9 Hz, 2H), 3.32 (t, J ¼ 4.71; found: C, 31.40; H, 1.98; N, 14.35.
7.1 Hz, 2H), 2.32 (t, J ¼ 7.14 Hz, 2H), 1.77–1.69 (m, 2H), 1.46–
1.38 (m, 2H). 13C NMR (101 MHz, DMSO) d 173.0, 144.8, 142.1,
Molecular modeling
131.8, 128.5, 123.5, 122.9, 115.7, 106.7, 51.2, 49.5, 32.5, 29.0,
27.0. HPLC-MS: m/z 645 [M]+, tR: 3.93 min, method B. Anal. On one hand, since no molecular model was available for the
calcd for C15H15Br4N7O2 requires C, 27.93; H, 2.34; Br, 49.56; N, structure of HDAC1 at the beginning of the project, an
15.20; O, 4.96; found: C, 28.50; H, 2.25; N, 14.85.
homology model was built using SWISS MODEL web server72–74
N-Hydroxy-3-(4-(2-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl) and was assessed by superimposition to other type I HDAC
ethyl)-1H-1,2,3-triazol-1-yl)propanamide (5e). The reaction of crystallized structures. The H++ web server75 which relies on the
4a (30 mg, 0.14 mmol) with 2b (75 mg, 0.15 mmol) afforded, AMBER76 force eld parameters and nite difference solutions
aer ash chromatography (Hex/AcOEt 1 : 3) and further to the Poisson–Boltzmann equation was used to calculate the
hydroxamate deprotection, 5e as a white solid (63 mg, 73%), mp protonation state of tritable groups such as the imidazole side
173–175 ꢁC. 1H NMR (400 MHz, DMSO) d 10.48 (br s, 1H), 8.82 chains of the histidine residues located in the catalytic site:
(br s, 1H), 7.80 (s, 1H), 5.05 (t, J ¼ 7.2 Hz, 2H), 4.49 (t, J ¼ 6.7 Hz, His140 doubly protonated, His141 protonated in Nd and His178
2H), 3.44 (t, J ¼ 6.7 Hz, 2H), 2.55 (t, J ¼ 7.2 Hz, 2H). 13C NMR protonated in N3. However, aer all the computational work in
(101 MHz, DMSO) d 165.8, 142.5, 142.2, 125.6, 123.1, 113.6, 56.4, this project was nished, a crystal structure of the complex of
45.5, 32.7, 25.6. HPLC-MS: m/z 618 [M ꢂ H]+, tR: 3.34 min, acetic acid-bound HDAC1 with the regulatory protein MTA1 was
method B. Anal. calcd for C13H11Br4N7O2 requires C, 25.31; H, published under the PDB code 4BKX.77 The amino acid
1.80; Br, 51.81; N, 15.89; O, 5.19; found: C, 25.14; H, 1.75; N, sequence of the crystallized HDAC1 started at Arg8 and ended at
16.01.
Ala376, whereas the modelled one started at Arg8 and ended at
N-Hydroxy-4-(4-(2-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl) Leu373, only three amino acids less than the crystal structure,
ethyl)-1H-1,2,3-triazol-1-yl)butanamide (5f). The reaction of 4b which did not alter the overall structure of the modelled
(40 mg, 0.18 mmol) with 2b (93 mg, 0.19 mmol) afforded, aer protein. Both structures were superimposed using PyMOL to
ash chromatography (Hex/AcOEt 1 : 5) and further hydrox- assess any possible differences that would make imperative to
amate deprotection, 5f as a white solid (91 mg, 83%), mp 175– repeat the modelling work. The overall alignment of the a-
1
ꢁ
177 C. H NMR (400 MHz, DMSO) d 10.40 (br s, 1H), 7.86 (s, carbons of the matching amino acids presented an RMS of
˚
1H), 5.07 (t, J ¼ 7.2 Hz, 2H), 4.28 (t, J ¼ 6.6 Hz, 2H), 3.44 (t, J ¼ 0.386 A and visual inspection of the overall structure and the
7.1 Hz, 2H), 2.07–1.78 (m, 4H). 13C NMR (101 MHz, DMSO) binding site showed no remarkable differences that would
d 168.0, 142.5, 142.3, 125.5, 122.9, 113.6, 56.4, 48.8, 28.9, 25.91, compromise the modelled results (ESI Fig. S7†). Following these
25.7. HPLC-MS: m/z 632 [M + H]+, tR: 9.92 min, method A. Anal. inspections we resolved not to repeat the modelling work as the
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results would not be substantially different. The numbering of
the amino acids of the model is equivalent to the one of the
crystal structure in 4BKX.
Determination of CK2a inhibition
The reaction mixture (20 mL) for determination of human CK2a
(prepared as described elsewhere)86 activity contained: peptide
substrate (100 mM, RRRADDSDDDDD from Aldrich), Tris–HCl
pH 7.5 (20 mM), MgCl2 (15 mM), 2-mercaptoethanol (6 mM) and
g[32P] ATP (100 mM) and the compound (50 mM) in 5% DMSO.
Aer 30 min of incubation at 37 ꢁC, 10 mL of every assay mixture
was spotted onto a square (1 cm ꢃ 1 cm) of Whatman P81 paper
and dried for 15 min. Next, squares were immersed in cold 0.5%
phosphoric acid, and washed 3 times, for 15 min each. Then the
squares were washed with 96% EtOH and dried for 30 min at 40
ꢁC. The radioactivity was quantied by BECKMAN LS6500
scintillation counter. The activity was calculated as the
On the other hand, the structure of CK2 bound to a non-
hydrolysable ATP analogue in complex with a Mg2+ ion (PDB
code 1DAW) was used to build the kinase–bound complexes.
The H++ web server predicted the side chain of His160, located
at the entrance on the binding site to be doubly protonated. The
missing residues and side chains were added and the macro-
molecule geometries rened by using Protein Preparation
module in Maestro. The ligands (compounds 5c, 5e) were built
using Maestro LigPrep module (http://www.Schrodinger.com).
The pKa of the tritable groups were calculated using the Sparc
pKa online calculator78 and the non-protonated form of the
hydroxamate was used for the docking calculations. The Glide
module79–81 was used to perform the docking calculations. For
the HDAC1 complexes the centre of the box was positioned on
the catalytic zinc ion present in the active site, while for CK2 it
was located at the centre of the non-hydrolysable ATP analogue
of the crystal structure. The box size was set up to enclose the
ligand-binding domain to ensure a proper exploration of the
binding poses. The docking procedure was performed with XP
(extra precision) mode, and a van der Waals radii scale factor of
1.0/0.8 for receptor and ligand, respectively. The best-obtained
result for each ligand was considered for analysis of the
ligand–receptor interactions and subsequent molecular
modelling simulations.
percentage of incorporated
[
32P]. The concentration of
compound that provided 50% inhibition of enzymatic activity
(IC50) was determined by semi-logarithmic dose–response plots
(Graph Pad Prism 5.0 for Windows, Graph Pad Soware Inc.,
San Diego, California, USA, 2007).
HDAC1 inhibition assay
In vitro HDAC1 inhibition was performed using HDAC1 Fluo-
rimetric Drug Discovery Assay Kit from Enzo Life Sciences, Inc.
The reaction was prepared in HDAC assay buffer (50 mM Tris–
HCl, pH ¼ 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg
mLꢂ1 BSA). HDAC1 enzyme (0.1 mg per well) was incubated at 37
ꢁC with 5 mM uorogenic substrate at indicated concentrations
of inhibitor diluted in 5% DMSO. Reactions were stopped aer
60 min with the developer, and the plate was incubated at RT for
45 min, followed by measuring the uorescence (Ex. 360 nm,
Em. 460 nm, Synergy Mx, BioTek).
For the MD simulations the charge distribution for the
ligands studied was obtained by tting the quantum
mechanically calculated (RHF/6-31G**//RHF/3-21G*) molec-
ular electrostatic potential (MEP) of the geometry-optimized
molecules to a point charge model, as implemented in
Gaussian 03 (Gaussian, Inc., Wallingford, CT). The general
AMBER (http://ambermd.org/) force eld (GAFF) was used to
assign bonded and nonbonded parameters (parm03) to the
In vitro studies on mammalian cell lines
ligand atoms. Each HDAC1 molecular system was immersed in Human T-leukemia cells of Jurkat line and human breast
a truncated octahedron containing ꢄ10 000 TIP3P water adenocarcinoma cells of MCF-7 line were obtained from cell
molecules82 and 4 Clꢂ ions,83 while each CK2 system was culture collection of Institute of Cancer Research, Vienna
immersed in a truncated octahedron containing ꢄ13 000 Medical University (Vienna, Austria). Human keratinocytes of
TIP3P water molecules and 9 Clꢂ ions to achieve system elec- HaCat line were obtained from Ludwig Institute for Cancer
troneutrality. The sander and pmemd modules of the Research (Uppsala, Sweden). Murine leukemia cells of L1210
AMBER12 suite (http://ambermd.org/) were used for the line and human embryonic kidney cells of 293T line were ob-
restrained and unrestrained MD simulations, respectively. tained from cell culture collection of R.E. Kavetsky Institute of
Periodic boundary conditions were applied and electrostatic Experimental Pathology, Oncology and Radiobiology, National
interactions were treated using the smooth particle mesh Academy of Sciences of Ukraine (Kyiv, Ukraine). Cells were
Ewald method84 with a grid spacing of 1 A. The cutoff distance cultured in the RPMI medium supplemented with 10% fetal calf
ꢀ
for the non-bonded interactions was 9 A, the SHAKE algo- serum (Sigma Chemical Co., St. Louis, USA), 50 mg mLꢂ1
85
ꢀ
rithm was applied to all bonds, and an integration step of 2.0 streptomycin (Sigma Chemical Co., St. Louis, USA), 50 units per
fs was used throughout. Aer an initial energy minimization of mL penicillin (Sigma Chemical Co., St. Louis, USA) in 5% CO2-
ꢁ
the water molecules and counter-ions, the system was heated containing humidied atmosphere at 37 C.
to 300 K in 25 ps aer which the solvent was allowed to
For experiments, cells were seeded into 24-well tissue culture
redistribute around the positionally restrained solute for 220 plates (Greiner Bio-one, Germany). Stock solutions (2 mM) of
ps. Aer this time, they were allowed to move freely so as to each CK2 inhibitor in DMSO (99.5% pure, Sigma, USA) were
explore the mutual adaptation between ligand and the protein. prepared, and additionally dissolved in serum-free culture
Snapshots from each 10 ns MD trajectory were collected every medium (RPMI for leukemia cells and DMEM for carcinoma
20 ps for further analysis carried out with the ptraj module of cells) prior to addition to cell culture. Final concentration of
AMBER to monitor the stability of the complexes.
DMSO in cell culture was 0.5% or less. Cytotoxicity studies on
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Jurkat and MCF-7 cells revealed no statistically signicant point for further development of antitumor drugs targeting two
toxicity of 0.5% DMSO solution on these cell lines. relevant enzymes involved in the same signaling pathway, as an
Cytotoxic effect of antitumor drugs was studied under the attractive alternative to classical combination therapy.
light microscope (Evolution 300, Delta Optical, Poland) aer
cell staining with trypan blue dye (0.1%).
Acknowledgements
FITC-conjugated Annexin V (BD Pharmingen, USA) and
propidum iodide (Sigma, USA) double staining were performed This work was supported by the Spanish Ministry of Science and
to detect early apoptotic events under treatment of Jurkat cels by Innovation (CTQ2011-24741 and CTQ2014-52604 R). We thank
CK2-HDAC inhibitors. In 24 h aer the addition of 5c and 5e Airbus Military for fellowships to M. P. Grants to J. H. T., C. C.
´
Jurkat cells were centrifuged at 2000 rpm, washed twice with 1ꢃ and K. F. from Fundacion Universitaria San Pablo CEU are also
PBS, and incubated for 15 min in Annexin V binding buffer (BD acknowledged. We thank also Synthelia Organics SL. for HPLC-
Pharmingen, USA) containing 1/20 volume of FITC-conjugated MS experiments.
Annexin V solution and PI (20 PI 20 of FITC-conjugated
training 1x PBS, and incubated for 15 min in Annexin V. Cyto-
morphological investigations were performed on Zeiss Axi-
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